miR-19 promotes osteosarcoma progression by targeting SOCS6

Accepted Manuscript miR-19 promotes osteosarcoma progression by targeting SOCS6 Zhengwen Sun, Qingxia Liu, Huanyu Hong, Haiguang Zhang, Guoqing Zhang PII:

S0006-291X(17)31964-2

DOI:

10.1016/j.bbrc.2017.10.002

Reference:

YBBRC 38619

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 3 September 2017 Accepted Date: 1 October 2017

Please cite this article as: Z. Sun, Q. Liu, H. Hong, H. Zhang, G. Zhang, miR-19 promotes osteosarcoma progression by targeting SOCS6, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.10.002. 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 miR-19 promotes osteosarcoma progression by targeting SOCS6 Zhengwen Sun1, Qingxia Liu2, Huanyu Hong1, Haiguang Zhang1, Guoqing Zhang1* Address: 1. Department of Surgery, Yantai mountain hospital, Yantai city, Shandong, 264000, China

264000, China *Corresponding authors. E-mail: [email protected]

SC

Running Title：miR-19 promotes osteosarcoma progression

RI PT

2. Maternity and Child Care Centers, Yantai mountain hospital, Yantai city, Shandong,

EP

TE D

M AN U

Abstract microRNAs (miRNAs) play critical roles in cancer development and progression. This study investigated the effects of miR-19 in human osteosarcoma (OS) development. Here, we showed that miR-19 was frequently upregulated in OS tissues and cell lines. Moreover the expression of miR-19 was associated with TNM stage, metastasis, size and poor overall survival. Mechanistically, miR-19 dramatically suppressed OS growth in vitro and in vivo. Bioinformatics analyses predicted that SOCS6 is a potential target gene of miR-19 in OS, which was confirmed by luciferase-reporter assay. We also found that SOCS6 expression was downregulated and negatively correlated with miR-19 expression in OS tissues clinically. Moreover, ectopic SOCS6 could reverse miR-19 induced OS growth. Finally, JAK2/STAT3 signaling pathway involves miR-19/SOCS6-mediated OS progression. Together, our data provide important evidence for miR-19 mediated SOCS6 in OS growth and revealed miR-19/SOCS6/JAK2/STAT3 pathway as a potential therapeutic strategy for OS patients

AC C

Key words: miR-19, SOCS6, JAK2/STAT3 pathway, osteosarcoma, growth Introduction Osteosarcoma is the most common bone malignancy encountered in childhood and adolescence[1], with rapid progression, high metastatic potential and poor clinical prognosis. Efforts to elucidate the underlying causes of osteosarcoma and to develop more effective therapies have thus far met with only limited success [2]. microRNAs (miRNAs), as a class of short, non-coding RNAs (~22 nt), known to negatively regulate their target genes through directly binding with the 3′untranslated regions (UTRs) of target mRNAs [3]. More and more evidences demonstrate that miRNAs plays critical roles in various biological processes, including cell proliferation, apoptosis, differentiation[2], dysregulated miRNAs also observed in osteosarcoma, regulating the initiation and progression of osteosarcoma. For example, the low expressed miR-486 was observed in 40 osteosarcoma tissues, and exogenetic miR-486 repressed osteosarcoma

ACCEPTED MANUSCRIPT

TE D

M AN U

SC

RI PT

progression[3]. miR-126 expression was evidently downregulated in osteosarcoma cell lines and osteosarcoma tissues, overexpression of miR-126 significantly inhibited cell proliferation, migration, invasion[4]. miR-100, miR-101, miR-126 and miR-195 also reported as oncogene and downregulated in osteosarcoma [4,5,6,7]. Moreover, miR-107, miR-373, miR-543 and miR-574 were significantly up-regulated and contributes to tumour growth in osteosarcoma[8,9,10,11]. MicroRNA-19 (miR-19), a key oncogenic component of the polycistronic miR-17∼92 cluster, confers tumorigenicity in multiple myeloma[12], retinoblastoma[13] and pancreatic tumor[14]. High serum miR-19a expression correlates with worse prognosis of patients with non-small cell lung cancer[15] and MicroRNA-19 induced EMT of lung cancer[16]. miR-19, as the key oncogenic, promote leukaemogenesis in Notch1-induced T-cell acute lymphoblastic leukaemia (T-ALL)[17]. miR-19 promotes the migration and invasiveness of breast cancer, and that miR-19 expression positively correlates with breast cancer aggressiveness[18]. However, the expression and role of miR-19 in osteosarcoma development and progression remains unclear. SOCS6 is a member of the suppressor of cytokine-induced signaling (SOCS) family, which are well-known negative regulators of cytokine receptor signaling[19]. Recently, SOCS6 was demonstrated to play critical roles in tumorigenesis and tumor development in different cancers, including gastric cancer[20], prostate cancer[21], colorectal carcinoma[22], non-small cell lung cancer [23] and gastric cancer[24]. However, the involvement of SOCS6 in osteosarcoma has not been elucidated. In this study, miR-19 was shown to be frequently upregulated in OS tissues and might act as an oncogene. miR-19 markedly induces OS cell growth by targeting SOCS6 via JAK2/STAT3 pathway.

AC C

EP

MATERIALS AND METHODS Cell lines, plasmids and miRNAs The Human OS cell lines (U2OS, HOS, Saos-2 and MG-63) and the normal human osteoplastic cell line (NHOst) were obtained from the Chinese Academy Medical Science (Beijing, China). The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37°C with 5% CO2. The has-miR-19 mimics, has-miR-19 inhibitor and corresponding negative controls were synthesized from RiBoBo (Guangzhou, China). The pcDNA-SOCS6 plasmid were purchased form Genechem Co.,Ltd. (Shanghai, China). The OS cell lines U2OS and MG-63 were infected with lenti-miR-19 or lenti-NC, according to the manufacturer’s instructions, and stable cells were isolated by flow cytometry to sort EGFP-positive cells. Tumor samples A total of 50 resected OS specimens and the paired normal bone tissue (NT) were collected from Yantai mountain hospital. Written informed consent was obtained from all study participants. All the tissues were immediately stored in liquid nitrogen until use. None of the patients received chemotherapy or radiotherapy prior to surgery. Tissue sample collection and use protocols were approved by the ethical review committees of the Xiangya Hospital Ethic Committee of Central South University.

ACCEPTED MANUSCRIPT

RI PT

Quantitative real-time PCR Toral RNA was extracted from human osteosarcoma specimen and cells using Trizol solution (Sigma-Aldrich, MO), and then 3 µg RNA was converted to cDNA and for qRT-PCR and was performed using SYBR Premix Ex Taq II (Takara, Japan) on the CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories, CA). and fold changes were calculated by relative quantification (2-△△Ct).. The miR- 19 expression was normalized to that of U6; the SOCS6 expression level was normalized to that of GAPDH.

SC

Cell proliferation assay U2OS and MG-63 cells were transfected with miR-19 mimics or inhibitors for 24 h, and the MTT (tetrazoliumsalt 3-[4,5-dimethylthiazole-2-yl]- 2,5-diphenyltetrazolium bromide) was used to detected the proliferation ability as previously described[5].

M AN U

Colony formation assay In order to evaluate colony formation, 1000 transfected OS cells were plated into a 6-well plate and cultured in DMEM supplemented with 10% FBS for two weeks. Then the cells were fixed and stained with methanol for 20 min, followed by 0.5% crystal violet for 15 min. The colonies with >50 cells/colony were quantified using an inverted microscope (IX83; Olympus Corporation, Japan).

EP

TE D

Flow cytometry analysis The treated U2OS and MG-63 cells were centrifuged at 1,200×g for 5 min, and then resuspended using PBS containing 70% ethanol, RNaseA (0.5 mg/ml) and propidium iodide (0.1 mg/ml), and immediately analyzed by flow cytometry (Beckman Coulter, Fullerton, CA). Data were analyzed with the Flowjo software (Tree Star Corp, Ashland, OR) and values were expressed as the mean and standard deviation of three independent experiments.

AC C

Western blotting Cells were washed twice with PBS and then harvested and lysed on ice for 30 min in lysis buffer containing protease inhibitors. And then, the protein was separated by 10% SDS-PAGE. Subsequently, proteins were separated on SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad, Hercules, USA). And then, the membranes were incubated with IL 6R anti-body (Abcam, Southampton, UK), VEGF anti-body and GAPDH (Bioworld Technology [Nanjing, China]). The protein complex was detected with enhanced chemiluminescence reagents (Pierce IL, USA). Luciferase reporter assay The 3′ UTR of SOCS6 containing miR-19 binding site was amplified and cloned to pGL3 vector to obtain the SOCS6-WT (wild type). Moreover, we mutated the binding site of SOCS6 and coned to pGL3 to obtain the SOCS6-Mut (mutant type) by a Site-Directed Mutagenesis Kit (SBS Genetech). U2OS, MG-63 and HEK293T cells were cotransfected with the SOCS6-WT/SOCS6-Mut vectors and miR-19 or the miRNA control. Luciferase

ACCEPTED MANUSCRIPT activity values were determined using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI).

SC

RI PT

Xenograft tumor model 6-week-old specific-pathogen-free (SPF) nude mice were randomly divided into 2 groups (5 mice per group). All the experiments were approved by the Medical Experimental Animal Care and Use Commission of The Yantai mountain hospital and all efforts were performed to relieve animal suffering. 5×10 6 MG-63 cells silenced miR-19 or NC were subcutaneously injected subcutaneously into the flank region of nude mice to establish the osteosarcoma xenograft model. The weight of Xenograft tumor mice was measured once every five days. A total of 35 days after inoculation, the animals were killed and the xenografts were isolated, the weight (g) and volume (mm3) of the xenografts were determined.

TE D

M AN U

Statistical analysis All data were analyzed by SPSS version 18.0 statistical software (SPSS, Chicago, IL, USA). Measurement data were expressed by mean ± standard deviation. The t test was used for comparisons between two groups. One-way analysis of variance (One-Way ANOVA) was applied for comparisons between multiple groups by followed by Turkey multiple comparison post-hoc analysis. A linear regression analysis was used, and bivariate correlations were calculated by Spearman's Rank Correlation Coefficients. Survival curves were delineated employing Kaplan-Meier method, and the differences were calculated using log-rank test. Statistical significance was concluded at *P < 0.01; #represents no statistical significance.

AC C

EP

RESULTS miR-19 expression is frequently upregulated and correlated with OS progression miR-19 was previously reported as an oncogenic and overexpressed in various cancers[12,13,14]. However, the expression and role of miR-19 in osteosarcoma remains unclear. Herein, we measured the expression levels of miR-19 in 42 OS tissues and 4 OS cell lines. Notably, miR-19 expression was dramatically upregulated in OS tissues compared with the paired normal bone tissue (non-tumor) by qRT-PCR (Figure 1A). We then determined the relationship between miR-19 and tumor metastasis of OS tissue according to their metastatic status. As shown in Figure 1B, miR-19 expression in the metastatic osteosarcoma tissues was significantly higher than that in nonmetastatic tissues. The clinical association analysis indicated that miR-19 expression was positively correlated with TNM stage, metastasis and size (Table 1). Kaplan-Meier analysis indicated that low miR-19 expression was associated with poorer overall survival (log-rank test, P=0.001, Figure 1C). Finally, we analyzed the levels of miR-19 in four OS cell lines and human osteoblastic cell line (NHOst). In accordance with the potential anti-tumour function of miR-19 in OS tissues, miR-19 expression was also upregulated in OS cell lines (U2OS, HOS, Saos-2 and MG-63) and the normal human osteoplastic cell line (NHOst) (Figure 1D). Together, our data suggest that miR-19 might function as oncogene and promote OS development.

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

miR-19 suppressed osteosarcoma growth in vitro and vivo To comparatively analyze tumor-relevant functional effects of the miR-19 in OS progression, lenti-miR-19-inhibitor was used to silence miR-19 expression and the effects of miR-19 on OS growth were demonstrated in vitro and vivo. Firstly, miR-19 mimics was used to force miR-19 expression, while miR-19 inhibitor was used to silence miR-19 expression (Figure 2A). Interesting, MTT experiments showed that ectopic miR-19 greatly enhanced the growth rates, whereas silenced miR-19 significantly inhibited the proliferation in both U2OS and MG-63 cells (Figure 2B). The pro-proliferation function of miR-19 was further confirmed using colony formation assays (Figure 2C). Additionally, cell-cycle assays showed that overexpressed miR-19 lead to the reduction of G1-S arrest and an increase of S phase, whereas silenced miR-19 presented the opposite phenotype in OS cells (Figure2D). These results were further supported by tumor formation in vitro. MG-63 cell stably silencing miR-19 were inoculated into nude mouse. The tumor volumes were measured every week, and tumor weight detected at the fifth week after cell transplantation. The results demonstrated that compared with the control, miR-19 knockdown significantly repressed the tumorigenesis (Figure2E). Together, these data clearly indicate that miR-19 functions as an oncomiR in OS.

AC C

EP

TE D

MiR-19 decreases SOCS6 expression by directly binding to its 3ʹ-UTR miRNAs were reported to involve in tumorgenesis by repressing its target gene directly. Therefore, we characterized the functional effects and target genes of miR-19 in OS. Herein, we used Targetscan (http://www.targetscan.org) and miRanda to predict target gene of miR-19. As shown in Figure 3A, SOCS6 has a putative target sequence for miR-19 and identified as a direct target gene of miR-19 in OS. We use a luciferase reports to further confirm this speculation. We transfected putative binding sequences of wild-type (WT), mutant-type (Mut) of SOCS6, miR-19 mimics or miR-control into U2OS and MG-63 cells. As speculated, miR-19 evidently inhibited the luciferase activity in WT group, but failed to affect the luciferase activity in SOCS6 Mut group (Figure 3B). Furthermore, we detected the effects of miR-19 on SOCS6 expression in both U2OS and MG-63 cells. We found that forced expression of miR-19 inhibited SOCS6 expression levels, while a strong enhancement of SOCS6 expression can be observed in miR-19 silenced group (Figure 3C). Finally, we measured the expression levels of SOCS6 in OS tissues and cell lines. Notably, SOCS6 expression was dramatically downregulated in OS tissues and cell lines compared with the paired normal bone tissue (NT) and NHOst cell line(Figure 3D and E)., whereas the relationship between miR-19 and SOCS6 was inversely in OS (Figure 3F). These results suggest that miR-19 regulated SOCS6 expression by directly target the 3’UTR of SOCS6 in OS cells. miR-19 regulated osteosarcoma growth by target SOCS6 To further explore the biological effects of SOCS6 in miR-19 induced OS growth, we used lentivirus miR-19 to overexpress miR-19 in OS cell and then transfect with SOCS6 plasmid in these cell to reverse SOCS6 expression (Figure 4A). As estimated, SOCS6 plasmid evidently reversed miR-19-repressed SOCS6 expression. And then, MTT assay,

ACCEPTED MANUSCRIPT colony formation assays and cell-cycle assays were used to demonstrate whether SOCS6 is responsible for miR-19-induced growth. As shown in Figure4B-D, SOCS6 evidently revised miR-19-induced cell proliferation, colony formation and increase of S phase in both U2OS and MG-63 cells. Collectively, these data indicate that SOCS6 is responsible for the tumor growth s induced by miR-19 overexpression in OS.

M AN U

SC

RI PT

miR-19/ SOCS6 regulates JAK2/STAT3 signalling pathway in osteosarcoma JAK2/STAT3 pathway are critical signaling involved in the development and homeostasis in mammals and recent has been reported to involves in the pathogenesis of various cancers including osteosarcoma[25,26]. Therefore, we analyzed whether Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) pathway plays important role in miR-19/SOCS6 induced OS growth. In the present study, western blot analysis was performed to assess the expression level of total JAK2 and STAT3, and the expression level of the phosphorylated forms of JAK2 and STAT3. As shown in Figure 4E, miR-19 significantly induced JAK2/STAT3 pathway, which was reversed by overexpressed SOCS6. Together, these results indicated that miR-19 regulate tumor growth via JAK2/STAT3 signaling pathway.

TE D

Discussion Studies are currently focusing on miRNAs, dysregulated in human cancers, which function as oncomiRs or anti-oncomiRs and related to cell growth, development, and differentiation of various cancers. As a part of our ongoing effort for the identification of natural products with anti-cancer effects in vitro and in vivo, we present here first time that miR-19 upregulated in OS tissues and cell line, directly repressing SOSC6 expression, which regulates OS growth through JAK2/STAT3 pathway.

AC C

EP

miR-17~ 92, also called OncomiR-1, is one of the earliest described oncogenic miRNAs [14,27]and has been implicated in a variety of cancer contexts by impairing tumor growth and survival[12,28,29]. Previous observations also indicated that miR-19 is a key oncogenic component of the miR-17-92 cluster[30], is frequently upregulated in several cancers including breast cancer[31], lung cancer[16], Colorectal Cancer[32] and so on. However, little was known about the functional role and mechanistic action of miR-19 in OS. In our current study, we found that miR-19 was upregulated in OS tumor samples and cell lines. The clinical association analysis showed that the increased miR-19 expression positively correlated with metastases, size, and TNM stage. Moreover, miR-19 was closely associated with prognosis and the low level of miR-19 may serve as an independent biomarker for poor prognosis in OS. More importantly, functional experiments revealed that miR-19 induces OS growth in vivo and in vitro. These results indicated that indicated that miR-19 could act as a tumor suppressor in OS. It is well known that miRNAs perform their biological functions by repressing its target mRNAs at post-transcriptional level via binding the 3’UTR of mRNA directly. This study first time revealed the important role of miR-19 in OS, therefore, understanding the

ACCEPTED MANUSCRIPT

EP

Acknowledgments None

TE D

M AN U

SC

RI PT

molecular regulation of miR-19 imply potential mechanism of OS development and contribute to novel therapeutic strategies. Herein, the bioinformational method predicted and luciferase reporter assay confirmed that SOSC6 is a direct target of miR-19 in OS. SOCS6 is a member of the suppressor of SOCS family and was demonstrated to play critical roles in tumorigenesis and tumor development in several cancers[33,34]. In this study, low expression of SOCS6 was observed in OS tissue and cell lines. We also analyzed the relationship between SOCS6 and miR-19 in OS. We found that SOCS6 expression is negatively correlated with miR-19 in OS tissue, moreover, overexpressed SOCS6 reversed miR-19 induced OS growth. In the past years, several signal pathways have been identified that are critical for tumorgenesis. The signal transducers and activators of transcription 3 (STAT3) is a latent transcription factor and the STAT3 phosphorylation is mediated through the activation of non-receptor protein tyrosine kinases family of Janus-like kinase (JAK). The JAK2/STAT3 pathway has shown to have roles in the oncogenesis in several cancers and recent represent an important therapeutic target of OS[35]. For example, 4-parvifuran and Cucurbitacin B emerge the therapeutic role for OS through regulating the JAK2/STAT3 pathway in osteosarcoma cells[26,36]. Park et al. and Liu et al. also revealed the antitumor activity of 4-Methoxydalbergione and Pterostilbene against human osteosarcoma cells by inhibiting the JAK2/STAT3 signaling pathway[25,37]. In the present study, miR-19 evidently induced the phosphorylation of JAK2 and the phosphorylation of STAT3 which demonstrated that miR-19 regulated tumor growth via JAK2/STAT3 signaling pathway. Taken together, the current study provides the first evidence that miR-19b repress SOCS6 expression directly to promote tumorigenesis of OS via JAK2/STAT3 signaling pathway. This finding promotes our understanding of OS progression and may provide great therapeutic potential in the diagnosis and treatment of OS.

AC C

Conflicts of Interest The authors declare no conflict of interest.

ACCEPTED MANUSCRIPT References [1] X.H. Sun, X.L. Geng, J. Zhang, C. Zhang, miRNA-646 suppresses osteosarcoma cell metastasis by downregulating fibroblast growth factor 2 (FGF2), Tumour Biol 36 (2015) 2127-2134. [2] X.M. Xu, J.C. Qian, Z.L. Deng, Z. Cai, T. Tang, P. Wang, K.H. Zhang, J.P. Cai, Expression of miR-21, miR-31, miR-96 and miR-135b is correlated with the clinical parameters of colorectal cancer, Oncol Lett 4 (2012) 339-345. [3] M. He, G. Wang, L. Jiang, C. Qiu, B. Li, J. Wang, Y. Fu, miR-486 suppresses the development of

RI PT

osteosarcoma by regulating PKC-delta pathway, Int J Oncol 50 (2017) 1590-1600.

[4] R. Jiang, C. Zhang, G. Liu, R. Gu, H. Wu, MicroRNA-126 inhibits proliferation, migration, invasion and EMT in osteosarcoma by targeting ZEB1,

(2017).

[5] Q. Xiao, Y. Yang, Q. An, Y. Qi, MicroRNA-100 suppresses human osteosarcoma cell proliferation and chemo-resistance via ZNRF2, Oncotarget (2017).

[6] R. Jiang, C. Zhang, G. Liu, R. Gu, H. Wu, MicroRNA-101 inhibits proliferation, migration and invasion

SC

in osteosarcoma cells by targeting ROCK1, Am J Cancer Res 7 (2017) 88-97.

[7] Q. Qu, X. Chu, P. Wang, MicroRNA-195-5p suppresses osteosarcoma cell proliferation and invasion by suppressing naked cuticle homolog 1, Cell Biol Int 41 (2017) 287-295.

M AN U

[8] H. Zhang, X. Guo, X. Feng, T. Wang, Z. Hu, X. Que, Q. Tian, T. Zhu, G. Guo, W. Huang, X. Li, MiRNA-543 promotes osteosarcoma cell proliferation and glycolysis by partially suppressing PRMT9 and stabilizing HIF-1alpha protein, Oncotarget 8 (2017) 2342-2355. [9] Y. Liu, Z. Cheng, F. Pan, W. Yan, microRNA-373 promotes growth and cellular invasion in osteosarcoma cells by activation of PI3K/AKT-Rac1-JNK pathway: The potential role in spinal osteosarcoma, Oncol Res (2016).

[10] R. Jiang, C. Zhang, G. Liu, R. Gu, H. Wu, MicroRNA-107 Promotes Proliferation, Migration, and

TE D

Invasion of Osteosarcoma Cells By Targeting Tropomyosin 1, Oncol Res (2017). [11] H. Xu, X. Liu, J. Zhou, X. Chen, J. Zhao, miR-574-3p acts as a tumor promoter in osteosarcoma by targeting SMAD4 signaling pathway, Oncol Lett 12 (2016) 5247-5253. [12] L. Chen, C. Li, R. Zhang, X. Gao, X. Qu, M. Zhao, C. Qiao, J. Xu, J. Li, miR-17-92 cluster microRNAs confers tumorigenicity in multiple myeloma, Cancer Lett 309 (2011) 62-70.

EP

[13] K. Conkrite, M. Sundby, S. Mukai, J.M. Thomson, D. Mu, S.M. Hammond, D. MacPherson, miR-17~92 cooperates with RB pathway mutations to promote retinoblastoma, Genes Dev 25 (2011) 1734-1745.

AC C

[14] B. Quattrochi, A. Gulvady, D.R. Driscoll, M. Sano, D.S. Klimstra, C.E. Turner, B.C. Lewis, MicroRNAs of the mir-17~92 cluster regulate multiple aspects of pancreatic tumor development and progression, Oncotarget (2017).

[15] Q. Lin, T. Chen, Q. Lin, G. Lin, J. Lin, G. Chen, L. Guo, Serum miR-19a expression correlates with worse prognosis of patients with non-small cell lung cancer, J Surg Oncol 107 (2013) 767-771.

[16] J. Li, S. Yang, W. Yan, J. Yang, Y.J. Qin, X.L. Lin, R.Y. Xie, S.C. Wang, W. Jin, F. Gao, J.W. Shi, W.T. Zhao, J.S. Jia, H.F. Shen, J.R. Ke, B. Liu, Y.Q. Zhao, W.H. Huang, K.T. Yao, D.J. Li, D. Xiao, MicroRNA-19 triggers epithelial-mesenchymal transition of lung cancer cells accompanied by growth inhibition, Lab Invest 95 (2015) 1056-1070. [17] K.J. Mavrakis, A.L. Wolfe, E. Oricchio, T. Palomero, K. de Keersmaecker, K. McJunkin, J. Zuber, T. James, A.A. Khan, C.S. Leslie, J.S. Parker, P.J. Paddison, W. Tam, A. Ferrando, H.G. Wendel, Genome-wide RNA-mediated interference screen identifies miR-19 targets in Notch-induced T-cell acute lymphoblastic leukaemia, Nat Cell Biol 12 (2010) 372-379.

ACCEPTED MANUSCRIPT [18] J. Zhou, X. Zhang, H. Shi, C. Fan, MiR-19 regulates breast cancer cell aggressiveness by targeting profilin 1, FEBS Lett (2017). [19] N.N. Kabir, J. Sun, L. Ronnstrand, J.U. Kazi, SOCS6 is a selective suppressor of receptor tyrosine kinase signaling, Tumour Biol 35 (2014) 10581-10589. [20] Q. Wu, G. Luo, Z. Yang, F. Zhu, Y. An, Y. Shi, D. Fan, miR-17-5p promotes proliferation by targeting SOCS6 in gastric cancer cells, FEBS Lett 588 (2014) 2055-2062. [21] J.G. Zhu, Q.S. Dai, Z.D. Han, H.C. He, R.J. Mo, G. Chen, Y.F. Chen, Y.D. Wu, S.B. Yang, F.N. Jiang, W.H. association in prognosis, Mol Cell Biochem 381 (2013) 51-59.

RI PT

Chen, Z.L. Sun, W.D. Zhong, Expression of SOCSs in human prostate cancer and their [22] E. Letellier, M. Schmitz, K. Baig, N. Beaume, C. Schwartz, S. Frasquilho, L. Antunes, N. Marcon, P.V. Nazarov, L. Vallar, J. Even, S. Haan, Identification of SOCS2 and SOCS6 as biomarkers in human colorectal cancer, Br J Cancer 111 (2014) 726-735.

[23] X. Xue, Y. Liu, Y. Wang, M. Meng, K. Wang, X. Zang, S. Zhao, X. Sun, L. Cui, L. Pan, S. Liu, MiR-21 SOCS6, and PTEN, Oncotarget 7 (2016) 84508-84519.

SC

and MiR-155 promote non-small cell lung cancer progression by downregulating SOCS1, [24] R.H. Lai, Y.W. Hsiao, M.J. Wang, H.Y. Lin, C.W. Wu, C.W. Chi, A.F. Li, Y.S. Jou, J.Y. Chen, SOCS6,

M AN U

down-regulated in, inhibits cell proliferation and colony formation, Cancer Lett 288 (2010) 75-85.

[25] K.R. Park, H.M. Yun, T.H. Quang, H. Oh, D.S. Lee, Q.S. Auh, E.C. Kim, 4-Methoxydalbergione suppresses growth and induces apoptosis in human osteosarcoma cells in vitro and in vivo xenograft model through down-regulation of the JAK2/STAT3 pathway, Oncotarget 7 (2016) 6960-6971.

[26] Z.R. Zhang, M.X. Gao, K. Yang, Cucurbitacin B inhibits cell proliferation and induces apoptosis in

TE D

human osteosarcoma cells via modulation of the JAK2/STAT3 and MAPK pathways, Exp Ther Med 14 (2017) 805-812.

[27] P. Mu, Y.C. Han, D. Betel, E. Yao, M. Squatrito, P. Ogrodowski, E. de Stanchina, A. D'Andrea, C. Sander, A. Ventura, Genetic dissection of the miR-17~92 cluster of microRNAs in Myc-induced B-cell lymphomas, Genes Dev 23 (2009) 2806-2811.

EP

[28] L. Hong, M. Lai, M. Chen, C. Xie, R. Liao, Y.J. Kang, C. Xiao, W.Y. Hu, J. Han, P. Sun, The miR-17-92 cluster of microRNAs confers tumorigenicity by inhibiting oncogene-induced senescence, Cancer Res 70 (2010) 8547-8557.

AC C

[29] J. Grillari, M. Hackl, R. Grillari-Voglauer, miR-17-92 cluster: ups and downs in cancer and aging, Biogerontology 11 (2010) 501-506.

[30] V. Olive, M.J. Bennett, J.C. Walker, C. Ma, I. Jiang, C. Cordon-Cardo, Q.J. Li, S.W. Lowe, G.J. Hannon, L. He, miR-19 is a key oncogenic component of mir-17-92, Genes Dev 23 (2009) 2839-2849.

[31] X. Zhang, H. Yu, J.R. Lou, J. Zheng, H. Zhu, N.I. Popescu, F. Lupu, S.E. Lind, W.Q. Ding, MicroRNA-19 (miR-19) regulates tissue factor expression in breast cancer cells, J Biol Chem 286 (2011) 1429-1435. [32] D. Cellura, K. Pickard, S. Quaratino, H. Parker, J.C. Strefford, G.J. Thomas, R. Mitter, A.H. Mirnezami, N.J. Peake, miR-19-Mediated Inhibition of Transglutaminase-2 Leads to Enhanced Invasion and Metastasis in Colorectal Cancer, Mol Cancer Res 13 (2015) 1095-1105. [33] X. Qiu, J. Zheng, X. Guo, X. Gao, H. Liu, Y. Tu, Y. Zhang, Reduced expression of SOCS2 and SOCS6 in hepatocellular carcinoma correlates with aggressive tumor progression and poor prognosis, Mol Cell Biochem 378 (2013) 99-106.

ACCEPTED MANUSCRIPT [34] H.Y. Lin, R.H. Lai, S.T. Lin, R.C. Lin, M.J. Wang, C.C. Lin, H.C. Lee, F.F. Wang, J.Y. Chen, Suppressor of cytokine signaling 6 (SOCS6) promotes mitochondrial fission via regulating DRP1 translocation, Cell Death Differ 20 (2013) 139-153. [35] J. Yan, Q. Wang, K. Zou, L. Wang, E.B. Schwartz, J.R. Fuchs, Z. Zheng, J. Wu, Inhibition of the JAK2/STAT3 signaling pathway exerts a therapeutic effect on osteosarcoma, Mol Med Rep 12 (2015) 498-502. [36] H.M. Yun, K.R. Park, T.H. Quang, H. Oh, J.T. Hong, Y.C. Kim, E.C. Kim, 4-parvifuran inhibits

RI PT

metastatic and invasive actions through the JAK2/STAT3 pathway in osteosarcoma cells, Arch Pharm Res 40 (2017) 601-609.

[37] Y. Liu, L. Wang, Y. Wu, C. Lv, X. Li, X. Cao, M. Yang, D. Feng, Z. Luo, Pterostilbene exerts antitumor activity against human osteosarcoma cells by inhibiting the JAK2/STAT3 signaling pathway,

AC C

EP

TE D

M AN U

SC

Toxicology 304 (2013) 120-131.

ACCEPTED MANUSCRIPT

EP

Table 1

TE D

M AN U

SC

RI PT

Figure 1. MiR-19 expression in clinical OS tissues and cell lines. A, the levels of miR-543 in 42 pairs of OS and adjacent noncancerous tissues using qRT-PCR. B, Kaplan-Meier analysis indicated that low miR-19 expression was associated with poorer overall survival. C, qRT-PCR shows that miR-19 significantly upregulated in OS cell lines. *P < 0.01. Figure 2. MiR-19 promotes OS cell growth in vitro and in vivo. A, The effect of miR-19 mimics and inhibitors on miR-19 expression. B, The effect of miR-19 OS cell growth by using the MTT assay. C, The role of miR-19 in colony formation in OS. D, Flow cytometry analysis detected OS cells in G1, S, and G2/M phase. E, The role of miR-19 in tumor formation in a mice xenograft model. The results are presented as means ± SD. Statistical significance was concluded at *P < 0.01. Figure 3. MiR-19 decreases SOCS6 expression by directly binding to its 3ʹ-UTR. A, The putative target sequence for miR-19 on the 3’ UTR of SOCS6. B, luciferase reporter assay revealed the target role of miR-19 on the 3’ UTR of SOCS6. C. The effects of miR-19 on SOCS6 expression. D, The expression levels of SOCS6 in OS tissue and paired normal bone tissue. E, The expression levels of SOCS6 in OS cells. F, The relationship of miR-19 and SOCS6 in OS tissue. The results are presented as means ± SD. Statistical significance was concluded at *P < 0.01. Figure 4. The effects of miR-19/ SOCS6 on the JAK2/stAt3 pathway. SOCS6 plasmid reversed miR-19-repressed SOCS6 expression in OS cells. B, SOCS6 involved miR-19-induced cell proliferation by using MTT assay. C, SOCS6 involved miR-19-induced colony formation using colony formation assays. D, SOCS6 involved miR-19-regulated cell-cycle by using cell-cycle assays . E, miR-19/ SOCS6 regulates JAK2/STAT3 signalling pathway in osteosarcoma. The results are presented as means ± SD. Statistical significance was concluded at *P < 0.01 vs control group，**P < 0.01 vs miR-19 mimics group.

AC C

Relationship between miR-19 and clinical characteristics of osteosarcoma patients miRNA-19 expression (N) P-values Factor Characteristic Lower Higher Gender

Age (years) TNM stage Metastases

Recurrence

Male

7

20

Female

7

16

< 18

6

17

> = 18

8

19

I

8

12

II~III

6

24

Lung

6

20

Other

4

6

No

4

10

Yes

10

11

p = 0.430 p = 0.579 p = 0.024* p = 0.021*

p = 0.012**

ACCEPTED MANUSCRIPT No Tumor maximum diameter (cm)

4

25

1.98 ± 0.22

3.54 ± 0.23

P = 0.007∆

*

p < 0.05, p < 0.01, Chi-square test. ∆ P < 0.01, student's t test.

AC C

EP

TE D

M AN U

SC

RI PT

**

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

1. miR-19 was upregulated and SOCS6 was downregulated in OS. 2. miR-19 dramatically suppressed OS growth in vitro and in vivo. 3. SOCS6 is a direct target gene of miR-19 in OS. 4. miR-19 regulated SOCS6/JAK2/STAT3 pathway in OS.