Mechanism of KDM5C-Mediated H3K4me3 Demethylation of HOXC-AS3 in the Proliferation of Colorectal Cancer Cells

Abstract

Colorectal cancer (CRC) ranks as the third most prevalent malignancy and the second leading cause of cancer-related mortality worldwide. This study explored the role of lysine-specific histone demethylase 5C (KDM5C) in CRC progression. Expression levels of KDM5C, HOXC-AS3, and discs large MAGUK scaffold protein 4 (DLG4) were evaluated. Following the KDM5C knockdown, cell proliferation assays were conducted. The recruitment of KDM5C and histone H3 lysine 4 tri-methylation (H3K4me3) to the HOXC-AS3 promoter region was investigated. Furthermore, the subcellular distribution of HOXC-AS3 was assessed using nuclear-cytoplasmic fractionation and RNA fluorescence in situ hybridization (RNA-FISH). The interactions between HOXC-AS3, YTH domain-containing protein 1 (YTHDC1), and DLG4 were detected. The stability of DLG4 mRNA was evaluated, and the functional roles of HOXC-AS3 and DLG4 in CRC cells were examined through combined experimental analyses. KDM5C expression was elevated in CRC cells, whereas HOXC-AS3 and DLG4 levels were notably reduced. Silencing KDM5C resulted in suppressed cell proliferation. Mechanistically, KDM5C inhibited HOXC-AS3 expression by demethylating H3K4me3 at its promoter. HOXC-AS3 promoted DLG4 mRNA stability by recruiting the RNA-binding protein YTHDC1. Combined experimental results indicated that overexpression of HOXC-AS3 or DLG4 reduced the inhibitory effect of KDM5C downregulation on CRC cells. In conclusion, KDM5C promotes CRC cell proliferation by demethylating H3K4me3, repressing HOXC-AS3 expression. The reduced HOXC-AS3 levels impair the recruitment of YTHDC1, leading to decreased DLG4 expression.

Keywords: DLG4; HOXC‐AS3; KDM5C; YTHDC1; colorectal cancer.

FIGURE 1

Overexpression of KDM5C in CRC, inhibition of CRC cell proliferation through KDM5C downregulation. (A, B) RT‐qPCR and Western blot were used to detect the expression of KDM5C in different cells. KDM5C siRNA (si‐KDM5C) was transfected into HCT116 and SW480 cells, with transfection of NC siRNA (si‐NC) as a negative control. (C, D) RT‐qPCR and Western blot were used to detect KDM5C expression in cells. (E) Cell proliferation was assessed using CCK‐8 assay. (F) Colony formation assay was performed to assess cell proliferation. The experiments were independently repeated three times, and the data are presented as the mean ± standard deviation. Multiple group comparisons in panel A–B were analyzed using one‐way ANOVA. In comparison, multiple group comparisons in panels C–F were analyzed using two‐way ANOVA, followed by Tukey's multiple comparisons test. **p < 0.01.

FIGURE 2

Suppresses of HOXC‐AS3 expression through KDM5C‐mediated H3K4me3 demethylation. (A) The enrichment of KDM5C and H3K4me3 on the HOXC‐AS3 promoter was analyzed by ChIP. (B, C) RT‐qPCR was used to detect HOXC‐AS3 expression in cells. The experiments were independently repeated three times, and the data are presented as the mean ± standard deviation. Multiple group comparisons in panel B were analyzed using one‐way ANOVA. Multiple group comparisons in panels A and C were analyzed using two‐way ANOVA, followed by Tukey's multiple comparisons test. **p < 0.01.

FIGURE 3

Alleviation of the inhibitory effect of KDM5C downregulation on CRC cell proliferation through downregulation of HOXC‐AS3. HOXC‐AS3 siRNA (si‐HOXC‐AS3) was transfected into HCT116 and SW480 cells, with transfection of NC siRNA (si‐NC) as a negative control. (A) RT‐qPCR was used to detect HOXC‐AS3 expression in cells. (B) Cell proliferation was assessed using CCK‐8 assay. (C) Colony formation assay was performed to assess cell proliferation. The experiments were independently repeated three times, and the data are presented as the mean ± standard deviation. Multiple group comparisons in panels A–C were analyzed using two‐way ANOVA, followed by Tukey's multiple comparisons test. **p < 0.01.

FIGURE 4

Recruitment of YTHDC1 by HOXC‐AS3 and promotion of DLG4 expression. (A, B) Subcellular fractionation and RNA FISH were performed to analyze the subcellular localization of HOXC‐AS3. (C) Multiple databases were used to predict proteins that can bind to HOXC‐AS3, and the intersection was taken. (D, E) RIP and RNA pull‐down were used to analyze the binding of HOXC‐AS3 with YTHDC1. (F) Interaction of YTHDC1 with DLG4 mRNA was predicted. (G) RIP was performed to analyze the binding of YTHDC1 to DLG4 mRNA. (H–K) RT‐qPCR and Western blot were used to detect the DLG4 expression in different cells. (L, M) RT‐qPCR and Western blot were used to detect the transfection efficiency of YTHDC1 in cells; (N) After treatment with actinomycin D, RT‐qPCR was used to detect DLG4 expression in cells. The experiments were independently repeated three times, and the data are presented as the mean ± standard deviation. Multiple group comparisons in panels H–I were analyzed using one‐way ANOVA. In comparison, multiple group comparisons in panels D, G, and J–N were analyzed using two‐way ANOVA, followed by Tukey's multiple comparisons test. *p < 0.05, **p < 0.01.

FIGURE 5

Alleviation of the inhibitory effect of KDM5C downregulation on CRC cell proliferation through downregulation of DLG4. DLG4 siRNA (si‐DLG4) was transfected into HCT116 and SW480 cells, with transfection of NC siRNA (si‐NC) as a negative control. (A, B) RT‐qPCR and Western blot were used to detect the expression of DLG4 in cells. (C) Cell proliferation was assessed using CCK‐8 assay. (D) Colony formation assay was performed to assess cell proliferation. The experiments were independently repeated three times, and the data are presented as the mean ± standard deviation. Multiple group comparisons in panels A–D were analyzed using two‐way ANOVA, followed by Tukey's multiple comparisons test. **p < 0.01.

FIGURE 6

Mechanism of KDM5C in CRC cell proliferation. KDM5C‐mediated demethylation of H3K4me3 inhibits HOXC‐AS3 expression, reducing the recruitment of YTHDC1 by HOXC‐AS3, therefore decreasing DLG4 expression and ultimately promoting CRC cell proliferation.