Screening And Analysis Of Circular RNA In The Proliferative Vascular Smooth Muscle Cells

Objective: Circular RNA（circRNA）is a class of ring endogenous noncoding RNA with no 5’ end cap and 3’ terminal poly（A）tail structure formed by covalent bond.Compared with linear RNA,it is more stable without being affected by RNA exonuclease,deadenosine and cap removing.It has been proved that circRNA is involved in various biological processes such as proliferation,apoptosis and senescence.The abnormal proliferation and apoptosis of vascular smooth muscle cells（VSMCs）is an important pathological event of vascular remodeling disease.It is unkown whether circRNA participates in the regulation of proliferation of VSMCs.In this study,we used gene chip technology to screen the expression profiles of circRNA and mRNA in human aortic smooth muscle cells（HASMCs）,and to predict the function of circRNA based on acting as micro RNA sponge or coding proteins.Then,we use loss of function and gain of function anylasis to explore the effect of circRNA on the proliferation and apoptosis of HASMCs.Methods:1 The expression profiles of circRNA and mRNA detected by microarrayThe expression profiles of circRNA and mRNA in the proliferative HASMCs induced by PDGF and quiescent HASMCs induced by serum starvation were anylysed using microarray.We screened the differentially expressed circRNA and encoding RNA（mRNA）with statistically significant expression（fold change ≥ 2or ≤-2,P<0.05）as the targets of the follow-up study.2 Validation of differentially expressed circRNA by q RT-PCRThirteen differentially expressed circRNA were amplified by quantitative real-time polymerase chain reaction（qRT-PCR）using divergent primers in the proliferative HASMCs and quiescent HASMCs.Relative expression levels were calculated by 2-△△Ct method.3 Bioinformatics analysis of differentially expressed circRNA and mRNAAccording to the enrichment score,significant items were selected.GO analysis contains molecular function,biological processes and cellular component of the differentially expressed circRNA and mRNA.The most significant pathways were chosen in pathway analysis,which the differentially expressed circRNA and mRNA are mainly involved in,to predict the potential function of circRNA.Based on the RNA sequencing data of AGO2 immunoprecipitation,differentially expression circRNA which could bind to AGO2 and may act as micro RNA sponge were identified.Then,five circRNAs were selected to construct the circRNA-micro RNA-mRNA interaction network.The correlation of expression levels between the differentially expressed circRNA and the expression of their corresponding linear mRNA was analyzed.Moreover,the circRNA which have a potential function for coding protein were screened by the circRNADb platform.4 Has_circ₀040705 and has_circ₀001304 were knocked down by si RNA in HASMCs.PCNA,SM22a,Bax,QKI were detected by Western blot.The apoptosis of HASMCs was analyesd by flow cytometry.5 Construction of circRNA expression vectorThe has_circ₀001304 and has_circ₀040705 were amplified by PCR.The PCR product was digested with Eco R I & Xba I and inserted into the pc DNA3₁（+）CircRNA Mini Vector.The recombinants were verified by digesting and sequencing.Results: 1 Using gene chip snalysis,we found that 66 circRNA and 3,019 mRNA were up-regulated,68 circRNA and 701 mRNA down-regulated in the proliferative HASMCs compared with quiescent HASMCs.2 q RT-PCR validated that 12 of 13 circRNA expression signitures were in accordance with the gene chip data.3 GO analysis demonstrated that the mRNA related with differentially expressed circRNA participate in a variety of biological activities,such as protein transport.Pathway analysis showed the most significant two pathways including AMPK and Micro RNAs in cancer.For the differentially expressed mRNA,GO analysis denoted the terms,such as negative regulation of cell cycle arrest.In addition,pathway analysis contained numerous pathways related to information transmission and proliferation,such as MAPK.Through analysis,we found that 78 differentially expressive circRNA could bind to AGO2 and could play a role of micro RNA sponge.Hsa_circ₀000079,hsa_circ₀002579,hsa_circ₀004872,hsa_circ₀004872,hsa_circ₀006371 and hsa_circ₀040705 were selected to construct the circRNA-micro RNA-mRNA interaction network.The results showed that the differentially expressed genes,such as C2orf44,IGF2BP1,HMGA2 and KLF7,were co-expressed with the differentially expressive circRNA which provided an important clue for explore the regulatory target of circRNA.It showed that there was no correlation between circRNA and the corresponding linear mRNA expression.Through the circRNADb platform,24 kinds of circRNA containing ribosomal entry sequence and open reading frame were screened which suggested that these circRNA could encode proteins.4 Has_circ₀040705 and has_circ₀001304 were silenced by si RNA in HASMCs.Western Blot analysis showed that PCNA significantly decreased.Meanwhile,SM22α and Bax significantly increased.Flow cytometry analysis showed that knock down of has_circ₀040705 or has_circ₀001304 could promote apoptosis of HASMCs.In addition,the knock down of has_circ₀001304 significantly inhibited the expression of QKI protein.5 The eukaryotic expression vector of has_circ₀001304 and has_circ₀040705 were successfully constructed by sequencing analysis.Conclusion:1 CircRNA is differentially expressed in proliferative HASMCs compared with quiescent HASMCs.CircRNAs may take function via acting as mi RNAs sponge or encoding proteins;2 Has_circ₀040705 and has_circ₀001304 can promote the proliferation and inhibit apoptosis of HASMCs.They would become the potential targets for the VSMC proliferative diseases.