J Huazhong Univ Sci Technol［Med Sci］ 34(6):889-895,2014 DOI 10.1007/s11596-014-1369-y J Huazhong Univ Sci Technol［Med Sci］ 34(6):2014

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Effect of MicroRNA-101 on Proliferation and Apoptosis of Human Osteosarcoma Cells by Targeting mTOR Song LIN (林 松)1†, Nan-nan SHAO (邵楠楠)2†, Lei FAN (范 磊)1, Xiu-cai MA (马秀才)1, Fei-fei PU (浦飞飞)1, Zeng-wu SHAO (邵增务)1# 1 Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China 2 Department of Radiology, Henan Cancer Hospital, Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou 450008, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2014

Summary: Studies have proved that microRNA-101 (miR-101) functions as a tumor suppressor and is associated with growth and apoptosis of various human cancers. However, the role of miR-101 in osteosarcoma and the possible mechanism by which miR-101 affects the tumor growth and apoptosis have not been fully elucidated. In this study, we found that the expression of miR-101 was down-regulated in osteosarcoma tissues and Saos-2 cell line as compared with that in adjacent non-neoplastic bone tissues and the osteoblastic cell line. To better characterize the role of miR-101 in osteosarcoma, we used a gain-of-function analysis by transfecting human osteosarcoma cell line Saos-2 with chemically synthesized miR-101 mimics. The results showed that overexpression of miR-101 inhibited the proliferation and promoted the apoptosis of Saos-2 cells. Meanwhile, bioinformatic analysis demonstrated that mTOR gene was a direct target of miR-101. Overexpression of miR-101 significantly decreased the expression of mTOR at both mRNA and protein levels in Saos-2 cells, consequently inhibiting Saos-2 cells proliferation and promoting cells apoptosis in an mTOR-dependent manner. Taken together, these data suggest that miR-101 may act as a tumor suppressor, which is commonly downregulated in both osteosarcoma tissues and cells. mTOR plays an important role in mediating miR-101 dependent biological functions in osteosarcoma. Reintroduction of miR-101 may be a novel therapeutic strategy by down-regulating mTOR expression. Key words: miR-101; mTOR; osteosarcoma; proliferation; apoptosis

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Osteosarcoma is the most common primary malignant bone tumor in children and young adults, which usually arises in the metaphysis of long bones[1, 2] and accounts for approximately 60% of malignant bone tumors in the first two decades of life[3]. Despite the use of combinational chemotherapy and advanced surgery, survival in osteosarcoma patients is still unsatisfactory. The 5-year survival rate for osteosarcoma patients with localized disease is approximately 65%[4], and most of them died of metastases eventually[5]. Additionally, chemotherapy is accompanied by many early and late side effects. Therefore, it is necessary to explore the potential molecular mechanisms of osteosarcoma progression and identify effective molecular targets for the treatment of osteosarcoma. MicroRNAs (miRNAs) are a class of endogenous small noncoding regulatory RNAs, approximately 22 nucleotides in length, which are known to regulate gene expression by binding to the 3' untranslated region (3'-UTR) of the complementary mRNA sequence, resulting in repressing translation or decreasing the stability of mRNAs[6, 7]. Growing evidence suggests that de                                                              

Song LIN, E-mail: [email protected]; Nan-nan SHAO, E-mail: [email protected] † Both authors contributed equally to this work. # Corresponding author, E-mail: [email protected]

regulation of miRNAs may contribute to many types of human diseases, including cancers. Several studies have shown that miRNAs may act as oncogenes or cancer suppressors, which exert a pivotal function in tumorigenesis[8, 9]. These data indicate that miRNAs may become ideal targets for cancer treatment and may provide new insights into the molecular mechanisms underlying tumorigenesis. MicroRNA-101 (miR-101) belongs to a family of miRNAs and emerging evidence suggests it is a tumor-suppressive miRNA[10–12]. Most of the studies indicated that miR-101 is obviously under-expressed in different types of human malignancies and displays a suppressive effect on cellular proliferation, apoptosis, and metastasis[10, 12–15]. mTOR is a protein kinase involved in the PI3K/Akt signaling pathway with a central role in the control of cell growth, proliferation, apoptosis, and metabolism[16–18]. It has been shown that mTOR is the target gene for miR-101, and up-regulation of miR-101 decreases the expression of mTOR and enhances cisplatin-induced apoptosis in hepatocellular carcinoma cells[14]. However, the expression and biological role of miR-101 and mTOR have not been totally elucidated in osteosarcoma. Therefore, it is important to further study the function of miR-101 and to explore the possible association between miR-101 and mTOR in osteosarcoma. In this study, we demonstrated that miR-101 was

890 downregulated in osteosarcoma tissues and cells, which regulated the expression of mTOR. Further investigation revealed that enforced expression of miR-101 or knockdown of mTOR could inhibit the proliferation of osteosarcoma cells and promote their apoptosis. These results suggest that miR-101 may act as a tumor suppressor and can be a novel candidate gene for the therapy of osteosarcoma. 1 MATERIALS AND METHODS 1.1 Patients and Tissue Samples Totally 23 pairs of human osteosarcoma specimens and adjacent non-neoplastic bone tissues were available from our department between March 2012 and October 2013. All patients provided informed consents for the use of their tissues before surgery. After surgical removal, the tissues were snap-frozen in liquid nitrogen and stored at –80ºC until use for qRT-PCR and Western blotting. This study was approved by the Research Ethics Committee of Union Hospital, Wuhan, China. All specimens were treated and made anonymously according to the ethical and legal standards. 1.2 Cell Lines and Culture Human osteosarcoma cell line Saos-2 and human osteoblastic cell line hFOB 1.19 were kindly provided by the Orthopedic Laboratory of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (China). Saos-2 cells were cultured in RPMI 1640 medium (Wuhan Boster, China) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37ºC. HFOB 1.19 cells were cultured in Dulbecco’s modified Eagle medium/F12 (DMEM) (Invitrogen, USA) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator with 5% CO2 at 33.5ºC. 1.3 Luciferase Reporter Assay An online program Target-Scan (release 6.2) was used for predicting the target of miR-101. The miR-101 targeted gene was evaluated by using a luciferase reporter assay in Saos-2 cells. The putative miR-101 complementary site in the 3'-UTR of mTOR mRNA (NM_004958; 3'-UTR: 131-137), or mutant sequence was cloned into the pGL3 luciferase reporter vector (Promega, USA). Saos-2 cells were cultivated in 24-well plates and were co-transfected using FuGENE (Roche Applied Science, USA) with 100 ng of pGL3-mTOR-miR-101 constructs, 10 ng miR-101 mimics or NC mimics, and 2 ng pRL-SV-40 RLuci vector (Promega, USA). Forty-eight h after transfection, cells were washed with PBS, then lysed and detected by using the dual-luciferase reporter assay system (Promega, USA) and bioluminescence detector Victor3V (Perkin-Elmer, USA) according to the manufacturer’s instructions. 1.4 qRT-PCR Assay The miR-101 and mTOR expression levels were evaluated using qRT-PCR. The analysis was performed according to the similar protocol of our previous study[9]. Briefly, total RNA from the frozen specimens and cultured cells was isolated using the TRIzol kit (Invitrogen, USA) according to the manufacturer’s protocol. The integrity and purity of total RNA were verified by UV

J Huazhong Univ Sci Technol［Med Sci］ 34(6):2014

spectrophotometry and gel-electrophoresis on formaldehyde dematuration gel. For miRNA quantitation, cDNA was synthesized with specific miRNA primers using the TaqMan microRNA Reverse Transcription kit (Applied Biosystems, USA). For mRNA quantitation, cDNA was synthesized using the PrimeScript RT reagent kit (Takara, China). The resulting cDNAs were quantified using SYBR Green (Takara, China) with an ABI 7500 fast real-time PCR system (Applied Biosystems, USA). The relative expression of miRNA and mRNA was evaluated using the 2−ΔΔCT method and normalized to the small nuclear RNA U6 and β-actin, respectively. The qPCR primers, obtained from Invitrogen (China), had the following sequences: miR-101 (forward: 5'-TACAGTACTGTGATAACTGAA-3'; reverse: 5'-TGGTGTCGTGGAGTCG-3'); U6 (forward: 5'-GCTTCGGCAGCACATATACTAAAAT-3'; reverse: 5'-CGCTTCACGAATTTGCGTGTCAT-3'); mTOR (forward: 5'-CGCTGTCATCCCTTTATCG-3'; reverse: 5'-ATGCTCAAACACCTCCACC-3'); β-actin (forward: 5'-TCACCCACACGTGCCCATCTCGA-3'; reverse: 5'-CAGCGGAACCGCTCATTGCCAATGG-3'). All experiments were repeated three times. 1.5 Western Blot Analysis Samples and cells were collected and lysed in RIPA buffer (Beytime, China). The total protein concentration was estimated using the BCA protein assay kit (KeyGEN, China). Equal amounts of protein (30 μg) were resolved with 10% SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes, and blocked for 0.5 h by blocking buffer (Beytime, China). The membranes were incubated with specific primary antibodies (mTOR, Cell Signaling, USA, 1:2000; GAPDH, CoWin Biotech, USA, 1:2000) overnight at 4ºC and then followed by incubation with horseradish-peroxidase-conjugated secondary antibody (Santa Cruz Biotech, USA). Blots were visualized by ECL (Pierce, USA). Experiments were done at least three times and the bands were analyzed using Quantity One analyzing system (Bio-Rad, USA). 1.6 miRNA and siRNA Transfection The human miR-101 duplex mimics, negative control oligonucleotide duplex mimics, mTOR siRNA and scramble control siRNA were designed and provided by Ribobio (China), and transfected into cells using LipofectamineTM2000 (Invitrogen, USA) according to the manufacturer’s instructions. At 24 h after transfection, cells transfected with miRNA mimics or siRNA were used for subsequent experiments including proliferation and apoptosis assays. 1.7 Cell Proliferation Assay The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to assay cells proliferation according to the manufacturer’s instructions. Briefly, Saos-2 cells were seeded at 5×103 per well in 96-well plates and transfected. At 48 h after transfection, 20 μL MTT (5 mg/mL, Sigma, USA) were added into each well for additional 4 h incubation at 37ºC. Then, the culture medium was discarded and 100 μL of dimethyl sulfoxide (DMSO, Sigma, USA) was added into each well. The absorbance (A) of each sample was recorded at 570 nm by a microplate reader (Bio-Rad, USA). The experiment was repeated for three times and the results

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J Huazhong Univ Sci Technol［Med Sci］ 34(6):2014

were described as a ratio of A570 nm with miR-101 mimics versus its corresponding control. 1.8 Apoptosis Assay Apoptosis was analyzed using the annexin V-FITC/PI apoptosis double staining kit (KeyGEN Biotech, China) according to the manufacturer’s instructions. Briefly, Saos-2 cells were seeded at a density of 5×105 cells/mL in six-well plates and transfected. At 48 h after transfection, the cells were harvested and suspended in 100 μL annexinV binding buffer, and then stained with 5 μL annexin V-FITC and 5 μL PI at room temperature for 15 min in the dark. The stained cells were analyzed immediately by flow cytometry. 1.9 Statistical Analysis The software of SPSS version 13.0 for Windows was used for statistical analysis. Data were expressed as ±s for three independent experiments. Comparisons between two groups were made by Student’s t-test. Statistical significances of mean difference among multiple groups were performed with analysis of variance (ANOVA) followed by Dunnett’s t-test. A P value