The Bioinformatics Analysis And The Preliminary Study Of Long Non-coding RNAs In Human Foamy Virus Infected Cells

BackgroundHuman foamy virus(HFV)is a complex and unique retrovirus with the longest genomes among retroviruses.Current researches have demonstrated that there are no cases of HFV infection with clinical symptoms in animals or humans.Instead,HFV is maintained asymptomatically in a lifelong infection in the host.Because of its non-pathogenic nature and capacity for packing large transgenes,HFV has great promise as a vector for gene therapy in the future.Long non-coding RNAs(lncRNAs)are considered as RNA transcripts with no evident protein-coding potential.These years,some studies have indicated that lncRNAs have diverse biological functions and differentially express in many diseases.At the same times,lncRNAs play vital functions during viral infections.However,there is no literature on how lncRNAs have functions in HFV infection.ObjectiveThe aim of this research is to explore differential expressions of lncRNAs during HFV infected 293T cells.Further,we reveal the functions of lncRNAs in HFV infections through predictions of biological function,pathway analysis and possible target genes in cis-acting or trans-acting manner.And we chose specific lncRNAs to preliminary study their functions.Methods1.We sent three samples of HFV infected 293T cells and three samples of HFV uninfected 293T cells to biological company(Genenery Bio-tech,Shanghai,China)to make the next-generation RNA high-throughput sequencing.Data in RNA-seq was classified and annotated and data of differentially expressed lncRNAs and mRNAs was made statistics and analysis.2.Utilizing qRT-PCR assays verified six differentially expressed lncRNAs selected from RNA-seq in HFV infected 293T cells and HT1080 cells in order to demonstrate the reliability of RNA-seq.Then,secondary structures of six lncRNAs were predicted by RNAfold bioinformatical software.3.Make use of bioinformatical methods to explore and figure out the data of GO/GOTM fuctional analysis and KEGG pathway analysis in RNA-seq.Classify the data of lncRNAs cis-acting and trans-acting regulatory networks in RNA-seq.4.Two differentially expressed lncRNAs,lnc-RP5-1086D14.3.1-1:1(lnc-RP5)and NONHSAG000101(lnc-NONH),were selected to design and screen their effective siRNAs and to constructe their over-expressed plasmids.qRT-PCR assays and western blot assays tested the effects of them to HFV transactivator Tas.Luciferase assays tested the effects of them to HFV internal promoter(IP).5.Through bioinformatics software RNA22v2,we respectively predicted specific mircoRNAs which could be targeted by lnc-RP5 or lnc-NONH.And qRT-PCR assays tested the effects of microRNAs wchich were influenced by over-expressed plasmids and siRNA of these two IncRNAs.6.Use over-expressed plasmids and siRNA of lnc-RP5 to demonstrate the effects to Notchl in mRNA and protein levels by qRT-PCR assays and western blot assays.Further,we demonstrated effects of Notch1 protein to HFV IP and transactivator Tas through luciferase assays,qRT-PCR assays and western blot assays.Results1.In HFV infected 293T cells,most differentially expressed IncRNAs are less than lOkb in length and comprise 20 or fewer exons.The distribution of the lncRNA classes,as determined by RNA-seq,is 36.1%sense lncRNAs,2.3%antisense lncRNAs,2.3%intronic lncRNAs,0.5%enhancer IncRNAs,4.1%sRNA host lncRNAs,5.5%bidirectional IncRNAs,and 49.3%intergenic IncRNAs.2.Our results show that 11,336 lncRNAs are differential expression,including 4,729 upregulated IncRNAs and 6,588 downregulated lncRNAs.In addition,61,367 mRNAs are differential expression,comprising 30,133 upregulated mRNAs and 31,220 downregulated mRNAs.(Note:some differentially expressed IncRNAs with low quality were not tested their fold chages.)3.qRT-PCR assays have demonstrated that two lncRNAs(NONHSAG000101 and ENSG00000204054)were upregulated and four lncRNAs(ENSG00000227254,ENSG00000247095,NONHSAT135354,and lnc-RP5-1086D14.3.1-1:1)were downregulated in HFV-infected 293T cells,which conformed with results in RNA-seq.Secondary structures of these six lncRNAs contain many stem-loop structures.4.GO functional analysis of differentially expressed IncRNAs enriched in"negative regulation of the homeostatic process","intracellular","N-acylmannosamine kinase activity" and so on.KEGG pathway analysis of differentially expressed IncRNAs mainly enriched in "pathways of cancer" and so on.409 differentially expressed IncRNAs could target protein genes in cis-acting manners and 637 differentially expressed IncRNAs could target protein genes in a trans-acting manner.5.lnc-RP5 and lnc-NONH could promote the expression of HFV transactivator Tas and activity of HFV internal promoter(IP).6.lnc-RP5 and lnc-NONH could respectively promote the expression of miR-129-5p and miR-34c-5p.7.MiR-129-5p and miR-34c-5p could promote the activity of HFV internal promoter(IP).8.lnc-RP5 could repress the expression of Notchl gene and Notchl protein could repress the activity of HFV IP and the expression of HFV transactivator Tas.Conclusion1.We use the next-generation RNA high-throughput sequencing to first identify and analyze differentially expressed lncRNAs during HFV infected 293T cells.And we make predictions of biological functions,related pathways and cis-acting or trans-acting regulatory networks about these differentially expressed IncRNAs.2.lnc-RP5 could promote the expression of HFV Tas through miR-129-5p/Notchl/HFV IP axis.3.lnc-NONH could promote the expression of HFV Tas through miR-34c-5p/HFV IP axis.