Effects Of MiR-769-3p On The Proliferation And Migration Ability Of KSHV-infected Nerve Cells By Regulating MTOR

Objective: The disease caused by KSHV infection is the most common cause of death in AIDS patients,and the neurological symptoms of patients and our previous research have found that KSHV can infect nerve cells.The main objective of this study was to elucidate the role of mTOR and its upstream regulatory molecule miR-769-3p in KSHV-infected nerve cells(SK-RG)and its influence on the virus.Methods: 1.The expression of mTOR in SK-RG cells was verified and interfered with based on the up-regulation of mTOR expression found by the previous transcriptome sequencing:(1)The expression level of mTOR in SH-SY5 Y and SK-RG cells was checked by qRT-PCR and Western blot.It was used to verify the transcriptome sequencing results;(2)mTOR interference fragments were constructed to interfere with mTOR expression in SK-RG cells;(3)qRT-PCR and Western blot were used to detect the interference efficiency of mTOR.2.The effects of mTOR on the proliferation and migration of SK-RG cells were analyzed:(1)MTT and plate cloning assays were used to check the changes in the proliferation ability of SK-RG cells after mTOR interference,and the variations of the cell cycle and cyclins were detected by the flow cytometry and Western blot.(2)The scratch and Transwell assay was used to detect the migration ability of cells after interference with mTOR,and the expression of migration proteins MMP2 and MMP9 were detected.(3)m RNA expression levels of LANA,RTA,K8.1 and v-GPCR of KSHV virus genes were detected by qRT-PCR.3.To analyze and screen miRNAs that may regulate mTOR and verify:(1)To predict miRNAs that may negatively regulate mTOR by Targetscan and miRanda,and to screen out candidate miRNAs according to the scoring grade,further screening was performed by qRT-PCR and Western blot;(2)Two genotypes of mTOR wild type and mutant were constructed according to the binding sites of miR-769-3p and mTOR 3’-UTR region predicted by Targetscan.The targeted regulation of miR-769-3p on mTOR 3’-UTR region was detected by the double luciferase reporter assay.(3)miR-769-3p was overexpressed in SK-RG cells through mimics,and the overexpression efficiency of miR-769-3p was detected by qRT-PCR.The expression levels of mTOR and p-mTOR after miR-769-3p was checked by Western blot.4.To study the effects of miR-769-3p on the proliferation and migration ability of SK-RG cells:(1)MTT and plate cloning assays was used to test the cell proliferation ability after overexpression of miR-769-3p in SK-RG cells,the variations of cell cycle and the cyclin were checked by flow cytometry and Western blot.(2)The scratche and transwell assays were used to detect variations of cell migration ability after overexpression of miR-769-3p,and the level of migration-related proteins MMP2 and MMP9 were detected.(3)The m RNA expression level of KSHV virus gene was detected by qRT-PCR.5.mTOR activator 3BDO was used in SK-RG cells overexpressing miR-769-3p for rescue experiments:(1)MTT assay and Western blot assay was used to detect the proliferative ability and cyclins level after reactivation of mTOR in miR-769-3p overexpressing SK-RG cells;(2)The transwell assay was used to detect the migration ability of cells after the treatment of activators.Western blot was used to detect the level of migration-related proteins.Results: 1.(1)qRT-PCR and Western blot confirmed that the expression of mTOR in SK-RG cells was higher than that in SH-SY5 Y cells.After mTOR interference,m RNA and protein levels of mTOR in SK-RG cells were significantly decreased(P<0.05).2.(1)After interference with mTOR in SK-RG cells,the proliferation and migration ability of cells were reduced,and the cell cycle was intercepted in the G0/G1 phase,while the levels of CDK4,CDK5,cyclin D1,MMP2 and MMP9 were reduced,and the expression of P27 was increased(P<0.05).(2)mTOR interference inhibited the m RNA expression of KSHV gene LANA,RTA,K8.1 and v-GPCR(P<0.05).3.The results of double luciferase reporter gene experiment and Western blot indicated that miR-769-3p can negativly regulate the mTOR expression.4.(1)After overexpression of miR-769-3p in SK-RG cells,the proliferation and migration ability of cells were reduced,and the cell cycle was intercepted in G0/G1 phase.The expression levels of CDK4,CDK5,cyclin D1,MMP2 and MMP9 were reduced,and the expression of P27 was increased(P<0.05).(2)Overexpression of miR-769-3p could inhibit the m RNA expression of KSHV gene LANA,RTA,K8.1 and v-GPCR(P<0.05).5.After the treatment of mTOR activator in SK-RG cells overexpressing miR-769-3p,cell proliferation and migration ability were enhanced,the expression levels of cyclin CDK4 and migration protein MMP2 were increased,and the expression of P27 was decreased(P<0.05).Conclusion: 1.The expression of mTOR is up-regulated in SK-RG cells and mTOR interference can inhibit the proliferation and migration of SK-RG cells as well as the expression of virus-related genes.2.miR-769-3p can inhibit SK-RG cell proliferation,migration and expression of virus-related genes by negatively regulating mTOR.3.miR-769-3p may be a candidate molecular drug for inhibiting the malignant progression of KSHV-infected nerve cells.