HCMV-IE86 Affects Glioma Cells By Promoting HnRNP A2/B1-mediated Alternative Splicing Of Bcl-x

ObjectHuman cytomegalovirus(HCMV)is a ubiquitous pathogen that can cause disease in immunocompromised people.The ie2 gene of HCMV is expressed immediately after viral infection,and is translated into IE86 protein,which plays an important role in regulating viral replication,and studies have shown that IE86 is involved in the malignant transformation of gliomas,but its pathogenic mechanism has not been clearly defined set forth.HnRNP A2 / B1 is a splicing factor that participates in regulating the selective splicing process of multiple genes,such as apoptosis-related gene bcl-x.HnRNP A2 / B1 has been shown to be overexpressed in many tumor cells,including glioma cell U251.Glioma is one of the most common malignant primary malignant brain tumors,originating in the central nervous system.Previous studies have demonstrated that IE86 can promote the malignant proliferation of glioma cells,but the mechanism is not yet clear.In this study,U251 glioma cells were selected to establish U251 cells capable of stably exogenously overexpressing HCMV-IE86,in-depth exploration of the molecular mechanism of HCMV-IE86 affecting glioma,and the splicing factor hnRNP A2 / B1 mediate The guiding role is to find another target for the treatment of gliomas infected with HCMV.Method1.Transfect pEGFP / IE86 into U251 cells with liposomes,and extract m RNA and protein,RT-PCR and Western blot to identify whether IE86 is exogenously overexpressed.2.Identify the components of IE86 immune complex in U251 cells by immunoprecipitation combined with mass spectrometry.3.Use RT-PCR to identify transgenic ie2 mice,extract the mRNA and protein of mouse brain tissue,and detect the differences of hnRNP A2 / B1 expression by RT-PCR and Western Blot in ie2 positive mice and negative mice,exogenously overexpress IE86 and normal U251 cells.4.In U251 cells,suppress the expression of hnRNP A2 / B1 gene by shRNA,and detect the mRNA and protein levels of hnRNP A2 / B1 after knockdown.5.RT-PCR,Western blot detect bcl-x splicing after knocking down hnRNP A2 / B1 in exogenously overexpressing IE86 cells.6.Flow cytometry,CCK8,etc.were used to detect the proliferation and apoptosis ability of hnRNP A2 / B1 cells knocked down in exogenously overexpressing IE86 cells and the expression of caspase3 by Western Blot.Result1.Successful establishment of U251 cell line capable of stably exogenously overexpressing IE86.2.Identification of IE86 immune complex components,8 splicing factors that interact with IE86.3.Compared with negative mice,the expression level of hnRNP A2 / B1 in ie2-positive mice is higher.Similarly,the expression level of hnRNP A2 / B1 in exogenously overexpressing IE86 cells is higher than that of normal U251 cells.4.Successful knockdown of hnRNP A2 / B1 by shRNA in U251 cells,and the expression level of hnRNP A2 / B1 was significantly reduced after knockdown.5.U251 cells with knocking down of hnRNP A2 / B1,compared with normal U251 cells,increased the expression of bcl-xs/Bcl-xS and decreased the expression of bcl-xl/Bcl-xL.However,down-regulating hnRNP A2 / B1 in IE86-positive cells significantly reduced the effect of IE86 on bcl-xs / bcl-xl,Bcl-xS/Bcl-xL expression compared with IE86-positive cells.6.After knocking down hnRNP A2 / B1,compared with normal U251 cells,cell proliferation decreased,apoptosis increased,and caspase3 expression increased.And down-regulating hnRNP A2 / B1 in cells exogenously overexpressing IE86,compared with cells exogenously overexpressing IE86,significantly reduced cell proliferation rate,increased apoptosis rate,and increased caspase3 expression.ConclusionsIE86 interacts with hnRNP A2/B1,and IE86 can promote the proliferation of glioma cells and inhibit apoptosis by promoting the expression of hnRNP A2/B1.