The Preliminary Study Of The Inhibition Of HIV-1 Replication By Long Noncoding RNA GAS5

Background:The human immunodeficiency virus(HIV)is a main pathogen of acquired immunodeficiency syndrome(AIDS)resulting in decreasing immune function.AIDS has been aroused worldwide concern due to the lack of specific vaccines or complete cure drugs.Long noncoding RNAs(lncRNAs)are non-protein coding transcript longer than 200 base pairs(bp)and being considered to be key regulators that involved in various biological processes that play plays a key role in the growth and development,such as species evolution,individual development,cancer,immune responses and etc.What deserves to be noticed is that IncRNAs abnormal expression is linked with human health's significant diseases closely.Previous studies have showed that host derived IncRNAs are differentially expressed after a variety of virus infections,including HIV-1 infection,suggesting IncRNA may have involved in regulation of virus replication.However,so far,lncRNA growth arrest-specific transcript 5(GAS5)is only found to be down-regulation in HIV-1 infection,there is no literature on how GAS5 participating in HIV-1 replication.Thus,we investigate the effects of GAS5 on HIV-1 replication,may provide novel biomarkers of antiviral drugs or potential targets for new therapeutics of AIDS.Objectives:To explore the function of GAS5 in HIV-1NL4-3 infection,with the aim to inhibit HIV-1 replication and provide experimental basis for AIDS research and therapeutics.Methods:We select the differentially expressed lncRNAs GAS5 from the microarray on literature by RT-PCR verification.pcDNA3.1(-),pcDNA3.1(-)-GAS5,GAS5-siRNA-1/2,NC-siRNA,HIV-1 provirus plasmid pNL4-3.Luc.R-E-,Renilla luciferase(pRL-TK)and helper plasmids pVSV-G were co-transfected into Jurkat or 293T cells after 48h of transfection,a dual-luciferase reporter assay was performed to test the efficacy of GAS5 on HIV-1NL4-3;The mRNA levels of HIV-1NL4-3 gag and pol were detected by RT-PCR in Jurkat or 293T cells;And western blotting was also performed to measure the HIV-1 p24 and Tat proteins in Jurkat or 293T cells.We use the bioinformatics software(Genomica,starBase v2.0)for prediction of RNA secondary structure and interaction with miR-873.The binding sequence were chemically synthesized and cloned into pmirGLO vector according to the instructions of the pmirGLO vector(pmirGLO-miR-873-WT,pmirGLO-miR-873-Mut).To preliminary assess the efficiency of the binding sequence,the GAS5 was co-transfected with pmirGLO-miR-873-WT or pmirGLO-miR-873-Mut into 293T cells.The luciferase activity was measured by a dual-luciferase reporter assay after 48h of transfection.Then,pcDNA3.1(-),pcDNA3.1(-)-GAS5,GAS5-siRNA-1/2,NC-siRNA were co-transfected into Jurkat or 293T cells,after 48h of transfection,miR-873 level was determined by RT-PCR verification.Similarity to the method above,pHAGE-hsa-miR-873 and miR-873-Inhibitor were co-transfected into Jurkat or 293T cells with pNL4-3.Luc.R-E-,etc.After 48h of transfection,a dual-luciferase reporter assay was performed to test the efficacy of miR-873 on HIV-1NL4-3;The mRNA levels of HIV-1NL4-3 gag and pol were measured by RT-PCR in Jurkat or 293T cells;Western blotting was also performed to detect the HIV-1 p24 proteins in Jurkat or 293T cells.Results:(1)GAS5 plays a negative regulatory role in the process of HIV-1NL4-3 replication.(2)GAS5 inhibit miR-873 expression.(3)miR-873 can promote HIV-1NL4-3 replication.Conclusions:GAS5 inhibit HIV-1 NL4-3 infection and it may be by acting as a ceRNA targetedly suppressing miR-873.