| Objective: Human cytomegalovirus(HCMV)is a neurotropic virus.Once the human body is infected with the virus,it will remain latent in the nervous system for life.Recent studies have shown that latent infection of HCMV can affect the development of nervous system tumors by regulating host cell micro RNA(miRNA).According to related reports,miR-409-3p plays an important regulatory role in colon cancer,bladder cancer,osteosarcoma and other diseases.Whether miR-409-3p plays a regulatory role in malignant glioma after HCMV infection,currently It’s not clear yet.ZEB1 is a transcription factor.ZEB1 plays a vital role in regulating the survival,differentiation,proliferation,senescence and apoptosis of cancer cells.And it is closely related to tumor cell migration,invasion and metastasis.At present,there are few studies on the mechanism of ZEB1 in malignant glioma.This study will focus on the effect of HCMV infection on micro RNA-409-3p/ZEB1 in glioma cells,and explore the molecular mechanism of viral infection regulating the migration of glioma cells.Methods: This study analyzed the transcriptome sequencing results after HCMV infection,and explored the differences in the expression levels of miR-409-3p before and after infection.Then verified the expression of miR-409-3p in glioma cell lines U251,T98,and U87 cells after infection with HCMV.At the same time,Targetscan was used to predict the bioinformatics of miR-409-3p target genes,and the zinc finger binding protein was screened out.1(ZEB1)is a potential downstream target gene of miR-409-3p,The wildtype(Wt)and mutant(Mut)dual luciferase vectors were used for binding site verification,followed by transfection of HCMV-infected U87 cells with miR-409-3p mimics,miR-409-3p-NC,and sh ZEB1,Real-time quantitative RT-PCR(q RT-PCR)detects changes in ZEB1 gene expression level,Western-Blot detects changes in ZEB1 protein expression level,The expression level of the ZEB1 gene were detected by real-time quantitative RT-PCR(q RTPCR),The expression level of the ZEB1 protein were detected by the Western-Blot,and The migration capacity of U87 cells were measured by comparing the rate of wound healing in cell scratches and the number of cell migrations in the Transwell cell migration experiment.Results: Transcriptome sequencing data showed that miR-409-3p was significantly down-regulated in HCMV-infected U87 cells(P <0.05).Quantitative RT-PCR(q RT-PCR)results showed that miR-409-3p was down-regulated in HCMV infected U87,U251,and T98 glioma cell lines(P <0.05),while ZEB1 was significantly up-regulated(P <0.05).Data provided by the Bioinformatics website showed that miR-409-3p could interact with the 3’-UTR of ZEB1,and the luciferase activity in cells transfected with wild-type ZEB13’-UTR and miR-409-3p is significantly lower than that of the mutant(P <0.01).The Wound Healing assay and Transwell migration assay showed that in the recovery experiment of overexpressing miR-409-3p and ZEB1,the overexpression of miR-409-3p inhibited the migration ability of U87 cells(P <0.05),while the recovery of ZEB1 expression could restored the migration ability of U87 cells(P <0.05).The results of q RT-PCR and Western Blot showed that the expression of ZEB1 in the overexpression group of miR-409-3p decreased(P <0.05),on the other hand,the expression of ZEB1 in the ZEB1 recovery group was restored..It was confirmed that miR-409-3p regulates the migration of glioma cells through ZEB1.Conclusion: In this study,we demonstrated the targeting relationship between miR-409-3p and ZEB1 gene.In HCMV infected U87 cells,HCMV induced miR-409-3p downregulation and then up-regulation of ZEB1,which enhanced the migration ability of U87 cells.The expression changes of miR-409-3p caused by HCMV infection play an important regulatory role in the course of glioma. |
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