Screening And Analysis Of TRNA Derived Small RNAs Related To Vascular Smooth Muscle Cell Proliferation And Preliminary Exploration Of Its Regulatory Mechanism

Objective:Abnormal proliferation of vascular smooth muscle cells(VSMCs)contributes to vascular remodelling diseases.tRNA-derived small RNAs(tsRNAs),a new type of non-coding RNAs(ncRNAs),regulate a variety of biological processes,especially cell proliferation.However,the function of tsRNAs in VSMC proliferation has not been fully elucidated.In this study,we performed high-throughput RNA sequencing on proliferative and quiescent human aortic smooth muscle cells(HASMCs)to analyse tsRNA expression profile.Real-time quantitative polymerase chain reaction(qRT-PCR)was performed to validate that the expression of the differentially expressed tsRNAs(DEtsRNAs)and detects its distribution in the nucleus and cytoplasm.Based on the miRNA-like function of tsRNAs,from the two perspectives of the nucleus(activating/silencing transcription by targeting promoter)and cytoplasm(inhibiting translation by targeting mRNA),predict the target genes of tsRNAs,construct the interaction networks,and use Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway to analyze the function of target genes.Finally,two tsRNAs,AS-tDR-000067 and AS-tDR-000076,were selected as the research objects for functional demonstration and target gene identification.Methods:1.Analysis of the expression profile of tsRNAs by high-throughput RNA sequencingHigh-throughput RNA sequencing technology was performed to analyze the expression profiles of tsRNAs in the three sets of proliferative and quiescent HASMCs.Proliferative HASMCs were induced by fetal bovine serum(FBS)and smooth muscle cell growth supplement(SMCGS).Quiescent HASMCs were induced by FBS and SMCGS starvation.tsRNAs with a statistical difference and more than 2.0 fold change(fold change≥ 2 or<-2,P<0.05)were selected for further study.2.Validation of differentially expressed AS-tDR-001370,AS-tDR-000067,AS-tDR-009512,and AS-tDR-000076 by qRT-PCRThe expression levels of AS-tDR-001370,AS-tDR-000067,AS-tDR-009512,and AS-tDR-000076 in proliferative HASMCs and quiescent HASMCs were detected by qRT-PCR.2-△△Ct method was applied to calculate the fold change of expression of tsRNAs.3.Validation of the distribution of AS-tDR-001370,AS-tDR-000067,AS-tDR-009512,and AS-tDR-000076 in the nucleus and cytoplasmThe nuclear and cytoplasmic components of proliferative HASMCs were isolated using the Nuclear/Cytosol Fractionation Kit,and small RNAs were extracted separately and reverse transcribed.The proportions of AS-tDR-001370,AS-tDR-000067,AS-tDR-009512,and AS-tDR-000076 in the nucleus and cytoplasm were detected by qRT-PCR.4.Analysis of the interaction between AS-tDR-001370,AS-tDR-000067,AS-tDR-009512,and AS-tDR-000076 and promotersFrom the perspective of nuclear miRNA targeting gene promoters to regulate transcription,the target promoters of four tsRNAs(AS-tDR-001370,AS-tDR-000067,AS-tDR-009512,and AS-tDR-000076)were predicted by RNAhybrid and MiRanda.To further screen proliferation-related genes containing target promoters,Venn analysis was performed on predicted target promoters of four tsRNAs through differentially expressed mRNAs(DEmRNAs)in proliferative HASMCs(GSE77279)(fold change≥ 2 or ≤-2,P<0.05).tsRNA-promoter interaction networks were constructed using Cytoscape_v3.7.1.GO and KEGG pathway analyses were utilized to reveal the function of nuclear target genes.5.Analysis of interaction between AS-tDR-001370,AS-tDR-000067,AS-tDR-009512,and AS-tDR-000076 and mRNA Based on cytoplasmic miRNA targeting mRNA to regulate translation,downstream target mRNAs of four tsRNAs(AS-tDR-001370,AS-tDR-000067,AS-tDR-009512,and AS-tDR-000076)were predicted by TargetScan and MiRanda.Venn analysis between predicted target mRNAs and DEmRNAs in the proliferative HASMCs(GSE77279)(fold change ≤-1.5,P<0.05)was performed to further obtain target DEmRNAs.Based on circular RNA(circRNA)targeting miRNA to regulate its function,upstream target circRNAs of four tsRNAs were predicted by RNAhybrid.Venn analysis between predicted target circRNAs and differentially expressed circRNAs(DEcircRNAs)in the proliferative HASMCs(GSE77278)(fold change≥ 2 or ≤-2,P<0.05)was utilized to further enrich target DEcircRNAs.tsRNA-mRNA and circRNA-tsRNA interaction networks were constructed using Cytoscape_v3.7.1.GO and KEGG pathway analyses were utilized to reveal the function of cytoplasmic target genes.6.The effect of AS-tDR-000067 and AS-tDR-000076 on the proliferation of HASMCsSingle-stranded antisense oligonucleotides(ASO)were designed and synthesized and transfected into proliferative HASMCs to know down AS-tDR-000067 and AS-tDR-000076.The knockdown efficiency was verified by qRT-PCR,the proliferation activity of HASMCs was analysed by EdU fluorescence staining,and the expression level of target genes was detected by Western blot.7.hsa＿circ＿0001402 may regulate VSMC proliferation through the AS-tDR-000067-p53/mitofusin 2(MFN2)axisAccording to the circRNA-tsRNA interaction network,hsa＿circ＿0001402,which interacts with AS-tDR-000067,is selected for further analysis.Our previous work has proved that hsa＿circ＿0001402 can inhibit the proliferation of HASMCs,so it is speculated that hsa＿circ＿0001402 may regulate VSMC proliferation through the AS-tDR-000067-p53/MFN2 axis.The overexpression plasmid of hsa＿circ＿0001402 was transfected into proliferative HASMCs,and the expression level of p53 and MFN2 were detected by Western blot.8.Construction of reporter gene-related vectorThe reporter gene-related vector of hsa＿circ＿0001402-WT and MFN2-WT were constructed.According to the binding site of hsa＿circ＿0001402 and MFN2 in AS-tDR-000067,the wild-type sequence was chemically synthesized.The sticky ends of Xho I and Not I were introduced into the 5’ and 3’ ends of the wild-type sequences,respectively,and inserted into psiCHECKTM-2 Vector.After transformation,a single colony was picked and subjected to colony PCR,single enzyme digestion identification and sequencing analysis.Results:1.A total of 3,891 tsRNAs were identified by sequencing,of which 887 and 951 tsRNAs were up-regulated and down-regulated in proliferative HASMCs compared to quiescent HASMCs,respectively(fold change≥ 2 or ≤-2,P<0.05).2.Consistent with the sequencing,AS-tDR-001370,AS-tDR-000067,AS-tDR-009512,and AS-tDR-000076 were up-regulated in proliferative HASMCs.3.tsRNAs were distributed in the cytoplasm and nucleus of proliferative HASMCs,and the level of nuclear tsRNA was significantly higher than that of cytoplasm.4.Nuclear AS-tDR-001370,AS-tDR-000067,AS-tDR-009512,and AS-tDR-000076 might target 243,854,202,and 120 proliferation-related genes in a promoter targeted manner,respectively.GO and KEGG pathway analyses showed that four target promoters sets of proliferation-related genes involved multiple proliferation-related pathways.5.Cytoplasmic AS-tDR-001370,AS-tDR-000067,AS-tDR-009512,and AS-tDR-000076 might target 46,69,184,306 DEmRNAs and 21,71,37,53 DEcircRNAs,respectively.GO and KEGG pathway analyses reveal that the target DEmRNAs of 4 tsRNAs involved in multiple proliferation-related pathways.6.ASO can effectively reduce the expression levels of AS-tDR-000067 and AS-tDR-000076 in proliferative HASMCs.EdU fluorescence staining showed that knocking down AS-tDR-000067 and AS-tDR-000076 significantly inhibited the proliferation of HASMCs.Western blot analysis showed that knocking down AS-tDR-000067 can promote the expression of nuclear target gene p53 and cytoplasmic target gene MFN2,and knocking down AS-tDR-000076 can inhibit nuclear target gene insulin like growth factor 2 mRNA binding protein 2(IGF2BP2)expression,and promote the expression of cytoplasmic target gene p53.7.Overexpression of hsa＿circ＿0001402 in proliferative HASMCs significantly increases the protein levels of p53 and MFN2.8.The reporter gene plasmids of hsa＿circ＿0001402-WT and MFN2-WT were successfully constructed.Conclusions:1.During the proliferation of HASMCs,the expression levels of many tsRNAs changes.Bioinformatics analysis shows that tsRNAs may play a regulatory role in two ways:nuclear tsRNAs target gene promoter to regulate transcription;cytoplasmic tsRNAs target mRNA to regulate translation.It is the first hypothesis that circRNAs may target tsRNAs to regulate mRNA translation.2.AS-tDR-000067 and AS-tDR-000076 can promote the proliferation of HASMCs,and hopefully,become therapeutic targets for vascular remodelling diseases induced by abnormal proliferation of VSMC.