| Objective: The objective of this study is to investigate the effect of miR-34a-5p on the proliferation and migration of SH-SY5 Y neurons infected with Kaposi’s Sarcoma-associated Herpesvirus(KSHV),and detect the targeted regulation of miR-34a-5p on c-fos.Methods: 1.Verify the regulation of miR-34a-5p on c-fos expression in KSHV-infected SH-SY5 Y cells.(1)The KSHV-infected(SK-RG cells)and uninfected SH-SY5 Y cells were used to screen differentially expressed miRNAs by transcriptome sequencing and bioinformatic analysis.q RT-PCR was used to verify the level of miR-34a-5p in SH-SY5 Y and SK-RG.(2)The binding sites of miR-34a-5p and c-fos 3’-UTR were predicted by website Star Base2.0,the targeting relationship between miR-34a-5p and c-fos 3’-UTR was determined by dual luciferase reporter gene assay to analyze the mechanism of function.(3)The expression level of c-fos in SH-SY5 Y and SK-RG cells was detected by Western blot.(4)Lipofectamine 2000 was used to transfect miR-34a-5p mimics and miR-NC into SK-RG cells,respectively,q RT-PCR was used to detect the transfection effect.The expression level of c-fos after transfection of miR-34a-5p mimics and miR-NC was detected by Western blot.2.To detect the effect of miR-34a-5p/c-fos on the proliferation and migration ability of KSHV-infected neuronal cells.(1)After up-regulated the miR-34a-5p,cell proliferation ability was determined by MTT and plate clone assay,the cell cycle was assessed by flow cytometry analysis,and CDK4,CDK6,cyclin D1 levels were determined by Western blot analysis;the cell migration ability was detected by wound healing,transwell assay and the level of MMP2,MMP9.(2)Rescue assays were carried out by upregulating c-fos in miR-34a-5p-overexpressing SK-RG cells,the expression level of c-fos was detected by q RT-PCR;MTT and plate cloning were used to detect cell proliferation,the cell cycle was assessed by flow cytometry analysis and the expression levels of CDK4,CDK6 and cyclin D1 were detected by Western blot;the cell migration ability was detected by wound healing,transwell assay and the level of MMP2,MMP9.(3)Taq-man real-time PCR was used to detect the DNA copy number of KSHV virus.(4)q RT-PCR was used to detect the m RNA levels of LANA,RTA,K8.1 and v GPCR of KSHV virus genes.3.To study the effect of miR-34a-5p on the proliferation of KSHV-infected SH-SY5 Y cells xenograft tumor.(1)The xenograft tumor model of SK-RG nude mouse was constructed.(2)The effects of miR-34a-5p on tumor growth in vivo were examined by injecting miR-34a-5p agomir and miR-NC agomir.(3)Immunohistochemical detection of cfos,CDK6,and cyclin D1 levels in tumor tissues.(4)q RT-PCR was used to detect the m RNA levels of tissue section virus genes.Results: 1.(1)The results of transcriptome sequencing and q RT-PCR indicated the expression of miR-34a-5p in SK-RG was lower than that in SH-SY5 Y cells(P<0.05).(2)The results of dual luciferase reporter gene assay suggested that miR-34a-5p could regulate the expression of c-fos through the 3’-UTR region.Western blot results showed that miR-34a-5p could negatively regulate the expression of c-fos.2.(1)After overexpression of miR-34a-5p in SK-RG cells,the proliferation and migration of the cells were decreased(P<0.05),The cell cycle was arrested in G0/G1 phase and the expressions level of CDK4,CDK6 and cyclin D1 were decreased(P<0.05).(2)In the rescue experiment,overexpression of c-fos in miR-34a-5p upregulated SK-RG cells enhance the proliferation and migration ability of the cells(P<0.05).The percentage of cells in G0/G1 phase was decreased and the expression level of CDK4,CDK6 and cyclin D1 were elevated(P<0.05).(3)miR-34a-5p inhibited the expression of LANA,RTA,K8.1 and v GPCR and reduced KSHV viral DNA copy number(P<0.05),while c-fos overexpression reversed its effect.3.(1)The xenograft tumor model of SK-RG nude mice was successfully constructed and xenograft tumor experiments showed that miR-34a-5p inhibited tumor weight and volume in vivo.(2)Immunohistochemical results showed that miR-34a-5p could inhibit the expression of c-fos,CDK6 and cyclin D1.(3)miR-34a-5p inhibited the expression of LANA,RTA,K8.1 and v GPCR of KSHV virus genes in vivo(P<0.05).Conclusion:(1)The expression of miR-34a-5p was down-regulated in SK-RG cells and miR-34a-5p could regulate the expression of c-fos through the 3’-UTR region.(2)miR-34-5p can inhibit the proliferation,migration and viral gene expression of KSHV-infected nerve cells by regulating c-fos.(3)This study suggests that miR-34a-5p may be a candidate molecular drug for inhibiting the malignant progression of KSHVinfected nerve cells. |
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