Circ-154 Up-regulates MiR-154-5p To Inhibit The Proliferation And Autophagy Of Vascular Smooth Muscle Cells

Objective:Vascular smooth muscle cells(VSMCs)are one of the main cells types that constitute the vascular wall.Abnormal proliferation and autophagy of VSMCs are closely related to the occurrence and development of cardiovascular diseases,such as myocardial infarction,coronary heart disease,and heart failure.Micro RNAs(miRNAs)are a class of endogenous small RNAs about 22 nucleotides in length.Increasing studies have elucidated that miRNAs are involved in regulating physiological processes,such as cells proliferation,autophagy,and differentiation.Circular RNAs(circ RNAs),a class of non-coding RNAs with a covalent closed circular structure,which is not degradable by an exonuclease and is more stable than linear non-coding RNAs,function in the occurrence and development of cardiovascular diseases,such as hypertension,atherosclerosis,and diabetes.Small RNA sequencing was performed to screen differentially expressed small RNAs in proliferative human aortic vascular smooth muscle cells(HASMCs)and quiescent HASMCs,and it was shown that the miR-154-5p expression was significantly down-regulated in proliferative HASMCs.Subcellular localization analysis revealed that miR-154-5p was distributed in both the cytoplasm and nucleus of HASMCs.However,its function in HASMCs proliferation and autophagy remains unclear,so we predicted and identified potential target genes of miR-154-5p from the perspective of miRNAs-m RNA and miRNAs-promoter,respectively.In addition,co-expression analysis of miRNAs-circ RNAs showed that circ-154 was co-expressed with miR-154-5p,suggesting that circ-154 might regulate miR-154-5p expression.Herein,the molecular mechanism of miR-154-5p regulating HASMCs proliferation/autophagy and the regulatory relationship between circ-154 and miR-154-5p were demonstrated,to provide a novel insight to explore the mechanism of HASMCs proliferation and autophagy.Methods:1.Using qRT-PCR,miR-154-5p expression in the proliferative and quiescent HASMCs was detected,and miR-154-5p expression in the nucleus and cytoplasm of proliferative HASMCs was detected.2.miR-154-5p inhibited HASMCs proliferationBased on nuclear miRNAs targeting gene promoter to regulate transcription,RNAhybrid and Mi Randa were performed to screen the target gene PRKD2 of miR-154-5p in the nucleus.From the perspective of cytoplasmic miRNAs-targeted m RNA regulation of translation,a target gene HMGA2 of miR-154-5p in the cytoplasm was selected using a combination of miRTar Base,Target Scan and miRWalk.Both PRKD2 and HMGA2 are genes related to cells proliferation.Mi R-154-5p mimics were transfected into HASMCs,and expression of PRKD2,HMGA2,and PCNA were detected by Western blot,and effects of miR-154-5p mimics on HASMCs viability and proliferation were detected by CCK-8 and Ed U assays.3.miR-154-5p inhibited HASMCs autophagyBased on the regulation of translation by cytoplasmic miRNAs-targeted m RNA,the combination of miRTar Base,Target Scan,and miRWalk was performed to screen ATG7,a target gene of miR-154-5p in the cytoplasm.HASMCs were transfected with miR-154-5p mimics for 24 h,then treated with 5?M rapamycin for 24 h,and ATG7,LC3-II/I,and p62 expression were detected by Western blot.HASMCs were co-transfected with miR-154-5p mimics and RFP-GFP-LC3 plasmid for 24 h,then treated with 5?M rapamycin for 24 h,the autophagy flow was detected by confocal laser scanning microscopy.4.The circ-154 recombinant plasmid was constructed and its overexpression efficiency was verified by qRT-PCR.The circ-154recombinant plasmid or circ-154 si RNA was transfected into HASMCs,and the expression level of miR-154-5p was detected by qRT-PCR.5.circ-154 inhibited HASMCs proliferationThe circ-154 recombinant plasmid or circ-154 si RNA was transfected into HASMCs,and PRKD2,HMGA2 and PCNA expression levels were detected by Western blot.The circ-154 recombinant plasmid was transfected into HASMCs,and its effect on HASMCs viability and proliferation was tested by CCK-8 and Ed U assays.6.circ-154 inhibited autophagy in HASMCscirc-154 si RNA was transfected into HASMCs and ATG7,LC3-II/I and p62 expression levels were detected by Western blot.The circ-154recombinant plasmid was transfected into HASMCs,and 24 hours later,treatment was continued with 5?M rapamycin for 24 hours,and ATG7,LC3-II/I,and p62 expression levels were detected by Western blot.The circ-154 recombinant plasmid was co-transfected with RFP-GFP-LC3 plasmid into HASMCs,and 24 hours later,treatment was continued with 5?M rapamycin for 24 hours to detect changes in autophagic flow by confocal laser scanning microscopy.7.miR-154-5p binds the ATG7 3?-UTRAccording to the binding sites of ATG7 and miR-154-5p,wild-type(WT)and mutant(MT)sequences were chemically synthesized,and the sticky ends of Xho I enzyme and Not I enzyme were introduced at their 5'and 3'ends,respectively,and inserted into the psi CHECK^TM-2 Vector,and single colonies were picked after transformation for colony PCR,single digestion identification,and sequencing analysis.Dual-luciferase reporter assay was performed to verify whether ATG7 is a direct target gene of miR-154-5p.Results:1.miR-154-5p was down-regulated in proliferative HASMCs,miR-154-5p was distributed in the nucleus and cytoplasm,and the expression was higher in the nucleus.2.In HASMCs,miR-154-5p inhibited PRKD2,HMGA2 and PCNA protein expression;CCK-8 and Ed U results indicated that miR-154-5p inhibited HASMCs viability and proliferation.3.In HASMCs,miR-154-5p inhibited ATG7 and LC3-II/I protein expression and promoted p62 protein expression;autophagy double fluorescence assay indicated that miR-154-5p inhibited autophagy in HASMCs.4.circ-154 recombinant plasmid was successfully constructed;in HASMCs,circ-154 up-regulated miR-154-5p expression,while circ-154si RNA down-regulated miR-154-5p expression.5.In HASMCs,circ-154 up-regulated PRKD2,HMGA2 and PCNA protein expression,while circ-154 si RNA down-regulated PRKD2,HMGA2and PCNA protein expression;CCK-8 and Ed U results indicated that circ-154inhibited HASMCs viability and proliferation.6.In HASMCs,circ-154 down-regulated ATG7 and LC3-II/I protein expression and up-regulated p62 protein expression,while circ-154 si RNA up-regulated ATG7 and LC3-II/I protein expression and down-regulated p62protein expression;autophagy double fluorescence assay indicated that miR-154-5p inhibited autophagy in HASMCs.7.The construction of ATG7-WT and ATG7-MT reporter plasmids was confirmed by colony PCR,digestion identification and sequencing analysis,and the dual-luciferase reporter gene showed that ATG7 was a direct target gene of miR-154-5p.Conclusion:1.miR-154-5p inhibits the proliferation and autophagy of HASMCs by targeting HMGA2 and ATG7.2.circ-154 up-regulates miR-154-5p to inhibit the proliferation and autophagy of HASMCs.