BACH2 And FUS Interaction Promotes Malignant Progression Of Glioma Cells Via The Linc00589/microRNA-10b-5p/WWC3 Pathway

Objective:Glioma is a common malignant tumour in the human central nervous system.It has a poor prognosis and the current treatment options are limited.Thus,it is important to study the biological behaviour and mechanism of glioma cells to facilitate the development of better therapies.This article aimed to investigate whether the interaction between the transcription factor BACH2 and the RNA-binding protein FUS can regulate the malignant biological behavior of glioma cells,and further studied its underlying molecular mechanism.We further aimed to explore whether the interaction between BACH2 and FUS could down-regulate the expression of Linc00589 by enhancing its transcriptional repression effect on Linc00589;thereby attenuat the negative regulatory effect of Linc00589 on miR-10b-5p;furhter reduce the WWC3 expression by increasing miR-10b-5p binds to the 3'UTR of WWC3,block Hippo signaling pathway,and promote malignant biological behavior of glioma cells.Methods:Real-time quantitative PCR and Western blotting were performed to investigate the endogenous expression levels of BACH2,FUS,Linc00589,miR-10b-5p,WWC3 in glioma tissues and cells.In order to study the molecular mechanism of the interaction between BACH2 and FUS and its effect on the malignant behavior of glioma cells,stable knockdown of BACH2,FUS,over-expressing Linc00589,over-expressing and silencing miR-10b-5p and over-expressing and silencing WWC3 glioma cell lines U87 and U251 and co-transfecting knockdown of BACH2 and FUS,cotransfecting over-expressing Linc00589 and over-expressing or silencing miR-10b-5p,cotransfection over-expressing miR-10b-5p and over-expressing WWC3 or WWC3(non3'UTR)U87 and U251 cells were constructed.CCK-8,Transwell,and flow cytometry were used to measure the proliferation,migration,invasion,and apoptotic capacity of glioma cells,respectively.Western blotting was used to detect the changes in the phosphorylation level of YAP which is a key signal molecule of Hippo signaling pathway.Co-immunoprecipitation and GST pull-down experiments were used to detect the interaction between BACH2 and FUS.The intracellular localization of BACH2 and FUS and the nucleoplasmic distribution of YAP were detected by immunofluorescence.The dual luciferase gene reporting system was used to determine the targeted binding of Linc00589 and miR-10b-5p,as well as mi R-10b-5p and WWC3.Chromatin Immunoprecitation(ChIP)assays were conducted to ascertain the binding of BACH2 to the Linc00589 promoter region.Additionally,the in vivo effects of BACH2,FUS and Linc00589 alone or in combination were studied in nude mice experiments.Results: This study provided evidence that BACH2,FUS was up-regulated in glioma tissues and cells,whereas Linc00589 was down-regulated in glioma tissues and cells.Knockdown of BACH2,FUS or overexpression of Linc00589 significantly inhibited proliferation,migration and invasion of glioma cells,and promoted glioma cells apoptosis.BACH2 had a binding effect with the Linc00589 promoter region.BACH2 enhanced its transcriptional repression against Linc00589 by interacting with FUS.Coknockdown of BACH2 and FUS significantly up-regulated the expression of Linc00589.miR-10b-5p was up-regulated in glioma tissues and cells,and WWC3 is down-regulated.Knocking down miR-10b-5p or overexpressing WWC3 significantly inhibited proliferation,migration and invasion of glioma cells and promoted glioma cells apoptosis.Linc00589 targeted miR-10b-5p and overexpression of Linc00589 negatively regulates miR-10b-5p expression in a RISC-dependent manner.miR-10b-5p targeted WWC3 3'UTR region,and knockdown of miR-10b-5p up-regulated WWC3 mRNA and protein expression by attenuating its targeted binding to the WWC3 3'UTR region.Overexpression of WWC3 activated the Hippo signaling pathway by up-regulating the phosphorylation level of YAP and promoting the outflow of YAP from the nucleus to the cytoplasm,thereby inhibited the proliferation,migration and invasion of glioma cells and promoted glioma cells apoptosis.Moreover,we found that knockdown of BACH2,knockdown of FUS,and overexpression of Linc00589 or the combination of the three could inhibit the growth of subcutaneously transplanted tumors in nude mice and prolong the survival time.Furthermore,the combined use of the three resulted in the smallest tumorsvolume and the longest survival time in nude mice.Conclusions: 1.BACH2 interacted with FUS.BACH2 bound to Linc00589 and negatively regulated the expression of Linc00589,promoted the proliferation,migration and invasion of glioma cells and inhibited apoptosis.2.Linc00589 bound miR-10b-5p,thereby weakening the negative regulatory effect of miR-10b-5p on WWC3,inhibiting the proliferation,migration and invasion of glioma cells,and promoting apoptosis.3.miR-10b-5p targeted the 3'UTR of WWC3 mRNA and negatively regulated the expression of WWC3,reduced the phosphorylation level of YAP,promoted the entry of YAP into the nucleus,increased the proliferation,migration and invasion of glioma cells and inhibited apoptosis.4.The interaction between BACH2 and FUS played an important role in regulating the biological behavior of glioma cells through the Linc00589 / miR-10b-5p / WWC3 axis.