| Objective Screening β-glycerophosphate combined with L-ascorbic acid and dexamethasone-induced circRNA,miRNA,mRNA related to VSMC calcification,and study the role and mechanism of circRNA in promoting VSMC calcification.Methods1.Screening the optimal concentration for inducing VSMC calcification: use different concentrations(0,10,30,50)mmol/L β glycerophosphate combined with 50 μg/ml L-ascorbic acid and 100 nmol/L dexamethasone to stimulate VSMC for 14 d.The gene expression levels of RUNX2,BMP4 and BMP2 were detected by qPCR,and the protein expression levels of RUNX2,BMP4 and BMP2 were detected by Western blot.2.Screening of differentially expressed circRNA during VSMC calcification: VSMC was randomly divided into control group and calcification group.The calcification group was stimulated with 10 mmol/L β-glycerophosphate combined with 50 μg/ml L-ascorbic acid and 100 nmol/L dexamethasone for 14 d.The gene expression levels of RUNX2,BMP4,BMP2 were detected by qPCR,the protein expression levels of RUNX2,BMP4,BMP2 were detected by Western blot,and the calcium salt deposited in VSMC was observed by Alizarin Red S staining.High-throughput sequencing was used to screen the circRNA with significantly different expression levels in the calcification group compared with the control group.Agarose gel electrophoresis and PCR product sequencing were used to verify the primer sequences of the circRNA.The circRNA with the most significant difference in expression levels and stable expression was screened by qPCR.3.To investigate the regulation effect of circRNA overexpression on vascular calcification: circRNA overexpression plasmid was constructed and then transfected into VSMC,and the VSMC was divided into control group,circ-NC group(transfection independent sequence)and circ-GALK2 group(transfection circ-GALK2 sequence),and the transfection efficiency was verified by qPCR.VSMC was then divided into circ-NC group and circ-GALK2 group.After transfection for 48 h,the culture medium was changed,and the VSMC was stimulated with 10 mmol/L β-glycerophosphate combined with 50 μg/ m L L-ascorbic acid and 100 nmol/L dexamethasone for 14 d.The gene expression levels of RUNX2,BMP4 and BMP2 were detected by qPCR,the protein expression levels of RUNX2,BMP4 and BMP2 were detected by Western blot,and the calcium salt deposited in VSMC was observed by Alizarin Red S staining.4.Circ RNA targeted miRNA screening: Use miRanda software to predict the relationship between miRNA and differentially expressed circRNA in the miRBase database,and then use qPCR to detect the expression of miRNA in the control group,circ-NC group,and circ-GALK2 group.Divide VSMC into miR-134-3p-NC group(transfection irrelevant sequence),miR-134-3p-inhibitor group(transfection miR-134-3p inhibitor sequence),miR-134-3p-mimic group(transfection miR-134-3p mimic sequence),the culture medium was changed 48 h after transfection,and the expression of miR-134-3p was detected by qPCR.Then the VSMC was divided into miR-134-3p-NC group,miR-134-3p-mimic group,miR-134-3p-inhibitor group and circ-NC group,circ-GALK2 group,circ-GALK2 + miR-134-3p-mimic group(transfected circ-GALK2 sequence and miR-134-3p mimic sequence at the same time),48 h later,replaced with a new medium,and continued to use 10 mmol/L βglycerophosphate combined with 50 μg/ml L-ascorbic acid and 100 nmol/L dexamethasone stimulates VSMC for 14 d.The gene expression levels of RUNX2,BMP4 and BMP2 were detected by qPCR,and the protein expression levels of RUNX2,BMP4 and BMP2 were detected by Western blot.5.Screening of downstream target gene of miR-134-3p: Use miRDB and Target Scan database to screen the target genes that miR-134-3p may act on.VSMC was divided into miR-134-3p-NC group,miR-134-3p-mimic group and miR-134-3p-inhibitor group.After 48 h,the VSMC was replaced with new culture medium.VSMC was cultured with 10 mmol/L β-glycerophosphate combined with 50 μg/ m L L-ascorbic acid and 100nmol/L dexamethasone for 14 d,and the protein expression level of CD36 was detected by Western blot.VSMC was then divided into control group and calcification group.In calcification group,VSMC was stimulated with 10 mmol/L β-glycerophosphate combined with 50 μg/ m L L-ascorbic acid and 100 nmol/L dexamethasone for 14 d,and the protein expression of CD36 was detected by Western blot.VSMC was then divided into circ-NC group,circ-GALK2 group and circ-GALK2 + miR-134-3p-mimic group.After 48 h,the old culture medium was discarded.Then,VSMC was cultured with 10mmol/L β-glycerophosphate combined with 50 μg/ m L L-ascorbic acid and 100 nmol/L dexamethasone for 14 d,and the protein expression level of CD36 was detected by Western blot.Results1.Establishment of VSMC calcification model: qPCR results showed that the gene expression levels of RUNX2,BMP4 and BMP2 were up-regulated whenβ-glycerophosphate concentration was 10 mmol/L compared with the control group(P< 0.05).Western blot results showed that the protein expressions of RUNX2,BMP4 and BMP2 were significantly up-regulated when β-glycerophosphate concentration was 10mmol/L compared with the control group.Therefore,10 mmol/L β-glycerophosphate was selected as the best treatment concentration for subsequent experiments.2.Screening and validation of candidate circRNAs in VSMC: qPCR results showed that RUNX2,BMP4 and BMP2 gene expression levels were up-regulated in calcification group compared with control group(P < 0.05).Western blot results showed that the protein expression levels of RUNX2,BMP4 and BMP2 were up-regulated in calcification group compared with the control group.The results of Alizarin Red S staining showed that the calcium salt deposition of VSMC in calcification group was higher than that in control group.According to the results of high-throughput sequencing and differential expression analysis,46 circRNAs with significantly different expressions were screened in the calcification group compared with the control group.In the calcification group,15 circRNAs were up-regulated and 31 circRNAs were down-regulated.Subsequently,5 circRNAs with the most significant expression differences and stable expression were selected from the candidate circRNAs for verification.Agarose gel electrophoresis confirmed that the PCR products of 5circRNAs were consistent with the expected size.The qPCR results showed that the expression level of circ-GALK2 in VSMC of calcification group was significantly up-regulated.Sequencing results showed a fragment of circ-GALK2 cross-junction sequence.Therefore,circ-GALK2 was selected as the study object to explore its regulatory effect on VSMC calcification.3.Overexpression of circRNA promotes vascular calcification: The overexpression plasmid of circ-GALK2 was designed and constructed by Guangzhou Jisai Company.The qPCR results showed that compared with the control group and circ-NC group,the expression level of circ-GALK2 gene in the circ-GALK2 group was up-regulated(P <0.05),and there was no statistical difference in the expression level of circ-GALK2 gene between the control group and circ-NC group.qPCR results showed that compared with circ-NC group,gene expression levels of RUNX2,BMP4 and BMP2 were up-regulated in circ-GALK2 group(P < 0.05).Western blot results showed that compared with the circ-NC group,the protein expression levels of RUNX2,BMP4 and BMP2 were up-regulated in the circ-GALK2 group.The results of Alizarin Red S staining showed that the calcium salt deposition of VSMC in the circ-GALK2 group was increased compared with that in the circ-NC group.4.Circ-GALK2 targets miR-134-3p in VSMC: According to the results of bioinformatics analysis,the preliminary analysis of 5 miRNAs and qPCR results showed that compared with the control group and circ-NC group,the expression level of miR-134-3p in circ-GALK2 group was the most significantly down-regulated(P <0.05).Sequence analysis revealed the presence of miR-134-3p binding sites in circ-GALK2.The qPCR results showed that compared with the miR-134-3p-NC group,the expression level of miR-134-3p gene in the miR-134-3p-inhibitor group was down-regulated,while the expression level of miR-134-3p mimic was up-regulated(P <0.05).qPCR results showed that compared with miR-134-3p-NC group,the gene expression levels of RUNX2,BMP4 and BMP2 in miR-134-3p-mimic group were down-regulated.The gene expression levels of RUNX2,BMP4 and BMP2 in miR-134-3p-inhibitor group were up-regulated(P < 0.05).Western blot results showed that compared with the miR-134-3p-NC group,the expression levels of RUNX2,BMP4 and BMP2 in the miR-134-3p-mimic group were down-regulated.The expression levels of RUNX2,BMP4 and BMP2 proteins in miR-134-3P-inhibitor group were up-regulated.qPCR results showed that compared with circ-NC group,gene expression levels of RUNX2,BMP4 and BMP2 were up-regulated in circ-GALK2 group.Compared with circ-GALK2 group,the gene expression levels of RUNX2,BMP4 and BMP2 were down-regulated in circ-GALK2 + miR-134-3p-mimic group(P < 0.05).Western blot results showed that compared with the circ-NC group,the protein expression levels of RUNX2,BMP4 and BMP2 were up-regulated in the circ-GALK2 group.Compared with circ-GALK2 group,the expression levels of RUNX2,BMP4 and BMP2 proteins were down-regulated in circ-GALK2 + miR-134-3p-mimic group.5.CD36 is the downstream target gene of miR-134-3p: CD36 gene 3,there is a binding site for miR-134-3p on UTR.Western blot results showed that compared with the miR-134-3p-NC group,the expression level of CD36 protein in the miR-134-3p-mimic group was down-regulated,while the expression level of CD36 protein in the miR-134-3p-inhibitor group was up-regulated.Western blot results showed that the protein expression level of CD36 in the calcification group was up-regulated compared with the control group.Western blot results showed that compared with the circ-NC group,the expression level of CD36 protein in the circ-GALK2 group was up-regulated;compared with the circ-GALK2 group,the expression level of CD36 protein in the circ-GALK2 + miR-134-3p-mimic group was down-regulated.Conclusionβ-glycerophosphate combined with L-ascorbic acid and dexamethasone induced circ-GALK2 to promote the expression of CD36 by adsorbing miR-134-3p,thereby promoting VSMC calcification. |
PDF Full Text Request