KLF9 aggravates the cardiomyocyte hypertrophy in hypertrophic obstructive cardiomyopathy through the lncRNA UCA1/p27 axis

Abstract

Cardiac hypertrophy refers to an abnormal increase in the thickness of the heart muscle. Our study explores the role of Krüppel-like factor 9 (KLF9) in hypertrophic obstructive cardiomyopathy (HOCM)-induced cardiomyocyte hypertrophy, providing new targets for the treatment of HOCM. Cardiomyocytes were treated with isoproterenol (ISO). The levels of natriuretic peptide B (BNP)/natriuretic peptide A (ANP)/KLF9/long non-coding RNA urothelial carcinoma-associated 1 (lncRNA UCA1)/p27 were measured. Cell surface area and protein/DNA ratio were tested. The binding between KLF9 and the lncRNA UCA1 promoter and between zeste homologue 2 (EZH2) and lncRNA UCA1 was verified. The enrichment of histone H3 lysine 27 tri-methylation (H3K27me3) and EZH2 on the p27 promoter was analysed. ISO treatment increased KLF9 and lncRNA UCA1 expression and decreased p27 expression in cardiomyocytes. KLF9 knockdown inhibited ISO-induced cardiomyocyte hypertrophy, reduced ANP and BNP expression, and alleviated cardiomyocyte damage. KLF9 activated lncRNA UCA1 expression. LncRNA UCA1 recruited EZH2 to the p27 promoter region, increasing the enrichment of H3K27me3, thereby epigenetically suppressing p27 expression. LncRNA UCA1 overexpression or p27 downregulation reduced the protective effect of KLF9 downregulation on cardiomyocyte hypertrophy. In conclusion, KLF9 activates lncRNA UCA1 expression, and lncRNA UCA1 epigenetically suppresses p27 expression, thereby exacerbating cardiomyocyte hypertrophy in HOCM.

Keywords: EZH2; HOCM; KLF9; LncRNA UCA1; cardiomyocyte hypertrophy.

FIGURE 1

Downregulation of KLF9 alleviates cardiomyocyte hypertrophy. AC16 cells were induced with ISO to simulate HOCM. Additionally, KLF9 siRNA (si‐KLF9) was transfected into the cells, with si‐NC transfected as a negative control. (A, B) The expression of KLF9 in the cells was detected using RT‐qPCR and Western blot assay. (C) The mRNA levels of hypertrophy‐related genes ANP and BNP in the cells were measured using RT‐qPCR. (D) Cell surface area was assessed using crystal violet staining. (E) The protein/DNA ratio in the cells was determined. The independent experiments were repeated three times, and the data were presented as mean ± standard deviation. Comparisons among multiple groups in panels (A, B) and (D, E) were analysed by one‐way ANOVA, while two‐way ANOVA was used for panel C. Tukey's multiple comparisons test was used for post hoc analysis. *p < .05, **p < .01.

FIGURE 2

KLF9 is enriched on the promoter of lncRNA UCA1 to activate lncRNA UCA1 expression. (A) The binding sites between KLF9 and the lncRNA UCA1 promoter were predicted using the JASPAR database. (B) The enrichment of KLF9 in the lncRNA UCA1 promoter region was assessed by ChIP analysis. (C) Dual‐luciferase reporter assay was conducted to analyse the binding of KLF9 to the lncRNA UCA1 promoter. (D) The expression of lncRNA UCA1 in the cells was measured using RT‐qPCR. The independent experiments were repeated three times, and the data were presented as mean ± standard deviation. Comparisons among multiple groups in panel D were analysed by one‐way ANOVA, while two‐way ANOVA was used for panels (B, C). Tukey's multiple comparisons test was used for post hoc analysis. **p < .01.

FIGURE 3

Overexpression of lncRNA UCA1 alleviates the protective effect of KLF9 downregulation on cardiomyocyte hypertrophy. AC16 cells were induced with ISO to simulate HOCM. Additionally, UCA1 pcDNA3.1 (oe‐UCA1) was transfected into the cells, with oe‐NC transfected as a negative control. (A) The expression of lncRNA UCA1 in the cells was measured using RT‐qPCR. (B) The mRNA levels of hypertrophy‐related genes ANP and BNP in the cells were assessed using RT‐qPCR. (C) The protein/DNA ratio in the cells was determined. (D) Cell surface area was evaluated using crystal violet staining. The independent experiments were repeated three times, and the data were presented as mean ± standard deviation. Comparisons between two groups in panel A were analysed by t test, while comparisons among multiple groups in panels (C, D) were analysed by one‐way ANOVA, and two‐way ANOVA was used for panel B. Tukey's multiple comparisons test was used for post hoc analysis. **p < .01.

FIGURE 4

LncRNA UCA1 recruits EZH2 to the promoter region of p27, increasing the enrichment of H3K27me3 and epigenetically suppressing p27 expression. (A) Subcellular localization of UCA1 was examined using nuclear‐cytoplasmic fractionation assay. (B, C) The expression of p27 in the cells was measured using RT‐qPCR and Western blot. (D) RIP analysis was performed to investigate the binding of UCA1 and EZH2. EZH2 siRNA (si‐EZH2) was transfected into the cells, with si‐NC transfected as a negative control. (E, F) The expression of EZH2 and p27 in the cells was assessed using RT‐qPCR and Western blot assay. (G) ChIP analysis was conducted to evaluate the enrichment of EZH2 and H3K27me3 in the p27 promoter region. The independent experiments were repeated three times, and the data were presented as mean ± standard deviation. Comparisons between two groups in panel D were analysed by t‐test, while comparisons among multiple groups in panels (B, C) were analysed by one‐way ANOVA, and two‐way ANOVA was used for panels (E–G). Tukey's multiple comparisons test was used for post hoc analysis. **p < .01.

FIGURE 5

Downregulation of p27 attenuates the protective effect of KLF9 downregulation on cardiomyocyte hypertrophy. AC16 cells were induced with ISO to simulate HOCM. Additionally, p27 siRNA (si‐p27) was transfected into the cells, with si‐NC transfected as a negative control. (A, B) The expression of p27 in the cells was measured using RT‐qPCR and Western blot assay. (C) The mRNA levels of hypertrophy‐related genes ANP and BNP in the cells were assessed using RT‐qPCR. (D) Cell surface area was evaluated using crystal violet staining. (E) The protein/DNA ratio in the cells was determined. The independent experiments were repeated three times, and the data were presented as mean ± standard deviation. Comparisons among multiple groups in panels (A, B) and (D, E) were analysed by one‐way ANOVA, while two‐way ANOVA was used for panel C. Tukey's multiple comparisons test was used for post hoc analysis. **p < .01.