Experimental Study On Migration Invasion And Proliferation Of Osteosarcoma Cells Through Down-regulation MiR-17-5p

Background Osteosarcoma is a highly malignant tumor arising from mesenchymal cells,which is one of the most common malignant tumors in China and accounts for 17% of the primary bone tumors.The malignant tumor is characterized by the formation of a fully differentiated or poorly differentiated osteoid like tissue.Osteosarcoma often showed local invasion,invasive growth,be early metastasis.It is generally believed that osteosarcoma is divided into ordinary type,telangiectasia,periosteal appearance,periosteal type,low center type,small cell type and other atypical type.Each type has its own morphological characteristics.In recent years,the 5 year survival rate of patients with in situ and non-metastatic osteosarcoma has reached 60-70%.However,for the treatment of metastatic osteosarcoma and recurrent osteosarcoma,the results are still just passable.Of unknown etiology,gene mutation,cognitive level difference,lack of extensive tissue biomarkers,aggressive and a series of problems for making human biological characteristics of osteosarcoma still faces various challenges.At present.The research and exploration of osteosarcoma mainly focus on the molecular mechanism of the disease especially in the development of various molecular markers and therapeutic targets.Therefore,it is very important to find out the rules and mechanisms of the occurrence and development of osteosarcoma from the gene level and to search for new molecular markers with high specificity and high sensitivity.Micro RNAs(mi RNAs)is a kind of 18-25 bp in length,single stranded,non-encoding RNA,through transcriptional and post transcriptional levels,combined with the 3’UTR of m RNA,regulate the expression of many genes that influence the growth process of many kinds of tumor cells at the molecular level,which mediates tumor disease occurrence,development and prognosis.Recent reports have shown that mi R-17-5p is highly expressed in a variety of tumor cells.Such as Lung cancer,breast cancer,bladder cancer,rectal cancer,stomach cancer,liver cancer and osteosarcoma.However,the effect of mi R-17-5p on the physiological functions of osteosarcoma cells and the mechanism of its molecular biology have not been reported.Therefore,we synthesized the mi R-17-5p inhibitor(mi R-17-5p inhibitor),to explore the mi R-17-5p inhibitor on human osteosarcoma cell migration,invasion,proliferation,apoptosis and other cell physiological function Affect the mechanism and principles of its occurrence and development,and provide the theoretical basis for molecular biology of clinical diagnosis and treatment of osteosarcoma disease,and provide a new cognitive angle for this kind of tumor disease.Purpose This study was conducted to investigate the difference of mi R-17-5p and SRCIN1 gene expression between osteosarcoma and osteosarcoma tissues in osteosarcoma and osteosarcoma adjacent to osteosarcoma.The The effect of mi R-17-5p inhibitor gene on the migration,invasion,proliferation and apoptosis of osteosarcoma cells was observed by artificial culture of human osteosarcoma cell line MG63.The target protein of mi R-17-5p was explored Molecular level of the regulatory mechanism to explore new mi RNAs for the new mi RNA in the prevention and treatment of osteosarcoma to provide basic research support and new targets.Methods 1 To collected a total of 28 patients with osteosarcoma from the First Affiliated Hospital of Zhengzhou University(from January 2015 to September 2016)and their adjacent tissues were stored in the refrigerator at-80 DEG C.Using real-time fluorescence quantitative PCR(real-time quantitative PCR,RT-q PCR)method for detection of the 28 cases of osteosarcoma,the expression level of mi R-17-5p SRCIN1 and adjacent tissues of m RNA gene,and analyze the relationship between the two.The expression level of SRCIN1 protein was detected by Western blot(Western blot).2 To compare the m RNA expression level of mi R-17-5p and SRCIN1 gene in osteoblast h FOB1.19 and osteosarcoma cell(MG63)which was detected by RT-q PCR method 3 mi R-17-5p inhibitor agomir and mi R-17-5p negative control were synthesized and transfected into osteosarcoma cells MG63 by liposome transfection.The experimental groups were as follows: mi R-17-5p inhibitor group: transfected mi R-17-5p inhibitor agomir.Group NC: transfection mi R-17-5p inhibitor negative control.Group Blank: only transfection reagent liposome.4.CCK-8 assay was used to detect the cell growth and colony formation in three groups,and the effect of mi R-17-5p inhibitor on the proliferation of osteosarcoma cell line MG63 was observed.5 The effect of mi R-17-5p inhibitor on apoptosis of osteosarcoma cell line MG63 was detected by flow cytometry and Tunel assay.6 to observe the effect of mi R-17-5p inhibitor on the migration ability of osteosarcoma cell line MG63.7 the effect of mi R-17-5p inhibitor on the invasiveness of osteosarcoma cell line MG63 was observed by Transwell assay.8 Using Pic Tar,mi Rwalk,Target Scan,mi Randa and other bioinformatics software to predict the target of mi R-17-5p.According to the prediction results of construction of wild type 3-UTR region containing SRCIN1 and mutant recombinant luciferase reporter vector,respectively,with the mi R-17-5p agomir or mi R-17-5p negative control were transfected into human osteosarcoma cell line MG63 cells,and the use of Western blot method and dual luciferase reporter assay demonstrated that SRCIN1 is a target of mi R-17-5p.9.Western blot was used to detect the expression of SRCIN1 in the three groups.Results 1 Compared with the adjacent tissues,the expression of mi R-17-5p in osteosarcoma was significantly increased(P<0.01),the m RNA level of SRCIN1 was significantly reduced(P<0.01),and the expression of mi R-17-5p was negatively correlated with the level of SRCIN1 in m RNA.In addition,the expression of SRCIN1 in osteosarcoma was lower than that in adjacent tissues.2 The expression of mi R-17-5p in osteosarcoma cell line MG63 was significantly higher than that of osteoblast h FOB1.19(P<0.01),and the expression of SRCIN1 was significantly lower than that of h FOB1.19(m RNA)in osteoblasts(P<0.01).3 The mi R-17-5p expression in mi R-17-5p inhibitor transfected group was significantly lower than that in NC group and Blank group(P <0.01).The results showed that the expression level of mi R-17-5p was significantly decreased after transfection of mi S-17-5p inhibitor in osteosarcoma cells.4.In the CCK-8(Cell Counting Kit)cell proliferation assay,the absorbance of the osteosarcoma cells at OD450 was significantly decreased in the mi R-17-5P inhibitor group compared with the NC group and the Blank group.(P <0.05).Compared with NC group and Blank group,there was no significant difference between the two groups(P> 0.05).There was no significant difference between the two groups(P> 0.05).5.Flow cytometry and Tunel assay showed that the apoptotic rate of osteosarcoma cells transfected with mi R-17-5p inhibitor group was significantly higher than that of NC group and Blank group compared with NC group and Blank group,(P <0.01).The apoptotic rate of osteosarcoma cells between NC group and Blank group was not significant(P> 0.05).The difference was not significant(P> 0.05).6.The results of Transwell showed that the number of osteosarcoma cells transfected with mi R-17-5P inhibitor group was significantly lower than that of NC group and Blank group,the difference was statistically significant(P < 0.05).There was no significant difference in the number of cells between the NC group and the Blank group through the basement membrane(P> 0.05).7.The results of scratches showed that the migration ability of cells transfected with mi R-17-5p group was significantly lower than that of NC group and Blank group compared with NC group and Blank group.Compared with Blank group,the migration ability of mi R-17-5p group was similar,No significant difference.8.Western blot and dual luciferase reporter assay results suggest that SRCIN1 is a target of mi R-17-5p.9.Western blot results suggest that the transfection of mi R-17-5p inhibitor agomir,mi R-17-5p inhibitor group and NC group and Blank group,the expression of SRCIN1 was significantly higher than NC group and Blank group increased,while the NC group compared with Blank group,the protein expression of SRCIN1 cells had no difference.Conclusions 1.In osteosarcoma pathology and osteosarcoma cells,mi R-17-5p abnormal high expression,SRCIN1 ralatived low expression.2.mi R-17-5p plays a biological role by acting on the 3’UTR region of SRCIN1 and negatively regulating its expression level.3.In osteosarcoma cells,the down-regulation of mi R-17-5p can effectively inhibit the migration,invasion and proliferation of osteosarcoma cells and promote the apoptosis of osteosarcoma cells,and play an inhibitory effect on osteosarcoma.