Using Transcriptome Sequencing Technology To Screen Key Genes Related To HIV-1 Unsusceptibility From Heps Subjects

Objective To screen key genes associated with highly exposed and persistently seronegative (HEPS) of HIV-1 infection, and to reveal genetic background of HIV-1 unsusceptibility in HEPS subjects.Methods Spouse and steady sexual partners of HIV-1-infected individuals were recruited from Methadone Maintenance Treatment (MMT) clinics or HIV Voluntary Counseling & Testing(VCT) clinics in Nanning, Liuzhou, Qinzhou cities in Guangxi. HEPS subjects were then strictly selected according to the HEPS standards. Control subjects were selected in the counterpart locations from age-, gender- and ethnic-matched healthy individuals. Demographic and sexual behaviors characteristics were collected by qualified investigators.Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood samples. Total cellular RNA was extracted from PBMCs and then sent to Beijing Genomics Institute (BGI) for high-throughput sequencing (Hi-Seq 2000).Key genes that related to HIV-1 unsusceptibility in HEPS subjects were screened from differentially expressed genes of sequencing.Results1. Sequencing Quality. In both samples, curves of base A were overlapped with those of base T, and curves of base C were overlapped with those of base G.The proportions of bad quality (<20) bases were very low in both samples.Reads of both samples were equally distributed in every section of genes. Of the total genes(16963) of sample C01(control), 8313 (49%) have genomic coverage higher than 90% (90%-100%) and only 1451 (9%) have genomic coverage lower than 10% (007%-10%). For GH01(HEPS sample), 8775 genes (51%) have genomic coverage higher than 90% (90%-100%) and 1427 (8%) have genomic coverage lower than 10% (0%-10%). The sequencing quality was guaranteed with excellent equilibrium of the base, randomness of fragment interrupt and high coverage of genes, suggesting a welldone sequencing.2. Readout results. Compared with human reference gene sequence, there were 47191584 and 47062392 reads from sample C01 and GH01, respectively.Of the total reads, 33577371 (71.15%) and 31811486 (67.59%) reads were matched in C01 and GH01, respectively. Unique match reads of the two samples were higher than 65% and perfect match reads of the two samples were around 50%.3. Differentially expressed genes(DEGs). Totally, there were 1906 differentially expressed genes between GH1 and C01, with 1139 up-regulating genes and 767 down-regulating genes (FDR≤0.001 and ｜log2 Ratio｜>1) in GH01. There were 15 genes reported to be associated with HIV-1 unsusceptibility, including CCL2, KIR2DL1, KIR2DS2, KIR3DL1, KIR3DL2,KIR2DL2, CCL4, CCL3L1, CCL20, KIR2DL3, CCL3, KIR2DS1, KIR3DS1,KIR2DS4 and CCR9. Besides, some other genes were found to be related to HIV-1 replication but there were no reports to show they are related to HIV-1 unsusceptibility in HEPS subjects. We will focus on following genes, CCL2,CCL3, CCL3L1, CCL3L3, CCL4, CCL20, CXCL9, CXCL10, CCR4, CCR9,SIGLEC1, SIGLEC14, CH25H, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1,KIR3DL2, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, HLA-DRB3,HLA-DRB4, HLA-DRB5, HLA-DQA2, HLA-DQB2, HLA-G, APOBEC3A,APOBEC3B, etc, on the basis of their fold change and published researches.4. Enrichment analysis of GO function on the differentially expressed genes.The differentially expressed genes were enriched into 47 functional groups,including five groups related to cellular components, five groups related to molecular functions and 37 groups related to biological processes(p-value<0.05).The cellular components were mainly membrane and cell surface-related, and the molecular functions mainly involved in cytokines activity, receptor binding and molecular transducer activity. The biological processes associated with HIV-1 with a proportion of differential gene enrichment higher than 10% mainly include, biological regulation, regulation of biological process, regulation of cellular process, response to stimulus, signaling , cell communication, cellular response to stimulus, signal transduction, cell surface receptor signaling pathway, positive regulation of biological process, immune system process,positive regulation of cellular process , regulation of response to stimulus, etc.5. KEGG pathway analysis. Seven significant differential pathways(Q value<0.05) that associated with HIV-1 infection were found between two samples, including cytokine-cytokine receptor interaction pathway, antigen processing and presentation pathway, cell adhesion molecules (CAMs) pathway,natural killer cell mediated cytotoxicity pathway, phagosome pathway,complement and coagulation cascades pathway, focal adhesion pathway and B cell receptor signaling pathway pathway.Conclusion1. In this study, we used transcriptome sequencing technology to screen key genes related to HIV-1 unsusceptibility from HEPS subjects, and found a number of differentially expressed genes that were potentially related to HIV-1 unsusceptibility, including, Chemokines, KIRs, HLA, APOBEC3, SIGLECs,ISGs, etc. All of these data lay a solid foundation for the future research on specific mechanism(s) responsible to HIV-1 unsusceptibility in HEPS subjects.2. Go functional enrichment analysis and KEGG pathways analysis strongly support the view that the differential genes we screened have crucial role in HIV-1 unsusceptibility. Morever, the results also showed that immune-related biological processes and signaling pathways play an important role in HIV-1 unsusceptibility.3. In this study, we used the transcriptome sequencing technology to successfully screen a number of key genes that may affect HIV-1 unsusceptibility in HEPS subjects. The results show that transcriptome sequencing technology has developed to a powerful tool to screen differentially expressed genes and can be applied to more biological researches.