Long Noncoding RNA HLA Complex Group 18 Improves the Cell Proliferation of Myocardial Fibroblasts by Regulating the Hsa-microRNA-133a/Epidermal Growth Factor Receptor Axis

Abstract

Hsa-microRNA (has-miR)-133a inactivates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and suppresses the cell proliferation of myocardial fibroblasts by downregulation of the epidermal growth factor receptor (EGFR) expression. Bioinformatics analysis exhibits extended noncoding RNA HLA complex group 18 (lncRNA-HCG18) binds to hsa-miR-133a. The purpose of the current experiment is to explore whether lncRNA-HCG18 adsorbed hsa-miR-133a through sponging, resulting in decreased inhibition of hsa-miR-133a on EGFR and ultimately promoting the proliferation of myocardial fibroblasts. To verify and study the correlation and mechanism between lncRNA-HCG18, hsa-miR-133a, and their target genes. Firstly, after overexpression/silencing of lncRNA-HCG18 in myocardial fibroblasts, the level of hsa-miR-133a expression was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and the EGFR, ERK1/2, and p-ERK1/2 expression levels were assessed by Western blotting to confirm that upregulation of EGFR and p-ERK1/2 protein levels by overexpression of lncRNA-HCG18, siRNA lncRNA-HCG18 (siHCG18) reduced the EGFR and p-ERK1/2 protein levels. Then, the luciferase reporter gene system was used to verify that lncRNA-HCG18 regulated EGFR expression by inhibiting the function of the hsa-miR-133a regulatory target gene. Then, a RAP assay was used to confirm that lncRNA-HCG18 interacted with hsa-miR-133a. Finally, the analysis of CCK-8 results indicated that the cell proliferation of myocardial fibroblasts was significantly reduced by siHCG18 while reversed by overexpression of lncRNA-HCG18. Thus, lncRNA-HCG18 inhibited cell viability of cardiac fibroblasts via the hsa-miR-133a/EGFR axis, which was regarded as a regulator of cell proliferation of cardiac fibroblasts in cardiovascular diseases.

Figure 1

(a) The lncRNA-HCG18 expression was detected by PCR. (b) DNA electrophoresis map of lncRNA-HCG18 genes and their fragments identified by enzyme cutting after insertion into the expression vector. (c) LncRNA-HCG18 gene sequencing. (d) pcDNA3.1-flag-sequencing lncRNA-HCG18 cloning.

Figure 2

Silent expressions detection of lncRNA-HCG18 mRNA was evaluated by qRT-PCR. ^∗∗∗P < 0.001.

Figure 3

The hsa-miR-133a mRNA expressions of vecHCG18 and siHCG18 treated-cardiac fibroblasts were detected by qRT-PCR. ^∗∗∗P < 0.001.

Figure 4

The ERK1/2, p-ERK1/2, and EGFR protein levels of vecHCG18 and siHCG18 treated-cardiac fibroblasts were evaluated by western blot. ^∗∗∗P < 0.001.

Figure 5

Luciferase reporter gene system confirms that lncRNA-HCG18 regulates EGFR expression by inhibiting miR-133a-regulated target gene function. ^∗∗P < 0.01, ^∗∗∗P < 0.001.

Figure 6

RIP experiments verify the interaction between lncRNA-HCG18 and hsa-miR-133a.

Figure 7

Cell viability was evaluated using the CCK-8 assay. ^∗P < 0.05.