- Email: [email protected]
MMP2 Axis
Accepted Manuscript LncRNA ODRUL contributes to osteosarcoma progression through the miR-3182/ MMP2 axis Kun-Peng Zhu, Xiao-Long Ma, Chun-Lin Zhang PII:
S1525-0016(17)30311-8
DOI:
10.1016/j.ymthe.2017.06.027
Reference:
YMTHE 4410
To appear in:
Molecular Therapy
Received Date: 13 April 2017 Revised Date:
27 May 2017
Accepted Date: 29 June 2017
Please cite this article as: Zhu K-P, Ma X-L, Zhang C-L, LncRNA ODRUL contributes to osteosarcoma progression through the miR-3182/MMP2 axis, Molecular Therapy (2017), doi: 10.1016/ j.ymthe.2017.06.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
LncRNA ODRUL contributes to osteosarcoma progression through the miR-3182/MMP2 axis Kun-Peng Zhu1,2, Xiao-Long Ma1,2, Chun-Lin Zhang1,2 *
Tongji University, Shanghai 200072, PR China
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1. Department of Orthopaedic Surgery, Shanghai Tenth People’s Hospital Affiliated to
2.Institute of Bone Tumor Affiliated to Tongji University School of Medicine,
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Shanghai 200072, PR China
Kun-Peng Zhu, Xiao-Long Ma, and Chun-Lin Zhang contributed equally to this study and share first authorship. These authors are considered Co-first authors. *
Correspondent author: Chun-Lin Zhang, MD, Department of OrthopaedicSurgery,
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Shanghai Tenth People’s Hospital Affiliated to Tong ji University, 301, Yan-chang Middle Road, Shanghai 200072, China. E-mail:[email protected]. Fax:
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+86 13761904091.
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Running title: LncRNA ODRUL/ miR-3182/MMP2 axis in OS
1
ACCEPTED MANUSCRIPT Abstract Recent findings have shown that lncRNA dysregulation is involved in many cancers, including osteosarcoma (OS). In a previous study, we reported a novel lncRNA,
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ODRUL, that could promote doxorubicin resistance in OS. We now report the function and underlying mechanism of ODRUL in regulating OS progression. We show that ODRUL is up-regulated in OS tissues and cell lines, and correlates with
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poor prognosis. ODRUL knockdown significantly inhibits OS cell proliferation,
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migration, invasion and tumor growth in vitro and vivo by decreasing MMPs expression. A microarray screen combined with online database analysis showed that miR-3182 is up-regulated and MMP2 is down-regulated in sh-ODRUL-expressing MG63 cells and that miR-3182 harbors potential binding sites for ODRUL and the
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3′UTR of MMP2 mRNA. In addition, miR-3182 expression and function are inversely correlated with ODRUL expression in vitro and vivo. A luciferase reporter assay demonstrated that ODRUL could directly interact with miR-3182 and
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up-regulate MMP2 expression via its competing endogenous RNA activity on
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miR-3182 at the posttranscriptional level. Taken together, our study has elucidated the role of oncogenic lncRNA ODRUL in OS progression and may provide a new target in OS therapy.
Keywords: Osteosarcoma, LncRNA ODRUL, MiR-3182.
2
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Introduction Osteosarcoma (OS) is the most common primary malignant bone cancer in
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children and adolescents; it is highly aggressive and readily metastasizes to the lung at the first stage of diagnosis.1-2 Although great improvements in therapeutic strategies including radiotherapy, adjuvant chemotherapy, and wide tumor excision have been
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achieved, the overall prognosis remains poor for most patients with tumor recurrence
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or metastases.3 Thus, there is a need to identify the molecular mechanisms underlying osteosarcoma tumorigenesis and progression, and to discover specific biomarkers and therapeutic targets for osteosarcoma.4
Long noncoding RNAs (lncRNAs) are a novel class of RNA transcript of more
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than 200 nucleotides in length that lack protein coding potential.5 In recent years, accumulating evidence has demonstrated that lncRNAs are dys-regulated in many disease states, particularly in tumors, and play critical roles in the regulation of
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various pathophysiological processes, such as cell proliferation, apoptosis, necrosis,
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autophagy, and so forth.6-9 Recently, several lncRNAs have been reported to be involved in the OS progression. Some classical lncRNAs previously reported in various cancers such as CCAL10, HULC11-13, UCA114-15, HOTTIP16-17, HNF1A-AS118 were also shown to be up-regulated in OS, promote OS progression and correlate with poor prognosis of OS patients. However, the specific lncRNAs involved in OS pathogenesis and progression have not been clearly identified. In this study, we focused on the function and regulatory mechanism of a novel 3
ACCEPTED MANUSCRIPT lncRNA in osteosarcoma. In a previous study, we identified an osteosarcoma doxorubicin
resistance-related
up-regulated
lncRNA
(ODRUL),
through
a
high–throughput microarray screen19 and found that lncRNA ODRUL might act as a
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pro-doxorubicin-resistant molecule by inducing ABCB1 gene expression in osteosarcoma cells through siRNA meditated knockdown of its expression20. However, the roles of lncRNA ODRUL in OS progression remain unclearly defined. Based on
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the previous result, we further determined the vital role of lncRNA ODRUL in the
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proliferation, migration, and invasion of OS in vitro and in vivo. And we found that lncRNA ODRUL could act as a ceRNA sponge for miR-3182 to further promote OS progression through up-regulating MMP2 and lncRNA ODRUL may be a therapeutic target of OS.
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Results
LncRNA ODRUL was up-regulated in the osteosarcoma tissues and cell lines, correlated with lung metastasis and worse prognosis
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To test whether ODRUL play an important role in OS carcinogenesis, we first
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measured the expression level of ODRUL in five human OS cell lines and the normal osteoblast cell line hFOB1.19 by qRT-PCR. The results showed that ODRUL was significantly up-regulated in osteosarcoma cells compared with hFOB1.19 cells (Fig.1A). Of them, MG63 and 143B cells with a higher expression of ODRUL were chosen for further experiments.Then, ODRUL expression level was investigated in 80 pairs of OS and paracancerous tissues and the results showed that ODRUL was significantly up-regulated in OS tissues compared with normal tissues (Fig.1B) and 4
ACCEPTED MANUSCRIPT higher expression of ODRUL in the lung metastasis group at early stage than that in the lung non-metastasis group (Fig. 1C). We also found that patients with higher expression of ODRUL had shorter overall survival time than those with lower
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expression of ODRUL (Fig. 1D). ODRUL promoted OS cell proliferation, migration, invasion and tumor growth in vitro and vivo
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We further analyzed the effect of ODRUL on the proliferation, migration and
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invasion of MG63 and 143B cells. The stably transfected cell lines with overexpression or knockdown of ODRUL were established in MG63 and 143B cells (Fig.2A). Cell proliferation was measured by using CCK-8 and cell clone formation assay. Results showed that cell proliferation rate and colony formation ability in the
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ODRUL group were significantly higher compared with the ODRUL-NC group, whereas cell proliferation rate and colony formation ability in the sh-ODRUL group were obviously lower than that in the sh-NC group (Fig.2B and C). Meanwhile,
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transwell and wound healing assay demonstrated that the numbers of migrating and
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invading cells in the ODRUL group were significantly increased compared with the control group, whereas migration and invasion were significantly decreased in the sh-ODRUL group compared with the control group (Fig.2D and E). Then, the effects of ODRUL overexpression or knockdown on the tumor growth in vivo were further analyzed in tumor-bearing nude mice. MG63 cells transfected with ODRUL, ODRUL -NC, sh-ODRUL, or sh-NC were subcutaneously injected into BALB/c athymic nude mice, respectively. As was shown in the figure 2F, from 2th to 7th week, it is obvious 5
ACCEPTED MANUSCRIPT that tumors formed in the ODRUL group grew much faster compared with the ODRUL -NC group and the volumes of transplanted tumors and weights of nude mice were smaller in the sh-ODRUL group when compared with the sh-NC group
ODRUL
was
predominantly
localized
in
the
posttranscriptional expression of miR-3182 and MMP2
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(Fig.2G). cytoplasm
and
regulated
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To examine the subcellular localization of ODRUL, Cy3-labeled probes specific
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for ODRUL were used for RNA-FISH. This analysis confirmed ODRUL-specific staining was observed in the cytoplasm of MG63 and 143B cells, whereas nearly no staining was observed in the nucleus (Fig. 3A). Then qPCR of nuclear and cytoplasmic fractions of the two cells further validated that ODRUL was mainly
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located in the cytoplasm (Fig.3B), indicating its role of regulating the gene expression at the posttranscriptional level. Since MMPs are well known to be involved in migration and invasion of OS cells, we asked whether lncRNA ODRUL promoted OS
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cell migration and invasion through regulating the expression of MMPs. We then
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assessed the MMP2 and MMP expression by qPCR and WB in the MG63 and 143B cells with knockdown or overexpression of ODRUL. Interestingly, we found that the expression of MMP2 and MMP9 was inversely correlated with ODRUL (Fig.3C and D). These data suggested that ODRUL could positively regulate posttranscriptional expression of MMP2 and MMP9 in OS cells (data of MMP9 was shown in the Figure S1). Using bioinformatics databases (miRanda, starBase v2.0, DIANA-LncBase v2.0), 6
ACCEPTED MANUSCRIPT miRNAs that may both interact with ODRUL and the 3′UTR of MMP2 or MMP9 were predicted. According to the summarized analysis results in the three databases(data shown in the Table S1), we found that there were nine miRNAs hsa-miR-3127-3p,
hsa-miR-4498,
hsa-miR-3182,
hsa-miR-5001-5p,
hsa-miR-3202,
hsa-miR-580-3p,
hsa-miR-4446-3p,
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including
hsa-miR-6132,
and
hsa-miR-6836-5p, who had potential binding sites for lncRNA ODRUL and the
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3′UTR of MMP2 mRNA, whereas, there was only has-miR-4773 predicted to hold the
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potential binding sites for lncRNA ODRUL and the 3′UTR of MMP9 mRNA. Furthermore, miRNA and mRNA microarrays were used to screen differentially expressed miRNAs and mRNAs associated with ODRUL in the paired sh-ODRUL and sh-NC MG63 cells. Further bioinformatics analysis showed that the ten
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previously predicted miRNAs and MMP2 or MMP9 were all found in the screening differentially expressed miRNAs and mRNAs, of which the up-regulated hsa-miR-3182 had the fold-change of ten, the down-regulated MMP2 of five and
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MM9 of four, respectively (Fig.3E). qRT-PCR was then performed to study the
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interaction between the ten miRNAs expression levels in MG63 and 143B cells with ODRUL overexpression or knockdown. Our results revealed that miR-3182 was the most influenced and consistently negative with the expression of ODRUL, indicating that competing regulation relationship between the ODRUL and miR-3182 expression (Fig. 3F). Meanwhile, miR-3182 could also negatively regulate the mRNA expression of lncRNA ODRUL. ODRUL expression in the miR-3182-mimics group distinctly increased and in the miR-3182-inhibitor group decreased relative to the corresponding 7
ACCEPTED MANUSCRIPT NC group (Fig.3G). Further bioinformatics analysis showed the miRNA response element (MRE) between the sequence of ODRUL and miR-3182 (Fig.3H). Then we further found that miR-3182 could also negatively regulate the mRNA and protein
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expression of MMP2, verified by the results that inhibition of miR-3182 up-regulated and overexpression of miR-3182 down-regulated the expression of MMP2 (Fig.3I and J).
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ODRUL was directly targeted by miR-3182, further regulating the expression of
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MMP2 through competitively binding with miR-3182
The mRNA and protein expression of MMP2 in the group of ODRUL combined with miR-3182-inhibitor significantly increased and MMP2 expression in the sh-ODRUL combined with miR-3182-mimics group distinctly inhibited when
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compared with the corresponding NC group, which revealed that the expression of MMP2 was mutually suppressed by ODRUL and miR-3182 (Fig.4A and B). Besides, as was shown in Figure 4C, co-transfection of miR-3182 mimic and ODRUL
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expression vector showed that miR-3182 remarkably reduced cell invasion promoted
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by ODRUL. Meanwhile, co-transfection of miR-3182 inhibitor and sh-ODRUL showed that the miR-3182 remarkably rescued the invasive ability of MG63 and 143B cells inhibited by the knockdown of ODRUL. However, co-transfection of miR-3182 mimic and sh-ODRUL showed that the numbers of invasive cells significantly decreased compared with the only miR-3182 mimic or sh-ODRUL transfection and co-transfection of miR-3182 inhibitor and ODRUL expression vector had the opposite effects, which may demonstrate the antagonism role of regulation in the OS cell 8
ACCEPTED MANUSCRIPT invasion between ODRUL and miR-3182. Dual luciferase reporter assay was further conducted to validate the direct binding of lncRNA ODRUL and 3′UTR of MMP2 mRNA with miR-3182. The results
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demonstrated that the miR-3182 mimics remarkably reduced, but the miR-3182 inhibitor increased luciferase activities of the reporter plasmid containing the potential binding sequence of 3′UTR of MMP2 mRNA or lncRNA ODRUL (wild type, WT),
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but without obvious changes in the reporter plasmid containing mutated sequence
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(mutant type, MUT) (Fig.4D and E). Moreover, co-transfection of lncRNA ODRUL could rescue the decreased luciferase activity of WT-MMP2 treated with miR-3182 mimics (Fig.4F). On the contrary, the luciferase activities of the WT-MMP2 were enhanced by miR-3182 inhibition, which could be reversed by sh-ODRUL
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respectively (Fig.4G). These data illustrated that lncRNA ODRUL directly regulated MMP2 expression through competitively binding with miR-3182 as a miRNA sponge. Mir-3182 expression was inversely correlated with ODRUL and poor prognosis
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Based on above findings, miR-3182 may also play a role in OS carcinogenesis.
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We then tested miR-3182 level in OS cell lines and tissues and found that miR-3182 was significantly down-regulated in five osteosarcoma cell lines, compared with the normal osteoblast cell line (Fig.5A) and dramatically reduced in OS tissues compared with the matched paracancerous tissues (Fig.5B). A negative correlation was demonstrated in expression of miR-3182 and lncRNA ODRUL in OS cell lines and tissues, which further implied the endogenous competing relationship between them (Fig.5C and D). Subsequently, the expression of miR-3182 in the lung metastasis 9
ACCEPTED MANUSCRIPT group at early stage was obviously lower than that in the lung non-metastasis group (Fig.5E) and the patients with higher expression of miR-3182 had longer overall survival time than those with lower expression (Fig.5F). These results indicated a
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potential tumor-suppressing role of miR-3182 in the development of the OS, contrary to the role of lncRNA ODRUL.
Mir-3182 suppressed OS cell proliferation, migration, invasion and tumor growth in
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vitro and vivo
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MG63 and 143B cells stably transfected with miR-3182 mimics or inhibitor were established. CCK-8 and cell clone formation assay showed that the cell proliferation rate
and
colony
formation
ability
were
significantly
increased
in
the
miR-3182-inhibitor group but were significantly decreased in the miR-3182-mimics
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group, when compared to the NC group (Fig.6A and B).Meanwhile, transwell and wound healing assay demonstrated that the numbers of migrating and invading cells in the miR-3182-inhibitor group were significantly increased compared with the
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inhibitor-NC group, whereas migration and invasion were significantly attenuated in
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the miR-3182-mimics group (Fig. 6C and D). Besides, overexpression of miR-3182 in the MG63 cells and the NC were subcutaneously injected into BALB/c athymic nude mice respectively. Consistent with the results in vitro, the tumor volumes and nude mice weights of the miR-3182 overexpression group of MG63 cells were significantly smaller than that of the control group, which conformed to the in vivo imaging analysis (Fig.6E and F).. Discussion 10
ACCEPTED MANUSCRIPT Recent studies have revealed that lncRNAs could participate in the initiation and progression of various cancers[8], including breast cancer, gastric cancer, bladder cancer, osteosarcoma, and so on[21-22]. Understanding the molecular mechanism of
osteosarcoma
[23-24]
progression of OS
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lncRNAs may help to explore new promising therapeutic strategy for the treatment of . Actually, several lncRNAs have been reported to involve in the [25-27]
. For example, Liu SH
[28]
found that a novel antisense
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lncRNA SATB2-AS1 overexpresses in OS and increases cell proliferation and growth [26]
reported that lncRNA
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through affecting its conjugate gene, SATB2. Sun L
EWSAT1 promotes OS cell growth and metastasis through suppression of MEG3 expression. Chen F [27] found that lncRNA BCAR4 promotes OS progression through activating GLI2-dependent gene transcription.
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In the present study, we found that a novel lncRNA, ODRUL previously reported by us, was overexpressed in osteosarcoma tissues and cell lines. OS patients with high ODRUL expression showed worse prognosis when compared with those with low
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ODRUL expression, and ODRUL expression was an independent prognostic factor of
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OS patients with significant clinical meaning. The function of ODRUL was subsequently investigated in our study. Our data indicated that knockdown of ODRUL inhibited and overexpression of it promoted OS cell proliferation, migration and invasion, tumor growth both in vitro and vivo. Furthermore, we found that ODRUL regulated migration and invasion of OS cells through up-regulating the expression of matrix metalloproteinase II (MMP2), a key proteinase during cancer invasion. These results suggested that lncRNA ODRUL functions as an oncogene and plays a critical 11
ACCEPTED MANUSCRIPT role in OS progression. LncRNAs have been shown to widely regulate the gene expression at different kinds of levels, such as pre-transcription, transcription and post-transcription, which [29-31]
. LncRNAs located in the nuclei always
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mainly depend on its cellular location
play role in the level of pre-transcription or transcription[32], while cytoplasmic lncRNAs often function as a competing endogenous RNA and sponge miRNAs, thus
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regulating the expression of target mRNA at the post-transcription level[33]. In the
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study, we identified that ODRUL mainly located in the cytoplasm through RNA FISH and cell cytoplasm/nucleus fraction isolation assay in the qualitative and quantitative aspects, which may suggest ODRUL could exert its regulatory role at the post-transcription level. Then competing endogenous RNAs (ceRNA) mechanism was
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first considered.
For this posttranscriptional regulatory mechanism, lncRNAs should have miRNA responsive elements (MRE) and act as miRNA sponges to control endogenous
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miRNAs available for binding with their target mRNAs, thus reducing the repression
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of these mRNAs[34]. Actually, many lncRNAs have been shown to play role in the tumorgenesis and progression by interfering with the miRNA pathways as ceRNAs. For example, Guo G et al
[35]
found that lncRNA-BGL3 regulates Bcr-Abl-mediated
cellular transformation by acting as a competitive endogenous RNA. Liu D et al
[36]
reported that lncRNA SPRY4-IT1 sponges miR-101-3p to promote proliferation and metastasis of bladder cancer cells through up-regulating EZH2. Sun C et al
[37]
reported that lncRNA NEAT1 promotes non-small cell lung cancer progression 12
ACCEPTED MANUSCRIPT through regulation of miR-377-3p-E2F3 pathway. Besides, Zhou Q et al [38] reported that lncRNA PVT1 promotes osteosarcoma development by acting as a molecular sponge to regulate miR-195, further influencing the expression of the downstream
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genes, like BCL2, CCND1, and FASN. In our present study, we first found that lncRNA ODRUL could promote the OS cell migration and invasion through inducing the expression of MMP2 and MMP9.
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Further microarray and bioinformatics databases analysis were conducted to search
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the potential miRNAs that have potential binding sites between the lncRNA ODRUL and 3’UTR of MMP2 or MMP9 mRNA. Only one miRNA (has-miR-4773) was predicted to be possible to play role between lncRNA ODRUL and MMP9, but further expression validation and dual luciferase reporter gene assay defined the
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pathway(data not shown). However, nine miRNAs were predicted between ODRUL and MMP2 and one of them, miR-3182 was further demonstrated to directly target ODRUL and MMP2 in MG63 and 143B cells.
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MicroRNA-3182 was previously reported to be specifically sorafenib-induced in [39]
. However, there is
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colorectal cancer cells in response to sorafenib treatment
seldom report about miR-3182 in osteosarcoma. The present study displayed that miR-3182 was significantly decreased in OS tissues and cell lines and negatively correlated with the expression of ODRUL. MiR-3182 also could act as an independent prognostic factor of OS patients, with a longer survival time of higher expression. Furthermore, functional assay found that miR-3182 inhibited proliferation, migration and invasion of OS cells and tumor growth both in vitro and vivo. Besides, 13
ACCEPTED MANUSCRIPT knockdown of miR-3182 could rescue the effect of sh-ODRUL on OS cell invasion and the expression of MMP2. Meanwhile, overexpression of miR-3182 also could rescue the effect of overexpression ODRUL on cell invasion and MMP2, which may
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demonstrate the antagonism role of regulation in the OS cell invasion and MMP2 expression between ODRUL and miR-3182 .In addition, the direct binding of lncRNA ODRUL and 3′UTR of MMP2 mRNA with miR-3182 was further validated by dual
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luciferase reporter assay. Taken together, these data revealed that lncRNA ODRUL
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could effectively sponge miR-3182 to promote osteosarcoma progression through up-regulating MMP2.
In conclusion, we identify that highly expressed ODRUL is an oncogenic lncRNA that exerts a crucial role in the OS progression. Besides, our study sheds light
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on the role of lncRNA ODRUL/ miR-3182/MMP2 pathway in OS for the first time, and reveals that lncRNA ODRUL could sponge miR-3182 to promote osteosarcoma progression through up-regulating MMP2, thus probably providing a novel
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therapeutic target in OS.
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Method and Materials
Cell lines and culture conditions SaoS2, HOS, U2-OS, MG63 and 143B human osteosarcoma cell lines (American Type Culture Collection) were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco, Gran Island, NY, USA), 100 U/mL of penicillin and 100 µg/mL of streptomycin (Invitrogen). Normal osteoblast cells (hFOB1.19) obtained from the Chinese Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were 14
ACCEPTED MANUSCRIPT cultured in Ham’s F12/ DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. Clinical samples
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A total of 80 primary osteosarcoma patients who received the same chemotherapy regimen before surgery and underwent complete resection surgery at Shanghai Tenth Hospital between 2006 and 2015 were included in this study. The clinical parameters
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PCR assays and western blotting analysis
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of osteosarcoma patients in this study are presented in Table 1.
Total RNA was extracted from tissues and cells with Trizol reagent (TAKARA) according to the product description. All mRNAs and miRNAs were reverse transcribed according to the protocol of the PrimeScript® RT Master Mix Perfect
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Real Time (TAKARA). The primers were shown in Table S1.
Cells were collected and lysed using RIPA protein extraction reagent (Beyotime, Beijing, China) supplemented with a protease inhibitor cocktail (Roche, Pleasanton,
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control.
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CA,USA).Autoradiograms were quantified by densitometry using GAPDH as a
Plasmid construction and cell transfection MG63 and 143B cells were transiently transfected with shRNAs after being sowed into the 6-well plates overnight. A scrambled negative control, a plasmid overexpressing ODRUL, and an empty vector, were cultured as well using the Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA) and FuGENE® HD Transfection Reagent (Roche, Germany) according to the manufacturer’s 15
ACCEPTED MANUSCRIPT instructions, respectively. 48h after transfection, the cells were harvested to detect the overexpression or knockout efficiency via qRT-PCR. Two different shRNAs against ODRUL were designed and synthesized by GenePharma (Shanghai, China). The
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target sequences for the sh-ODRUL included: sh-ODRUL-1 and sh-ODRUL-2 with the former having the highest inhibition efficiency (sh-ODRUL mentioned in the article refers to sh-ODRUL-1). The synthetic ODRUL sequence (319bp) was
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designed sequences were shown in the Table S2.
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sub-cloned into the pEGFP-N1 plasmid vector followed by a sequencing analysis. The
CCK-8 assay
Cells were incubated in 10 % CCK-8 diluted in normal culture medium at 37 °C until visual color conversion occurred. Proliferation rates were determined at 24, 48, 72h
set at 570nM.
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after transfection. The absorbance of each well was measured with a microplate reader
Colony formation assay
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Cells were seeded in 6-well plates and were incubated for 24 h. The colonies were
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stained with crystal violet solution 14 days later. The colony number in each well was counted and calculated. Wound healing assay and cell invasion assay A total of stable transfected cells were seeded onto six-well plates and cultured overnight. Wounds were created by scratching cell layer with a sterile plastic pipette tips and washed with culture medium. Cells were further cultured with medium containing 1% FBS in 48h. 16
ACCEPTED MANUSCRIPT For the invasion assays, a 24-well transwell chamber with the upper chamber coated with Matrigel (BD Bioscience) was used. 1.0×105 cells in 100uL serum free DMEM medium were seeded in the top chamber, 500uL medium containing 10% FBS was
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placed to the lower chamber. After incubation for 48 h, cells on the upper membrane surface were wiped off using a cotton swab and the cells that had traversed the membrane were staining by crystal violet and counted.
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Xenograft transplantation
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Female nude (BALB/c) mice (4 weeks old) were purchased. Mice were divided into several groups according to the completely randomized method. MG63 cells stably expressing sh-ODRUL or NC were propagated and 1×107 cells were inoculated subcutaneously into the right side of the posterior flank of mice. Tumor growth was
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examined at the indicated time points and tumor volumes were measured. After 7 weeks, the mice were killed and tumors were removed and weighed. Intratumoral injection of Ad-sh-ODRUL (Ad-ODRUL) (2×109plaque-forming units [PFU]) or
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Ad-sh-NC was performed. Mice were photographed at the indicated times with
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anIVIS@ Lumina II system (Caliper Life Sciences, Hopkinton, MA). RNA-fluorescence in situ hybridization (FISH) Cy3-labeled ODRUL and DAPI-labeled U6 probes were obtained from Genepharma (Shanghai, China). RNA FISH were performed using fluorescent in situ hybridization kit according to the manufacturer's protocol (Thermo Fisher). Cell cytoplasm/nucleus fraction isolation Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher) were employed to 17
ACCEPTED MANUSCRIPT prepare cytoplasmic and nuclear extracts. RNAs extracted from each of the fractions were subjected to following RT–qPCR analysis to demonstrate the levels of nuclear control transcript (β-actin), cytoplasmic control transcript (U6), and ODRUL.
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Dual luciferase reporter assay MG63 cells were seeded at 3× 104 cells/well in 24-well plates and allowed to settle overnight. The next day, cells were co-transfected with pmirGLO-ODRUL-WT or
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-MUT reporter plasmids and miR-3182 mimic or inhibitor. 24h after transfection, the
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relative luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) and normalized against Renilla luciferase activity. Statistical analysis
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All statistical analyses were performed using SPSS 22.0 software (IBM). Data are presented as mean ± SEM .Differences between groups were analyzed using the Student’s t test or one-way ANOVA .Overall survival was calculated by Kaplan-Meier
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survival analysis and compared using the log-rank test. p values ＜ 0.05 were
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considered statistically significant. Conflicts of Interests: We declare that we have no conflicts of interest. Acknowledgments: This project was supported by a Grant from the National Natural Science Foundation of China (No.81572630), Shanghai Pujiang Program of Shanghai Science and Technology Commission (NO.13PJD023) and Shanghai Jiaotong University Medical-Engineering Cross Research Fund (NO.YG2012MS49). Author Contributions: ZKP and MXL carried out the molecular genetic studies. 18
ACCEPTED MANUSCRIPT MXL carried out the tumor-bearing nude mice assays. ZKP and ZCL participated in the design of the study and performed the statistical analysis. ZKP drafted the manuscript and ZCL helped to correct it.
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13. Yu X, Zheng H, Chan MT, Wu WK(2017). HULC: an oncogenic long non-coding RNA in human cancer. J Cell Mol Med 21:410-417. 14. Wen JJ, Ma YD, Yang GS, Wang GM(2017). Analysis of circulating long non-coding RNA UCA1 as potential biomarkers for diagnosis and prognosis of osteosarcoma. European review for medical and pharmacological sciences 21:498-503. 15. Li W, Xie P, Ruan WH(2016). Overexpression of lncRNA UCA1 promotes 20
ACCEPTED MANUSCRIPT osteosarcoma progression and correlates with poor prognosis. Journal of bone oncology 5:80-5. 16. Li Z, Zhao L, Wang Q(2016). Overexpression of long non-coding RNA HOTTIP
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17. Li F, Cao L, Hang D, Wang F, Wang Q(2015). Long non-coding RNA HOTTIP is
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up-regulated and associated with poor prognosis in patients with osteosarcoma.
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International journal of clinical and experimental pathology 8:11414-20. 18. Zhao H, Hou W, Tao J, Zhao Y, Wan G, Ma C, et al. (2016). Upregulation of lncRNA HNF1A-AS1 promotes cell proliferation and metastasis in osteosarcoma through activation of the Wnt/beta-catenin signaling pathway. American journal of
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19. Zhu KP, Zhang CL, Shen GQ, Zhu ZS(2015). Long noncoding RNA expression profiles of the doxorubicin-resistant human osteosarcoma cell line MG63/DXR and its
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parental cell line MG63 as ascertained by microarray analysis. International journal
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of clinical and experimental pathology 8:8754-73. 20. Zhang CL, Zhu KP, Shen GQ, Zhu ZS(2016). A long non-coding RNA contributes to doxorubicin resistance of osteosarcoma. Tumour biology: the journal of the International Society for Oncodevelopmental Biology and Medicine 37:2737-48. 21. Martens-Uzunova ES, Bottcher R, Croce CM, Jenster G, Visakorpi T, Calin GA(2014). Long noncoding RNA in prostate, bladder, and kidney cancer. European urology 65:1140-51. 21
ACCEPTED MANUSCRIPT 22. Gan L, Xu M, Zhang Y, Zhang X, Guo W(2015). Focusing on long noncoding RNA dysregulation in gastric cancer. Tumour biology: the journal of the International Society for Oncodevelopmental Biology and Medicine 36:129-41.
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23. Li Z, Yu X, Shen J(2016). Long non-coding RNAs: emerging players in osteosarcoma. Tumour biology: the journal of the International Society for Oncodevelopmental Biology and Medicine 37:2811-6.
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24. Chen R, Wang G, Zheng Y, Hua Y, Cai Z(2017). Long non-coding RNAs in
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osteosarcoma. Oncotarget . doi: 10.18632/oncotarget.14726.
25. Wang B, Su Y, Yang Q, Lv D, Zhang W, Tang K, et al. (2015). Overexpression of Long Non-Coding RNA HOTAIR Promotes Tumor Growth and Metastasis in Human Osteosarcoma. Molecules and cells 38:432-40.
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26. Sun L, Yang C, Xu J, Feng Y, Wang L, Cui T(2016). Long Noncoding RNA EWSAT1 Promotes Osteosarcoma Cell Growth and Metastasis Through Suppression of MEG3 Expression. DNA and cell biology 35:812-8.
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27. Chen F, Mo J, Zhang L(2016). Long noncoding RNA BCAR4 promotes
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osteosarcoma progression through activating GLI2-dependent gene transcription. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 37:13403-12. 28. Liu SH, Zhu JW, Xu HH, Zhang GQ, Wang Y, Liu YM, et al. (2017). A novel antisense long non-coding RNA SATB2-AS1 overexpresses in osteosarcoma and increases cell proliferation and growth. Molecular and cellular biochemistry. doi: 10.1007/s11010-017-2953-9. 22
ACCEPTED MANUSCRIPT 29. Ma L, Bajic VB, Zhang Z(2013). On the classification of long non-coding RNAs. RNA biology 10:925-33. 30. Kurokawa R(2011). Long noncoding RNA as a regulator for transcription.
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Progress in molecular and subcellular biology 51:29-41. 31. Bonasio R, Shiekhattar R(2014). Regulation of transcription by long noncoding RNAs. Annual review of genetics 48:433-55.
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32. Shafik A, Schumann U, Evers M, Sibbritt T, Preiss T(2016). The emerging
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epitranscriptomics of long noncoding RNAs. Biochimica et biophysica acta 1859:59-70.
33. Yoon JH, Abdelmohsen K, Gorospe M(2013). Posttranscriptional gene regulation by long noncoding RNA. Journal of molecular biology 425:3723-30.
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34. Tay Y, Rinn J, Pandolfi PP(2014). The multilayered complexity of ceRNA crosstalk and competition. Nature 505:344-52. 35. Guo G, Kang Q, Zhu X, Chen Q, Wang X, Chen Y, et al. (2015). A long
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noncoding RNA critically regulates Bcr-Abl-mediated cellular transformation by
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acting as a competitive endogenous RNA. Oncogene 34:1768-79. 36. Liu D, Li Y, Luo G, Xiao X, Tao D, Wu X, et al. (2017). LncRNA SPRY4-IT1 sponges miR-101-3p to promote proliferation and metastasis of bladder cancer cells through up-regulating EZH2. Cancer letters 388:281-91. 37. Sun C, Li S, Zhang F, Xi Y, Wang L, Bi Y, et al. (2016). Long non-coding RNA NEAT1 promotes non-small cell lung cancer progression through regulation of miR-377-3p-E2F3 pathway. Oncotarget 7:51784-814. 23
ACCEPTED MANUSCRIPT 38. Zhou Q, Chen F, Zhao J, Li B, Liang Y, Pan W, et al. (2016). Long non-coding RNA PVT1 promotes osteosarcoma development by acting as a molecular sponge to regulate miR-195. Oncotarget 7:82620-33.
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39. Pehserl AM, Ress AL, Stanzer S, Resel M, Karbiener M, Stadelmeyer E, et al. (2016). Comprehensive Analysis of miRNome Alterations in Response to Sorafenib
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Treatment in Colorectal Cancer Cells. Int. J. Mol. Sci.17:2011.
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Fig.1 LncRNA ODRUL was up-regulated in the osteosarcoma tissues and cell lines, correlated with lung metastasis and worse prognosis. (A) Expression level of ODRUL in five human OS cell lines and normal osteoblast cell line hFOB1.19. (B) Expression level of ODRUL in 80 pairs of OS and paracancerous tissues. (C)
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Expression level of ODRUL in OS tissues of lung metastasis and lung non-metastasis group at early stage. (D) OS patients with higher expression of ODRUL had shorter overall survival time than those with lower expression. Data are presented as mean ±
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SEM, *P < 0.05.
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Fig.2 ODRUL promoted OS cell proliferation, migration, invasion and tumor growth in vitro and vivo. (A) RT–qPCR analysis of the effect on knockdown or overexpression of the expression of ODRUL by sh-RNA or vector transfection. (B) CCK-8 assays were performed to examine cell proliferation rate of MG63 and 143B cells after knockdown or overexpression of ODRUL. (C) Clone formation assays were performed to examine cell vitality after transfection. (D) Transwell assays were performed to identify the capacity of cell invasion after transfection. (E) Wound 24
ACCEPTED MANUSCRIPT healing assays were performed to examine the capacity of cell migration after transfection. (F) General conditions and in vivo imaging of nude mice in the four groups when exposed to the same treatment. (G) The nude mice were sacrificed in the
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7th week. Tumors formed in the ODRUL group grew much faster compared with the ODRUL-NC group and the volumes of transplanted tumors and weights of nude mice were smaller in the sh-ODRUL group when compared with the sh-NC group. Data are
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presented as mean ± SEM, *P < 0.05.
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Fig.3 ODRUL was predominantly localized in the cytoplasm and regulated posttranscriptional expression of miR-3182 and MMP2. (A) Subcellular localization of ODRUL by RNA-FISH in the MG63 and 143B cells. Nuclei are stained blue (DAPI) and ODRUL is stained red. (B) Nuclear and cytoplasmic
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fractions assay further validated the subcellular localization of ODRUL. (C) LncRNA ODRUL regulated the mRNA level of MMP2 expression. (D) LncRNA ODRUL regulated the protein level of MMP2 expression. (E)miRNA and mRNA microarrays
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were used to screen differentially expressed miRNAs and mRNAs associated with
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ODRUL in the paired sh-ODRUL and sh-NC MG63 cells. (F) qRT-PCR was performed to study the interaction between the miRNAs expression levels with ODRUL overexpression or knockdown and only miR-3182 was consistently negatively with the expression of ODRUL.(G) qRT-PCR was performed to study the interaction between the ODRUL expression levels with miR-3182 overexpression or knockdown. (H) The miR-3182 response element (MRE) between the sequence of ODRUL and MMP2 by bioinformatics analysis. (I) miR-3182 negatively regulated 25
ACCEPTED MANUSCRIPT the mRNA level of MMP2 expression. (J) miR-3182 negatively regulated the protein level of MMP2 expression. Data are presented as mean ± SEM, *P < 0.05. Fig.4 ODRUL was directly targeted by miR-3182, further regulating the
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expression of MMP2 through competitively binding with miR-3182. (A)The mRNA expression of MMP2 was examined in the MG63 and 143B cells co-transfected with miR-3182 mimics or inhibitor, ODRUL vector or sh-ODRUL, or
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their NCs. (B) The protein expression of MMP2 was examined in the same cells
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previously described.(C) Transwell assays were performed to observe the biological behaviors of OS cells co-transfected with miR-3182 mimics or inhibitor, sh-ODRUL or ODRUL vector and their NCs. (D) WT and MUT sequences designed for the ODRUL and MMP2 according to their binding sites with miR-3182. (E)ODRUL
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luciferase activity assays. Wide type or mutant ODRUL was co-transfected with miR-3182 mimics or miR-3182 inhibitor, respectively. Mir-3182 mimics repressed, but miR-3182 inhibitor enhanced the luciferase activity of the WT-ODRUL reporter
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which including wild type sequence of ODRUL. There was no obvious change of the
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luciferase activity for the MUT-ODRUL reporter which containing mutant ODRUL sequence. (F) MMP2 3′UTR luciferase activity assays. Overexpression of ODRUL rescued the luciferase activity, which repressed by the transfection with miR-3182 mimics in the WT- MMP2 but not in the MUT- MMP2 reporter. (G) Down-regulation of ODRUL with sh-RNA reversed the luciferase activity, which enhanced by the transfection with miR-3182 inhibitor in the WT-MMP2 but not in MUT-MMP2 reporter. The normalized luciferase activity in the control group was set to 1. Data are 26
ACCEPTED MANUSCRIPT presented as mean ± SEM, *P < 0.05. Fig.5 miR-3182 expression was inversely correlated with ODRUL and poor prognosis. (A) Expression level of miR-3182 in five human OS cell lines and normal
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osteoblast cell line hFOB1.19. (B) Expression level of miR-3182 in 80 pairs of OS and paracancerous tissues. (C) The relative expression level of ODRUL and miR-3182 in five human OS cell lines and normal osteoblast cell line hFOB1.19. (D)
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The relative expression level of ODRUL and miR-3182 in 80 pairs of OS and
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paracancerous tissues. (E) Expression level of miR-3182 in OS tissues of lung metastasis and lung non-metastasis group at early stage. (F) OS patients with lower expression of miR-3182 had shorter overall survival time than those with higher expression. Data are presented as mean ± SEM, *P < 0.05.
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Fig.6 miR-3182 suppressed OS cell proliferation, migration, invasion and tumor growth in vitro and vivo. (A) CCK-8 assays were performed to examine cell proliferation rate transfected with miR-3182 mimics or inhibitor. (B) Clone formation
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assays were performed to examine cell vitality transfected with miR-3182 mimics or
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inhibitor. (C) Transwell assays were performed to identify the capacity of cell invasion after miRNA transfection. (D) Wound healing assays were performed to examine the capacity of cell migration after miRNA transfection. (E) General conditions and in vivo imaging of nude mice in the miR-3182 mimics and mimics-NC transfected groups when exposed to the same treatment. (F) The nude mice were sacrificed in the 7th week. Tumors formed in the miR-3182 mimics group grew slower compared with the mimics-NC group and the volumes of transplanted tumors were 27
ACCEPTED MANUSCRIPT smaller in the miR-3182 mimics group when compared with the mimics-NC group. Data are presented as mean ± SEM, *P < 0.05. Fig. S1 ODRUL positively regulated posttranscriptional expression of MMP2. (A)
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LncRNA ODRUL regulated the mRNA level of MMP2 expression. (B) LncRNA ODRUL regulated the protein level of MMP2 expression. Data are presented as mean
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± SEM, *P < 0.05.
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ACCEPTED MANUSCRIPT Table 1. Clinical parameters of osteosarcoma patients enrolled in this study Pathological characteristics
Cases (n)
ODRUL expression
P value
miR-3182 expression
P value
Male
52
12.42 ± 1.28
0.12
3.12 ± 0.28
0.15
Female
28
12.55 ± 1.15
≥25
25
12.74 ± 1.16
<25
55
12.92 ± 1.08
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Gender
3.28 ± 0.11
Age
3.13 ± 0.27
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0.13
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3.16 ± 0.24
0.07
Location
0.09
36
12.25 ± 1.15
3.15 ± 0.24
Proximal of Tibia
28
12.14 ± 1.06
3.16 ± 0.53
Other
16
12.34 ± 1.16
3.23 ± 0.17
48
No
32
23.62 ± 1.18 11.06 ± 0.34
<0.05
1.03 ± 0.17 4.54 ± 0.16
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Yes
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Distal of Femur
Lung Metastasis
0.22
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Data are presented as mean ± SEM.
1
<0.05
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S1525-0016(17)30311-8
DOI:
10.1016/j.ymthe.2017.06.027
Reference:
YMTHE 4410
To appear in:
Molecular Therapy
Received Date: 13 April 2017 Revised Date:
27 May 2017
Accepted Date: 29 June 2017
Please cite this article as: Zhu K-P, Ma X-L, Zhang C-L, LncRNA ODRUL contributes to osteosarcoma progression through the miR-3182/MMP2 axis, Molecular Therapy (2017), doi: 10.1016/ j.ymthe.2017.06.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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LncRNA ODRUL contributes to osteosarcoma progression through the miR-3182/MMP2 axis Kun-Peng Zhu1,2, Xiao-Long Ma1,2, Chun-Lin Zhang1,2 *
Tongji University, Shanghai 200072, PR China
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1. Department of Orthopaedic Surgery, Shanghai Tenth People’s Hospital Affiliated to
2.Institute of Bone Tumor Affiliated to Tongji University School of Medicine,
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Shanghai 200072, PR China
Kun-Peng Zhu, Xiao-Long Ma, and Chun-Lin Zhang contributed equally to this study and share first authorship. These authors are considered Co-first authors. *
Correspondent author: Chun-Lin Zhang, MD, Department of OrthopaedicSurgery,
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Shanghai Tenth People’s Hospital Affiliated to Tong ji University, 301, Yan-chang Middle Road, Shanghai 200072, China. E-mail:[email protected]. Fax:
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+86 13761904091.
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Running title: LncRNA ODRUL/ miR-3182/MMP2 axis in OS
1
ACCEPTED MANUSCRIPT Abstract Recent findings have shown that lncRNA dysregulation is involved in many cancers, including osteosarcoma (OS). In a previous study, we reported a novel lncRNA,
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ODRUL, that could promote doxorubicin resistance in OS. We now report the function and underlying mechanism of ODRUL in regulating OS progression. We show that ODRUL is up-regulated in OS tissues and cell lines, and correlates with
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poor prognosis. ODRUL knockdown significantly inhibits OS cell proliferation,
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migration, invasion and tumor growth in vitro and vivo by decreasing MMPs expression. A microarray screen combined with online database analysis showed that miR-3182 is up-regulated and MMP2 is down-regulated in sh-ODRUL-expressing MG63 cells and that miR-3182 harbors potential binding sites for ODRUL and the
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3′UTR of MMP2 mRNA. In addition, miR-3182 expression and function are inversely correlated with ODRUL expression in vitro and vivo. A luciferase reporter assay demonstrated that ODRUL could directly interact with miR-3182 and
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up-regulate MMP2 expression via its competing endogenous RNA activity on
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miR-3182 at the posttranscriptional level. Taken together, our study has elucidated the role of oncogenic lncRNA ODRUL in OS progression and may provide a new target in OS therapy.
Keywords: Osteosarcoma, LncRNA ODRUL, MiR-3182.
2
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Introduction Osteosarcoma (OS) is the most common primary malignant bone cancer in
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children and adolescents; it is highly aggressive and readily metastasizes to the lung at the first stage of diagnosis.1-2 Although great improvements in therapeutic strategies including radiotherapy, adjuvant chemotherapy, and wide tumor excision have been
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achieved, the overall prognosis remains poor for most patients with tumor recurrence
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or metastases.3 Thus, there is a need to identify the molecular mechanisms underlying osteosarcoma tumorigenesis and progression, and to discover specific biomarkers and therapeutic targets for osteosarcoma.4
Long noncoding RNAs (lncRNAs) are a novel class of RNA transcript of more
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than 200 nucleotides in length that lack protein coding potential.5 In recent years, accumulating evidence has demonstrated that lncRNAs are dys-regulated in many disease states, particularly in tumors, and play critical roles in the regulation of
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various pathophysiological processes, such as cell proliferation, apoptosis, necrosis,
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autophagy, and so forth.6-9 Recently, several lncRNAs have been reported to be involved in the OS progression. Some classical lncRNAs previously reported in various cancers such as CCAL10, HULC11-13, UCA114-15, HOTTIP16-17, HNF1A-AS118 were also shown to be up-regulated in OS, promote OS progression and correlate with poor prognosis of OS patients. However, the specific lncRNAs involved in OS pathogenesis and progression have not been clearly identified. In this study, we focused on the function and regulatory mechanism of a novel 3
ACCEPTED MANUSCRIPT lncRNA in osteosarcoma. In a previous study, we identified an osteosarcoma doxorubicin
resistance-related
up-regulated
lncRNA
(ODRUL),
through
a
high–throughput microarray screen19 and found that lncRNA ODRUL might act as a
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pro-doxorubicin-resistant molecule by inducing ABCB1 gene expression in osteosarcoma cells through siRNA meditated knockdown of its expression20. However, the roles of lncRNA ODRUL in OS progression remain unclearly defined. Based on
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the previous result, we further determined the vital role of lncRNA ODRUL in the
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proliferation, migration, and invasion of OS in vitro and in vivo. And we found that lncRNA ODRUL could act as a ceRNA sponge for miR-3182 to further promote OS progression through up-regulating MMP2 and lncRNA ODRUL may be a therapeutic target of OS.
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Results
LncRNA ODRUL was up-regulated in the osteosarcoma tissues and cell lines, correlated with lung metastasis and worse prognosis
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To test whether ODRUL play an important role in OS carcinogenesis, we first
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measured the expression level of ODRUL in five human OS cell lines and the normal osteoblast cell line hFOB1.19 by qRT-PCR. The results showed that ODRUL was significantly up-regulated in osteosarcoma cells compared with hFOB1.19 cells (Fig.1A). Of them, MG63 and 143B cells with a higher expression of ODRUL were chosen for further experiments.Then, ODRUL expression level was investigated in 80 pairs of OS and paracancerous tissues and the results showed that ODRUL was significantly up-regulated in OS tissues compared with normal tissues (Fig.1B) and 4
ACCEPTED MANUSCRIPT higher expression of ODRUL in the lung metastasis group at early stage than that in the lung non-metastasis group (Fig. 1C). We also found that patients with higher expression of ODRUL had shorter overall survival time than those with lower
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expression of ODRUL (Fig. 1D). ODRUL promoted OS cell proliferation, migration, invasion and tumor growth in vitro and vivo
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We further analyzed the effect of ODRUL on the proliferation, migration and
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invasion of MG63 and 143B cells. The stably transfected cell lines with overexpression or knockdown of ODRUL were established in MG63 and 143B cells (Fig.2A). Cell proliferation was measured by using CCK-8 and cell clone formation assay. Results showed that cell proliferation rate and colony formation ability in the
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ODRUL group were significantly higher compared with the ODRUL-NC group, whereas cell proliferation rate and colony formation ability in the sh-ODRUL group were obviously lower than that in the sh-NC group (Fig.2B and C). Meanwhile,
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transwell and wound healing assay demonstrated that the numbers of migrating and
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invading cells in the ODRUL group were significantly increased compared with the control group, whereas migration and invasion were significantly decreased in the sh-ODRUL group compared with the control group (Fig.2D and E). Then, the effects of ODRUL overexpression or knockdown on the tumor growth in vivo were further analyzed in tumor-bearing nude mice. MG63 cells transfected with ODRUL, ODRUL -NC, sh-ODRUL, or sh-NC were subcutaneously injected into BALB/c athymic nude mice, respectively. As was shown in the figure 2F, from 2th to 7th week, it is obvious 5
ACCEPTED MANUSCRIPT that tumors formed in the ODRUL group grew much faster compared with the ODRUL -NC group and the volumes of transplanted tumors and weights of nude mice were smaller in the sh-ODRUL group when compared with the sh-NC group
ODRUL
was
predominantly
localized
in
the
posttranscriptional expression of miR-3182 and MMP2
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(Fig.2G). cytoplasm
and
regulated
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To examine the subcellular localization of ODRUL, Cy3-labeled probes specific
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for ODRUL were used for RNA-FISH. This analysis confirmed ODRUL-specific staining was observed in the cytoplasm of MG63 and 143B cells, whereas nearly no staining was observed in the nucleus (Fig. 3A). Then qPCR of nuclear and cytoplasmic fractions of the two cells further validated that ODRUL was mainly
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located in the cytoplasm (Fig.3B), indicating its role of regulating the gene expression at the posttranscriptional level. Since MMPs are well known to be involved in migration and invasion of OS cells, we asked whether lncRNA ODRUL promoted OS
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cell migration and invasion through regulating the expression of MMPs. We then
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assessed the MMP2 and MMP expression by qPCR and WB in the MG63 and 143B cells with knockdown or overexpression of ODRUL. Interestingly, we found that the expression of MMP2 and MMP9 was inversely correlated with ODRUL (Fig.3C and D). These data suggested that ODRUL could positively regulate posttranscriptional expression of MMP2 and MMP9 in OS cells (data of MMP9 was shown in the Figure S1). Using bioinformatics databases (miRanda, starBase v2.0, DIANA-LncBase v2.0), 6
ACCEPTED MANUSCRIPT miRNAs that may both interact with ODRUL and the 3′UTR of MMP2 or MMP9 were predicted. According to the summarized analysis results in the three databases(data shown in the Table S1), we found that there were nine miRNAs hsa-miR-3127-3p,
hsa-miR-4498,
hsa-miR-3182,
hsa-miR-5001-5p,
hsa-miR-3202,
hsa-miR-580-3p,
hsa-miR-4446-3p,
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including
hsa-miR-6132,
and
hsa-miR-6836-5p, who had potential binding sites for lncRNA ODRUL and the
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3′UTR of MMP2 mRNA, whereas, there was only has-miR-4773 predicted to hold the
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potential binding sites for lncRNA ODRUL and the 3′UTR of MMP9 mRNA. Furthermore, miRNA and mRNA microarrays were used to screen differentially expressed miRNAs and mRNAs associated with ODRUL in the paired sh-ODRUL and sh-NC MG63 cells. Further bioinformatics analysis showed that the ten
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previously predicted miRNAs and MMP2 or MMP9 were all found in the screening differentially expressed miRNAs and mRNAs, of which the up-regulated hsa-miR-3182 had the fold-change of ten, the down-regulated MMP2 of five and
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MM9 of four, respectively (Fig.3E). qRT-PCR was then performed to study the
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interaction between the ten miRNAs expression levels in MG63 and 143B cells with ODRUL overexpression or knockdown. Our results revealed that miR-3182 was the most influenced and consistently negative with the expression of ODRUL, indicating that competing regulation relationship between the ODRUL and miR-3182 expression (Fig. 3F). Meanwhile, miR-3182 could also negatively regulate the mRNA expression of lncRNA ODRUL. ODRUL expression in the miR-3182-mimics group distinctly increased and in the miR-3182-inhibitor group decreased relative to the corresponding 7
ACCEPTED MANUSCRIPT NC group (Fig.3G). Further bioinformatics analysis showed the miRNA response element (MRE) between the sequence of ODRUL and miR-3182 (Fig.3H). Then we further found that miR-3182 could also negatively regulate the mRNA and protein
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expression of MMP2, verified by the results that inhibition of miR-3182 up-regulated and overexpression of miR-3182 down-regulated the expression of MMP2 (Fig.3I and J).
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ODRUL was directly targeted by miR-3182, further regulating the expression of
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MMP2 through competitively binding with miR-3182
The mRNA and protein expression of MMP2 in the group of ODRUL combined with miR-3182-inhibitor significantly increased and MMP2 expression in the sh-ODRUL combined with miR-3182-mimics group distinctly inhibited when
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compared with the corresponding NC group, which revealed that the expression of MMP2 was mutually suppressed by ODRUL and miR-3182 (Fig.4A and B). Besides, as was shown in Figure 4C, co-transfection of miR-3182 mimic and ODRUL
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expression vector showed that miR-3182 remarkably reduced cell invasion promoted
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by ODRUL. Meanwhile, co-transfection of miR-3182 inhibitor and sh-ODRUL showed that the miR-3182 remarkably rescued the invasive ability of MG63 and 143B cells inhibited by the knockdown of ODRUL. However, co-transfection of miR-3182 mimic and sh-ODRUL showed that the numbers of invasive cells significantly decreased compared with the only miR-3182 mimic or sh-ODRUL transfection and co-transfection of miR-3182 inhibitor and ODRUL expression vector had the opposite effects, which may demonstrate the antagonism role of regulation in the OS cell 8
ACCEPTED MANUSCRIPT invasion between ODRUL and miR-3182. Dual luciferase reporter assay was further conducted to validate the direct binding of lncRNA ODRUL and 3′UTR of MMP2 mRNA with miR-3182. The results
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demonstrated that the miR-3182 mimics remarkably reduced, but the miR-3182 inhibitor increased luciferase activities of the reporter plasmid containing the potential binding sequence of 3′UTR of MMP2 mRNA or lncRNA ODRUL (wild type, WT),
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but without obvious changes in the reporter plasmid containing mutated sequence
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(mutant type, MUT) (Fig.4D and E). Moreover, co-transfection of lncRNA ODRUL could rescue the decreased luciferase activity of WT-MMP2 treated with miR-3182 mimics (Fig.4F). On the contrary, the luciferase activities of the WT-MMP2 were enhanced by miR-3182 inhibition, which could be reversed by sh-ODRUL
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respectively (Fig.4G). These data illustrated that lncRNA ODRUL directly regulated MMP2 expression through competitively binding with miR-3182 as a miRNA sponge. Mir-3182 expression was inversely correlated with ODRUL and poor prognosis
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Based on above findings, miR-3182 may also play a role in OS carcinogenesis.
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We then tested miR-3182 level in OS cell lines and tissues and found that miR-3182 was significantly down-regulated in five osteosarcoma cell lines, compared with the normal osteoblast cell line (Fig.5A) and dramatically reduced in OS tissues compared with the matched paracancerous tissues (Fig.5B). A negative correlation was demonstrated in expression of miR-3182 and lncRNA ODRUL in OS cell lines and tissues, which further implied the endogenous competing relationship between them (Fig.5C and D). Subsequently, the expression of miR-3182 in the lung metastasis 9
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potential tumor-suppressing role of miR-3182 in the development of the OS, contrary to the role of lncRNA ODRUL.
Mir-3182 suppressed OS cell proliferation, migration, invasion and tumor growth in
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vitro and vivo
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MG63 and 143B cells stably transfected with miR-3182 mimics or inhibitor were established. CCK-8 and cell clone formation assay showed that the cell proliferation rate
and
colony
formation
ability
were
significantly
increased
in
the
miR-3182-inhibitor group but were significantly decreased in the miR-3182-mimics
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group, when compared to the NC group (Fig.6A and B).Meanwhile, transwell and wound healing assay demonstrated that the numbers of migrating and invading cells in the miR-3182-inhibitor group were significantly increased compared with the
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inhibitor-NC group, whereas migration and invasion were significantly attenuated in
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the miR-3182-mimics group (Fig. 6C and D). Besides, overexpression of miR-3182 in the MG63 cells and the NC were subcutaneously injected into BALB/c athymic nude mice respectively. Consistent with the results in vitro, the tumor volumes and nude mice weights of the miR-3182 overexpression group of MG63 cells were significantly smaller than that of the control group, which conformed to the in vivo imaging analysis (Fig.6E and F).. Discussion 10
ACCEPTED MANUSCRIPT Recent studies have revealed that lncRNAs could participate in the initiation and progression of various cancers[8], including breast cancer, gastric cancer, bladder cancer, osteosarcoma, and so on[21-22]. Understanding the molecular mechanism of
osteosarcoma
[23-24]
progression of OS
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lncRNAs may help to explore new promising therapeutic strategy for the treatment of . Actually, several lncRNAs have been reported to involve in the [25-27]
. For example, Liu SH
[28]
found that a novel antisense
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lncRNA SATB2-AS1 overexpresses in OS and increases cell proliferation and growth [26]
reported that lncRNA
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through affecting its conjugate gene, SATB2. Sun L
EWSAT1 promotes OS cell growth and metastasis through suppression of MEG3 expression. Chen F [27] found that lncRNA BCAR4 promotes OS progression through activating GLI2-dependent gene transcription.
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In the present study, we found that a novel lncRNA, ODRUL previously reported by us, was overexpressed in osteosarcoma tissues and cell lines. OS patients with high ODRUL expression showed worse prognosis when compared with those with low
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ODRUL expression, and ODRUL expression was an independent prognostic factor of
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OS patients with significant clinical meaning. The function of ODRUL was subsequently investigated in our study. Our data indicated that knockdown of ODRUL inhibited and overexpression of it promoted OS cell proliferation, migration and invasion, tumor growth both in vitro and vivo. Furthermore, we found that ODRUL regulated migration and invasion of OS cells through up-regulating the expression of matrix metalloproteinase II (MMP2), a key proteinase during cancer invasion. These results suggested that lncRNA ODRUL functions as an oncogene and plays a critical 11
ACCEPTED MANUSCRIPT role in OS progression. LncRNAs have been shown to widely regulate the gene expression at different kinds of levels, such as pre-transcription, transcription and post-transcription, which [29-31]
. LncRNAs located in the nuclei always
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mainly depend on its cellular location
play role in the level of pre-transcription or transcription[32], while cytoplasmic lncRNAs often function as a competing endogenous RNA and sponge miRNAs, thus
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regulating the expression of target mRNA at the post-transcription level[33]. In the
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study, we identified that ODRUL mainly located in the cytoplasm through RNA FISH and cell cytoplasm/nucleus fraction isolation assay in the qualitative and quantitative aspects, which may suggest ODRUL could exert its regulatory role at the post-transcription level. Then competing endogenous RNAs (ceRNA) mechanism was
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first considered.
For this posttranscriptional regulatory mechanism, lncRNAs should have miRNA responsive elements (MRE) and act as miRNA sponges to control endogenous
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miRNAs available for binding with their target mRNAs, thus reducing the repression
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of these mRNAs[34]. Actually, many lncRNAs have been shown to play role in the tumorgenesis and progression by interfering with the miRNA pathways as ceRNAs. For example, Guo G et al
[35]
found that lncRNA-BGL3 regulates Bcr-Abl-mediated
cellular transformation by acting as a competitive endogenous RNA. Liu D et al
[36]
reported that lncRNA SPRY4-IT1 sponges miR-101-3p to promote proliferation and metastasis of bladder cancer cells through up-regulating EZH2. Sun C et al
[37]
reported that lncRNA NEAT1 promotes non-small cell lung cancer progression 12
ACCEPTED MANUSCRIPT through regulation of miR-377-3p-E2F3 pathway. Besides, Zhou Q et al [38] reported that lncRNA PVT1 promotes osteosarcoma development by acting as a molecular sponge to regulate miR-195, further influencing the expression of the downstream
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genes, like BCL2, CCND1, and FASN. In our present study, we first found that lncRNA ODRUL could promote the OS cell migration and invasion through inducing the expression of MMP2 and MMP9.
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Further microarray and bioinformatics databases analysis were conducted to search
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the potential miRNAs that have potential binding sites between the lncRNA ODRUL and 3’UTR of MMP2 or MMP9 mRNA. Only one miRNA (has-miR-4773) was predicted to be possible to play role between lncRNA ODRUL and MMP9, but further expression validation and dual luciferase reporter gene assay defined the
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pathway(data not shown). However, nine miRNAs were predicted between ODRUL and MMP2 and one of them, miR-3182 was further demonstrated to directly target ODRUL and MMP2 in MG63 and 143B cells.
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MicroRNA-3182 was previously reported to be specifically sorafenib-induced in [39]
. However, there is
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colorectal cancer cells in response to sorafenib treatment
seldom report about miR-3182 in osteosarcoma. The present study displayed that miR-3182 was significantly decreased in OS tissues and cell lines and negatively correlated with the expression of ODRUL. MiR-3182 also could act as an independent prognostic factor of OS patients, with a longer survival time of higher expression. Furthermore, functional assay found that miR-3182 inhibited proliferation, migration and invasion of OS cells and tumor growth both in vitro and vivo. Besides, 13
ACCEPTED MANUSCRIPT knockdown of miR-3182 could rescue the effect of sh-ODRUL on OS cell invasion and the expression of MMP2. Meanwhile, overexpression of miR-3182 also could rescue the effect of overexpression ODRUL on cell invasion and MMP2, which may
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demonstrate the antagonism role of regulation in the OS cell invasion and MMP2 expression between ODRUL and miR-3182 .In addition, the direct binding of lncRNA ODRUL and 3′UTR of MMP2 mRNA with miR-3182 was further validated by dual
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luciferase reporter assay. Taken together, these data revealed that lncRNA ODRUL
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could effectively sponge miR-3182 to promote osteosarcoma progression through up-regulating MMP2.
In conclusion, we identify that highly expressed ODRUL is an oncogenic lncRNA that exerts a crucial role in the OS progression. Besides, our study sheds light
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on the role of lncRNA ODRUL/ miR-3182/MMP2 pathway in OS for the first time, and reveals that lncRNA ODRUL could sponge miR-3182 to promote osteosarcoma progression through up-regulating MMP2, thus probably providing a novel
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therapeutic target in OS.
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Method and Materials
Cell lines and culture conditions SaoS2, HOS, U2-OS, MG63 and 143B human osteosarcoma cell lines (American Type Culture Collection) were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco, Gran Island, NY, USA), 100 U/mL of penicillin and 100 µg/mL of streptomycin (Invitrogen). Normal osteoblast cells (hFOB1.19) obtained from the Chinese Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were 14
ACCEPTED MANUSCRIPT cultured in Ham’s F12/ DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. Clinical samples
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A total of 80 primary osteosarcoma patients who received the same chemotherapy regimen before surgery and underwent complete resection surgery at Shanghai Tenth Hospital between 2006 and 2015 were included in this study. The clinical parameters
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PCR assays and western blotting analysis
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of osteosarcoma patients in this study are presented in Table 1.
Total RNA was extracted from tissues and cells with Trizol reagent (TAKARA) according to the product description. All mRNAs and miRNAs were reverse transcribed according to the protocol of the PrimeScript® RT Master Mix Perfect
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Real Time (TAKARA). The primers were shown in Table S1.
Cells were collected and lysed using RIPA protein extraction reagent (Beyotime, Beijing, China) supplemented with a protease inhibitor cocktail (Roche, Pleasanton,
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control.
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CA,USA).Autoradiograms were quantified by densitometry using GAPDH as a
Plasmid construction and cell transfection MG63 and 143B cells were transiently transfected with shRNAs after being sowed into the 6-well plates overnight. A scrambled negative control, a plasmid overexpressing ODRUL, and an empty vector, were cultured as well using the Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA) and FuGENE® HD Transfection Reagent (Roche, Germany) according to the manufacturer’s 15
ACCEPTED MANUSCRIPT instructions, respectively. 48h after transfection, the cells were harvested to detect the overexpression or knockout efficiency via qRT-PCR. Two different shRNAs against ODRUL were designed and synthesized by GenePharma (Shanghai, China). The
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target sequences for the sh-ODRUL included: sh-ODRUL-1 and sh-ODRUL-2 with the former having the highest inhibition efficiency (sh-ODRUL mentioned in the article refers to sh-ODRUL-1). The synthetic ODRUL sequence (319bp) was
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designed sequences were shown in the Table S2.
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sub-cloned into the pEGFP-N1 plasmid vector followed by a sequencing analysis. The
CCK-8 assay
Cells were incubated in 10 % CCK-8 diluted in normal culture medium at 37 °C until visual color conversion occurred. Proliferation rates were determined at 24, 48, 72h
set at 570nM.
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after transfection. The absorbance of each well was measured with a microplate reader
Colony formation assay
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Cells were seeded in 6-well plates and were incubated for 24 h. The colonies were
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stained with crystal violet solution 14 days later. The colony number in each well was counted and calculated. Wound healing assay and cell invasion assay A total of stable transfected cells were seeded onto six-well plates and cultured overnight. Wounds were created by scratching cell layer with a sterile plastic pipette tips and washed with culture medium. Cells were further cultured with medium containing 1% FBS in 48h. 16
ACCEPTED MANUSCRIPT For the invasion assays, a 24-well transwell chamber with the upper chamber coated with Matrigel (BD Bioscience) was used. 1.0×105 cells in 100uL serum free DMEM medium were seeded in the top chamber, 500uL medium containing 10% FBS was
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placed to the lower chamber. After incubation for 48 h, cells on the upper membrane surface were wiped off using a cotton swab and the cells that had traversed the membrane were staining by crystal violet and counted.
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Xenograft transplantation
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Female nude (BALB/c) mice (4 weeks old) were purchased. Mice were divided into several groups according to the completely randomized method. MG63 cells stably expressing sh-ODRUL or NC were propagated and 1×107 cells were inoculated subcutaneously into the right side of the posterior flank of mice. Tumor growth was
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examined at the indicated time points and tumor volumes were measured. After 7 weeks, the mice were killed and tumors were removed and weighed. Intratumoral injection of Ad-sh-ODRUL (Ad-ODRUL) (2×109plaque-forming units [PFU]) or
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Ad-sh-NC was performed. Mice were photographed at the indicated times with
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anIVIS@ Lumina II system (Caliper Life Sciences, Hopkinton, MA). RNA-fluorescence in situ hybridization (FISH) Cy3-labeled ODRUL and DAPI-labeled U6 probes were obtained from Genepharma (Shanghai, China). RNA FISH were performed using fluorescent in situ hybridization kit according to the manufacturer's protocol (Thermo Fisher). Cell cytoplasm/nucleus fraction isolation Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher) were employed to 17
ACCEPTED MANUSCRIPT prepare cytoplasmic and nuclear extracts. RNAs extracted from each of the fractions were subjected to following RT–qPCR analysis to demonstrate the levels of nuclear control transcript (β-actin), cytoplasmic control transcript (U6), and ODRUL.
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Dual luciferase reporter assay MG63 cells were seeded at 3× 104 cells/well in 24-well plates and allowed to settle overnight. The next day, cells were co-transfected with pmirGLO-ODRUL-WT or
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-MUT reporter plasmids and miR-3182 mimic or inhibitor. 24h after transfection, the
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relative luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) and normalized against Renilla luciferase activity. Statistical analysis
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All statistical analyses were performed using SPSS 22.0 software (IBM). Data are presented as mean ± SEM .Differences between groups were analyzed using the Student’s t test or one-way ANOVA .Overall survival was calculated by Kaplan-Meier
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survival analysis and compared using the log-rank test. p values ＜ 0.05 were
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considered statistically significant. Conflicts of Interests: We declare that we have no conflicts of interest. Acknowledgments: This project was supported by a Grant from the National Natural Science Foundation of China (No.81572630), Shanghai Pujiang Program of Shanghai Science and Technology Commission (NO.13PJD023) and Shanghai Jiaotong University Medical-Engineering Cross Research Fund (NO.YG2012MS49). Author Contributions: ZKP and MXL carried out the molecular genetic studies. 18
ACCEPTED MANUSCRIPT MXL carried out the tumor-bearing nude mice assays. ZKP and ZCL participated in the design of the study and performed the statistical analysis. ZKP drafted the manuscript and ZCL helped to correct it.
1.
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34. Tay Y, Rinn J, Pandolfi PP(2014). The multilayered complexity of ceRNA crosstalk and competition. Nature 505:344-52. 35. Guo G, Kang Q, Zhu X, Chen Q, Wang X, Chen Y, et al. (2015). A long
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ACCEPTED MANUSCRIPT 38. Zhou Q, Chen F, Zhao J, Li B, Liang Y, Pan W, et al. (2016). Long non-coding RNA PVT1 promotes osteosarcoma development by acting as a molecular sponge to regulate miR-195. Oncotarget 7:82620-33.
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39. Pehserl AM, Ress AL, Stanzer S, Resel M, Karbiener M, Stadelmeyer E, et al. (2016). Comprehensive Analysis of miRNome Alterations in Response to Sorafenib
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Treatment in Colorectal Cancer Cells. Int. J. Mol. Sci.17:2011.
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Fig.1 LncRNA ODRUL was up-regulated in the osteosarcoma tissues and cell lines, correlated with lung metastasis and worse prognosis. (A) Expression level of ODRUL in five human OS cell lines and normal osteoblast cell line hFOB1.19. (B) Expression level of ODRUL in 80 pairs of OS and paracancerous tissues. (C)
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Expression level of ODRUL in OS tissues of lung metastasis and lung non-metastasis group at early stage. (D) OS patients with higher expression of ODRUL had shorter overall survival time than those with lower expression. Data are presented as mean ±
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SEM, *P < 0.05.
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Fig.2 ODRUL promoted OS cell proliferation, migration, invasion and tumor growth in vitro and vivo. (A) RT–qPCR analysis of the effect on knockdown or overexpression of the expression of ODRUL by sh-RNA or vector transfection. (B) CCK-8 assays were performed to examine cell proliferation rate of MG63 and 143B cells after knockdown or overexpression of ODRUL. (C) Clone formation assays were performed to examine cell vitality after transfection. (D) Transwell assays were performed to identify the capacity of cell invasion after transfection. (E) Wound 24
ACCEPTED MANUSCRIPT healing assays were performed to examine the capacity of cell migration after transfection. (F) General conditions and in vivo imaging of nude mice in the four groups when exposed to the same treatment. (G) The nude mice were sacrificed in the
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7th week. Tumors formed in the ODRUL group grew much faster compared with the ODRUL-NC group and the volumes of transplanted tumors and weights of nude mice were smaller in the sh-ODRUL group when compared with the sh-NC group. Data are
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presented as mean ± SEM, *P < 0.05.
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Fig.3 ODRUL was predominantly localized in the cytoplasm and regulated posttranscriptional expression of miR-3182 and MMP2. (A) Subcellular localization of ODRUL by RNA-FISH in the MG63 and 143B cells. Nuclei are stained blue (DAPI) and ODRUL is stained red. (B) Nuclear and cytoplasmic
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fractions assay further validated the subcellular localization of ODRUL. (C) LncRNA ODRUL regulated the mRNA level of MMP2 expression. (D) LncRNA ODRUL regulated the protein level of MMP2 expression. (E)miRNA and mRNA microarrays
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were used to screen differentially expressed miRNAs and mRNAs associated with
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ODRUL in the paired sh-ODRUL and sh-NC MG63 cells. (F) qRT-PCR was performed to study the interaction between the miRNAs expression levels with ODRUL overexpression or knockdown and only miR-3182 was consistently negatively with the expression of ODRUL.(G) qRT-PCR was performed to study the interaction between the ODRUL expression levels with miR-3182 overexpression or knockdown. (H) The miR-3182 response element (MRE) between the sequence of ODRUL and MMP2 by bioinformatics analysis. (I) miR-3182 negatively regulated 25
ACCEPTED MANUSCRIPT the mRNA level of MMP2 expression. (J) miR-3182 negatively regulated the protein level of MMP2 expression. Data are presented as mean ± SEM, *P < 0.05. Fig.4 ODRUL was directly targeted by miR-3182, further regulating the
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expression of MMP2 through competitively binding with miR-3182. (A)The mRNA expression of MMP2 was examined in the MG63 and 143B cells co-transfected with miR-3182 mimics or inhibitor, ODRUL vector or sh-ODRUL, or
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their NCs. (B) The protein expression of MMP2 was examined in the same cells
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previously described.(C) Transwell assays were performed to observe the biological behaviors of OS cells co-transfected with miR-3182 mimics or inhibitor, sh-ODRUL or ODRUL vector and their NCs. (D) WT and MUT sequences designed for the ODRUL and MMP2 according to their binding sites with miR-3182. (E)ODRUL
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luciferase activity assays. Wide type or mutant ODRUL was co-transfected with miR-3182 mimics or miR-3182 inhibitor, respectively. Mir-3182 mimics repressed, but miR-3182 inhibitor enhanced the luciferase activity of the WT-ODRUL reporter
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which including wild type sequence of ODRUL. There was no obvious change of the
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luciferase activity for the MUT-ODRUL reporter which containing mutant ODRUL sequence. (F) MMP2 3′UTR luciferase activity assays. Overexpression of ODRUL rescued the luciferase activity, which repressed by the transfection with miR-3182 mimics in the WT- MMP2 but not in the MUT- MMP2 reporter. (G) Down-regulation of ODRUL with sh-RNA reversed the luciferase activity, which enhanced by the transfection with miR-3182 inhibitor in the WT-MMP2 but not in MUT-MMP2 reporter. The normalized luciferase activity in the control group was set to 1. Data are 26
ACCEPTED MANUSCRIPT presented as mean ± SEM, *P < 0.05. Fig.5 miR-3182 expression was inversely correlated with ODRUL and poor prognosis. (A) Expression level of miR-3182 in five human OS cell lines and normal
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osteoblast cell line hFOB1.19. (B) Expression level of miR-3182 in 80 pairs of OS and paracancerous tissues. (C) The relative expression level of ODRUL and miR-3182 in five human OS cell lines and normal osteoblast cell line hFOB1.19. (D)
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The relative expression level of ODRUL and miR-3182 in 80 pairs of OS and
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paracancerous tissues. (E) Expression level of miR-3182 in OS tissues of lung metastasis and lung non-metastasis group at early stage. (F) OS patients with lower expression of miR-3182 had shorter overall survival time than those with higher expression. Data are presented as mean ± SEM, *P < 0.05.
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Fig.6 miR-3182 suppressed OS cell proliferation, migration, invasion and tumor growth in vitro and vivo. (A) CCK-8 assays were performed to examine cell proliferation rate transfected with miR-3182 mimics or inhibitor. (B) Clone formation
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assays were performed to examine cell vitality transfected with miR-3182 mimics or
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inhibitor. (C) Transwell assays were performed to identify the capacity of cell invasion after miRNA transfection. (D) Wound healing assays were performed to examine the capacity of cell migration after miRNA transfection. (E) General conditions and in vivo imaging of nude mice in the miR-3182 mimics and mimics-NC transfected groups when exposed to the same treatment. (F) The nude mice were sacrificed in the 7th week. Tumors formed in the miR-3182 mimics group grew slower compared with the mimics-NC group and the volumes of transplanted tumors were 27
ACCEPTED MANUSCRIPT smaller in the miR-3182 mimics group when compared with the mimics-NC group. Data are presented as mean ± SEM, *P < 0.05. Fig. S1 ODRUL positively regulated posttranscriptional expression of MMP2. (A)
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LncRNA ODRUL regulated the mRNA level of MMP2 expression. (B) LncRNA ODRUL regulated the protein level of MMP2 expression. Data are presented as mean
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± SEM, *P < 0.05.
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ACCEPTED MANUSCRIPT Table 1. Clinical parameters of osteosarcoma patients enrolled in this study Pathological characteristics
Cases (n)
ODRUL expression
P value
miR-3182 expression
P value
Male
52
12.42 ± 1.28
0.12
3.12 ± 0.28
0.15
Female
28
12.55 ± 1.15
≥25
25
12.74 ± 1.16
<25
55
12.92 ± 1.08
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Gender
3.28 ± 0.11
Age
3.13 ± 0.27
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0.13
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3.16 ± 0.24
0.07
Location
0.09
36
12.25 ± 1.15
3.15 ± 0.24
Proximal of Tibia
28
12.14 ± 1.06
3.16 ± 0.53
Other
16
12.34 ± 1.16
3.23 ± 0.17
48
No
32
23.62 ± 1.18 11.06 ± 0.34
<0.05
1.03 ± 0.17 4.54 ± 0.16
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Yes
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Distal of Femur
Lung Metastasis
0.22
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Data are presented as mean ± SEM.
1
<0.05
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