The Study Of The Role And Molecular Mechanism Of MiR-548c-3p On Proliferation, Migration And Invasion In Osteosarcoma

Background:Osteosarcoma is the most common primary malignant tumor, which is often found in adolescents and children. High incidence and fast progression of osteosarcoma lead to high malignany and distant metastases, which resulted in low long-term suvival rate. Statistics show that the 5-year survival rate in patients only accept amputation is about 16%. Osteosarcoma often occurs in metaphysis of limb long bone. It also occurs in other areas such as ilium and spine. Surgical amputation is the major treatments for early osteosarcoma. However, the shortness of this treatment is obvious and includs big surgical trauma, high morbidity, and low long-term survival rate. With the progress of surgery and the introduction of preoperative and postoperative chemotherapy with novel concept, the clinical remission rate and long-term survival rate of osteosarcoma obtains big improvement. However, despite the improvement of surgery and adjuvant chemotherapy, clinical prognosis is still very poor. Statistical data obtained from 2015 pateints in China showed the disease-free survival rate was 56.0%; the relapse-free survival rate was 60.0%; the recurrence rate was 9.1%; lung metastasis rate was 24.8%. The reasons that caused poor prognosis are pulmonary metastasis and drug resistance. The molecular mechanisms about initiation and progression of osteosarcoma is unclear yet. Revealing molecular mechanisms is crucial for diagnosis and therapy of osteosarcoma.MiRNAs is a bunch small noncoding RNA which was found recently. miRNA play an important role in initiation and progression of tumor. They can bind to 3’ untranslated regions of oncogenes and tumor supprssor genes and caused downregualtion of the genes. Previous study indicates that dysregulation of miRNAs is closely related to proliferation, metastasis and prognosis of osteosarcoma. miR-143 is abnormally downregulated in osteosarcoma. It inhibits proliferation and promotes apoptosis of osteosarcoma cells by modulating expression of BCL-2. In addition, miR-143 regulates migration and invasion and mediates chemoresistance of osteosarcoma cells by targeting matrix metalloproteinase-13 (MMP-13). miR-199a-3p is an other key regulator in initiation and progression of osteosarcoma. Recent studies reveal many miRNAs which plays an important role in initiation and progression of osteosarcoma. Unfortunately, no miRNA have been use in therapy of osteosarcoma. Therefore, it’s crucial to identify new miRNAs which plays an important role in initiation and progression of osteosarcoma, and clearify the mechanism abhout how miRNA regulate downstream targets, which will provide new target and strategy for therapy of osteosarcoma.Integrin subunit alpha V (ITGAV) belongs to the integrin subunit alpha family. It binds to multiple ligands in extracellular matrix via arg-gly-asp sequence (R-G-D) of these ligands, and involves in cell to cell and cell to extracellular matrix adhesion. Recent studies showed ITGAV plays an important role in ageogensis, migration and invasion of tumor. ITGAV binds to extracellular ligands and promotes secretion and activation of matrix metalloproteinase, which result in activation and migration of vascular endothelial cells through a series of cascade reaction. ITGAV inhibits the apoptosis of endothelial cells and induce angiogenesis by interacting with bFGF and VEGF synergistically. Numerous studies implicated that expression level of ITGAV is important for diagnosis, treatment and prognosis of tumor. ITGAV expression level is closely related to the progress of colon cancer and the malignant degree of brain glioma, and associated with differentiation and lymph node metastases of squamous cell carcinoma of larynx and hypopharynx. Overexpression of ITGAV often indicates poor prognosis of cancer. Immunohistochemical results show strong positive expression of ITGAV in osteosarcoma tissues. Moreover, expression of ITGAV is closely associated with the malignancy degree and clinical stage of osteosarcoma. Expression of ITGAV decreased in osteosarcoma tissues sensitive to chemotherapy. Conversely, expression of ITGAV didn’t decreased in thoes tissues not sensitive to chemotherapy. These data indicates expression of ITGAV is not only serve as predictor of malignancy degree, but also as indicator that help determine the effect of chemotherapy in osteosarcoma.In our previous study, we screen miRNAs which differentially expressed in Osteosarcoma by microchips. Our data showed there is significant difference in expression of miR-548c-3p between osteosarcoma tissues and paracancerous tissues. miR-548d induce apoptosis and cell cycle arrest in tumor cells, which result in inhibition of proliferation. In breast cancer, miR-548c-3p binds to 3’UTR of ECSH1 and leads to downregulation of ECSH1, which results in inhibition of proliferation of breast cancer cells. So far the effects of miR-548c-3p on cell proliferation, colony formation, migration, invasion, cell cycle and apoptosis of osteosarcoma are not clear.Aims(1) To illuminate expression profile of miR-548c-3p in osteosarcoma and its effects on proliferation, colony formation, migration, invasion, cell cylce and apotosis of osteosarcoma cells.(2) To reveal the molecular mechanisms of miR-548c-3p involving initiation and progression of osteosarcoma by targeting ITGAV.MethodsThe first part:expresssion profile of miR-548c-3p and effects of miR-548c-3p on proliferation, colony formation, migration, invasion, cell cycle and apoptosis of osteosarcoma.First, quantitative real-time PCR (QPCR) was performed to examine expression of miR-548c-3p in osteosarcoma cell lines, tumor tissues and paracancerous tissues. Statistical methods was used to analyze difference between expression of miR-548c-3p in osteosarcoma cell lines, tumor tissues and paracancerous tissues, and to determine expression patterns of miR-548c-3p in osteosarcoma tissues and cells.Second, osteosarcoma cell line SAOS2 was used in this work, in which expression of miR-548c-3p was relatively low. miR-548c-3p mimics was synthetise and transfected into SAOS2 cells. Cells was divided into three group:group control (lipofectamine only), group NC (lipofectamine and NC), group miR-548c-3p (lipofectamine and miR-548c-3p mimics). quantitative real-time PCR (QPCR) was performed to examine expression of miR-548c-3p in SAOS2 cells and determine the transfection efficiency of miR-548c-3p mimics.Third, NC or miR-548c-3p mimics was transfected into SAOS2 cells respectively. Cells was divided into three group:group control (lipofectamine only), group NC (lipofectamine and NC), group miR-548c-3p (lipofectamine and miR-548c-3p mimics). CCK8 assays was performed to examine cell proliferation at oh,24h,48h,72 and 96h after transfection.Fourth, NC or miR-548c-3p mimics was transfected into SAOS2 cells respectively. Cells was divided into three group:group control (lipofectamine only), group NC (lipofectamine and NC), group miR-548c-3p (lipofectamine and miR-548c-3p mimics). Colony formation assays, PI staining and PI/AnexinV double staining were performed to examine colony formation, cell cycle and apoptosis of SAOS2 cells.Fifth, NC or miR-548c-3p mimics was transfected into SAOS2 cells respectively. Cells was divided into three group:group control (lipofectamine only), group NC (lipofectamine and NC), group miR-548c-3p (lipofectamine and miR-548c-3p mimics). Transwell assays was performed to examine migration and invasion of SAOS2 cells.Sixth, NC or miR-548c-3p mimics was transfected into SAOS2 cells respectively. Cells was divided into three group:group control (lipofectamine only), group NC (lipofectamine and NC), group miR-548c-3p (lipofectamine and miR-548c-3p mimics). Transfected cells were injected into the armpit of nude mice. Tumor bulges was found in the injection position 10 days after the injection. Tumor sizes was measure every three or four days. Growth cure was generated based on data of tumor size. The effect of miR-548c-3p on tumor growth was evalued based on growth cure of tumor.The second part:the molecular mechanism of miR-548c-3p involving initiation and progression of osteosarcoma by targeting ITGAVFirst, online program Targetscan7.0 (http://www.targetscan.org/) and miRbase (http://www.mirbase.org/) were used to identify target gene of miR-548c-3p.Second, total RNA of osteosarcoma tissues and paracancerous tissues were extracted and transcriped into cDNA. mRNA expressions of ITGAV in osteosarcoma tissues and paracancerous tissues were examined by QPCR. Total proteins of osteosarcoma tissues and paracancerous tissues were extracted and protein expressions of ITGAV were examined by westernblot.Third, miR-548c-3p mimics, NC mimics, miR-548c-3p inhibitor, and NC inhibitor were synthetise and transfected into SAOS2 cells using lipofactamine, which resulted in up- or down-regulation of miR-548c-3p. Cells was divided into three group:group miR-548c-3p mimics, group NC mimics, group miR-548c-3p inhibitor and group NC inhibitor. QPCR and westernblot were used to examine mRNA and protein expressions of ITGAV, respectively.Fourth, miR-548c-3p mimics, NC mimics, miR-548c-3p inhibitor, and NC inhibitor were transfected into SAOS2 cells with reporter constructs containing wild type or mutated 3’UTR of ITGAV. Luciferase assays were performed to determine whether miR-548c-3p binds to 3’UTR of ITGAV.Fifth, siRNA targeted ITGAV was synthetise and transfected into SAOS2 cells. QPCR and westernblot were used to examine mRNA and protein expressions of ITGAV, respectively. It will help determine the transfection efficiency of siRNA.Sixth, CCK8 assays and colony formation assays were performed to examine effects of silecing ITGAV on cell proliferation and colony formation of SAOS2 cells.Seventh, PI staining was performed to examine effects of silecing ITGAV on cell cycle of SAOS2 cells.Eighth, PI/AnexinV double staining were performed to examine effects of silecing ITGAV on apoptosis of SAOS2 cells.ResultsThe effects of miR-548c-3p on prolifearion, colony formation, migration, invasion, cell cycle and apoptosis of osteosarcoma.(1) qPCR results indicated expression of miR-548c-3p in specimens of osteosarcoma were lower than thoes in corresponding non-cancerous tissues (p<0.0001).(2) qPCR results indicated expression of miR-548c-3p in osteosarcoma cell lines 143B(P=0.0036), SaoS2(P=0.0007) and HOS(P=0.0091) were lower than thoes in osteoblast cell line OB3.(3) CCK8 results showed osteosarcoma cells transfected with miR-548c-3p grew slower than thoes transfected with NC, which indicated miR-548c-3p inhibited proliferation of osteosarcoma cells.(4) Colony formation assays showed osteosarcoma cells transfected with miR-548c-3p developed less colonies than thoes transfected with NC (p<0.0001), which indicated miR-548c-3p inhibited the ability of colony formation in osteosarcoma cells.(5) The cell cycle assays showed percentage of cells in G2/M phase in miR-548c-3p group were higher than thoes in NC group(P<0.0001); percentage of cells in S phase in miR-548c-3p group were lower than thoes in NC group(P=0.003); percentage of cells in G1 phase in miR-548c-3p group were similar with thoes in NC group(P=0.4165).(6) The cell apoptosis assays showed no significant difference were observed between control group and NC group; apoptosis rate in miR-548c-3p group were higher than thoes in control group and NC group(P<0.001).(7) Transwell assays showed no significant difference were observed between the migration and invasion abilities of control group and NC group; migration and invasion abilities of miR-548c-3p group were lower than thoes in control group and NC group(P<0.001).(8) The xenograft assays showed miR-548c-3p group developed smaller tumors than control group and NC group; tumors in miR-548c-3p group grew much slower than thoes in control group and NC group.The molecular mechanisms underlying inhibitory effectes of miR-548c-3p on cell proliferation in osteosarcoma(1) qPCR results indicated expression of ITGAV in specimens of osteosarcoma were higher than thoes in corresponding non-cancerous tissues(P<0.0001).(2) qPCR results showed expression of ITGAV in miR-548c-3p mimics group were lower than thoes in NC mimics group(P<0.0001); expression of ITGAV in miR-548c-3p inhibitor group were higher than thoes in NC inhibitor group(P<0.0001).(3) Bioimformatic analysis reveals 3’UTR of ITGAV contains binding cite of miR-548c-3p. Results of luciferase assays shows relative luciferase activity of group miR-548c-3p is lower than thoes of group NC in wild type ITGAV 3’UTR transfection experiments (P=0.0002). In mutated ITGAV 3’UTR transfection experiments, there is no significant difference between group miR-548c-3p and group NC (P=0.3635). These data indicates miR-548c-3p binds to’GAUUUUU’in 3’UTR of ITGAV and causes downregulation of ITGAV in osteosarcoma cells. this is one of the mechanisms underlying inhibitory effects of miR-548c-3p on cell proliferation.(4) CCK8 results showed osteosarcoma cells transfected with ITGAV siRNA grew slower than thoes transfected with NC(P<0.0001), which indicated ITGAV siRNA inhibited proliferation of osteosarcoma cells.(5) Colony formation assays showed osteosarcoma cells transfected with ITGAV siRNA developed less colonies than thoes transfected with NC(P<0.0001), which indicated ITGAV siRNA inhibited the ability of colony formation in osteosarcoma cells.(6) The cell cycle assays showed percentage of cells in G2/M phase in ITGAV siRNA group were higher than thoes in NC group(P<0.0001); percentages of cells in G0/G1 and S phase in ITGAV siRNA group were lower than thoes in NC group(P<0.0001).(7) The cell apoptosis assays showed apoptosis rate of ITGAV siRNA group were higher than thoes in NC group(P<0.0001).Conclusion(1) miR-548c-3p is down-regulated in specimens of osteosarcoma and in osteosarcoma cell lines 143B, SaoS2 and HOS. The expression profile of miR-548c-3p is consistent with the profile of tumor suppressor genes.(2) Overexpression of miR-548c-3p inhibits cell proliferation in osteosarcoma cells, which indicates the important role of miR-548c-3p in regulation mecanism of proliferation.(3) Exogenerous expression of miR-548c-3p inhibits migration and invasion of osteosarcoma cells, which indicates the important role of miR-548c-3p in regulation mecanism of migration and invasion.(4) Exogenerous expression of miR-548c-3p induces apoptosis in osteosarcoma cells, which indicates miR-548c-3p modulate cell proliferation by inducing apoptosis.(5) Bioimformatic analysis reveals 3’UTR of ITGAV contains binding cite of miR-548c-3p. Our data shows miR-548c-3p binds to ’GAUUUUU’ in 3’UTR of ITGAV and causes downregulation of ITGAV in osteosarcoma cells, which is one of the mechanisms underlying inhibitory effects of miR-548c-3p on cell proliferation.(6) Results of CCK8 assays and colony formation assays indicates downregulation of ITGAV inhibits cell proliferation and colony formation of osteosarcoma, which is consistent with effects of miR-548c-3p on proliferation and colony formation.(7) Cell cycle analysis show downregulation of ITGAV induces G1 arrest in osteosarcoma, which is consistent with effects of miR-548c-3p on cell cycle.(8)Cell apoptosis assays show downregulation of ITGAV increases apoptosis in osteosarcoma, which is consistent with effects of miR-548c-3p on apoptosis. In conclusion, our results reveal the expression profile and biological function of miR-548c-3p in osteosarcoma. We also elucidate the molecular mechanism about how miR-548c-3p regulate its downstream target ITGAV. Our study help strenghen the understanding of molecular mechanism about initiation and progression of osteosarcoma, which may provide novel target and new strategy for osteosarcoma therapy.