MicroRNA-148a-3p Promotes Human Glioma Cell Migration And Invasion By Targeting ST18

Objective:Nowadays,glioma is a common primary brain malignancy in central nervous system diseases.Due to its characteristics of high recurrence rate,high fatality rate,poor prognosis,and et al.,the current treatment program for glioma at home and abroad is basically a comprehensive treatment program of surgical treatment and adjuvant radiotherapy and chemotherapy.Although patients received such treatment,the survival rate and recurrence rate of glioma after surgery are still not optimistic.In this study,microRNA(miRNA)miR-148a-3p was selected as the research object,and Suppression of tumorigenicity 18(ST18)was screened and verified as its potential target gene in glioma,meanwhile,which effects of ST18 gene regulated by miR-148a-3p on the biological functions of Glioblastomamultiforme(GBM)were investigated.It lays a theoretical foundation for providing new directions and ideas for clinical targeted therapy of GBM in the future.Methods:1.Bioinformatics databases(TargetScan,miRanda,miRBase)was used to screen the potential target gene of miR-148a-3p: Thetarget gene prediction databases commonly used in bioinformatics at present.were used to find out the potential target gene of miR-148a-3p.The target genes of miR-148a-3p studied in GBM field were excluded by literature reading,and the target gene ST18 with high possibility was selected as the target gene of this study.2.Real-time fluorescence quantitation PCR(qRT-PCR)that Western Blotting were chosen to verify the expression level of ST18 gene in each GBM cell line: qRT-PCR and Western Blotting were chosen to verify the protein and mRNAexpression level of ST18 in immortalized glial cell(HA1800)and glioma cell lines(U87 and U251),and the mRNA and protein expression levels of ST18 gene in glioma cell lines under different miR-148a-3p expression conditions(overexpression or inhibition)were detected.3.Detection of the targeting relationship between miR-148a-3p and ST18 gene by luciferase reporter assay:In HEK-293 T cells,luciferase reporter gene assay was used to verify the targeting relationship between miR-148a-3p and ST18 gene.4.Transwell migration and invasion assay and scratch assay were used to detect the biological function of miR-148a-3pregulating ST18 gene on the migration and invasion of glioma cells: In glioma cell lines U87 and U251,the effect of miR-148a-3p regulating ST18 gene on the migration ability of glioma cells was detected by scratch test and Transwell migration test.Transwell invasion assay was used to detect the effect of miR-148a-3p regulating ST18 gene on the invasion ability of glioma cells.Results:1.The target gene was successfully screened,and ST18 was one of the potential target genes of miR-148a-3p: According to the data from various bioinformatics databases,ST18 is the downstream targeted regulatory target gene of miR-148a-3p.2.ST18 gene was down-regulated in glioma cells: The result of qRT-PCR showed that the mRNA expression level of ST18 gene in glioma cell lines(U87,U251,and T98G)was significantly lower than that in immortalized glial cells(HA1800).3.miR-148a-3p targeted regulation of ST18 gene: After overexpression or inhibition of miR-148a-3p,the mRNA and protein expression of ST18 gene in glioma cell lines(U87,U251)were detected by qRT-PCR and Western blotting,respectively.It was found that the mRNA and protein expression levels of ST18 gene in glioma cells with overexpression of miR-148a-3p were significantly lower than those in the control group.When miR-148a-3p expression was inhibited,the mRNA and protein expression levels of ST18 gene in each glioma cell lines were significantly higher than those in the control group.In addition,the fluorescence intensity test results of HEK-293 T cells transfected with reporter plasmid showed that the fluorescence intensity of ST18-WT + miR-148 a experimental group was significantly decreased compared with that of ST18-WT+ miR-148a-NC group,then the difference was significant(P<0.05).It was shown that ST18 was the direct target gene of miR-148 a in glioma cells.4.miR-148 a targeted the regulation of ST18 gene to promote the migration ability of glioma cells:In glioma cells U87 and U251,scratch experiments were carried out after overexpression of ST18 gene,and it was found that the overexpression of ST18 gene could significantly reduce the migration ability of glioma cells.In addition,the cell migration ability was detected by Transwell migration assay,and it was found that overexpression of ST18 could reduce the migration ability of glioma cells.Moreover,overexpression of ST18 gene and miR-148a-3p can significantly restore themigration ability of glioma cells.It has been shown that miR-148a-3p can promote the migration of glioma cells by targeting the regulation of ST18 gene.5.miR-148a3 p targeted the regulation of ST18 gene to promote the invasion ability of glioma cells:In glioma cells U87 and U251,the cell invasion ability was detected by Transwell invasion assay.The results showed that overexpression of ST18 could reduce the invasion ability of glioma cells.Moreover,overexpression of ST18 gene and miR-148a-3p can significantly restore the invasion ability of glioma cells.It has been shown that miR-148 a can promote the invasion ability of glioma cells by targeting the regulation of ST18 gene.Conclusion:1.ST18 is one of the target genes of miR-148a-3p.2.miR-148a-3p targeting regulation of ST18 promoted gliomacells migration and invasion.