The Effects Of MiR-100 On Proliferation,Invasion And Migration Of Osteosarcoma Cell

BackgroundOsteosarcoma?OS?is the most common primary malignant bone tumor,mainly affectsadolescents and children,characterized by high malignancy and early metastasis,it seriously affects the physical and mental health of patients.Although OS is treated with neoadjuvant chemotherapy,surgery,radiotherapy and other comprehensive treatments,the patient's 5-year survival rate has increased to 60-80%,However,there are still many patients who have the risk of local recurrence and metastasis and poor prognosis.Recurrence and metastasis are important factors affecting survival rate of OS patients,but,the mechanism about the occurrence and metastasis of OS is not yet clear,therefore,it is necessary to explore the specific mechanism of invasion and metastasis of OS and further improve the survival rate of patients.miRNA?microRNAs?are closely related to the occurrence and development of tumors,and participate in the growth,differentiation,metastasis and apoptosis of various tumors,there were few studies on the correlation between miRNA and OS.It has been found that miR-100 is low expression in many malignant tumors and inhibits the proliferation and metastasis of malignant tumors,preliminary experiment has confirmed that miR-100 is low expression in OS cell lines,and we need to further clarify the effects of miR-100 on proliferation,invasion and metastasis of OS cell.ObjectiveTo clarify the expression level of miR-100 in OS cell,to investigate the effects of overexpressionof miR-100 on proliferation,invasion and migration of OS cell,to provide theoretical basis and new method for gene targeting therapy of OS.Methods1.The expression levels of miR-100 in OS cell lines HOS,U-2OS,Saos-2,MG-63 and normalosteoblast NHOst were detected by qRT-PCR.2.The miR-100 mimics and miR-100 NC were respectively transfect to the OS cell line MG-63 by liposome 2000 transient transfect technology to construct the intervention model of OS cell,and the cell model was divided into miR-100 group?miR-100 mimics?,miR-100 NC group?miR-100 NC?,control group?liposome 2000?;the expression levels of miR-100 in miR-100group,miR-100 NC group,control group were detected by qRT-PCR and to verify the transfection efficiency of miR-100.3.CCK-8 proliferation assay was respectively conducted after transfection 0h,24h,48h,72h and 96h,the absorbance of wavelength at 450nm was determined by ELIASA,cell proliferation ability was compared between the miR-100 group,the miR-100 NC group and control group at different time points.4.After the cell model was successfully constructed,Transwell invasion assay was conducted in a small chamber containing matrigel,the field of vision is counted under the light microscope,the changes of cell invasion ability were compared between the miR-100 group,the miR-100 NC group and control group.5.After the cell model was successfully constructed,Transwell migration assay was conducted in a small chamber containing non-matrigel,the field of vision is counted under the light microscope,the changes of cell migration ability were compared between the miR-100 group,the miR-100NC group and control group.Results1.We use the qRT-PCR detected the expression levels of miR-100 and found the OS cell linesHOS?P=0.004?,U-2OS?P=0.000?,Saos-2?P=0.017?and MG-63?P=0.000?were significantly lower than that of the normal osteoblast NHOst,there was a statistical difference,the result showed that miR-100 was low expression in OS cell lines compared with the NHOst.2.The results of transfection experiment in OS cell line MG-63 showed that the expression level of miR-100 group was significantly higher than that of the miR-100 NC group and control group,there was a statistical difference between the miR-100 group and the miR-100 NC group?P=0.000?,there was a statistical difference between the miR-100 group and the control group?P=0.000?,there was no statistical difference between the miR-100 NC group and the control group?P=0.992?,the model of miR-100 transfected with OS was successfully constructed.3.The CCK-8proliferation assay found that the CCK-8 OD_450nm values were roughly close between the miR-100 group,miR-100 NC group and control group after transfection 0h time point,and there was no statistical difference between the three groups?P=0.439?;the CCK-8 OD_450nm50nm value of miR-100 group was lower than that of the miR-100 NC group and control group after transfection 24h,48h,72h,96h time points,there was a statistical difference between the miR-100 group and the miR-100 NC group?P<0.05?,there was a statistical difference between the miR-100 group and the control group?P>0.05?,there was no statistical difference between the miR-100 NC group and the control group?P>0.05?;compared with miR-100 NC group,control group,the proliferation curve of miR-100 group was significantly lower,it was shown that the overexpression of miR-100 inhibit the proliferation of OS cell.4.Transwell invasion assay found that the number of permeable basement membrane cells of miR-100 group was significantly lower than that of the miR-100 NC group and control group,there was a statistical difference between the miR-100 group and the miR-100 NC group?P=0.000?,there was a statistical difference between the miR-100 group and the control group?P=0.000?;the number of cells were roughly close between the miR-100 NC group and the control group,there was no statistical difference between the two groups?P=0.904?,it was suggested that the overexpression of miR-100 inhibit the invasion ability of OS cell.5.Transwell migration assay found that the number of cells entering the lower chamber of miR-100 group was significantly lower than that of the miR-100 NC group and control group,there was a statistical difference between the miR-100 group and the miR-100 NC group?P=0.000?,there was a statistical difference between the miR-100 group and the control group?P=0.000?;the number of cells were roughly the same between the miR-100 NC group and the control group,there was no statistical difference between the two groups?P=0.880?,it was suggested that the overexpression of miR-100 inhibit the migration ability of OS cell.Conclusion1.miR-100 was low expression in OS cell lines compared with the normal osteoblast NHOst.2.Overexpression of miR-100 inhibited the proliferation,invasion and migration of OS cell in OS cell lines.3.miR-100 can be a targeted gene for the treatment of OS,and there is a new method to treat OS.