Hmgcs2 is the hub gene in diabetic cardiomyopathy and is negatively regulated by Hmgcs2, promoting high glucose-induced cardiomyocyte injury

Abstract

Background: Diabetic cardiomyopathy (DCM) represents a major cause of heart failure and a large medical burden worldwide. This study screened the potentially regulatory targets of DCM and analyzed their roles in high glucose (HG)-induced cardiomyocyte injury.

Methods: Through GEO database, we obtained rat DCM expression chips and screened differentially expressed genes. Rat cardiomyocytes (H9C2) were induced with HG. 3-hydroxy-3-methylglutarylcoenzyme A synthase 2 (Hmgcs2) and microRNA (miR)-363-5p expression patterns in cells were measured by real-time quantitative polymerase chain reaction or Western blot assay, with the dual-luciferase assay to analyze their binding relationship. Then, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, lactate dehydrogenase assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, enzyme-linked immunosorbent assay, and various assay kits were applied to evaluate cell viability, cytotoxicity, apoptosis, inflammation responses, and oxidative burden.

Results: Hmgcs2 was the vital hub gene in DCM. Hmgcs2 was upregulated in HG-induced cardiomyocytes. Hmgcs2 downregulation increased cell viability, decreased TUNEL-positive cell number, reduced HG-induced inflammation and oxidative stress. miR-363-5p is the upstream miRNA of Hmgcs2. miR-363-5p overexpression attenuated HG-induced cell injury.

Conclusions: Hmgcs2 had the most critical regulatory role in DCM. We for the first time reported that miR-363-5p inhibited Hmgcs2 expression, thereby alleviating HG-induced cardiomyocyte injury.

Keywords: GEO database chip; diabetic cardiomyopathy; high glucose; hmgcs2; miR-363-5p.

Figure 1

Identification of differentially expressed genes in GSE6880 and GSE5606. (A, B) Heat maps of differentially expressed genes in GSE6880 and GSE5606, the x‐coordinate represents sample number, tree diagram, and the upper right bars represent color gradation. (C, D) Intersections of upregulated and downregulated DEGs in two chips. (E) Map of candidate gene interaction network, each ellipse represents a gene, and lines between genes indicate interactions between genes. (F) The top 10 genes with the highest degree value in gene interaction network map, the x‐coordinate represents degree value, the greater degree value of the gene indicates it has more interacting genes and it is more core.

Figure 2

Hmgcs2 downregulation inhibits HG‐induced cell injury. H9C2 cells were transfected with Hmgcs2 siRNA (si‐Hmgcs2), with cells transfected with si‐NC as the negative control, followed by high glucose induction. (A) Cell viability was examined by MTT assay. (B) LDH release of each cell group. (C) Cell apoptosis was assessed by TUNEL assay (scale bar = 50 μm). (D) mRNA levels of Hmgcs2 were determined by RT‐qPCR. (E) Expression levels of Hmgcs2 were determined by Western blot assay. Cell experiments were performed in triplicate. N = 3. Data in panel D (left) were analyzed by the t test, and data in panels A–E were analyzed by one‐way ANOVA, followed by Tukey's multiple comparison test. **p < .01.

Figure 3

Hmgcs2 downregulation inhibits HG‐induced cell inflammation and oxidative stress. (A–C) Contents of TNF‐α, IL‐6, and IL‐1β were determined by ELISA. (D) ROS levels of each cell group; E: MDA levels of each cell group. (F) SOD levels of each cell group. N = 3. Data in panels A–F were analyzed by one‐way ANOVA, followed by Tukey's multiple comparisons test. **p < .01.

Figure 4

miR‐363‐5p is the upstream miRNA of Hmgcs2. (A) The prediction and intersection of upstream miRNAs of Hmgcs2. (B) Prediction of the binding site of Hmgcs2 and miR‐363‐5p. (C) The targeted binding of Hmgcs2 to miR‐363‐5p was tested by the dual‐luciferase assay. (D) mRNA levels of miR‐363‐5p were determined by RT‐qPCR. (E, F) Expression levels of Hmgcs2 after miR‐363‐5p upregulation were determined by RT‐qPCR and Western blot assay. N = 3. Data in panels D, E, and F were analyzed by the t test, and data in panel C were analyzed by one‐way ANOVA, followed by Tukey's multiple comparison test. *p < .05, **p < .01.

Figure 5

miR‐363‐5p downregulation attenuates HG‐induced cell injury. H9C2 cells were transfected with miR‐363‐5p mimic (miR‐mimic), with cells transfected with miR‐NC as the negative control, followed by HG induction. (A, B) Expression levels of Hmgcs2 were determined by RT‐qPCR and Western blot assay. (C) Cell viability was tested by MTT assay. (D) LDH release of each cell group. (E) Cell apoptosis was evaluated by TUNEL assay (scale bar = 50 μm). (F) Contents of TNF‐α, IL‐6, and IL‐1β were determined by ELISA; G: ROS levels of each cell group. (H) MDA levels of each cell group. (I) SOD levels of each cell group. N = 3. Data in panels A–I were analyzed by one‐way ANOVA, followed by Tukey's multiple comparison test. *p < .05, **p < .01.