HCMV Ie2 Affects The Migration Of Glioblastoma By Mediating The Different Splicing Patterns Of RON Through HnRNP A2B1

Objective: Human cytomegalovirus(HCMV)belongs to the subfamily of ?-herpesvirus,which is a linear double-stranded DNA virus.In the early stage of infection,HCMV mainly expresses two proteins including IE72(ie1-encoded)and IE86(ie2-encoded).IE86 is a glycosylated protein composed of 579 amino acids,which plays an important role in the occurrence and development of malignant glioma.Malignant glioma is the most common intracranial tumor with a high degree of malignancy and a poor prognosis.Most of them show invasive growth and cannot be completely removed by surgery.Cobbs et al.first noted the association of HCMV infection with human glioblastomas.However,it is unknown whether IE86 plays a key role in inducing the migration of malignant glioma.Heterogeneous nuclear ribonucleoproteins(hnRNPs)is an RNA-binding protein and hnRNP A2B1 is a member of its family.Recent studies demonstrate that hnRNP A2B1 protein is highly expressed in tissue specimens of malignant glioma and is associated with malignant glioma classification.RON is a proto-oncogene whose alternatively spliced variants participate in several tumor processes,such as cell migration,apoptosis,and epithelial-to-mesenchymal transition(EMT).? Ron(also known as Ron ? 165)is generated by skipping exon 11.Overexpression of ? Ron leads to the loss of epithelial phenotype,increasing cell motility and invasiveness.So far,the role of hnRNP A2 B1 in glioblastoma development remains poorly understood.This study investigated the effects of ie2 on the expression of hnRNP A2B1 in gliomas and the migration mechanism of malignant glioma.Methods: Co-immunoprecipitation(CO-IP)was used to investigated which proteins interact with IE86 protein in U87 malignant glioma(MG)cells.Using liquid chromatography mass spectrometry(LC-MS / MS)technology and database search to find interacting proteins.The expression level of splicing factor hnRNP A2B1 was measured by Quantitative Real-time Polymerase Chain Reaction(qRT-PCR)and Western Blot in in the brain tissues of ie2 transgenic mice and C57 mice(negative control),and U87 MG and U87 MG transfected with pEGFP-N3-ie2 plasmid.Using QRT-PCR and Western Blot,we assessed the expression of hnRNP A2B1 after pPR244-shRNA knockdown.Wound healing assay and Transwell migration assay were used to evaluate the changes of U87 MG migration activity after transfected of pEGFP-N3-ie2 plasmid and knockdown of hnRNPA2B1.The splicing changes of RON were detected by RT-PCR after transfected of pEGFP-N3-ie2 plasmid and knockdown of hnRNP A2B1.Results: In U87 MG cells,seven proteins interacted with IE86,which are hnRNP A2B1,hNRNP A1,hnRNP C,hnRNP M,SYNCRIP,hnRNP K and hnRNP H1.The most abundant protein detected was hnRNP A2B1.qRT-PCR and Western Blot results showed that much higher expression of hnRNP A2B1 in HCMV· ie2 transgenic mice brain compared to that in C57 wild type(wt)mice(P<0.01),and the expression of hnRNP A2B1 in U87 MG transfected with pEGFP-N3-ie2 plasmid was higher than in U87MG(P< 0.01).HnRNP A2B1 expression was effectively knocked down by pPR244-shRNA in the three groups,and the mRNA and protein expression of hnRNP A2B1 decreased compared with the U87-NC group(P<0.01).Wound healing assay suggested that the migration ability of the pEGFP-N3-ie2-U87 group was increased(P< 0.05)and the migration ability of the sh1-A2B1-U87 group was reduced(P< 0.05)compared with the U87-NC group.The results of Transwell migration assay manifested that the migration ability of pEGFP-N3-ie2-U87 group increased(P< 0.01),while that of sh1-A2B1-U87 group decreased(P< 0.01),and shRNA could partially offset the promotion of pEGFP-N3-ie2.RT-PCR results showed that compared with U87-NC group,the splicing of RON in pEGFP-N3-ie2-U87 group was enhanced and the ratio of ? Ron / Ron was increased(P< 0.05);the ratio of ? Ron / Ron was decreased in sh1-A2B1-U87 group(P< 0.05).Conclusion: In this study,IE86 protein interacts with hnRNP A2B1 protein in U87 MG cells.hnRNP A2B1 is highly expressed in the HCMV·ie2 transgenic mice model.The expression level of hnRNP A2B1 was increased in U87 MG cell line transfected with pEGFP-N3-ie2 plasmid.In addition,hnRNP A2B1 knockdown in U87 MG cells inhibited tumor migration,and this effect might be mediated by hnRNP A2B1 through affecting the splicing patterns of RON.Our data suggested that HCMV· ie2 promotes the migration of glioblastoma by mediating the different splicing patterns of RON through hnRNP A2B1.