PVT1 Modulates Proliferation And Migration Of BMSCs In C6 Cell Microenvironment Through Regulating MiR-134-5p

Aim:This study was aimed to explore the effect of glioma C6 cell microenvironment on the proliferation and migration ability of BMSCs and the role and mechanism of long noncoding RNA PVT1 in these changes.Methods:Part Ⅰ: BMSCs were indirectly co-cultured with glioma C6 cells to simulate the glioma C6 cell microenvironment through 6-well transwell chamber.The experiments were divided into co-cultured group(BMSCs co-cultured with C6 cells)and control group(BMSCs cultured individually).CCK8,flow cytometry assay and soft agar colony formation assay were conducted to detect the proliferation,cell cycle and colony formation ability of the two group of cells.Wound healing assay as well as transwell migration assay was carried out to detect the migration ability of the two group of cells.Mi R-134-5p expression in the two of cells was detected by stem loop RT-qPCR.Lnc RNAs up-regulated in gliomas were looked up by searching the previous researches.Then RNA22 v2 micro RNA target detection software was used to predict the lncRNAs that may target miR-134-5p.RT-qPCR was carried out to select the differentially expressed lncRNA between normal BMSCs and co-cultured BMSCs,and the candidate lncRNA was selected for further study.Part Ⅱ: Specific siRNA against PVT1 was transfected to co-cultured BMSCs to knockdown PVT1 expression.RT-qPCR was used to measure the efficiency of si-PVT1.The effect of PVT1 on the proliferation,colony formation,cell cycle and migration ability of co-cultured BMSCs were detected by CCK8,soft agar colony forming assay,flow cytometry assay,wound healing assay and transwell migration assay.Part Ⅲ: The expression of miR-134-5p in si-PVT1 group and si-NC group was measured by stem loop RT-qPCR.Dual luciferase reporter assay was conducted to test the binding between PVT1 and miR-134-5p.The proliferation,colony formation,cell cycle and migration ability of co-cultured BMSCs which were transfected with si-PVT1 or co-transfected with si-PVT1 and miR-134-5p inhibitor were detected using the same methods as part Ⅱ.The m RNA of miR-134-5p and STAT3 were explored by RT-qPCR and the protein of STAT3 was explored by WB.Results:Part Ⅰ: The proliferation ability of co-cultured BMSCs was significantly enhanced compared with normal BMSCs.In co-cultured BMSC group,colony sphered in soft agar.The expression of miR-134-5p in co-cultured BMSCs was down-regulated in comparison with normal BMSCs.PVT1,H19 and XIST were predicted to have binding sites with miR-134-5p.RT-qPCR results showed that only PVT1 was up-regulated in co-cultured BMSCs,so PVT1 was chosen for further study.Part Ⅱ: RT-qPCR showed that PVT1 was significantly down-regulated after transfected with si-PVT1 in co-cultured BMSCs.Compared with si-NC group,cell proliferation,colony formation and migration abilities all inhibited in si-PVT1 group.Part Ⅲ: Mi R-134-5p was upregulated in si-PVT1 group detected by RT-qPCR.The relative luciferase activity did not reduce in the GP-miRGLO-PVT1-WT plasmid and miR-134-5p mimics co-transfected group in comparison with GP-miRGLO-PVT1-MUT and miR-134-5p mimics co-transfected group.The proliferation,colony formation and migration ability of co-cultured BMSCs co-transfected with si-PVT1 and miR-134-5p inhibitor were significantly enhanced compared with si-PVT1 group.Mi R-134-5p was down-regulated in si-PVT1 and miR-134-5p inhibitor co-transfected group compared with si-PVT1 group;STAT3m RNA and protein was down-regulated in si-PVT1 group,and the miR-134-5p inhibitor reversed the decreases in STAT3 m RNA and protein levels.Conclusion:1.The proliferation,colony formation and migration abilities of co-cultured BMSCs was significantly enhanced;2.The expression of miR-134-5p was down-regulated and PVT1up-regulated in co-cultured BMSCs compared with normal BMSCs.PVT1 positively regulated the proliferation,colony formation and migration abilities of co-cultured BMSCs.3.PVT1 promoted the proliferation,colony formation and migration abilities of co-cultured BMSCs by positively regulate STAT3 expression through inhibiting miR-134-5p.