MicroRNA-541 promotes the proliferation of vascular smooth muscle cells by targeting IRF7

Abstract

MiRNAs play crucial roles in abnormal proliferation and invasion of VSMCs. However, the roles and mechanisms of miRNAs in VSMCs are not fully understood. In our study, we demonstrated that PDGF-BB and serum induced proliferation of VSMCs led to the upregulation of miR-541. We also showed that overexpression of miR-541 promoted VSMC proliferation and invasion. In addition, Interferon regulatory factor 7 (IRF7) was found to be a potential target of miR-541 and upregulation of IRF7 could inhibit VSMC proliferation. Restored expression of miR-541 promoted IRF7-inhibited VSMCs proliferation. In conclusion, these findings suggest that inhibitors targeting miR-541 or its specific downstream molecules may be therapeutic strategy for VSMC growth-related diseases.

Keywords: IRF7; Vascular smooth muscle cells; miR-541; microRNA.

Figure 1

miR-541 was up-regulated by PDGF-BB and FBS in VSMCs. A. The CCK8 proliferation assay has shown that PDGF-BB can induce the VSMCs proliferation. B. qRT-PCR analysis was performed to the levels of miR-541 in VSMCs after PDGF-BB treatment. PDGF-BB can enhance the expression of miR-541. U6 snRNA was used as internal control. C. The CCK8 proliferation assay has shown that (fetal bovine serum) FBS can induce the VSMCs proliferation. D. qRT-PCR was performed to the levels of miR-541 in VSMCs after PBS treatment. FBS can enhance the expression of miR-541. U6 snRNA was used as internal control. *p<0.05, and **p<0.01, *** p<0.001.

Figure 2

Overexpression of miR-541 promoted the proliferation of VSMCs. A. qRT-PCR analysis of miR-541 in VSMCs upon transfection of miR-541 mimics. The expression of miR-541 in VSMCs transfected with miR-541 mimics was up-regulated. U6 snRNA was used as internal control. B. The CCK8 proliferation assay has shown that miR-541 overexpression can induce the VSMCs proliferation. C. qRT-PCR analysis of miR-541 in VSMCs upon transfection of miR-541 inhibitor. The expression of miR-541 in VSMCs transfected with miR-541 inhibitors was down-regulated. U6 snRNA was used as internal control. D. The CCK8 proliferation assay has shown that miR-541 downregulation can inhibit the VSMCs proliferation. E. qRT-PCR analysis of PCNA in VSMCs upon transfection of miR-541 mimics, inhibitor, scramble or control. F. Western blot analysis of PCNA in VSMCs upon transfection of miR-541 mimics, inhibitors, or scramble or control. *p<0.05, and **p<0.01, *** p<0.001.

Figure 3

Overexpression of miR-541 promoted the invasion of VSMCs. A. Invasion analysis of VSMCs after treatment with miR-541 mimics, inhibitors or scramble or control. B. The relative ratio of invasive cells per field is shown, *p<0.05, **p<0.01, and ***p<0.001.

Figure 4

miR-541 targets IRF7 in VSMCs. A. Schematic representation of IRF7 3’UTR showing the putative miR-541 target site. B. Relative luciferase activity of the indicated IRF7 reporter construct in VSMCs is shown. Firefly luciferase values were normalized to Renilla luciferase activity and plotted as relative luciferase activity. C. RT-PCR analysis was performed to examine the effects of miR-541 mimic on IRF7 expression in VSMCs. Ectopic expression of miR-541 significantly decreased IRF7 transcripts. GAPDH was used as internal control. D. Western blotting was performed to examine the effect of miR-541 mimic on the expression of IRF7. GAPDH was also detected as a loading control. E. Inhibition of miR-541 promoted the protein expression of IRF7. F. RT-PCR analysis was performed to examine the effects of miR-541 inhibitor on IRF7 expression in VSMCs. ***p<0.001.

Figure 5

Overexpression of IRF7 can inhibit VSMCs proliferation. A. qRT-PCR analysis was performed to the levels of IRF7 in VSMCs after PDGF-BB treatment. PDGF-BB can inhibit the expression of IRF7. GAPDH was used as internal control. B. qRT-PCR analysis was performed to the levels of IRF7 in VSMCs after FBS treatment. FBS can inhibit the expression of IRF7. GAPDH was used as internal control. C. Western blotting was performed to examine the effects of pcDNApcDNA-IRF7 on the expression of IRF7. GAPDH was also detected as a loading control. D. The CCK8 proliferation assay has shown that IRF7 can inhibit the VSMCs proliferation. E. IRF7 overexpression depressed the PCNA mRNA expression. GAPDH was used as internal control. F. IRF7 overexpression inhibited the PCNA protein expression. GAPDH was also detected as a loading control. *p<0.05, ** p<0.01, and ***p<0.001.

Figure 6

Restoration of miR-541 promoted IRF7-inhibited VSMCs proliferation. A. The cell growth in VSMCs co-transfected with either miR-541 mimic or scramble and pcDNA-IRF7 or pcDNAempty vector using CCK-8 proliferation assay. B. The mRNA expression of PCNA in VSMCs co-transfected with either miR-541 mimic or scramble and pcDNA-IRF7 or pcDNA empty vector using qRT-PCR analysis. GAPDH was used as internal control. C. The protein expression of PCNA in VSMCs co-transfected with either miR-541 mimic or scramble and pcDNA-IRF7 or pcDNA empty vector using western blot analysis. GAPDH was also detected as a loading control. *p<0.05, ** p<0.01, and ***p<0.001.