HNRNPA2/B1 Regulates Apoptosis-related Gene PLK3 Expression And BIM Alternative Splicing In Breast Cancer Cells

Objectives:As a splicing regulator,Previous studies found HNRNP A2/B1 overexpression in breast cancers associated with various physiological processes of cells.Our studies are to illustrate HNRNP A2/B1 regulates apoptosis-related gene PLK3 expression and BIM alternative splicing in breast cancer cells.Methods:We knocked out HNRNP A2/B1 by CRISPSR-Cas9 technique in MCF-7 and MDA-MB-231 cell,combined transcriptome sequencing analysis with RTPCR to verify the expression of apoptosis-related gene.On one hand,RNA immunoprecipitations were performed from MDA-MB-231 or MCF-7 cell to show whether HNRNP A2/B1 bind to the pre-mRNA of apoptosis-related gene.On the other hand,we detected whether the apoptosis-related genes were spliced by RT-PCR.Finally,we detect the effect of these genes on cell-related phenotypes through CCK8 assay,transwell and flow cytometry.Tunel method was used to detect the apoptosis of mouse xenografts of MCF-7 before and after HNRNP A2/B1 knocked out.Then cells were treated with different concentrations of hydrogen peroxide to verify the apoptosis rate before and after HNRNP A2/B1 knocked out.And we also overexpressed HNRNP A2/B1 to detect its effection on apoptosis.RESULTS:Knocked-out of HNRNP A2/B1 in breast cancer cells inhibited the expression of Pro-apoptotic proteins PLK3,overexpression of hnRNP A2 promoted the expression of PLK3 protein.The result of RNA immunoprecipitation showed that HNRNP A2/B1 protein could bind to the pre-mRNA of Pro-apoptotic genes PLK3 and BIM,and splicing the exon E125 of BIM gene.Overexpression of PLK3 protein in MCF-7 cells promotes its apoptosis,inhibits its proliferation and migration.The apoptosis rate of HNRNP A2/B1 knocked-out group of MCF-7 nude mice subcutaneous xenografts was lower than that of the mock group,and the expression of cleaved-caspase3 was decreased.The apoptotic rate of HNRNP A2/B1 knocked-out cells was higher than that of not knocked out when treated with concentration of hydrogen peroxide.Overexpression HNRNP A2/B1 suppressed MCF-7 cell apoptosis.Conclusion:HNRNP A2/B1 protein regulates PLK3 expression and can bind to PLK3 pre-mRNA.PLK3 can promote apoptosis and inhibits its proliferation and migration of breast cancer cell MCF-7.The HNRNP A2/B1 protein splicing exon E125 by binding to the pre-mRNA of the BIM gene.HNRNP A2/B1 protein can suppress the apoptosis of MCF-7 and MDA-MB-231 cells.hnRNPA2/B1 regulates apoptosis-related gene PLK3 expression and BIM alternative splicing in breast cancer cells.