The Effects Of MiR-124 On Proliferation And Invasion Of Human Osteosarcoma MG-63 Cell Lines

Objective:Osteosarcoma(OS)is the most common primary malignant bone tumor in Department of orthopedics.Osteosarcoma is characterized by originated from mesenchymal tissue,and producing spindle cell like osteoid tissue.It is mostly found in 15-25 years old children and adolescents.Osteosarcoma mostly occurred in vascularized metaphysis,and 50% of the total number of cases occurred in the distal femur and proximal tibial.Osteosarcoma has strong local destruction,high disability rate,high mortality and high incidence of blood transfer.Therefore,the researchers pay more attention to the osteosarcoma.With the rapid development of new technology and the continuous improvement of treatment,the 5-year survival rate of osteosarcoma patients in the primary site has reached about 70%.However,due to the high degree of malignancy and poor prognosis of osteosarcoma,the long-term survival opportunities for patients with metastasis or recurrence are still less than20%.Therefore,further elucidating the molecular mechanism of osteosarcoma cell proliferation and metastasis is of great guiding significance for the prevention of osteosarcoma and the development of related drugs.Mi RNA is an endogenous non coding single strand small molecule RNA,which is composed of 19~25 nucleotides and is highly conserved in the population.There are a lot of evidences have showed that the expression of mi RNA in lung cancer,prostate cancer,liver cancer,breast cancer,gastric cancer and other tumor tissues had abnormal expression,the overexpression or inhibiting the expression are closely related to tumor development,invasion,metastasis and chemotherapy resistance.Mi R-124 is a highly conserved mi RNA,which is low in a variety of tumor tissues,such as liver cancer,breast cancer,cervical cancer and melanoma.In the study of thyroid papillary carcinoma cells,mi R-124 was found that up regulation of mi R-124 expression could reduce the invasion and metastasis of papillary thyroid carcinoma cells,and the regulation of IQGAP1 expression might be one of the mechanisms that inhibit the invasion and metastasis of papillary thyroid carcinoma cells.But the exact biological function of mi R-124 in osteosarcoma is not very clear.Signal transducer and activator of transcription factor 3(STAT3)is an important signal transduction factor of JAK/STAT signaling pathway,activation of STAT3 could form homo or hetero dimer,which transferring external signals to the nucleus,and regulating the transcription of the corresponding DNA by combining with the corresponding DNA cis acting element.STAT3 participates in a variety of important biological processes in the body,such as cell proliferation,apoptosis,invasion,angiogenesis,and immune escape.More and more experiments have shown that STAT3 is a proto oncogene and is overexpressed in a variety of tumor tissues.However,in osteosarcoma,whether STAT3 is abnormal and whether it is a downstream target of mi R-124 is a problem to be discussed.We detect the expression of mi R-124 in osteosarcoma,Changes of proliferation and invasiveness of osteosarcoma MG-63 cells after mi R-124 inhibitors or mi R-124 mimics was transfected and expression changes of STAT3 protein,to investigate the effect and mechanism of mi R-124 on the proliferation and invasion of osteosarcoma MG-63 cells.Methods:1.A total of 27 cases of osteosarcoma and adjacent normal bone tissues were collected from department of pathology in the First Affiliated Hospital of China Medical University from January 2013 to June 2016,and all specimens were diagnosed as osteosarcoma by pathologist in our hospital.The human normal osteoblast line h FOB1.19 and osteosarcoma cell line MG-63 were purchased from the Shanghai Institute of cell research,CAS.mi R-124 mimics,inhibitors and negative control were purchased from Shanghai genepharma Co.Ltd.,lipofectamine2000 transfection reagent,Trizol reagent and RIPA lysate was purchased from Invitrogen company,STAT3 monoclonal antibody was purchased from Cell company Signaling,Transwell small chamber and Matrigel glue were purchased from America BD company.Ultra low temperature refrigerator(Japan SANYO);fluorescence microscope(Japan Nikon);UV spectrophotometer(German Eppendorf);fluorescence quantitative PCR instrument ABI7500(ABI company);Biofuge Stratos high speed refrigerated centrifuge(German Heraeus company);PCR instrument(American Beckman company).2.The normal osteoblast line h FOB1.19 and osteosarcoma cell MG-63 were cultured,and the cells of the logarithmic growth period were transfected according to the instructions of Lipofectamine2000.Real-time fluorescence quantitative PCR was used to detect the expression of mi R-124 in osteosarcoma tissue and adjacent normal bone tissue specimens.Immunohistochemical method was used to detect the expression of STAT3 in osteosarcoma tissue and adjacent normal bone tissue.The MTT assay was used to detect the proliferation of MG-63 cells after transfection of mi R-124 mimics or inhibitors.The transwell assay was used to detect the changes of proliferation and invasion of MG-63 cells after transfection of mi R-124 mimics or inhibitors.Western blot method was used to detect the expression of STAT3 protein of MG-63 cells after transfection of mi R-124 mimics or inhibitor.The experimental data were analyzed with SPSS 18 statistical software,and Single factor analysis of variance was used in each group.Results: 1.The results of real time PCR showed that the expression of mi R-124 in osteosarcoma tissue was significantly lower than that of normal tissue adjacent to cancer,P<0.01.2.The results of immunohistochemical showed that the mean optical density of STAT3 expression in osteosarcoma tissues was(0.58 + 0.11),which was significantly higher than that in normal tissues adjacent to cancer(0.19+0.06),P<0.01.3.Compared with the control group,the viability of MG-63 cells transfected with mi R-124 mimics was decreased significantly,while the activity of MG-63 cells transfected with mi R-124 inhibitors was increased significantly,P<0.01.4.Compared with the control group,the number of MG-63 cells through the artificial basement membrane Matrigel glue and microporous membrane was decreased significantly after mi R-124 mimics was transfected;and the number of MG-63 cells through the artificial basement membrane Matrigel glue and microporous membrane was increased significantly after mi R-124 inhibitor was transfected,P<0.01.5.Compared with the control group,the expression of STAT3 in MG-63 cells was significantly decreased after mi R-124 mimics was transfected,while the expression of STAT3 was increased significantly after mi R-124 inhibitor was transfected,P<0.01.Conclusions: The expression of mi R-124 was down regulated in osteosarcoma,and mi R-124 could inhibit the proliferation and invasion of osteosarcoma MG-63 cells,and its mechanism might be related to the down-regulation of STAT3 expression.