MicroRNA-198 inhibited tumorous behaviors of human osteosarcoma through directly targeting ROCK1

Biochemical and Biophysical Research Communications xxx (2016) 1e9

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MicroRNA-198 inhibited tumorous behaviors of human osteosarcoma through directly targeting ROCK1 Shilian Zhang*, Yuehua Zhao, Lijie Wang Department of Pediatrics, Affiliated Hospital of Weifang Medical University, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 February 2016 Accepted 9 March 2016 Available online xxx

Osteosarcoma is an aggressive primary sarcoma of bone and occurs mainly in adolescents and young adults. The prognosis of OS remains poor, and most of them will die due to local relapse or metastases. The discovery of microRNAs provides a new possibility for the early diagnosis and treatment of OS. Thus, the aim of this study was to explore the expression and functions of microRNA-198 (miR-198) in osteosarcoma. The expression levels of miR-198 were determined by qRT-PCR in osteosarcoma tissues and cell lines. Cell proliferation assays, migration and invasion assays were adopted to investigate the effects of miR-198 on tumorous behaviors of osteosarcoma cells. The results showed that miR-198 expression levels were lower in osteosarcoma tissues and cell lines. In addition, low miR-198 expression levels were correlated with TNM stage and distant metastasis. After miR-198 mimics transfection, cell proliferation, migration and invasion were significantly suppressed in the osteosarcoma cells. Furthermore, ROCK1 was identified as a novel direct target of miR-198 in osteosarcoma. These findings suggested that miR-198 may act not only as a novel prognostic marker, but also as a potential target for molecular therapy of osteosarcoma. © 2016 Elsevier Inc. All rights reserved.

Keywords: microRNA-198 Osteosarcoma Growth Metastasis ROCK1

1. Introduction Osteosarcoma (OS), the most common primary malignant bone tumor, is an aggressive primary sarcoma of bone and occurs mainly in adolescents and young adults, comprising 2.4% of all malignancies in pediatric patients, and ~20% of all primary bone cancers [1e4]. Previous studies indicated that OS arises from primitive mesenchymal bone-forming cells that undergo aberrant alterations in the differentiation program [5]. In the past decade, development in the therapy of OS has improved the prognosis of patients, leading to an overall 5-year survival rate of ~70%. However, the prognosis of OS remains poor and most of them will die due to local relapse or metastases after surgical resection and intensive-chemotherapy [6,7]. The initiation and progression of OS is rather complicated by its extreme rearranged genome, lack of precursor lesions, and high genetic instability [8]. Accumulated evidences have validated a number of tumor-related signaling pathways and specific mediators of OS pathogenesis, progression, and prognosis. However, the

* Corresponding author. Shilian Zhang , Department of Pediatrics, Affiliated Hospital of Weifang Medical University. No.2428, Yuhe Road, Weifang, Shandong 2610410, China. E-mail address: [email protected] (S. Zhang).

molecular mechanisms underlying the formation and development of OS remains poorly-understood [9]. Therefore, it is urgent to elucidate the potential mechanism that mediates the initiation and progression of OS, and develop better prognosis, new therapeutic targets and approaches for OS treatment. microRNAs (miRNAs) are a group of small, non-coding, single strand and endogenous RNAs, ranging between 16 and 27 nucleotides in length [10]. Since their discovery nearly 20 years ago, bioinformatics and biological studies have identified more than 1000 miRNAs and could regulate thousands of human proteinencoding genes [11]. miRNA is important in the regulation of their target mRNAs [12]. They bind to the 30 untranslated regions (30 UTRs) of target mRNAs in a base pairing manner and induce their degradation or transcriptional repression [13]. Through this mechanism of posttranscriptional gene regulation, miRNAs play critical roles in a various biological processes in cancers, including cell proliferation, cell cycle, migration, invasion, apoptosis, and metastasis [14]. miRNAs have been demonstrated to act as tumor suppressors or oncogenes in human cancers, which is dependent on the roles of their target mRNAs [15,16]. The deregulated of miRNAs has been identified to be correlated with the progression and prognosis in various kinds of human cancers; however, the abnormal expression of miRNAs and their functions in

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Please cite this article in press as: S. Zhang, et al., MicroRNA-198 inhibited tumorous behaviors of human osteosarcoma through directly targeting ROCK1, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.040

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tumorigenesis are still largely unknown [17]. In this study, we found lower levels of miR-198 in OS tissues and cell lines than in paired adjacent non-tumor bone tissue and normal osteoblast cell line, respectively. In addition, low miR-198 expression levels were correlated with TNM stage and distant metastasis. The roles of the miR-198 in OS progression and the underlying molecular mechanisms were also investigated. Moreover, ROCK1 was validated as a direct target gene of miR-198 in OS. miR-198 suppressed the OS development via the down-regulation of ROCK1. Our results illustrated the roles of miR-198 in regulating the proliferation, migration and invasion of the OS cells, and suggested a potential therapeutic target for the treatment of OS. 2. Material and methods 2.1. Clinical tissue samples and cell lines This study was approved by the Ethics Committee of Affiliated Hospital of Weifang Medical University. A total of 76 clinical samples were obtained from patients with OS and paired adjacent nontumor bone tissue at Affiliated Hospital of Weifang Medical University. Those patients who received chemo- or radio-therapy were excluded from this study. All tissues were snap-frozen in liquid nitrogen for further analysis. Human OS cell lines (HOS, MG63, G293, SAOS2, U2OS), normal osteoblast cell line (hFOB) and HEK293T cell line were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). All cells were cultured at 37  C under 5% (v/v) CO2 in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY). The medium was supplemented with 10% (v/v) fetal bovine serum(FBS; Gibco), 100 U/ml penicillin (Gibco) and 100 U/ml streptomycin (Gibco). 2.2. Transfection Cells were seeded in 6-well plates and cultured for overnight. miR-198 mimics, corresponding negative control (NC), the siRNAs targeting ROCK1 (si-ROCK1) and corresponding negative control (si-NC) were synthesized and purified by GenePharma (Shanghai, China). ROCK1 overexpressed plasmid (pCDNA3.1-ROCK1) and blank vector pCDNA3.1 were purchased from Chinese Academy of Sciences (Changchun, China). Cells were transfected with these oligonucleotide using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction. 2.3. RNA isolation and quantitative real-time reverse transcriptionPCR (qRT-PCR) TRIzol reagent (Invitrogen) was used to isolate total RNA from tissues or cells. Total RNA was used to synthesize cDNA with PrimeScript reverse transcription-PCR kit (TaKaRa, Shiga, Japan), and subjected to quantitative real-time PCR. The relative expression levels of miR-198 were measured using a SYBR PrimeScript miRNA quantitative real-time polymerase chain reaction Kit (TaKaRa) following to the manufacturer's instructions, with U6 as an internal control. ROCK1 mRNA expression levels were quantified using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), with GADPH as an internal control. The reaction was performed on ABI 7500 Sequence Detection System (ABI, Vernon, CA, USA).

Cells were counted and seeded in 96-well plates in triplicates at a density of 4000 cells/well. Cell proliferation was measured at 24, 48, 72 and 96 h after transfection. 10 ml CCK8 solution was plated into each well of the 96-well plates. After incubation at 37  C for 2 h, the absorbance at 450 nm was measured with a microplate reader. Each assay was performed in triplicate and repeated three times. 2.5. Migration and invasion assays Cell migration assay was performed using 24-well transwell chambers containing 8-mm-pore-diameter polycarbonate membrane (Corning, New York, NY, USA). 200 ml cell suspension containing 4  104 cells was added into the upper chamber, and 500 ml culture medium containing 20% (v/v) FBS was added to the lower chamber. After incubation at 37  C under 5% (v/v) CO2 for 24 h, the non-filtered cells were gently removed with a cotton swab, and the migrated cells were fixed with 100% methanol, stained with 0.5% crystal violet and washed with phosphate-buffered saline (PBS; Gibco). The migrated cell number was counted under the microscope. The cell invasion assay was performed in a parallel manner, except that the transwell chambers were coated with Matrigel (BD Biosciences, San Jose, CA, USA). 2.6. Luciferase report assay The pMIR-ROCK1-30 UTR wild-type (Wt) and pMIR-ROCK1mutant-type (Mut) reporter vectors were synthesized and purified by GenePharma. Cells were seeded in 24-well plates at a density of 60% influence. After incubation overnight, luciferase reporter vectors, and miR-198 mimics or NC were transfected into cells using Lipofectamine 2000. Firefly and Renilla luciferase activities were detected by using Dual-Luciferase Reporter Assay System (Promega, Madison, WI, Germany) following to the manufacturer's instructions, 48 h after transfection. All transfection assays were carried out in triplicate.

30 UTR

2.7. Western blot Seventy-two hours after transfection, the total proteins from the transfected cells were extracted by ice-cold RIPA lysis buffer supplemented with 1 mM proteinase inhibitor PMSF (Sigma, St. Louis, MO, USA). The protein concentration was quantified with a BCA assay kit (Beyotime, Shanghai, China). Equal amounts of protein was separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride (PVDF, Millipore, Bedford, MA, USA) membrane, and then blocked with 5% non-fat milk in Tris-buffered saline. The membranes were incubated with primary antibodies, mouse antihuman monoclonal ROCK1 antibody (sc-365628; Santa Cruz Biotechnology, CA, USA) and mouse anti-human monoclonal GADPH antibody (sc-69778; Santa Cruz Biotechnology, CA, USA), at 4  C overnight. The membranes were washed and subsequently probed with secondary antibody, goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1/4000 dilution for 1 h at room temperature. Proteins were visualized with chemiluminescent detection system (ECL; Beyotime). GAPDH was used as internal control. 2.8. Statistical analysis

2.4. Cell proliferation assays The effect of miR-198 on OS cell proliferation was determined by using Cell Counting Kit-8 (CCK8; Dojindo, Kumamoto, Japan) assay.

The results were expressed as mean ± S.D. All data was analyzed using SPSS 13.0 statistical software (SPSS Inc, Chicago, IL, USA). Differences were considered significant at P value less than 0.05.

Please cite this article in press as: S. Zhang, et al., MicroRNA-198 inhibited tumorous behaviors of human osteosarcoma through directly targeting ROCK1, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.040

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3. Results 3.1. miR-198 was downregulated in OS tissues and cell lines

Table 1 Correlation between of miR-198 expression levels with clinicopathological features in osteosarcoma patients. Clinicopathological features

To gain insight into the biological roles of miR-198 in OS, miR198 expression levels in 76 paired OS tissues and adjacent nontumor bone tissues were measured. We found that miR-198 was significantly downregulated in OS tissues compared with that in paired adjacent non-tumor bone tissues (Fig. 1A, P < 0.05). Then, the relative expression levels of miR-198 in five OS cell lines and normal osteoblast cell line hFOB were detected by qRT-PCR. Results showed that miR-198 expression levels were lower in all five OS cell lines than in hFOB (Fig. 1B, P < 0.05). HOS and U2OS cell lines expressed lower miR-198 levels in five OS cell lines. Considering this, HOS and U2OS cells were chosen to perform following experiments.

3.2. Correlation between miR-198 expression levels and clinicopathologic features in patients with OS To explore whether miR-198 expression was correlated with clinicopathological features in OS patients, statistical analysis was performed. The results showed that decreased expression levels of miR-198 were correlated with TNM stage (P ¼ 0.040) and distant metastasis (P ¼ 0.001). However, there was no correlation between miR-198 expression levels and other clinicopathological factors, including gender, age, anatomical location and tumor size Table 1.

3

Gender Male Female Age < 40years 40 years Anatomical location Tibia/femur Elsewhere Tumor size (cm) <5 cm 5 cm TNM stage I-II III þ IV Distant metastasis Yes No

Case number

miR-198 expression Low

High

39 37

20 24

19 13

47 29

25 19

22 10

48 28

31 13

17 15

49 27

28 16

21 11

37 39

17 27

20 12

37 39

29 15

8 24

P Value

0.231

0.290

0.122

0.858

0.040

0.001

3.3. miR-198 suppressed OS cell proliferation, migration and invasion To investigate the effect of miR-198 in OS cell proliferation, HOS and U2OS cells were transfected with miR-198 mimics or NC. Transfection efficiency was assessed by qRT-PCR. As shown in Fig. 2A, miR-198 was strongly upregulated in HOS and U2OS cells transfected with miR-198 mimics (P < 0.05). Cell proliferation assays revealed that over expression of miR198 suppressed the proliferation of HOS and U2OS cells compared to NC (Fig. 2B, P < 0.05). Then, migration and invasion assays were conducted to analyze the effects of miR-198 on migration and invasion abilities of OS cells. We found that ectopic expression of miR198 induced a significant decrease in migration and invasion abilities of HOS and U2OS cells (Fig. 2C, P < 0.05). All these data demonstrated the tumor suppressive roles of miR-198 in OS. 3.4. ROCK1 was a direct target of miR-198 To explore the potential target of miR-198, miRanda (http:// www.microrna.org) and TargetScan 7.0 (http://www.targetscan. org/) were used to predicate miR-198 target genes. Among these candidate targets, ROCK1 attracted our attention (Fig. 3A). To validate whether miR-198 could directly target 30 UTR of ROCK1, luciferase reporter assays were performed. After cotransfection with the luciferase report vectors and miR-198 mimics or NC, over expression of miR-198 by miR-198 mimics resulted in a significant downregulation in the luciferase activities of the pMIR-ROCK1-30 UTR Wt (Fig. 3B, P < 0.05), whereas pMIRROCK1-30 UTR Mut blocked this effect. Furthermore, qRT-PCR and western blot was adopted to investigate the regulatory effects of miR-198 on ROCK1 expression at mRNA and protein levels, respectively. As shown in Fig. 3C and D, miR-198 overexpression significantly suppressed ROCK1 expression at both mRNA and protein levels in HOS and U2OS cells (P < 0.05). These findings suggested that ROCK1 was a direct target of miR-198 in OS.

Fig. 1. miR-198 expression was decreased in OS tissues and cell lines. (A) The expression levels of miR-198 in 76 paired OS tissues and adjacent non-tumor bone tissue. (B) The levels of miR-198 in five OS cell lines and normal osteoblast cell line hFOB were detected by qRT-PCR. *P < 0.05.

3.5. Downregulation of ROCK1 mimicked functions with miR-198 overexpression in OS cells The roles of ROCK1 on OS cell proliferation, migration and

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shown in Fig. 4C, transfection of si-ROCK1 in HOS and U2OS cells repressed cell migration and invasion, in comparison with si-NC (P < 0.05). These findings suggested that miR-198 inhibited OS cell proliferation, migration and invasion through downregulation of ROCK1. 3.6. Restoration of ROCK1 reversed the effects of miR-198 overexpression in OS cells To further determine whether the roles of miR-198 in OS were mediated by ROCK1, pCDNA3.1-ROCK1 or blank vector (pCDNA3.1) was transfected into HOS and U2OS cells. The transfection efficiency was assessed by western blot. As shown in Fig. 5A, ROCK1 was significantly increased in HOS and U2OS cells after transfection with pCDNA3.1-ROCK1 (P < 0.05). Then, cell proliferation assays, migration and invasion assays were performed in HOS and U2OS cells transfected with miR-198 mimics with/without pCDNA3.1-ROCK1. The results showed that restoration of ROCK1 dramatically reversed the tumor-suppressive roles of miR-198 overexpression on OS cell. proliferation (Fig. 5B, P < 0.05), migration and invasion (Fig. 5C, P < 0.05). These results indicated that miR-198 acted as a tumor suppressor in OS by directly targeting ROCK1. 4. Discussion

Fig. 3. ROCK1 was a direct target of miR-198. (A) The seed sequence of miR-198 targets the 30 UTR of ROCK1 (Wt and Mut). (B) Luciferase report assay in HEK293T cells cotransfected with luciferase report vectors and miR-198 mimics or NC. (C) ROCK1 mRNA expression levels were detected by qRT-PCR in HOS and U2OS cells transfected with the miR-198 mimics or NC. (D) ROCK1 expression was measured by Western blot in HOS and U2OS cells transfected with the miR-198 mimics or NC. *P < 0.05.

invasion were evaluated. Transfection efficiency of si-ROCK1 was determined by western blot. As shown in Fig. 4A, transfection of siROCK1 obviously reduced the expression levels of ROCK1 in HOS and U2OS cells (P < 0.05). To assess the effects of ROCK1 on OS cell proliferation, cell proliferation assays were performed. Results showed that transfection of si-ROCK1 decreased HOS and U2OS cells proliferation, comparing to si-NC (Fig. 4B, P < 0.05). In addition, we also examined the effects of ROCK1 on OS cell migration and invasion. As

OS is the most common primary malignant bone tumor occurring in childhood and adolescence, with high degree of malignancy and high mortality [18]. However, the molecular mechanisms of OS carcinogenesis and progression remains elusive. Increasing studies have demonstrated that the deregulation of miRNAs in cancers is a most common phenomena. Moreover, miRNAs are contributed to the growth and metastasis of several human cancers, also including OS [19e21]. Therefore, identification of specific miRNAs involved in growth and metastasis can provide new insight for diagnostic, prognostic biomarkers and therapeutic targets of OS. This study showed that miR-198 played suppressive roles in inhibiting the growth and metastasis of OS cells. First, we demonstrated that the expression levels of miR-198 were low in OS tissues and cell lines compared with paired adjacent non-tumor bone tissues and normal osteoblast cell line hFOB, respectively. In addition, low miR-198 expression levels were correlated with TNM stage and distant metastasis. Then, our data revealed that enforced miR-198 expression inhibited OS cell proliferation, migration and invasion in vitro. Moreover, a novel functional link between miR198 and ROCK1 in the context of OS development was demonstrated. All these results suggested that miR-198 played a substantial role in OS inhibition and may serve as a therapeutic target in OS patients. Several reports have indicated that miR-198 was downregulated in several kinds of human cancers, including gastric cancer [22], lung adenocarcinoma [23,24], non-small-cell lung cancer [25], colorectal cancer [26], and hepatocellular carcinoma [27]. However, in pancreatic ductal adenocarcinoma [28] and esophageal cancer [29], it was upregulated in tumor tissues. In this study, we found that miR-198 was downregulated in OS tissues and cell lines. Our work expanded the studies of miR-198 expression in human cancers. These contradiction results also indicated that miRNAs expression has tissue specificity. The expression of miR-198 was demonstrated to associate with

Fig. 2. Effects of miR-198 on OS cell proliferation, migration and invasion. (A) After transfection with miR-198 mimics or NC, miR-198 expression in HOS and U2OS cells was detected by qRT-PCR. (B) Proliferation of HOS and U2OS cells was significantly decreased after transfection with miR-198 mimics, compared to NC. (C) The migration and invasion abilities of HOS and U2OS cells transfected with miR-198 mimics or NC were evaluated by migration and invasion assays. *P < 0.05.

Please cite this article in press as: S. Zhang, et al., MicroRNA-198 inhibited tumorous behaviors of human osteosarcoma through directly targeting ROCK1, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.040

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Fig. 4. Effects of ROCK1 on OS cell proliferation, migration and invasion. (A) After transfection with si-ROCK1 or si-NC, ROCK1 in HOS and U2OS cells was detected by western blot. (B) Proliferation of HOS and U2OS cells was significantly decreased after transfection with si-ROCK1, compared to si-NC. (C) The migration and invasion abilities of HOS and U2OS cells transfected with si-ROCK1 or si-NC were evaluated by migration and invasion assay. *P < 0.05.

clinicopathological features in cancers. For instance, in gastric cancer, decreased miR-198 expression was obviously correlated

with larger tumor size, deeper invasion depth, positive lymph node metastasis, advanced tumor-node-metastasis (TNM) stage, and

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Fig. 5. Restoration of ROCK1 reversed the effects of miR-198 overexpression in OS cells. (A) After transfection with pCDNA3.1-ROCK1 or pCDNA3.1, ROCK1 levels in HOS and U2OS cells were measured by western blot. Restoration of ROCK1 dramatically reversed the tumor-suppressive roles of miR-198 overexpression on OS cell proliferation (B), migration and invasion (C).*P < 0.05.

shorter overall survival. Moreover, multivariate regression analysis identified low miR-198 expression as an independent predictor of poor survival in patients with gastric cancer [22]. Wu et al. also found that, in lung adenocarcinoma [23], its low expression was associated with TNM stage and lymph node metastasis. In esophageal cancer, high miR-198 expression levels had a significant correlation with survival time and that patients with a higher

expression of miR-198 had a shorter survival time. Moreover, cox multi-factor model analysis showed that miR-198 expression has a significantly association with patient prognosis, tumor length, and survival time [29]. These studies suggested that miR-198 may act as diagnostic and prognostic biomarkers for these cancers. Previous studies also showed that miR-198 was involved in biological processes in cancers. Wu et al. [23] and Yang et al. [25]

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reported that miR-198 inhibited proliferation and induced apoptosis in lung adenocarcinoma and in non-small-cell lung cancer, respectively. Wang and his colleagues [26] confirmed that miR-198 repressed tumor proliferation and metastasis in colorectal cancer. In hepatocellular carcinoma, Tan et al. [27] showed that miR-198 suppressed migration and invasion abilities in a c-MET dependent manner. Consistent with these finding, we demonstrated that miR-198 acted as a suppressive roles in OS growth and metastasis. These data suggested that miR-198 may be a therapeutic target in cancers. Identification of miR-198 targets is critical for understanding its roles in tumorigenesis, and also is essential for exploring novel therapeutic targets. Previous studies indicated that several target genes of miR-198 have been identified, such as SHMT1 in lung adenocarcinoma [23], FUT8 in colorectal cancer [26], FGFR1 in nonsmall-cell lung cancer [25], and HGF/c-MET pathway in hepatocellular carcinoma [27]. However, the detail molecular mechanism responsible for the roles of miR-198 in OS remains unclear. In this study, ROCK1 was identified as a novel target of miR-198. Bioinformatics analysis revealed that the 30 UTR of ROCK1 has a complementary site for the seed region of miR-198. In addition, we showed that miR-198 directly bound to the 30 UTR of ROCK1, which contains a miR-198-binding sites using a luciferase reporter assay. Moreover, qRT-PCR and western blot analysis indicated that miR198 over expression decreased ROCK1 expression in OS cells. Furthermore, ROCK1 underexpression by si-ROCK1 could mimics the effects of miR-198 overexpression in OS cells. Finally, restoration of ROCK1 reversed the effects of miR-198 overexpression in OS cells. Rho-associated kinase (ROCK) is an essential downstream effector of the Rho small GTPase, which acts as a molecular switch that binds GTP (active) and GDP (inactive) to regulate cell survival, proliferation and cytoskeleton organization, inducing alterations in cell shape/morphology, invasion and movement [30e32]. ROCK1, located at chromosome 18 (18q11.1) [33], has been found upregulated in various types of human cancers, and related to cancer progression, metastasis and poor prognosis [34e36]. Liu et al. reported that ROCK1 expression levels were also higher in OS tissues, and knockdown of ROCK1 inhibited proliferation and enhanced apoptosis in OS cells, suggesting ROCK1 could be a target for OS [37]. In addition, ROCK1 has been reported to be regulated by several miRNAs, such as mIR-144 in OS [38], miR-340 in breast cancer [39], miR-335 in hepatocellular carcinoma [40], miR-124 in gastric cancer [41] and so on. Taken together, miR-198/ROCK1 based targeted therapy could be a new therapeutic strategies for OS patients. In conclusion, our study suggested that miR-198 may function as a tumor suppressor via directly targeting ROCK1 in OS, and the abnormal expression of miR-198 might be a key factor in OS growth and metastasis. Disclosure The authors declare that they have no conflict of interest related to the publication of this manuscript. References [1] N. Marina, M. Gebhardt, L. Teot, R. Gorlick, Biology and therapeutic advances for pediatric osteosarcoma, Oncol. 9 (2004) 422e441. [2] S.S. Bielack, N. Marina, S. Ferrari, L.J. Helman, S. Smeland, J.S. Whelan, G.H. Reaman, Osteosarcoma: the same old drugs or more? J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 26 (2008) 3102e3103 author reply 3104e3105. [3] A. Longhi, C. Errani, M. De Paolis, M. Mercuri, G. Bacci, Primary bone osteosarcoma in the pediatric age: state of the art, Cancer Treat. Rev. 32 (2006) 423e436. [4] R. Gorlick, C. Khanna, Osteosarcoma, J. Bone Mineral Res. Off. J. Am. Soc. Bone

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Please cite this article in press as: S. Zhang, et al., MicroRNA-198 inhibited tumorous behaviors of human osteosarcoma through directly targeting ROCK1, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.040