CircHECTD1 promoted MIRI-associated inflammation via inhibiting miR-138-5p and upregulating ROCK2

Abstract

Myocardial ischemia-reperfusion injury (MIRI) was often observed after surgeries, causing a lot of suffering to patients. Inflammation and apoptosis were critical determinants during MIRI. We conveyed experiments to reveal the regulatory functions of circHECTD1 in MIRI development. The Rat MIRI model was established and determined by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. We analyzed cell apoptosis using TUNEL and flow cytometry. Proteins expression was evaluated by western blot. The RNA level was determined by qRT-PCR. Secreted inflammatory factors were analyzed by ELISA assay. To predict the interaction sequences on circHECTD1, miR-138-5p, and ROCK2, bioinformatics analysis was performed. Dual-luciferase assay was used to confirm these interaction sequences. CircHECTD1 and ROCK2 were upregulated in the rat MIRI model, while miR-138-5p was decreased. CircHECTD1 knockdown alleviated H/R-induced inflammation in H9c2 cells. Direct interaction and regulation of circHECTD1/miR-138-5p and miR-138-5p/ROCK2 were confirmed by dual-luciferase assay. CircHECTD1 promoted H/R-induced inflammation and cell apoptosis by inhibiting miR-138-5p. miR-138-5p alleviated H/R-induced inflammation, while ectopic ROCK2 antagonized such effect of miR-138-5p. Our research suggested that the circHECTD1-modulated miR-138-5p suppressing is responsible for ROCK2 activation during H/R-induced inflammatory response, providing a novel insight into MIRI-associated inflammation.

Keywords: ROCK2; circHECTD1; inflammation; ischemia/reperfusion injury; miR-138-5p.

FIGURE 1

Enhanced CircHECTD1 and ROCK2 and decreased miR‐138‐5p were detected in the rat MIRI model. (A) TTC staining of sham and MIRI rat model. Infarction size was analyzed and displayed in columns. (B) TUNEL assay of sham and MIRI rat model. The percentage of apoptosis myocytes was analyzed and displayed in columns. Tissues of the hearts were subjected to assays in C, D, E: (C) CircHECTD1 level was detected by qRT‐PCR. (D) The miR‐138‐5p level was detected by qRT‐PCR. (E) qRT‐PCR analysis of ROCK2 in sham and MIRI rat model. (F) qRT‐PCR of circHECTD1 in control or H/R‐treated H9c2 cells. (G) MiR‐138‐5p level in H9c2 cells was analyzed using qRT‐PCR. (H) qRT‐PCR analysis of ROCK2 in control or H/R‐treated H9c2 cells. (I) Western blot analysis of ROCK2 in control or H/R‐treated H9c2 cells. Data were analyzed using student's t‐test, *p < 0.05, **p < 0.01.

FIGURE 2

Knockdown of circHECTD1 reduced H/R‐induced inflammatory response of cardiomyocytes. H9c2 cell was treated with H/R and transfected with sh‐NC or sh‐circHECTD1. (A) qRT‐PCR analysis of circHECTD1 in H9c2 cells. (B) qRT‐PCR analysis of miR‐138‐5p and ROCK2. (C) Western blot analysis of ROCK2 expression. (D) Flow cytometry analysis of apoptosis. Apoptosis rates were analyzed and displayed in columns. (E) RNA levels of IL‐6, TNF‐α, and IL‐1β were analyzed by qRT‐PCR. (F) ELISA analysis of IL‐6, TNF‐α, and IL‐1β in H9c2 cells. Data were analyzed using one‐way ANOVA with the Tukey HSD post‐hoc test, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

CircHECTD1 acted as a sponge of miR‐138‐5p. (A) The interaction region of circHECTD1 and miR‐s138‐5p was analyzed by starBase and found that there were complementary binding sites between them. (B) H9c2 cells were transfected with circHECTD1 mutants and miR‐138‐5p mimics, inhibitors, or NC as indicated. A dual luciferase reporter assay was then performed. (C) Cells were harvested by RIP lysis buffer and incubated with human anti‐Ago2 antibody or normal IgG for RIP assay. RNA was detected by qRT‐PR and the relative enrichment level was analyzed. (D) The CircHECTD1 probe was labeled by biotin and incubated with RNA extracted from H9c2 cells. MiR‐138‐5p was detected by qRT‐PCR and the relative enrichment level was analyzed. (E) qRT‐PCR analysis of miR‐138‐5p in H9c2 cells transfected with sh‐circHECTD1 or sh‐NC. Data were analyzed using one‐way ANOVA with the Tukey HSD post‐hoc test, **p < 0.01, ***p < 0.001.

FIGURE 4

CircHECTD1 regulates H/R‐induced inflammation through miR‐138‐5p. H9c2 cells were transfected with sh‐circHECTD1, sh‐NC, miR‐138‐5p inhibitor or NC inhibitor (A) qRT‐PCR analysis of circHECTD1 and miR‐138‐5p as indicated. (B) Apoptosis analysis of cells in H9c2 cells. (C) qRT‐PCR analysis of TNF‐α, IL‐6 and IL‐1β in H9c2 cells. (D) ELISA analysis of TNF‐α, IL‐6, and IL‐1β in H9c2 cells. Data were analyzed using one‐way ANOVA with the Tukey HSD post‐hoc test, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

MiR‐138‐5p interacted with the mRNA of ROCK2 and decreased its protein expression. (A) Interaction region between miR‐138‐5p and ROCK2 was analyzed by starBase. (B) H9c2 cells transfected with ROCK2 mutants and miR‐138‐5p mimics, inhibitors, or NC as indicated. Dual luciferase analysis was performed 48 hours after transfection. (C) qRT‐PCR analysis of miR‐138‐5p in H9c2 cells. (D) qRT‐PCR analysis of ROCK2 mRNA in H9c2 cells transfected with miR‐138‐5p mimics, inhibitor, or NC as indicated. (E) Western blot analysis of ROCK2 in H9c2 cells. (F) qRT‐PCR analysis of ROCK2 mRNA in H9c2 cells. (G) Western blot analysis of ROCK2 in H9c2 cells. Data were analyzed using one‐way ANOVA with the Tukey HSD post‐hoc test, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

miR‐138‐5p attenuated H/R‐induced myocardial inflammatory response through ROCK2. (A) Apoptosis analysis of H9c2 cells transfected with miR‐138‐5p, pcDNA3.1‐ROCK2, or pcDNA3.1‐NC. (B) Expression of IL‐6, TNF‐α, and IL‐1β in H9c2 cells were detected by qRT‐PCR. (C) ELISA analysis of TNF‐α, IL‐6, and IL‐1β in H9c2 cells. Data were analyzed using one‐way ANOVA with the Tukey HSD post‐hoc test, *p < 0.05, **p < 0.01, ***p < 0.001.