The Regulatory Effect Of Hsa_circ₀001402 On The Proliferation Of Vascular Smooth Muscle Cells And Its Diagnostic Value In Unstable Angina

Objective:Vascular smooth muscle cells（VSMCs）are one of the main cell types that make up the vascular wall and play a vital role in maintaining the normal physiological function of blood vessels.Abnormal proliferation of VSMCs is one of the causes of many vascular remodeling diseases,such as atherosclerosis,hypertension and thoracic aortic aneurysms.Circular RNA（circ RNA）has a covalently closed circular structure,which is not easily degraded by the exonuclease and is more stable than linear RNA.Circ RNA can be secreted into exosomes and is a potential diagnostic biomarker for the disease.Our group previously identified many differentially-expressed circ RNAs（DEcirc RNAs）between proliferative human aortic smooth muscle cells（HASMCs）and quiescent HASMCs by microarray,and hsa_circ₀001402 is one of the significantly down-regulated DEcirc RNAs.However,the regulatory effect of hsa_circ₀001402 on VSMC proliferation remains unclear.This study demonstrated the mechanism of hsa_circ₀001402 in regulating VSMC proliferation and its potential as a plasma diagnostic biomarker for unstable angina（UA）,to provide a potential therapeutic target for VSMC proliferative cardiovascular disease and a potential plasma diagnostic biomarker for UA.Methods:1. Quantitative real-time polymerase chain reaction（qRT-PCR）validation of hsa_circ₀001402The expression levels of hsa_circ₀001402 in proliferative HASMC and quiescent HASMC were detected by qRT-PCR,and the fold change was calculated by the 2^-ΔΔCt method.2. Bioinformatics analysis of hsa_circ₀001402The micro RNAs（miRNAs）sponged by hsa_circ₀001402 were predicted by circ Bank,Circ Interactome,and miRDB databases.Differentially-expressed m RNAs（DEm RNAs）targeted by miRNAs were predicted from 3′-UTR and CDS targeting perspectives using a combination of miRTar Base,Target Scan,miRWalk,and RNAhybrid databases.Through Gene Ontology（GO）and pathway analyses,functional analysis of DEm RNAs co-expressed with hsa_circ₀001402 was conducted to determine the potential role of hsa_circ₀001402 in VSMC regulation.3.Construction of vectors related to hsa_circ₀001402Construction of the recombinant plasmid pcDNA3.1（+）Circ RNA Mini-402.The primers were designed according to the sequence of hsa_circ₀001402.Meanwhile,the Eco R I restriction site was introduced at the 5′end of the primers,and the Xba I restriction site was introduced at the 3′end of the primers.The total RNA of HASMCs was extracted for RT-PCR amplification.The amplified products were then purified by the Gel Extraction purification after agarose gel electrophoresis.After double enzyme digestion,the products were inserted into the expression vector of pcDNA3.1（+）Circ RNA Mini Vector and then transformed to perform colony PCR,double enzyme digestion validation and sequencing analysis.Construction of reporter gene-related vector.The wild-type and mutant-type sequences containing the sticky ends of Xho I and Not I were chemically synthesized and inserted into the psi CHECK^TM-2 Vector to obtain four recombinant plasmids（hsa_circ₀001402-WT,hsa_circ₀001402-MT,FKBPL-WT,and FKBPL-MT）.The validation method is the same as the above.4.Regulation of hsa_circ₀001402 on VSMC proliferationThe recombinant plasmid pcDNA3.1（+）Circ RNA Mini-402 was transfected into VSMCs.Firstly,the overexpression efficiency of the plasmid was validated by qRT-PCR;secondly,the influence of the plasmid on the cell viability of VSMCs was detected by Cell Counting Kit（CCK-8）;finally,the expression level of the plasmid related proteins was detected by Western blot.5.Regulation of hsa-miR-183-5p on VSMC proliferationHsa-miR-183-5p mimics were transfected into VSMCs.The expression levels of related proteins were detected by Western blot.6.Detection of hsa_circ₀001402 in plasma of UA patientsThe expression levels of hsa_circ₀001402 in plasma of UA patients and healthy people were detected by qRT-PCR,and the fold change was calculated by the 2^-ΔΔCt method.Results:1.Hsa_circ₀001402 was downregulated in proliferative HASMCs.2.Through bioinformatics analysis,it was found that hsa_circ₀001402might sponge 69 miRNAs.Target DEm RNAs were predicted for miRNAs from the perspectives of 3′-UTR and CDS targeting,and these target DEm RNAs were analyzed by GO and pathway analyses.It was found that hsa-miR-183-5p may target 87 DEm RNAs,and GO and pathway analyses indicated that hsa-miR-183-5p may regulate VSMC proliferation through cluster of differentiation 44（CD44）and FK506 binding protein like（FKBPL）.It was also found that hsa-miR-545-3p may target 157 DEm RNAs,and GO and pathway analyses indicated that hsa-miR-545-3p may regulate VSMC proliferation through low-density lipoprotein receptor-related protein 1（LRP1）.3. The expression vector of hsa_circ₀001402 and the related reporter gene vector were successfully constructed.4. The recombinant plasmid pcDNA3.1（+）Circ RNA Mini-402 could effectively overexpress hsa_circ₀001402.Hsa_circ₀001402 reduced the cell viability of VSMCs.The expression levels of CD44,FKBPL,LRP1,and cyclin-dependent kinase inhibitor 1A（p21）were increased by hsa_circ₀001402 in VSMCs,and the expression level of proliferating cell nuclear antigen（PCNA）was decreased.5. In VSMCs,hsa-miR-183-5p decreased the expression levels of CD44,FKBPL,and p21,and increased the expression levels of PCNA.6. The expression level of hsa_circ₀001402 in UA patients was significantly down-regulated.Conclusions:1.Hsa_circ₀001402 was down-regulated in proliferative HASMCs,and overexpression of hsa_circ₀001402 inhibited VSMC proliferation.The mechanism was speculated to be:hsa_circ₀001402,as a ce RNA molecule,up-regulated the expression of CD44 and FKBPL by sponging hsa-miR-183-5p,and up-regulated the expression of LRP1 by sponging hsa-miR-545-3p.2.Hsa_circ₀001402 was significantly down-regulated in plasma of UA patients compared with healthy subjects.Hsa_circ₀001402 is a potential plasma diagnostic biomarker for UA.