MiR-21-5p Influences Gliomas Progressions Via Regulating S100A10

Background:Glioma is an aggressive central nervous system tumor with a poor prognosis.The therapeutic targets and prognostic biomarkers have not been fully elucidated.S100A10 is involved in a variety of biological characteristics of malignant tumors,but it’s biological functions and the relationships with glioma pathological grade,treatment and prognosis of glioma are still unclear.In this study,we studied the function of S100A10 in the glioma progression,exploring whether S100A10 was correlated to the prognosis of glioma patients,and the influence of S100A10 on the malignant characteristics of glioma which are regulated by miR-21-5p.It might provide a new method in investigating the biological characteristics of glioma and providing a predictive indicators for the prognosis of glioma patients.Methods:1.The expression and clinical information data of S100A10 were downloaded from public databases(TCGA,CGGA and GEPIA2).The gene expression information,clinical significance and follow-up information of patients were obtained to identify the differential expression of S100A10 and it’s prognostic value.2.Tumor and peri-tumor tissues from 79 patients diagnosed with primary glioma of different grades and normal brain tissues from 6 patients with brain injury were collected and made into tissue chips for pathological HE staining and immunohistochemical staining to analyze the differences of S100A10 expression levels between tumors and peri-tumor as well as different grades gliomas.3.Real-time fluorescence quantitative PCR(qPCR)was used to detect the difference of S100A10 mRNA expression levels between glioma cell lines and normal glioma cells.Western blotting was performed to detect the difference of S100A10 protein expressions between different grades of glioma and normal brain tissues.4.the expression of S100A10 was knocked down by siRNA.CCK-8 cell proliferation assay,wound healing assay,transwell and flow cytometry were used to detect the proliferation,migration,invasion and apoptosis of glioma cells in the knock down group and the control group.Western blotting was performed to detect the changes of protein markers expressing in stem cells and epithelial mesenchymal transformation in U87 and U251 glioma cells after knocking down S100A10.5.The upstream micro RNA(miR-21-5p)of S100A10 was predicted by the online prediction website of miRNA target genes.Double luciferase assay was performed to identify the binding site of miR-21-5p in the downstream target gene in order to confirm whether S100A10 was a direct binding target of miR-21-5p.Western blotting was used to detect the influence of up or down expression of miR-21-5p on downstream target gene S100A10.6.Small interfering RNA of S100A10(si-S100A10)was co-transfected with miR-21-5p inhibitor into U87 and U251 cell lines to detect whether inhibiting S100A10 expression could reverse the effect of miR-21-5p inhibitor on the biological function of U87 and U251 glioma cells.Flow cytometry,Transwell,and western blotting were performed for analysis these effects.Results:1.Database analysis indicated that the expressions of S100A10 in glioma tissues were higher than that in the peri-tumor tissues.S100A10 higher expression predicted worse prognosis and was negatively correlated with the overall survival time of glioma patients.2.The results of q PCR and WB showed that S100A10 mRNA and protein expressions in glioma tissues were significantly increased compared to that in the peri-tumor tissues.In comparison with normal glial cells,the expression level of S100A10 protein in glioma cell lines was also significantly increased.3.The results of CCK-8 proliferation assay showed that down-regulated S100A10 expression could significantly inhibit the proliferation of U87 and U251 cells.The results of wound healing assay and transwell showed that interfering with S100A10 expression could significantly inhibit cell migration and invasion ability.The results of flow cytometry showed that knockdown of S100A10 expression could increase the apoptosis rate of U87 and U251 glioma cells.4.The results of WB showed that interference with S100A10 expression could reduce the expression of Nestin and CD133,the two surface markers of glioma stem cells;The expression of Vimentin,a marker of epithelial mesenchymal transformation,decreased,while the expression of E-cadherin increased.5.Dual luciferase assay confirmed that S100A10 was the downstream target gene of miR-21-5p,by which the expression of S100A10 is affected.Inhibiting the expression of miR-21-5p could up-regulate the expression of S100A10,while over-expressing miR-21-5p can down-regulate the expression of S100A10.6.The results of CCK-8 proliferation assay and flow cytometry assay showed that inhibition of miR-21-5p expression could promote the cell proliferation and reduce the cell apoptosis rate of U87 and U251 glioma cells;However,overexpression of miR-21-5p inhibited proliferation and increased cell apoptosis rate in U87 and U251 glioma cells.7.The results of transwell invasion assay and flow cytometry assay showed that reducing the expression of S100A10 significantly reversed the promoting effects of miR-21-5p inhibitor on the invasion of U87 and U251 glioma cells,and reversed the inhibitory effect of miR-21-5p inhibitor on the apoptosis of U87 and U251 glioma cells.Conclusion:Over-expressions of S100A10 in glioma tissues and glioma cells were associated with malignant glioma cell characteristics.Down-regulating S100A10 expression inhibited the proliferation,invasion,migration and epithelial and mesenchymal transformation of glioma cells,as well as reduced the expression of glioma stem cells makers.S100A10 was a new prognostic predictor among glioma patients.The expression of S100A10 was affected by miR-21-5p,of which S100A10 is the downstream target gene.