The Role Of Long Non-coding RNA (lncRNA-ITGA2) In The Functional Regulation Of Vascular Smooth Muscle Cell And The Underlying Mechanisms

Background:Atherosclerosis(AS)is the most important mechanism of cardiovascular and cerebrovascular diseases and has become one of the most serious diseases that threaten human health.Vascular smooth muscle cells(VSMCs)are the main cellular components that constitute the structure of the blood vessel wall.The excessive proliferation and migration are pivotal pathological basis of atherosclerosis,in-stent restenosis,pulmonary hypertension and other vascular proliferative diseases.Platelet-derived growth factor-BB(PDGF-BB)is a potent proliferative agent for VSMCs,but the specific mechanism of PDGF-BB signaling pathway involved in biological processes such as proliferation and migration of VSMCs is not elusive.Genomics studies have shown that mammalian genomes have more than 90% transcripts,but only 2% can transcribe into protein-coding genes,and about 98% are transcribed into non-coding RNAs(ncRNAs).Long non-coding RNA(lncRNA),defined as non-coding transcripts more than 200 nucleotides,can regulate gene expression at multiple levels,including genomic imprinting,chromatin modification,transcriptional activation and regulation.A large number of research results have been reported on how lncRNA plays a regulatory role in tumor development.However,in the process of cardiovascular disease development,the functional study of lncRNA is still in its infancy and deserves further investigation.We used the lncRNAs microarray technology to screen the target lncRNA in human aortic-VSMCs(HA-VSMCs)proliferation model treated with PDGF-BB(20ng/mL).Through bioinformatics analysis and subsequent qRT-PCT technology,we identified a novel lncRNA that was significantly up-regulated in the HA-VSMCs proliferation model,named lncRNA-ITGA2.The full-length sequence of lncRNA-ITGA2 was successfully obtained by RACE and its subcellular localization in HA-VSMCs was mainly located in the nucleus,dectected by cytoplasmic separation technique and RNA-FISH.We used small interfering RNA(siRNA)and adenovirus transfection technology to achieve knockdown and overexpression of lncRNA-ITGA2 in HA-VSMCs,observe changes in biological functions.After knock-down of lncRNA-ITGA2 expression,RNA sequencing(RNA-seq)has been performed to predict possible downstream target genes and signaling pathways.In this study,we will examine the biological function of lncRNA-ITGA2 in the proliferation and migration in HA-VSMCs and in the pathogenesis of atherosclerosis and look into the role of lncRNA in coronary heart disease.Part ? Identification and Characterization of the Long Non-coding RNA lncRNA-ITGA2Methods:(1)Candidate differentially expressed lncRNA was obtained by bioinformatic analysis of lncRNAs microarray.(2)The expression level of the target lncRNA in HA-VSMCs proliferation model was determined by qRT-PCR.(3)The full-length transcript sequence of the target lncRNA was obtained by RACE experiment,and the coding potential was predicted by the open-database.(4)PCR assay to detect the expression of lncRNA-ITGA2 in human cell lines.(5)The subcellular localization localization of the target lncRNA was determined by nuclear and cytoplasmic separation experiments and RNA-FISH experiments.Results:(1)Expression profile analysis revealed that HA-VSMCs treated with or without PDGF-BB displayed unique mRNA and lncRNA espression profile.(2)The expression level lncRNA-ITGA2 was significantly increased in PDGF-BB and FBS-induced HA-VSMCs proliferation model,determined by qRT-PCR.(3)The full-length transcript sequence of lncRNA-ITGA2 was successfully determined by RACE.In addition,online open database revealed that lncRNA-ITGA2 does not have protein coding ability.(4)lncRNA-ITGA2 is selectively highly expressed in HA-VSMCs(5)Nuclear and Cytoplasmic separation experiments and RNA-FISH experiments revealed that lncRNA-ITGA2 is mainly localized in the nucleus.Conclusion: A novel lncRNA,named lncRNA-ITGA2,related to the proliferation of HA-VSMCs was obtained by lncRNAs microarray and biosignal analysis.In addition,subsequent qRT-PCR revealed that the expression of lncRNA-ITGA2 was significantly increased in PDGF-BB and FBS induced HA-VSMCs proliferation model.Part ? lncRNA-ITGA2 regulates proliferation and migration of human aortic vascular smooth muscle cellsMethods:(1)The knockdown and overexpression efficiency of lncRNA-ITGA2 expression by siRNA and adenovirus was detected by qRT-PCR analysis.(2)we use CCK-8 and EdU assay to detect the HA-VSMCs proliferation ability after depletion or overexpression of lncRNA-ITGA2 gene leves.(3)we use Annexin V-FITC apoptosis detection kit to detect the HA-VSMCs apoptosis ability after depletion or overexpression of lncRNA-ITGA2 gene leves.(4)we use cell cycle kit to detect the HA-VSMCs cycle phase distribution after depletion or overexpression of lncRNA-ITGA2 gene leves.(5)The migration ability of HA-VSMCs after transfected with lncRNA-ITGA2 siRNA or adenovirus were determined by cell scratch healing experiments and transwell chamber assay.(6)Western blot analysis was performed to detect the effect of lncRNA-ITGA2 knockdown or overexpression on apoptosis and cycle-related proteins in HA-VSMCsResults:(1)Transfection of Si-lncRNA-ITGA2 and Ad-lncRNA-ITGA2 in HA-VSMCs can effectively knock down and overexpress the expression level of lncRNA-ITGA2.(2)CCK-8 and EdU assay showed that knockdown or overexpression of lncRNA-ITGA2 can inhibit and increase the proliferation of HA-VSMCs,under both basal or PDGF-BB stimulated conditions.(3)Apoptotic flow assay and western blot ananlysis showed that knockdown the expression of lncRNA-ITGA2 significantly promoted the apoptosis of HA-VSMCs,and overexpression of lncRNA-ITGA2 had no significant effect on apoptosis.(4)Cell cycle detection revealed that knockdown or overexpression of lncRNA-ITGA2 can inhibit or accelerate cell cycle progression,respectively.(5)Cell scratch healing experiments and transwell chamber experiments showed that knockdown or overexpression of lncRNA-ITGA2 can inhibit or enhance the cell migration ability under both quiescent or PDGF-BB stimulated condidtions,respectively.Conclusion: lncRNA-ITGA2 promoted the proliferation and migration of HA-VSMCs and accelerated cell cycle progression.Part ? lncRNA-ITGA2 promotes the proliferation and migration of HA-VSMCs by regulating the expression of ITGA2,and accelerates the process of atherosclerosis Methods:(1)Prediction of downstream target genes and signaling pathways that may be regulated by lncRNA-ITGA2 by RNA-seq analysis.(2)Detection of lncRNA downstream gene and signaling pathway molecule expression by qRT-PCR,western blot and Immunofluorescence analysis.(3)Detection of lncRNA-ITGA2 after transfected with ITGA siRNA or adenovirus to clarify the upstream and downstream regulatory relationship between lncRNA-ITGA2 and ITGA2 molecules.(4)We performed dual luciferase assay to detect the effect of lncRNA-ITGA2 on ITGA2 promotor activity.(5)CHIRP-DNA experiments were used to explore whether lncRNA-ITGA2 can regulate the transcriptional activity of ITGA2 by directly binding its complementary sequences in the genome.(6)ChIP-seq and ChIP-qPCR experiments were used to elucidate the relationship between H3K27 ac and the transcriptional activity of ITGA2.(7)CCK-8 and cell scratch healing assay were used to elucidated the.effects of co-transfection of lncRNA-ITGA2 and ITGA2 on the proliferation and migration of HA-VSMCs.(8)Immunohistochemistry and western blot ananlysis were used to detect the expression changes of ITGA2 in arterial vascular tissue from CHD patients and Non-CHD group.qRT-PCR assay analysis showd the expression level lncRNA-ITGA2 from peripheral blood sample between CHD and Non-CHD group.Results:(1)We predicted that the ITGA2 molecule might be the target gene of lncRNA-ITGA2 after RNA-seq analysis.(2)Knockdown or overexpression of lncRNA-ITGA2 expression down-regulate or up-regulate ITGA2 mRNA and protein levels,respectively,and knockdown of lncRNA-ITGA2 expression can significantly inhibit PDGF-BB-induced FAK phosphorylation.(3)PDGF-BB can significantly upregulate the mRNA and protein level of ITGA2 molecule.(4)Knockdown or overexpression of ITGA2 molecules had no effect on lncRNA-ITGA2 expression level.(5)Dual luciferase reporter gene experiments demonstrated that lncRNA-ITGA2 has no direct binding ability to the ITGA2 promoter,but after constructing the lncRNA-ITGA2 sequence onto a fluorescent vector containing the ITGA2 promoter,dual luciferase reporter gene experiments demonstrated that lncRNA-ITGA2 can regulate ITGA2 transcriptional activity through an enhancer-like effect.(7)The results of CHIRP-DNA experiments showed that lncRNA-ITGA2 has a clear binding site in its genomic complementary region and the binding DNA level can be upregulated after overexpressed lncRNA-ITGA2.(6)ChIP-seq experiments showed that the binding peak of H3K23 ac in ITGA2 promoter region after overexpression the expression level of lncRNA-ITGA2 was significantly increased,and the subsequent experimental results were further verified by qRT-PCR.(8)CCK8 assay and cell scratch healing experiments showed that knockdown or overexpression of ITGA2 molecules can significantly reduce or enhance the proliferation and migration of HA-VSMCs,but co-transfection of Ad-lncRNA-ITGA2 or Si-linRNA-ITGA2 can restore or inhibit the proliferation and migration ability of HA-VSMCs.(9)Both Western blot and immunohistochemistry demonstrated that the expression level of ITGA2 in vascular tissue samples of CHD group was significantly higher than that of the Non-CHD group.qRT-PCR experiments showed that the expression level of lncRNA-ITGA2 in peripheral blood of CHD group was significantly higher than that of Non-CHD group.Conclusion:(1)lncRNA-ITGA2 was significantly up-regulated in the PDGF-BB-induced vascular smooth muscle cell proliferation model.(2)lncRNA-ITGA2 can act as an important regulatory factor to promote the proliferation and migration of HA-VSMCs by regulating the expression of ITGA2 and its signaling pathways protein levels.(3)lncRNA-ITGA2 synergistically increases the transcriptional activity of ITGA2 in the nucleus through binds to its genomic complementary region and up-regulates the H3K27 ac acetylation level in the ITGA2 promoter region.The two synergistically activate the ITGA2-FAK signaling pathway,promote the proliferation and migration of HA-VSMCs,and accelerate progression of atherosclerosis.