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Downregulation of lncRNA SNHG12 reversed IGF1R-induced osteosarcoma metastasis and proliferation by targeting miR-195-5p
Journal Pre-proofs Research paper Downregulation of lncRNA SNHG12 reversed IGF1R-induced osteosarcoma metastasis and proliferation by targeting miR-195-5p Ning Xu, Jiayuan Xu, Zhuan Zuo, Yang Liu, Feng Yan, Chenglong Han PII: DOI: Reference:
S0378-1119(19)30804-2 https://doi.org/10.1016/j.gene.2019.144145 GENE 144145
To appear in:
Gene Gene
Received Date: Revised Date: Accepted Date:
31 May 2019 23 September 2019 24 September 2019
Please cite this article as: N. Xu, J. Xu, Z. Zuo, Y. Liu, F. Yan, C. Han, Downregulation of lncRNA SNHG12 reversed IGF1R-induced osteosarcoma metastasis and proliferation by targeting miR-195-5p, Gene Gene (2019), doi: https://doi.org/10.1016/j.gene.2019.144145
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Downregulation of lncRNA SNHG12 reversed IGF1R-induced osteosarcoma metastasis and proliferation by targeting miR-195-5p Authors: Ning Xu, Jiayuan Xu, Zhuan Zuo, Yang Liu, Feng Yan, Chenglong Han✉ Ning Xu: [email protected] Jiayuan Xu: [email protected] Zhuan Zuo: [email protected] Yang Liu: [email protected] Feng Yan: [email protected] Chenglong Han: [email protected] Affiliations: Department of Orthopedic Surgery, The First Affiliated Hospital of Harbin Medical University, No. 23, Youzheng St, Nangang, Harbin, Heilongjiang, 150001, China
✉ Corresponding
author:
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Abstract Long non-coding RNA SNHG12 (lncSNHG12) plays important roles in the onset and progression of various cancers. However, the role of lncSNHG12 in osteosarcoma (OS) remains unclear. Therefore, the aim of the present study was to determine the function of lncSNHG12 in OS. A bioinformatics website was used to predict the downstream targets of lncSNHG12. In addition, qRT-PCR was employed to assess lncSNHG12 expression in OS cells. Cell migration and proliferation in vitro were verified using the transwell migration, clone formation, and CCK8 assays. Tumor metastasis and xenograft formation were monitored in nude mice with or without downregulation of lncSNHG12. The results show that lncSNHG12 was upregulated in OS cell lines. Downregulation lncSNHG12 suppressed the metastasis and proliferation both in vitro and in vivo. Also, lncSNHG12 downregulation suppressed the expression of insulin growth factor 1 receptor (IGF1R) expression through sponging miR-195-5p, which was verified with the luciferase reporter assay and rescue experiments. These findings suggest that downregulation of lncSNHG12 may suppress aggressive OS phenotypes. Moreover, lncSNHG12 silencing inhibited OS metastasis and growth by targeting the miR-195-5p/IGF1R axis, which represents a candidate marker and target for OS treatment and management.
Keywords: miR-195-5p, lncRNA SNHG12, IGF1R, osteosarcoma, proliferation, invasion
Introduction Osteosarcoma (OS) is a primary malignant bone neoplasm mainly of adolescents and children that is associated with a significantly high rate of cancer-related mortality (Damron et al., 2007; Nie and Peng, 2018). Metastasis, which occurs in 25%–30% of OS patients, negatively effects the efficacy of treatment (Tabone et al., 1994; Krishnamurthy et al., 2018). Therefore, the prognosis of OS patients is often poor. So, it is meaningful to clarify the molecular mechanisms that underlie the process of OS metastasis to develop novel treatment strategies. Long noncoding RNAs (lncRNAs) belong to a family of noncoding RNA that have >200 nucleotides, which are crucial to cell differentiation and epigenetic regulation. LncRNA dysfunction is highly associated with the pathogenesis of various human cancers (Evans et al., 2016; Lorenzen and Thum, 2016; Luo and Chen, 2016; Wan et al., 2017). Previous investigations have inferred that lncRNAs are of indispensable importance in the pathogenesis of OS and that lncRNA MALAT1, TUG1, SNHG12, and HULC have indispensable functions in promoting the progression of OS (Dong et al., 2015; Sun et al., 2015; Ju et al., 2018). Yet, the impact of lncRNA SNHG12 on the biological behavior of OS remains unknown. Therefore, the aim of the present study was to elucidate the roles and molecular mechanisms of lncRNA SNHG12 in OS. MicroRNAs (miRNAs) are tiny non-coding RNA molecules, 20–22 nucleotides in length, which post-transcriptionally regulate mRNA expression by interacting with the 3′-untranslated regions (3′-UTRs) (Calin and Croce, 2006). Several miRNAs act as suppressor and tumor-promoting genes, which regulate the migration, invasion, and proliferation of cancer cells (Zhang et al., 2007; Liu et al., 2012). Former studies have found that the associations between miRNAs and lncRNAs are essential to cancer progression (Zhang et al., 2013). Moreover, lncRNA SNHG12 was reported to modulate cancer progression by competitively binding to miRNA (Long et al., 2018; Zhou et al., 2018). However, the function of lncRNA SNHG12 in OS remains unknown. The results of the current investigation illustrated that lncRNA SNHG12
functions as a tumor promoter. We further showed that lncRNA SNHG12 knockdown might inhibit insulin growth factor 1 receptor (IGF1R)-mediated OS proliferation and migration through the regulation of miR-195-5p. The results of the present study also provide reference values for the essential functions of lncRNA SNHG12 in OS metastasis, which could potentially lead to the use of lncRNA SNHG12 as a molecular target for oncotherapy.
Materials & Methods Ethics statement Twelve BALB/c nude mice (age, 4 weeks; body weight, 15–20 g) were purchased from SLAC Laboratory Animal Co. Ltd (Shanghai, China). The study protocol was approved by the Ethics Committee of Huashan Hospital affiliated with Fudan University (Shanghai, China).
Transfection and cell culture Four OS cell lines (i.e., 143B, MG-63, SJSA1, and MHM) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultivated at 37 °C in Dulbecco's modified Eagle’s medium (DMEM; Gibco Laboratories, Gaithersburg, MD, USA) supplemented with 100 U/mL of penicillin, 10% fetal bovine serum (FBS; Gibco Laboratories), 100 mg/mL of streptomycin under an atmosphere of 5% CO2/95% air. Human hFOB 1.19 osteoblasts were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultivated in F12 medium containing 10% FBS. After culturing in Lipofectamine 2000 reagent (Invitrogen Corporation, Carlsbad, CA, USA), the 143B and MG-63 cells were transfected with small interfering RNAs (silncSNHG12), miR-195-5p mimics, negative controls (NCs), miR-195-5p inhibitors, and an IGF1R overexpression vector. LncSNHG12-silenced and lentiviral-stabilized 143B cells were used to validate the effects of lncSNHG12 in vivo.
Cell-counting Kit-8 (CCK8) assay
The cells were cultivated in the wells of 96-well plates at a density of 2,000 cells/well. At 0, 24, 48, and 72 h, 10 µL of CCK8 reagent (Dojindo Labs, Kumamoto, Japan) were added to each well. After incubation for an additional 1 h, the absorbance at 450 nm was measured using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
RNA extraction and real-time quantitative polymerase chain reaction (qRT-PCR) Total RNA was extracted from tumor cells or tissues using the Native Protein Purification Kit and the mirVana™ PARIS™ Kit (both, Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s protocol. After dissolving in 100 mL of RNase-free water, the concentration and purity of all RNA samples were determined at an absorbance ratio of 260/280 nm. Complementary DNA (cDNA) was synthesized using the ImProm-II™ Reverse Transcription System (Promega Corporation, Madison, WI, USA) in accordance with the manufacturer’s instructions. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed with SYBR Green to detect mRNA levels, which were quantified by the 2-ΔΔCt method. For miR-195-5p analyses, the miR-195-5p stem-loop RT primer was 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACG CCAAT-3'
and
miR-195-5p
5'-CGTAGCAGCACAGAAAT-3'
was
amplified
(sense)
and
with
the
primer
pair
5'-GTGCAGGGTCCGAGGT-3'
(antisense). The primer pair 5'-TCTGGTGATCGAGGACTTCC-3' (sense) and 5'-ACCTCCTCAGTATCACACACT-3' (antisense) was used for amplification of lncRNA SNHG12. U6 was employed as an internal miRNA control and was amplified with the primer pair 5'-CTCGCTTCGGCAGCACA-3' (sense) and 5'-AACGCTTCACGAATTTGCGT-3'
(antisense).
Glyceraldehyde
3-phosphate
dehydrogenase (GAPDH) was used as an internal lncRNA control and amplified with the
primer
pair
5'-TGACTTCAACAGCGACACCCA-3'
(sense)
and
5'-CACCCTGTTGCTGTAGCCAAA-3' (antisense). Each sample was examined in triplicate and transformed with the relevant lncRNA SNHG12 or miR-195-5p
expression level according to that of GAPDH or U6, respectively. The starBase v3.0 platform was applied to predict the lncRNA/miRNA target genes. The association between miR-195-5p and IGF1R was determined using the TargetScan web server(http://www.targetscan.org/).
Migration assay Cell migration was determined in a 24-well transwell chambers with 8-μm pore membranes (BD Biosciences, Franklin Lakes, NJ, USA). In brief, 1 × 105 cells were placed in the upper chamber with 200 μL of serum-free medium, while the lower chamber contained 500 μL of complete medium. After 24 h of culturing, the cells in the lower chamber were fixed with 4% paraformaldehyde for 30 min and then stained with crystal violet for 10 min.
Tumor xenograft formation In total, the right flank of each mouse was injected with 2 × 107 viable wild-type (WT) or sh-lncSNHG12 143B cells, as described in our previous report (Yang et al., 2018b). The size of the tumors was measured every 5 days with the use of Vernier calipers and the tumor volume was calculated with the equation: volume = 0.5 × width2 × length. All mice were euthanatized at 30 days after implantation and the tissues were harvested for qRT-PCR analyses.
Western blot analysis Proteins of the lysed tissues or cells were isolated by 10% sodium dodecyl sulfate polyacrylamide-gel electrophoresis (Invitrogen Corporation) and then transferred to polyvinylidene fluoride membranes (EMD Millipore Corporation, Billerica, USA). Primary antibodies against IGF1R and GAPDH were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Chemiluminescence reagents (Cell Signaling Technology, Inc., Beverly, MA, USA) were utilized to visualize immunoreactive proteins.
Luciferase activity assay The mutant (MUT)/WT-type lncRNA SNHG12 and MUT/WT-type IGF1R 3'UTR including the binding site regarding miR-195-5p were subcloned to a basic pGL3 vector (Promega Corporation). After culturing in the wells of 6-well plates for 48 h (1 × 105 cells/well), HEK-293T cells were transiently transfected with MUT/WT-type lncRNA SNHG12 or the MUT/WT-type IGF1R 3'UTR segment vector and then co-transfection with 20 nM miR-195-5p mimics or the control. The cells were harvested at 48 h after transfection. The Dual Luciferase Reporter Assay System (Promega Corporation) was used to detect luciferase activity as previously reported (Zhang et al., 2017).
Colony-forming unit The OS cells were cultivated in full DMEM for 14 days and then stained with 0.1% crystal violet. The number of visible colonies was counted manually.
Immunohistochemical (IHC) analyses IHC analysis was performed as previously described (Torres et al., 2007). Briefly, antibodies against Ki67 (Thermo Fisher Scientific, Inc., Rockford, USA) were used to detect the abundance of replicated cells. Three different high power fields were used to quantify Ki67 positivity of each section.
Statistics analyses Data are presented as the mean ± standard deviation (SD). GraphPad Prism software (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA) was used to identify significant differences among groups. A probability (p) value of ≤ 0.05 was considered statistically significant.
Results Downregulation of lncRNA SNHG12 (lncSNHG12) suppressed OS growth in vivo
The qRT-PCR results confirmed that lncSNHG12 expression was dramatically upregulated in the OS cell lines MHM, 143B, SJSA1, and MG-63, as compared with normal hFOB 1.19 OS cells (Figure 1A), suggesting abnormal expression of lncSNHG12 relative to OS progression. To verify the role of lncSNHG12 in the progression of OS, lentiviral-stabilized and lncSNHG12-silenced (sh-lncSNHG12) 143B cells or a sh-NC were constructed. The results demonstrated that expression of lncSNHG12 significantly decreased after lncSNHG12 downregulation, as compared with the control and NC (Figure 1B). The result also showed that lncSNHG12 silencing reduced the tumor weight and volume, as compared to the NC group of nude mice xenografts (Figure 1C–E). Immunohistochemical detection showed that lncSNHG12 silencing decreased the number of Ki67-positive cells in the tumor tissues (Figures 1F and 1G). Statistical analyses suggested that miR-195-5p might be the target of ncSNHG12 and further qRT-PCR analysis verified that miR-195-5p expression was enhanced after downregulation of lncSNHG12 (Figure 1H). However, western blot detection found that IGF1R expression in tumors decreased in the lncSNHG12 silenced group, as compared with the NC group (Figures 1I and 1J). Increased evidence has shown that downregulation of IGF1R can suppress tumor progression (Liu et al., 2018; Zhang et al., 2018). Together, these findings suggest that downregulation of lncSNHG12 suppressed OS growth, probability via the regulation of miR-195-5p/IGF1R.
lncSNHG12 silencing inhibited the migration and proliferation of OS cells through regulation of the miR-195-5p/IGF1R axis in vitro. Both 143B and MG-63 cells were employed to explore the relationship among lncSNHG12, miR-195-5p, and IGF1R. The qRT-PCR analysis results verified that lncSNHG12 expression was downregulated after transfection with siRNA against lncSNHG12 (silncSNHG12). Treatment with the specific inhibitor miR-195-5p or transfection
with
vectors
overexpressing
IGF1R
had
no
effect
on
silncSNHG12-induced silencing of lncSNHG12 (Figures 2A and 2B), suggesting that both miR-195-5p and IGF1R were downstream of lncSNHG12. The results also
showed that lncSNHG12 silencing promoted the upregulation of miR-195-5p in both 143B and MG-63 cells, while treatment with the specific inhibitor miR-195-5p reversed the effects of silncSNHG12. IGF1R overexpression had no effect on the promotion of miR-195-5p-induced lncSNHG12 silencing (Figures 2C and 2D), suggesting that IGF1R was downstream of miR-195-5p. The results of western blot analysis showed that lncSNHG12 silencing suppressed IGF1R expression, while treatment with the miR-195-5p inhibitor or transfection with vectors overexpressing IGF1R rescued the expression of IGF1R (Figures 2E and 2F), suggesting that IGF1R was the target of miR-195-5p. The results of the CCK8 and cloning formation assays illustrated that lncSNHG12 silencing suppressed the proliferation of MG-63 and 143B cells, while IGF1R overexpression and miR-195-5p inhibition rescued the proliferation of both cell types even after silencing of lncSNHG12 (Figures 2G–K). The result of flow cytometry detection found that downregulation of lncRNA SNHG12 increased the chemosensitivity of 143B cells to cisplatin, but simple lncRNA SNHG12 silence can not increased the apoptosis rate (Supplement. 1). The results of the transwell migration assay showed that lncSNHG12 silencing suppressed the migration of both 143B and MG-63 cells, while IGF1R overexpression and miR-195-5p inhibition rescued the migratory abilities of both cell lines even after lncSNHG12 silencing (Figures 2L–N). In this regard, these results verified that downregulation of lncSNHG12 suppressed metastasis and proliferation of tumor cells by regulation of miR-195-5p/IGF1R signaling.
IGF1R overexpression reversed the inhibitory effect of miR-195-5p on cell migration and growth in vitro To verify the association between miR-195-5p and IGF1R, both MG-63 and 143B cells were transfected with miR-195-5p mimics, which were integrated with or without vectors overexpressing IGF1R. The qRT-PCR results showed that miR-195-5p expression was highly increased after transfection with the miR-195-5p mimic, while IGF1R overexpression had no effect on miR-195-5p expression (Figures 3A and 3B). The results of western blot analyses validated that miR-195-5p
overexpression suppressed IGF1R expression, which was recovered after transfection with vectors overexpressing IGF1R (Figures 3C and 3D). The results of the CCK8 and cloning formation assays validated that miR-195-5p overexpression suppressed the proliferation of both MG-63 and 143B cells, while IGF1R overexpression rescued the miR-195-5p suppressive effects (Figures 3E–I). As a measure of the effects of miR-195-5p and IGF1R on metastasis, the transwell migration assay results illustrated that
miR-195-5
overexpression
suppressed
cell
migration,
while
IGF1R
overexpression promoted the migration of both MG-63 and 143B cells (Figures 3J– L). These findings illustrate that expression of miR-195-5 suppressed tumor cell metastasis and proliferation via the suppression of IGF1R.
The associations among miR-195-5p, lncSNHG12, and IGF1R In regard to the associations among miR-195-5p, lncSNHG12, and IGF1R, lncSNHG12 interacted with miR-195-5p. The luciferase reporter assay was employed to determine if miR-195-5p is a candidate target of ncSNHG12 (Figure 4A). The results showed that lncSNHG12 inhibited the luciferase activities of the WT cell, but not the MUT cells (Figure 4B). To determine if IGF1R is a target of miR-195-5p, statistics analyses were utilized to verify if miR-195-5p directed the associations of the 3'-UTR of IGF1R and found that IGF1R expression was suppressed at the mRNA level (Figure 4C). The results of the luciferase reporter assay verified that miR-195-5p inhibited the luciferase activity of the WT cells (Figure 4D). These findings showed that silencing of lncSNHG12 inhibited the growth and metastasis of OS cells by targeting the miR-195-5p/IGF1R axis.
Discussion Increasing evidence has confirmed that lncRNAs have important functions in gene imprinting, the immune response, cell differentiation, the occurrence of human diseases, tumor formation, and other biological functions (Esteller, 2011; Guttman and Rinn, 2012). In the present study, lncSNHG12 expression was increased in OS
cells. LncSNHG12 was initially identified as an oncogene that promotes the development and onset of cancers of the central nervous system (Lei et al., 2018), stomach (Yang et al., 2018a), lung (Wang et al., 2017b), and breast (Wang et al., 2017a). Also, knockdown of lncSNHG12 suppressed the growth of OS cells in vivo. The results of the in vitro experiment verified that downregulation of lncSNHG12 suppressed the migration and proliferation of OS cells by promoting miR-195-5p expression. Treatment with the miR-195-5p inhibitor rescued the migration and proliferation of OS cells even after downregulation of lncSNHG12. Fluorescein staining also showed that lncSNHG12 decreased miR-195-5p levels by interacting with miR-195-5p. As a miR-15 family member, the function of miR-195-5p has been verified in various cancers of the thyroid, liver, central nervous system, and breast (Zhu et al., 2015; Wang et al., 2017c; Shou et al., 2019). The results of a previous study suggested that miR-195-5p was highly downregulated during the progression of OS (Qu et al., 2017). Meanwhile, the results of the current study found that miR-195-5p overexpression might suppress the migration and proliferation of OS cells via downregulation of IGF1R. Overexpression of IGF1R decreased the inhibitory effect of miR-195-5p on the proliferation and migration of OS cells. Fluorescein staining also confirmed that miR-195-5p was associated with the IGF1R 3'-UTR and suppressed IGF1R expression at the mRNA level. IGF1R is frequently overexpressed in tumors of the colon, prostate, rectum, breast, thyroid, and ovary, and regulates the proliferation and survival of tumor cells (Riedemann and Macaulay, 2006; Zorea et al., 2018). Moreover, IGF1R signaling functions in tumor cell adhesion and motility, thereby contributing to the metastasis of tumor cells (Wang et al., 2017d), suggesting that IGF1R is a potential target in tumor cells. In summary, the findings of the current study provide evidence that lncSNHG12 has important roles in the progression of OS. Downregulation of lncRNA SNHG12 reversed the IGF1R-induced metastasis and proliferation of OS cells via targeting miR-195-5p. These results confirmed that the lncRNA SNHG12/miR-195-5p/IGF1R axis could serve as a new therapeutic design strategy for the treatment of OS.
Conflict of Interest None declared.
Acknowledgements The study was funded by the General Program of the Natural Science Foundation of Heilongjiang Province (grant no. H2018036), the Heilongjiang Province’s Scientific Research Project of the Health and Family Planning Commission (grant no. 2017-052), and the First Affiliated Hospital of Harbin Medical University Scientific Research Innovation Foundation (grant no.2017B021).
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Figure legends Figure 1. The role of lncRNA SNHG12 (lncSNHG12) in OS. (A) The results of qRT-PCR analysis were plotted to define the lncSNHG12 expression profiles in the OS cell lines SJSA1, MG-63, MHM, and 143B, and the normal osteoblast cell line hFOB 1.19. Data are presented as the mean ± SD.
***p
< 0.001 vs. hFOB 1.19 cells.
(B) qRT-PCR was employed to determine the lncSNHG12 expression patterns in adenovirus-transfected 143B cells (sh-lncSNHG12) and control transfected cells (sh-NC). Data are presented as the mean ± SD.
**p
< 0.01,
***p
< 0.001 vs. sh-NC.
(C) Sum of weekly mouse tumor volumes. Data are presented as the mean ± SD. **p < 0.01,
***p
< 0.001 vs. sh-NC. (D) Representative photographs of the formation of
tumors in xenografts of nude mice (n = 6). (E) Tumor weights were measured at 30 days after injection. Data are presented as the mean ± SD. ***p < 0.001 vs. sh-NC. (F and G) Immunohistochemical analysis of the proportion of Ki-67-positive cells. The
relative amounts of Ki-67-positive cells were calculated. Data are presented as the mean ± SD.
***p
< 0.001 vs. sh-NC. (H) qRT-PCR analysis of miR-195-5p
expression. Data are presented as the mean ± SD.
***p
< 0.001 vs. sh-NC. (I–J)
Western blot analysis of IGF1R expression in tumor tissues. Data are presented as the mean ± SD. ***p < 0.001 vs. sh-NC. NC, negative control.
Figure 2. Silencing of lncSNHG12 inhibited the proliferation and migration of OS cells by regulation of the miR-195-5p/IGF1R axis in vitro. (A–D) The qRT-PCR detection of lncSNHG12 (A-B) and miR-195-5p (C-D) expression after transfection with siRNA against lncSNHG12 combined with or without the miR-195-5p inhibitor and vectors overexpressing IGF1R in both MG-63 (A and C) and 143B (B and D) cells. Data are presented as the mean ± SD.
###p
< 0.001 vs. silncSNHG12.
***p
<
0.001 vs. NC. (E–F) Western blots showing IGF1R expression. Relative protein levels were analyzed and data are presented as the mean ± SD.
***p
< 0.001 vs. NC. ###p <
0.001 vs. silncSNHG12. (G and H) CCK8 assays were performed to assess cell proliferation. Data are presented as the mean ± SD.
***p
< 0.001 vs. NC. (I–K)
Cloning formation assay showing the proliferation of MG-63 and 143B cells. Data are presented as the mean ± SD. ***p < 0.001 vs. NC. ###p < 0.001 vs. silncSNHG12. (L– N) The migration of both MG-63 and 143B cells was determined using the transwell migration assay. Data are presented as the mean ± SD. *p < 0.05, ***p < 0.001 vs. NC. ###p
< 0.001 vs. silncSNHG12. GADPH, glyceraldehyde 3-phosphate dehydrogenase
(loading control); NC, normal control.
Figure 3. IGF1R overexpression reversed the miR-195-5p inhibitory effect on cell growth and migration in vitro. Both MG-63 and 143B cells were transfected with miR-195-5p mimics combined with or without vectors overexpressing IGF1R. (A and B) qRT-PCR detection of the expression of miR-195-5p in both MG-63 and 143B cells. Data are presented as the mean ± SD.
***p
< 0.001 vs. NC.
###p
< 0.001 vs.
mimic. (C and D) Western blot showing IGF1R expression in both MG-63 and 143B cells. Relative protein levels were analyzed and data are presented as the mean ± SD.
***p
< 0.001 vs. NC. ###p < 0.001 vs. mimic. (E and F) CCK8 assays were performed
to assess cell proliferation. Data are presented as the mean ± SD. ***p < 0.001 vs. NC. ##p
< 0.01,
###p
< 0.001 vs. mimic. (G–I) Cloning formation assay of cell
proliferation. Data are presented as the mean ± SD.
***p
< 0.001.
###p
< 0.001 vs.
mimic. (J–L) Cell migration ability was determined using the transwell migration assay. Data are presented as the mean ± SD.
***p
< 0.001 vs. NC.
###p
< 0.001 vs.
mimic. NC, negative control.
Figure 4. The interactions and relationships among miR-195-5p, lncSNHG12, and IGF1R. (A) The predicted lncSNHG12 miR-195-5p-binding sites. The MUT version of lncSNHG12 is also given. (B) Relative luciferase activity was defined at 48 h after transfection with the miR-195-5p mimic/NC or lncSNHG12 MUT/WT in 293T cells. Data are presented as the mean ± SD.
***p
< 0.001. (C) The predicted miR-223
binding sites with the 3'-UTR of FGFR2. The mutated version of the 3'-UTR-FGFR2 is given. (D) Relative luciferase activity was determined at 48 h after transfection with the miR-195-5p mimic/NC or 3'-UTR-IGF1R WT/MUT in 293T cells. Data are presented as the mean ± SD. ***p < 0.001. 1 Downregulation of lncSNHG12 suppressed aggressive OS metastasis and growth in both in vitro and in vivo experiment. 2 LncSNHG12 promotion OS metastasis and growth via targeting the miR-195-5p/IGF1R axis, which represents a candidate marker and target for OS treatment and management. lncSNHG12, Long non-coding RNA SNHG12; IGF1R, insulin growth factor 1 receptor; OS, osteosarcoma; lncRNAs, long noncoding RNAs; miRNAs, microRNAs; DMEM, dulbecco's modified Eagle’s medium; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0378-1119(19)30804-2 https://doi.org/10.1016/j.gene.2019.144145 GENE 144145
To appear in:
Gene Gene
Received Date: Revised Date: Accepted Date:
31 May 2019 23 September 2019 24 September 2019
Please cite this article as: N. Xu, J. Xu, Z. Zuo, Y. Liu, F. Yan, C. Han, Downregulation of lncRNA SNHG12 reversed IGF1R-induced osteosarcoma metastasis and proliferation by targeting miR-195-5p, Gene Gene (2019), doi: https://doi.org/10.1016/j.gene.2019.144145
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Downregulation of lncRNA SNHG12 reversed IGF1R-induced osteosarcoma metastasis and proliferation by targeting miR-195-5p Authors: Ning Xu, Jiayuan Xu, Zhuan Zuo, Yang Liu, Feng Yan, Chenglong Han✉ Ning Xu: [email protected] Jiayuan Xu: [email protected] Zhuan Zuo: [email protected] Yang Liu: [email protected] Feng Yan: [email protected] Chenglong Han: [email protected] Affiliations: Department of Orthopedic Surgery, The First Affiliated Hospital of Harbin Medical University, No. 23, Youzheng St, Nangang, Harbin, Heilongjiang, 150001, China
✉ Corresponding
author:
Tel: +86-13945186142 Fax: 0451-85555828
Abstract Long non-coding RNA SNHG12 (lncSNHG12) plays important roles in the onset and progression of various cancers. However, the role of lncSNHG12 in osteosarcoma (OS) remains unclear. Therefore, the aim of the present study was to determine the function of lncSNHG12 in OS. A bioinformatics website was used to predict the downstream targets of lncSNHG12. In addition, qRT-PCR was employed to assess lncSNHG12 expression in OS cells. Cell migration and proliferation in vitro were verified using the transwell migration, clone formation, and CCK8 assays. Tumor metastasis and xenograft formation were monitored in nude mice with or without downregulation of lncSNHG12. The results show that lncSNHG12 was upregulated in OS cell lines. Downregulation lncSNHG12 suppressed the metastasis and proliferation both in vitro and in vivo. Also, lncSNHG12 downregulation suppressed the expression of insulin growth factor 1 receptor (IGF1R) expression through sponging miR-195-5p, which was verified with the luciferase reporter assay and rescue experiments. These findings suggest that downregulation of lncSNHG12 may suppress aggressive OS phenotypes. Moreover, lncSNHG12 silencing inhibited OS metastasis and growth by targeting the miR-195-5p/IGF1R axis, which represents a candidate marker and target for OS treatment and management.
Keywords: miR-195-5p, lncRNA SNHG12, IGF1R, osteosarcoma, proliferation, invasion
Introduction Osteosarcoma (OS) is a primary malignant bone neoplasm mainly of adolescents and children that is associated with a significantly high rate of cancer-related mortality (Damron et al., 2007; Nie and Peng, 2018). Metastasis, which occurs in 25%–30% of OS patients, negatively effects the efficacy of treatment (Tabone et al., 1994; Krishnamurthy et al., 2018). Therefore, the prognosis of OS patients is often poor. So, it is meaningful to clarify the molecular mechanisms that underlie the process of OS metastasis to develop novel treatment strategies. Long noncoding RNAs (lncRNAs) belong to a family of noncoding RNA that have >200 nucleotides, which are crucial to cell differentiation and epigenetic regulation. LncRNA dysfunction is highly associated with the pathogenesis of various human cancers (Evans et al., 2016; Lorenzen and Thum, 2016; Luo and Chen, 2016; Wan et al., 2017). Previous investigations have inferred that lncRNAs are of indispensable importance in the pathogenesis of OS and that lncRNA MALAT1, TUG1, SNHG12, and HULC have indispensable functions in promoting the progression of OS (Dong et al., 2015; Sun et al., 2015; Ju et al., 2018). Yet, the impact of lncRNA SNHG12 on the biological behavior of OS remains unknown. Therefore, the aim of the present study was to elucidate the roles and molecular mechanisms of lncRNA SNHG12 in OS. MicroRNAs (miRNAs) are tiny non-coding RNA molecules, 20–22 nucleotides in length, which post-transcriptionally regulate mRNA expression by interacting with the 3′-untranslated regions (3′-UTRs) (Calin and Croce, 2006). Several miRNAs act as suppressor and tumor-promoting genes, which regulate the migration, invasion, and proliferation of cancer cells (Zhang et al., 2007; Liu et al., 2012). Former studies have found that the associations between miRNAs and lncRNAs are essential to cancer progression (Zhang et al., 2013). Moreover, lncRNA SNHG12 was reported to modulate cancer progression by competitively binding to miRNA (Long et al., 2018; Zhou et al., 2018). However, the function of lncRNA SNHG12 in OS remains unknown. The results of the current investigation illustrated that lncRNA SNHG12
functions as a tumor promoter. We further showed that lncRNA SNHG12 knockdown might inhibit insulin growth factor 1 receptor (IGF1R)-mediated OS proliferation and migration through the regulation of miR-195-5p. The results of the present study also provide reference values for the essential functions of lncRNA SNHG12 in OS metastasis, which could potentially lead to the use of lncRNA SNHG12 as a molecular target for oncotherapy.
Materials & Methods Ethics statement Twelve BALB/c nude mice (age, 4 weeks; body weight, 15–20 g) were purchased from SLAC Laboratory Animal Co. Ltd (Shanghai, China). The study protocol was approved by the Ethics Committee of Huashan Hospital affiliated with Fudan University (Shanghai, China).
Transfection and cell culture Four OS cell lines (i.e., 143B, MG-63, SJSA1, and MHM) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultivated at 37 °C in Dulbecco's modified Eagle’s medium (DMEM; Gibco Laboratories, Gaithersburg, MD, USA) supplemented with 100 U/mL of penicillin, 10% fetal bovine serum (FBS; Gibco Laboratories), 100 mg/mL of streptomycin under an atmosphere of 5% CO2/95% air. Human hFOB 1.19 osteoblasts were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultivated in F12 medium containing 10% FBS. After culturing in Lipofectamine 2000 reagent (Invitrogen Corporation, Carlsbad, CA, USA), the 143B and MG-63 cells were transfected with small interfering RNAs (silncSNHG12), miR-195-5p mimics, negative controls (NCs), miR-195-5p inhibitors, and an IGF1R overexpression vector. LncSNHG12-silenced and lentiviral-stabilized 143B cells were used to validate the effects of lncSNHG12 in vivo.
Cell-counting Kit-8 (CCK8) assay
The cells were cultivated in the wells of 96-well plates at a density of 2,000 cells/well. At 0, 24, 48, and 72 h, 10 µL of CCK8 reagent (Dojindo Labs, Kumamoto, Japan) were added to each well. After incubation for an additional 1 h, the absorbance at 450 nm was measured using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
RNA extraction and real-time quantitative polymerase chain reaction (qRT-PCR) Total RNA was extracted from tumor cells or tissues using the Native Protein Purification Kit and the mirVana™ PARIS™ Kit (both, Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s protocol. After dissolving in 100 mL of RNase-free water, the concentration and purity of all RNA samples were determined at an absorbance ratio of 260/280 nm. Complementary DNA (cDNA) was synthesized using the ImProm-II™ Reverse Transcription System (Promega Corporation, Madison, WI, USA) in accordance with the manufacturer’s instructions. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed with SYBR Green to detect mRNA levels, which were quantified by the 2-ΔΔCt method. For miR-195-5p analyses, the miR-195-5p stem-loop RT primer was 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACG CCAAT-3'
and
miR-195-5p
5'-CGTAGCAGCACAGAAAT-3'
was
amplified
(sense)
and
with
the
primer
pair
5'-GTGCAGGGTCCGAGGT-3'
(antisense). The primer pair 5'-TCTGGTGATCGAGGACTTCC-3' (sense) and 5'-ACCTCCTCAGTATCACACACT-3' (antisense) was used for amplification of lncRNA SNHG12. U6 was employed as an internal miRNA control and was amplified with the primer pair 5'-CTCGCTTCGGCAGCACA-3' (sense) and 5'-AACGCTTCACGAATTTGCGT-3'
(antisense).
Glyceraldehyde
3-phosphate
dehydrogenase (GAPDH) was used as an internal lncRNA control and amplified with the
primer
pair
5'-TGACTTCAACAGCGACACCCA-3'
(sense)
and
5'-CACCCTGTTGCTGTAGCCAAA-3' (antisense). Each sample was examined in triplicate and transformed with the relevant lncRNA SNHG12 or miR-195-5p
expression level according to that of GAPDH or U6, respectively. The starBase v3.0 platform was applied to predict the lncRNA/miRNA target genes. The association between miR-195-5p and IGF1R was determined using the TargetScan web server(http://www.targetscan.org/).
Migration assay Cell migration was determined in a 24-well transwell chambers with 8-μm pore membranes (BD Biosciences, Franklin Lakes, NJ, USA). In brief, 1 × 105 cells were placed in the upper chamber with 200 μL of serum-free medium, while the lower chamber contained 500 μL of complete medium. After 24 h of culturing, the cells in the lower chamber were fixed with 4% paraformaldehyde for 30 min and then stained with crystal violet for 10 min.
Tumor xenograft formation In total, the right flank of each mouse was injected with 2 × 107 viable wild-type (WT) or sh-lncSNHG12 143B cells, as described in our previous report (Yang et al., 2018b). The size of the tumors was measured every 5 days with the use of Vernier calipers and the tumor volume was calculated with the equation: volume = 0.5 × width2 × length. All mice were euthanatized at 30 days after implantation and the tissues were harvested for qRT-PCR analyses.
Western blot analysis Proteins of the lysed tissues or cells were isolated by 10% sodium dodecyl sulfate polyacrylamide-gel electrophoresis (Invitrogen Corporation) and then transferred to polyvinylidene fluoride membranes (EMD Millipore Corporation, Billerica, USA). Primary antibodies against IGF1R and GAPDH were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Chemiluminescence reagents (Cell Signaling Technology, Inc., Beverly, MA, USA) were utilized to visualize immunoreactive proteins.
Luciferase activity assay The mutant (MUT)/WT-type lncRNA SNHG12 and MUT/WT-type IGF1R 3'UTR including the binding site regarding miR-195-5p were subcloned to a basic pGL3 vector (Promega Corporation). After culturing in the wells of 6-well plates for 48 h (1 × 105 cells/well), HEK-293T cells were transiently transfected with MUT/WT-type lncRNA SNHG12 or the MUT/WT-type IGF1R 3'UTR segment vector and then co-transfection with 20 nM miR-195-5p mimics or the control. The cells were harvested at 48 h after transfection. The Dual Luciferase Reporter Assay System (Promega Corporation) was used to detect luciferase activity as previously reported (Zhang et al., 2017).
Colony-forming unit The OS cells were cultivated in full DMEM for 14 days and then stained with 0.1% crystal violet. The number of visible colonies was counted manually.
Immunohistochemical (IHC) analyses IHC analysis was performed as previously described (Torres et al., 2007). Briefly, antibodies against Ki67 (Thermo Fisher Scientific, Inc., Rockford, USA) were used to detect the abundance of replicated cells. Three different high power fields were used to quantify Ki67 positivity of each section.
Statistics analyses Data are presented as the mean ± standard deviation (SD). GraphPad Prism software (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA) was used to identify significant differences among groups. A probability (p) value of ≤ 0.05 was considered statistically significant.
Results Downregulation of lncRNA SNHG12 (lncSNHG12) suppressed OS growth in vivo
The qRT-PCR results confirmed that lncSNHG12 expression was dramatically upregulated in the OS cell lines MHM, 143B, SJSA1, and MG-63, as compared with normal hFOB 1.19 OS cells (Figure 1A), suggesting abnormal expression of lncSNHG12 relative to OS progression. To verify the role of lncSNHG12 in the progression of OS, lentiviral-stabilized and lncSNHG12-silenced (sh-lncSNHG12) 143B cells or a sh-NC were constructed. The results demonstrated that expression of lncSNHG12 significantly decreased after lncSNHG12 downregulation, as compared with the control and NC (Figure 1B). The result also showed that lncSNHG12 silencing reduced the tumor weight and volume, as compared to the NC group of nude mice xenografts (Figure 1C–E). Immunohistochemical detection showed that lncSNHG12 silencing decreased the number of Ki67-positive cells in the tumor tissues (Figures 1F and 1G). Statistical analyses suggested that miR-195-5p might be the target of ncSNHG12 and further qRT-PCR analysis verified that miR-195-5p expression was enhanced after downregulation of lncSNHG12 (Figure 1H). However, western blot detection found that IGF1R expression in tumors decreased in the lncSNHG12 silenced group, as compared with the NC group (Figures 1I and 1J). Increased evidence has shown that downregulation of IGF1R can suppress tumor progression (Liu et al., 2018; Zhang et al., 2018). Together, these findings suggest that downregulation of lncSNHG12 suppressed OS growth, probability via the regulation of miR-195-5p/IGF1R.
lncSNHG12 silencing inhibited the migration and proliferation of OS cells through regulation of the miR-195-5p/IGF1R axis in vitro. Both 143B and MG-63 cells were employed to explore the relationship among lncSNHG12, miR-195-5p, and IGF1R. The qRT-PCR analysis results verified that lncSNHG12 expression was downregulated after transfection with siRNA against lncSNHG12 (silncSNHG12). Treatment with the specific inhibitor miR-195-5p or transfection
with
vectors
overexpressing
IGF1R
had
no
effect
on
silncSNHG12-induced silencing of lncSNHG12 (Figures 2A and 2B), suggesting that both miR-195-5p and IGF1R were downstream of lncSNHG12. The results also
showed that lncSNHG12 silencing promoted the upregulation of miR-195-5p in both 143B and MG-63 cells, while treatment with the specific inhibitor miR-195-5p reversed the effects of silncSNHG12. IGF1R overexpression had no effect on the promotion of miR-195-5p-induced lncSNHG12 silencing (Figures 2C and 2D), suggesting that IGF1R was downstream of miR-195-5p. The results of western blot analysis showed that lncSNHG12 silencing suppressed IGF1R expression, while treatment with the miR-195-5p inhibitor or transfection with vectors overexpressing IGF1R rescued the expression of IGF1R (Figures 2E and 2F), suggesting that IGF1R was the target of miR-195-5p. The results of the CCK8 and cloning formation assays illustrated that lncSNHG12 silencing suppressed the proliferation of MG-63 and 143B cells, while IGF1R overexpression and miR-195-5p inhibition rescued the proliferation of both cell types even after silencing of lncSNHG12 (Figures 2G–K). The result of flow cytometry detection found that downregulation of lncRNA SNHG12 increased the chemosensitivity of 143B cells to cisplatin, but simple lncRNA SNHG12 silence can not increased the apoptosis rate (Supplement. 1). The results of the transwell migration assay showed that lncSNHG12 silencing suppressed the migration of both 143B and MG-63 cells, while IGF1R overexpression and miR-195-5p inhibition rescued the migratory abilities of both cell lines even after lncSNHG12 silencing (Figures 2L–N). In this regard, these results verified that downregulation of lncSNHG12 suppressed metastasis and proliferation of tumor cells by regulation of miR-195-5p/IGF1R signaling.
IGF1R overexpression reversed the inhibitory effect of miR-195-5p on cell migration and growth in vitro To verify the association between miR-195-5p and IGF1R, both MG-63 and 143B cells were transfected with miR-195-5p mimics, which were integrated with or without vectors overexpressing IGF1R. The qRT-PCR results showed that miR-195-5p expression was highly increased after transfection with the miR-195-5p mimic, while IGF1R overexpression had no effect on miR-195-5p expression (Figures 3A and 3B). The results of western blot analyses validated that miR-195-5p
overexpression suppressed IGF1R expression, which was recovered after transfection with vectors overexpressing IGF1R (Figures 3C and 3D). The results of the CCK8 and cloning formation assays validated that miR-195-5p overexpression suppressed the proliferation of both MG-63 and 143B cells, while IGF1R overexpression rescued the miR-195-5p suppressive effects (Figures 3E–I). As a measure of the effects of miR-195-5p and IGF1R on metastasis, the transwell migration assay results illustrated that
miR-195-5
overexpression
suppressed
cell
migration,
while
IGF1R
overexpression promoted the migration of both MG-63 and 143B cells (Figures 3J– L). These findings illustrate that expression of miR-195-5 suppressed tumor cell metastasis and proliferation via the suppression of IGF1R.
The associations among miR-195-5p, lncSNHG12, and IGF1R In regard to the associations among miR-195-5p, lncSNHG12, and IGF1R, lncSNHG12 interacted with miR-195-5p. The luciferase reporter assay was employed to determine if miR-195-5p is a candidate target of ncSNHG12 (Figure 4A). The results showed that lncSNHG12 inhibited the luciferase activities of the WT cell, but not the MUT cells (Figure 4B). To determine if IGF1R is a target of miR-195-5p, statistics analyses were utilized to verify if miR-195-5p directed the associations of the 3'-UTR of IGF1R and found that IGF1R expression was suppressed at the mRNA level (Figure 4C). The results of the luciferase reporter assay verified that miR-195-5p inhibited the luciferase activity of the WT cells (Figure 4D). These findings showed that silencing of lncSNHG12 inhibited the growth and metastasis of OS cells by targeting the miR-195-5p/IGF1R axis.
Discussion Increasing evidence has confirmed that lncRNAs have important functions in gene imprinting, the immune response, cell differentiation, the occurrence of human diseases, tumor formation, and other biological functions (Esteller, 2011; Guttman and Rinn, 2012). In the present study, lncSNHG12 expression was increased in OS
cells. LncSNHG12 was initially identified as an oncogene that promotes the development and onset of cancers of the central nervous system (Lei et al., 2018), stomach (Yang et al., 2018a), lung (Wang et al., 2017b), and breast (Wang et al., 2017a). Also, knockdown of lncSNHG12 suppressed the growth of OS cells in vivo. The results of the in vitro experiment verified that downregulation of lncSNHG12 suppressed the migration and proliferation of OS cells by promoting miR-195-5p expression. Treatment with the miR-195-5p inhibitor rescued the migration and proliferation of OS cells even after downregulation of lncSNHG12. Fluorescein staining also showed that lncSNHG12 decreased miR-195-5p levels by interacting with miR-195-5p. As a miR-15 family member, the function of miR-195-5p has been verified in various cancers of the thyroid, liver, central nervous system, and breast (Zhu et al., 2015; Wang et al., 2017c; Shou et al., 2019). The results of a previous study suggested that miR-195-5p was highly downregulated during the progression of OS (Qu et al., 2017). Meanwhile, the results of the current study found that miR-195-5p overexpression might suppress the migration and proliferation of OS cells via downregulation of IGF1R. Overexpression of IGF1R decreased the inhibitory effect of miR-195-5p on the proliferation and migration of OS cells. Fluorescein staining also confirmed that miR-195-5p was associated with the IGF1R 3'-UTR and suppressed IGF1R expression at the mRNA level. IGF1R is frequently overexpressed in tumors of the colon, prostate, rectum, breast, thyroid, and ovary, and regulates the proliferation and survival of tumor cells (Riedemann and Macaulay, 2006; Zorea et al., 2018). Moreover, IGF1R signaling functions in tumor cell adhesion and motility, thereby contributing to the metastasis of tumor cells (Wang et al., 2017d), suggesting that IGF1R is a potential target in tumor cells. In summary, the findings of the current study provide evidence that lncSNHG12 has important roles in the progression of OS. Downregulation of lncRNA SNHG12 reversed the IGF1R-induced metastasis and proliferation of OS cells via targeting miR-195-5p. These results confirmed that the lncRNA SNHG12/miR-195-5p/IGF1R axis could serve as a new therapeutic design strategy for the treatment of OS.
Conflict of Interest None declared.
Acknowledgements The study was funded by the General Program of the Natural Science Foundation of Heilongjiang Province (grant no. H2018036), the Heilongjiang Province’s Scientific Research Project of the Health and Family Planning Commission (grant no. 2017-052), and the First Affiliated Hospital of Harbin Medical University Scientific Research Innovation Foundation (grant no.2017B021).
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Figure legends Figure 1. The role of lncRNA SNHG12 (lncSNHG12) in OS. (A) The results of qRT-PCR analysis were plotted to define the lncSNHG12 expression profiles in the OS cell lines SJSA1, MG-63, MHM, and 143B, and the normal osteoblast cell line hFOB 1.19. Data are presented as the mean ± SD.
***p
< 0.001 vs. hFOB 1.19 cells.
(B) qRT-PCR was employed to determine the lncSNHG12 expression patterns in adenovirus-transfected 143B cells (sh-lncSNHG12) and control transfected cells (sh-NC). Data are presented as the mean ± SD.
**p
< 0.01,
***p
< 0.001 vs. sh-NC.
(C) Sum of weekly mouse tumor volumes. Data are presented as the mean ± SD. **p < 0.01,
***p
< 0.001 vs. sh-NC. (D) Representative photographs of the formation of
tumors in xenografts of nude mice (n = 6). (E) Tumor weights were measured at 30 days after injection. Data are presented as the mean ± SD. ***p < 0.001 vs. sh-NC. (F and G) Immunohistochemical analysis of the proportion of Ki-67-positive cells. The
relative amounts of Ki-67-positive cells were calculated. Data are presented as the mean ± SD.
***p
< 0.001 vs. sh-NC. (H) qRT-PCR analysis of miR-195-5p
expression. Data are presented as the mean ± SD.
***p
< 0.001 vs. sh-NC. (I–J)
Western blot analysis of IGF1R expression in tumor tissues. Data are presented as the mean ± SD. ***p < 0.001 vs. sh-NC. NC, negative control.
Figure 2. Silencing of lncSNHG12 inhibited the proliferation and migration of OS cells by regulation of the miR-195-5p/IGF1R axis in vitro. (A–D) The qRT-PCR detection of lncSNHG12 (A-B) and miR-195-5p (C-D) expression after transfection with siRNA against lncSNHG12 combined with or without the miR-195-5p inhibitor and vectors overexpressing IGF1R in both MG-63 (A and C) and 143B (B and D) cells. Data are presented as the mean ± SD.
###p
< 0.001 vs. silncSNHG12.
***p
<
0.001 vs. NC. (E–F) Western blots showing IGF1R expression. Relative protein levels were analyzed and data are presented as the mean ± SD.
***p
< 0.001 vs. NC. ###p <
0.001 vs. silncSNHG12. (G and H) CCK8 assays were performed to assess cell proliferation. Data are presented as the mean ± SD.
***p
< 0.001 vs. NC. (I–K)
Cloning formation assay showing the proliferation of MG-63 and 143B cells. Data are presented as the mean ± SD. ***p < 0.001 vs. NC. ###p < 0.001 vs. silncSNHG12. (L– N) The migration of both MG-63 and 143B cells was determined using the transwell migration assay. Data are presented as the mean ± SD. *p < 0.05, ***p < 0.001 vs. NC. ###p
< 0.001 vs. silncSNHG12. GADPH, glyceraldehyde 3-phosphate dehydrogenase
(loading control); NC, normal control.
Figure 3. IGF1R overexpression reversed the miR-195-5p inhibitory effect on cell growth and migration in vitro. Both MG-63 and 143B cells were transfected with miR-195-5p mimics combined with or without vectors overexpressing IGF1R. (A and B) qRT-PCR detection of the expression of miR-195-5p in both MG-63 and 143B cells. Data are presented as the mean ± SD.
***p
< 0.001 vs. NC.
###p
< 0.001 vs.
mimic. (C and D) Western blot showing IGF1R expression in both MG-63 and 143B cells. Relative protein levels were analyzed and data are presented as the mean ± SD.
***p
< 0.001 vs. NC. ###p < 0.001 vs. mimic. (E and F) CCK8 assays were performed
to assess cell proliferation. Data are presented as the mean ± SD. ***p < 0.001 vs. NC. ##p
< 0.01,
###p
< 0.001 vs. mimic. (G–I) Cloning formation assay of cell
proliferation. Data are presented as the mean ± SD.
***p
< 0.001.
###p
< 0.001 vs.
mimic. (J–L) Cell migration ability was determined using the transwell migration assay. Data are presented as the mean ± SD.
***p
< 0.001 vs. NC.
###p
< 0.001 vs.
mimic. NC, negative control.
Figure 4. The interactions and relationships among miR-195-5p, lncSNHG12, and IGF1R. (A) The predicted lncSNHG12 miR-195-5p-binding sites. The MUT version of lncSNHG12 is also given. (B) Relative luciferase activity was defined at 48 h after transfection with the miR-195-5p mimic/NC or lncSNHG12 MUT/WT in 293T cells. Data are presented as the mean ± SD.
***p
< 0.001. (C) The predicted miR-223
binding sites with the 3'-UTR of FGFR2. The mutated version of the 3'-UTR-FGFR2 is given. (D) Relative luciferase activity was determined at 48 h after transfection with the miR-195-5p mimic/NC or 3'-UTR-IGF1R WT/MUT in 293T cells. Data are presented as the mean ± SD. ***p < 0.001. 1 Downregulation of lncSNHG12 suppressed aggressive OS metastasis and growth in both in vitro and in vivo experiment. 2 LncSNHG12 promotion OS metastasis and growth via targeting the miR-195-5p/IGF1R axis, which represents a candidate marker and target for OS treatment and management. lncSNHG12, Long non-coding RNA SNHG12; IGF1R, insulin growth factor 1 receptor; OS, osteosarcoma; lncRNAs, long noncoding RNAs; miRNAs, microRNAs; DMEM, dulbecco's modified Eagle’s medium; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: