Korean J Physiol Pharmacol.  2022 May;26(3):183-193. 10.4196/kjpp.2022.26.3.183.

Differentially expressed mRNAs and their upstream miR-491-5p in patients with coronary atherosclerosis as well as the function of miR-491-5p in vascular smooth muscle cells

• Ding H
• Pan Q
• Qian L
• Hu C

• KMID: 2529401
• DOI: http://doi.org/10.4196/kjpp.2022.26.3.183

Abstract

MicroRNAs (miRNAs) regulate gene expression and are biomarkers for coronary atherosclerosis (AS). A novel miRNA-mRNA regulation network of coronary AS still needs to be disclosed. The aim of this study was to analyze potential mRNAs in coronary AS patients and the role of their upstream miR-491-5p in vascular smooth muscle cells (VSMCs). We first confirmed top ten mRNAs according to the analysis from Gene Expression Omnibus database (GSE132651) and examined the expression levels of them in the plaques and serum from AS patients. Five mRNAs (UBE2G2, SLC16A3, POLR2C, PNO1, and AMDHD2) presented significantly abnormal expression in both plaques and serum from AS patients, compared with that in the control groups. Subsequently, they were predicted to be targeted by 11 miRNAs by bioinformatics analysis. Among all the potential upstream miRNAs, only miR-491-5p was abnormally expressed in the plaques and serum from AS patients. Notably, miR-491-5p overexpression inhibited viability and migration, and significantly increased the expression of contractile markers (α-SMA, calponin, SM22α, and smoothelin) in VSMCs. While silencing miR-491-5p promoted viability and migration, and significantly suppressed the expression of α-SMA, calponin, SM22α, and smoothelin. Overall, miR-491-5p targeted UBE2G2, SLC16A3, and PNO1 and regulated the dysfunctions in VSMCs.

Keyword

Coronary artery disease; miR-491-5p; PNO1; SLC16A3; UBE2G2

Figure

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  Fig. 1 Expression of 10 mRNAs in plaques and adjacent intimal tissues from atherosclerosis (AS) patients. (A) Expression levels of ten mRNAs (UBE2G2, SNORD45C, SNORD45A, SNORD45B, RABGGTB, SLC16A3, POLR2C, PNO1, CEMP1, AMDHD2) in plaques and adjacent intima from coronary AS patients were measured using RT-qPCR with pared t-test, as presented by the scatter diagram. (B) mRNAs (UBE2G2, SNORD45C, SNORD45A, SLC16A3, POLR2C, PNO1, CEMP1, AMDHD2) with significant difference were listed in the bar graph. *p < 0.05, **p < 0.01, NS indicates no significance.

• 

  Fig. 2 Expression of 10 mRNAs in serum from atherosclerosis (AS) patients and healthy controls. (A) Expression levels of ten mRNAs (UBE2G2, SNORD45C, SNORD45A, SNORD45B, RABGGTB, SLC16A3, POLR2C, PNO1, CEMP1, AMDHD2) in the serum of coronary AS patients and healthy patients were measured using RT-qPCR with unpaired t-test. (B) mRNAs (UBE2G2, SNORD45C, SNORD45A, SLC16A3, POLR2C, PNO1, CEMP1, AMDHD2) with significant difference were listed. *p < 0.05, **p < 0.01, ***p < 0.001, NS indicates no significance.

• 

  Fig. 3 Potential upstream miRNAs for the abnormally expressed mRNAs. (A) TargetScan predicted the potential upstream miRNAs of UBE2G2, SLC16A3, POLR2C, PNO1, and AMDHD2. (B) Expression levels of 11 miRNAs in plaques and adjacent intima from coronary AS patients were examined using RT-qPCR with pared t-test. (C) Expression levels of 11 miRNAs in the serum of coronary atherosclerosis (AS) patients and healthy patients were examined using RT-qPCR with unpaired t-test. *p < 0.05, **p < 0.01.

• 

  Fig. 4 MiR-491-5p negatively regulates UBE2G2, SLC16A3, PNO1 expression in vascular smooth muscle cells (VSMCs). (A) Overexpression efficiency of miR-491-5p in VSMCs was evaluated using RT-qPCR. (B) Expression levels of UBE2G2, SLC16A3, POLR2C, PNO1, and AMDHD2 in VSMCs transfected with miR-491-5p mimics and NC mimics were examined by RT-qPCR. (C) Protein levels of UBE2G2, SLC16A3, POLR2C, PNO1, and AMDHD2 in VSMCs transfected with miR-491-5p mimics and NC mimics were examined by Western blot. (D) RNA pull down assay was for detecting whether UBE2G2, SLC16A3, and PNO1 bind with miR-491-5p. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 5 MiR-491-5p inhibited viability, migration, and increased expression of contractile markers in vascular smooth muscle cells (VSMCs). (A) Cell counting kit 8 (CCK-8) revealed the viability in VSMCs with transfection of miR-491-5p mimics and NC mimics. (B) Wound healing was performed to detect the migration capacity of VSMCs with transfection of miR-491-5p mimics and NC mimics. (C) The protein levels of contractile markers (α-SMA, calponin, SM22α, and smoothelin) in VSMCs with transfection of miR-491-5p mimics and NC mimics were measured by Western blot. *p < 0.05, **p < 0.01.

• 

  Fig. 6 MiR-491-5p antagomir promotes viability, migration, and increased expression of contractile markers in vascular smooth muscle cells (VSMCs). (A) CCK-8 revealed the viability in VSMCs with transfection of NC antagomir, miR-491-5p antagomir, miR-491-5p antagomir+sh-NC, miR-491-5p antagomir+sh-UBE2G2, miR-491-5p antagomir+sh-SLC16A3, and miR-491-5p antagomir+sh PNO1. (B) Wound healing was performed to detect the migration capacity of VSMCs with transfection of NC antagomir, miR-491-5p antagomir, miR-491-5p antagomir+sh-NC, miR-491-5p antagomir+sh-UBE2G2, miR-491-5p antagomir+sh- SLC16A3, and miR-491-5p antagomir+sh PNO1. (C) The protein levels of contractile markers (α-SMA, calponin, SM22α, and smoothelin) in VSMCs with transfection of NC antagomir, miR-491-5p antagomir, miR-491-5p antagomir+sh-NC, miR-491-5p antagomir+sh-UBE2G2, miR-491-5p antagomir+sh- SLC16A3, and miR-491-5p antagomir+sh PNO1. *p < 0.05, **p < 0.01.

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