| Background:Herpes simplex virus type 1(HSV-1)is a widespread herpes virus in the world.Infected people may have skin lesions or even central neuropathy and other clinical symptoms.HSV-1 is mainly transmitted through oral contact(saliva),and the serum positive rate in the population can reach 80%.At present,there is no effective vaccine against HSV-1.Antiviral therapy is the main strategy for clinical control of HSV-1 infection,but there is a high drug resistance in people with low immunity.Therefore,HSV-1 virus infection and its treatment process still need to be further studied.piRNA(PIWI-interacting RNA)was first identified in the testis of Drosophila melanogaster.It is a kind of silent small RNA containing 24-31 nucleotides(nt).piRNA usually interacts with PIWI protein to induce target gene silencing through the formation of RNA-induced silencing complex(RISC).piRNA protects the genomes of most animal reproductive lines from the expression of transposons.In recent years,studies have shown that piRNA can fight RNA virus infection in mosquitoes.In addition,the expression and function of piRNA in mammalian virus Coxsackievirus B3(CVB3):and human rhinovirus(HRV)have also been studied.However,the expression of piRNA in the process of HSV-1 infection has not been studied in detail.Methods:In order to analyze the expression pattern of piRNA during HSV-1 staining,we used the RNA-seq dataset we studied earlier,numbered GSE102470,which was uploaded to the public database GEO(https://www.ncbi.nlm.nih.gov/geo/).First of all,through the use of a variety of health letter analysis software to control the quality of the original data.Then carry on a series of sequence alignment annotation to the effective data(clean reads),filter out other small RNA,and get the final piRNA sequence to be analyzed.Then the expression of piRNA was calculated by TPM(transcript per million),and the length of piRNA was counted.The differentially expressed piRNA was screened according to the criteria of p<0.05 and |log2FoldChange|≥1.In order to confirm the differentially expressed piRNA identified by RNA-seq analysis,the differentially expressed piRNA was verified by RNA extraction and real-time fluorescence quantitative PCR(RT-qPCR).The host target genes of differentially expressed piRNA were predicted by miRanda algorithm,and the functional enrichment analysis of GO and KEGG was carried out.The interaction between top 10 up-regulated piRNA and HSV-1 major genes was analyzed by RNAInter database.After that,the virus titer in the supernatant was determined by transfection piRNA mimic and CCID50.Results:After quality control,the RNA-seq data set shows that the effective data amount of each sample is 17.29M-21.36M,and the genome comparison rate is 95.37%-97.85%.After filtering and screening other non-piRNA sequences,it is found that the known piRNA comparison rate distribution is 0.65%to 3.59%,the length of piRNA is mainly distributed in 15-32nt.The results of piRNA differential expression analysis during HSV-1 infection showed that a total of 69 piRNAs were significantly different between the two groups.Among them,52 piRNAs were up-regulated and 17 were down-regulated.The results of RT-qPCR support the reliability of quantitative expression of piRNA in RNA-seq.The functional enrichment results of GO and KEGG showed that the differentially expressed piRNA target genes were involved in the biological processes such as virus infection and immune response.Four up-regulated piRNAs most likely to interact with the major genes of HSV-1 were screened,which were piR-hsa-28382(alias piR-36233),piR-hsa-11080(alias piR-48966),piR-hsa-23248(alias piR-33082)and piR-hsa-28190(alias piR-36041).The results of virus titer detection after piRNA mimics transfection showed that the virus titer of piRNA-hsa-28382 mimic transfection group was significantly lower than that of negative control group,while that of piRNA-hsa-28190 mimic group was significantly higher.Conclusion:HSV-1 infection of KMB17 cells can lead to changes in piRNA expression.In this study;a total of 69 differentially expressed piRNAs were detected,among which 52 were up-regulated and 17 were down-regulated.GO and KEGG functional enrichment analysis suggested that these differentially expressed piRNAs might be involved in viral infection and immune response,and piRNA-hsa-28382 might be involved in antiviral proliferation by silencing the expression of virus genes.Background:circular RNA(circRNA)is a type of non-coding RNA with circular structure produced by back splicing of linear mRNA precursors.circRNA can be divided into three main types,including exon circRNA intron circRNA and exon-intron circRNA.circRNA functions mainly through miRNA sponge interaction with proteins and regulation of gene expression.Studies have shown that circRNA may be related to tumor progression.Abnormal expression of circRNA has been detected in many cancer types in recent years,various viruses have been reported to produce circRNA.In our previous study,it was found that simian virus 40(SV40)would produce a new circRNA after infecting Vero cells,which we named circ-17kT.The existence of circ-17kT verified by Sanger sequencing,northern blot and other experiments,and its function was preliminarily analyzed.This project will further study the function of circ-17kT on the basis of the previous study.Methods:After SV40 infection,RNA extraction,reverse transcription PCR,PCR and agarose gel electrophoresis were performed to verify the expression of circ-17kT in Vero,CV-1 and MA104 cells.The reverse splicing sites of PCR products were detected by T vector ligation and Sanger sequencing.Then the RNase R resistance of the extracted RNA was analyzed.Then the subcellular localization of circ-17kT was detected by nuclear-cytoplasmic separation test and RT-qPCR assay.The RNA-binding proteins of circ-17kT were predicted by RBPDB database,and the possible circ-17kT-binding proteins were verified by RNA-binding protein immunoprecipitation(RIP).After that,the expression of circ-17kT binding protein SRSF10 was knocked down by transfection of siRNA to analyze the binding function of circ-17kT and SRSF10 protein in the process of SV40 infection.The expression of circ-17kT,17kT,VP1 and LT genes was detected by RT-qPCR and western blot,and the titer of SV40 in the supernatant was detected by CCID50.Results:After SV40 infection in Vero,CV-1 and MA104 cells,the expression of circ-17kT could be detected,and the reverse splicing site could be detected after Sanger sequencing.The results of RNase R resistance analysis showed that GAPDH,U6,VP1 and LT linear RNA did not have RNase R resistance,while circ-17kT had obvious RNase R resistance and was hardly digested and degraded.The results of nuclear-cytoplasmic separation experiment showed that circ-17kT was distributed in both nucleus and cytoplasm,and the distribution was similar.RIP assay showed that circ-17kT could bind to splicing factor SRSF10 protein.After knocking down the expression of SRSF10,the detection of circ-17kT and 17kT increased,but had no significant effect on the titer of virus and the expression of VP1 and LT.Conclusion:The expression of circ-17kT in SV40 infected cells is universal,and compared with other linear RNA,circ-17kT has obvious RNase R resistance.circ-17kT can bind to splicing factor SRSF10 protein and affect the expression of SV40 early gene 17kT.However,there are no experimental results to prove that the binding of circ-17kT and SRSF10 protein has a significant effect on the expression of SV40 VP1 and LT and the proliferation of virus. |
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