The efficacy of sorafenib against hepatocellular carcinoma is enhanced by 5-aza-mediated inhibition of ID1 promoter methylation

Abstract

Sorafenib resistance greatly restricts its clinical application in patients with hepatocellular carcinoma (HCC). Numerous studies have reported that ID1 exerts a crucial effect in cancer initiation and development. Our previous research revealed an inhibitory role of ID1 in sorafenib resistance. However, the upstream regulatory mechanism of ID1 expression is unclear. Here, we discovered that ID1 expression is negatively correlated with promoter methylation, which is regulated by DNMT3B. Knockdown of DNMT3B significantly inhibited ID1 methylation status and resulted in an increase of ID1 expression. The demethylating agent 5-aza-2'-deoxycytidine (5-aza) remarkably upregulated ID1 expression. The combination of 5-aza with sorafenib showed a synergistic effect on the inhibition of cell viability.

Keywords: DNA methylation; ID1; drug resistance; hepatocellular carcinoma; sorafenib.

Fig. 1

ID1 is downregulated in HCC. (A) The expression levels of ID1 in breast invasive cancer (BRCA), lung adenocarcinoma (LUAD), prostate adenocarcinoma (PRAD), and liver hepatocellular carcinoma (LIHC) were downloaded from UALCAN database. The box‐whisker plots present interquartile ranges (IQRs), including minimum, 1st quartile, median, 3rd quartile, and maximum values. Welch's t‐test estimated the significance of differences in expression levels between normal and primary tumors. ***P < 0.001, vs. the Normal group; (B) Western blot experiment was used to observe the different expression levels of ID1 in HepG2, SK‐Hep 1, and Hep3B. (C) The immunoblot band intensities were analyzed by the image j software. The ratio of the ID1 band intensity over the GAPDH band intensity in HepG2 was arbitrarily set at 1.0. The experiments were independently triplicated. Data are shown as mean ± SEM. Statistical differences between the two groups were examined by Student's t‐test. **P < 0.01, ***P < 0.001; (D) RT‐qPCR assay was conducted to compare the mRNA levels of ID1 in HepG2, SK‐Hep 1, and Hep3B. All reactions were performed in triplicate for each sample. Data are shown as mean ± SEM. Statistical differences between the two groups were examined by Student's t‐test. *P < 0.05, **P < 0.01.

Fig. 2

ID1 expression is correlated with promoter methylation. (A) Online software (http://www.urogene.org/methprimer/) prediction of CpG islands in the promoter region (−1500/+1) of ID1. The light blue areas on the map indicate the potential CpG islands. (B) MSP was used to analyze DNA methylation in the ID1 promoter region (left panel), M: Methylation; U: Unmethylation; The experiments were independently triplicated. Band intensity from MSP results was quantified by image j software. The band intensity in un‐methylation group was arbitrarily set at 1.0, data are shown as mean ± SEM. Statistical differences between the two groups were examined by Student's t‐test. *P < 0.05, **P < 0.01; (C–D) Effect of 5‐aza on the expression of ID1 at mRNA and protein levels were assessed by RT‐qPCR (C) and western blot (D), respectively. The experiments were independently triplicated, data are shown as mean ± SEM. Statistical differences between the two groups were examined by Student's t‐test. *P < 0.05, **P < 0.01, compared with the control group; (E) Three‐dimensional (3D) representation of 5‐aza in complex with ID1 with the highest binding energy of −5.2 kcal·mol⁻¹. ID1 dimeric interface is shown in green and orange ribbon. Molecular docking was performed using autodock vina v.1.1.2, and graphics were generated with pymol v2.4.1 software (San Carlos, California, USA).

Fig. 3

DNMT3B regulates ID1 expression. (A) Oncoporint from cbioportal showing genetic alterations in ID1, DNMT1, DNMT3A, and DNMT3B in HCC from TCGA (n = 372) (upper panel); The heatmap shows the mRNA expression levels [z‐score normalized log₂ (FPKM) values] and was generated through cBioPortal (low panel). (B) Correlation of ID1 mRNA expression with DNMT1, DNMT3A, and DNMT3B mRNA expression in HCC were analyzed through cBioPortal, the regression line (red line) depicts the linear association between the two gene expression levels. (C) The protein expression of ID1, DNMT1, DNMT3A, and DNMT3B in Hep3B cells was analyzed after transfection with small interfering RNA (siRNA) targeting DNMT1, DNMT3A, and DNMT3B, respectively. Representative western blotting bands (left panel) and the corresponding densitometric analysis (right panel) are shown. The ratio of the ID1/DNMT1/DNMT3A/DNMT3B band intensity over the GAPDH band intensity in NC group was arbitrarily set at 1.0. The experiments were independently triplicated, data are shown as mean ± SEM. Statistical differences between the two groups were examined by Student's t‐test. *P < 0.05, **P < 0.01, compared with the NC group. (D) MSP was performed to measure the methylation level of ID1 in Hep3B cells after transfection with siRNA targeting DNMT1, DNMT3A and DNMT3B, respectively. Bar chart showing the relative promoter methylation level of ID1 was significantly lower in the si‐DNMT3B group. The experiments were independently triplicated, data are shown as mean ± SEM. Statistical differences between the two groups were examined by Student's t‐test. **P < 0.01, compared with the NC group.

Fig. 4

Synergistic effect of 5‐aza on the cytotoxicity of sorafenib in HCC. HepG2 (A) or Hep3B (B) cells were incubated with sorafenib alone or combined with 4 μm of 5‐aza for 24 h. MTT assay was employed to observe the cell viability. The experiments were independently triplicated. Data are shown as mean ± SEM. Statistical differences between the two groups were examined by Student's t‐test. *P < 0.05, **P < 0.01, compared with sorafenib alone. The protein levels of ID1 and p‐AKT in HepG2 (C) or Hep3B (D) were detected by western blot. The immunoblot band intensities were analyzed by image j software. The ratio of the ID1 and p‐AKT band intensities over the GAPDH band intensity was arbitrarily set at 1.0. The experiments were independently triplicated, data are shown as mean ± SEM. Statistical differences between the two groups were examined by Student's t‐test. *P < 0.05, **P < 0.01.

Fig. 5

Down‐regulation of sorafenib on the ID1 promoter activity. (A) Continuous truncated ID1 promoter constructs were transfected into cells following sorafenib incubation to determine the relative activity of luciferase. The experiments were independently triplicated, data are shown as mean ± SEM. Statistical differences between the two groups were examined by Student's t‐test. ***P < 0.001; (B) Bar chart showing relative promoter methylation level of ID1 in cells with the presence or absence of sorafenib.