HDAC 1/4-mediated silencing of microRNA-200b promotes chemoresistance in human lung adenocarcinoma cells

Abstract

Chemoresistance is one of the most significant obstacles in lung adenocarcinoma (LAD) treatment, and this process involves genetic and epigenetic dysregulation of chemoresistance-related genes. Previously, we have shown that restoration of microRNA (miR)-200b significantly reverses chemoresistance of human LAD cells by targeting E2F3. However, the molecular mechanisms involved in the silencing of miR-200b are still unclear. Here we showed that histone deacetylase (HDAC) inhibitors could restore the expression of miR-200b and reverse chemoresistant phenotypes of docetaxel-resistant LAD cells. HDAC1/4 repression significantly increased miR-200b expression by upregulating histone-H3 acetylation level at the two miR-200b promoters partially via a Sp1-dependent pathway. Furthermore, silencing of HDAC1/4 suppressed cell proliferation, promoted cell apoptosis, induced G2/M cell cycle arrest and ultimately reversed in vitro and in vivo chemoresistance of docetaxel-resistant LAD cells, at least partially in a miR-200b-dependent manner. HDAC1/4 suppression-induced rescue of miR-200b contributed to downregulation of E2F3, survivin and Aurora-A, and upregulation of cleaved-caspase-3. HDAC1/4 levels in docetaxel-insensitive human LAD tissues, inversely correlated with miR-200b, were upregulated compared with docetaxel-sensitive tissues. Taken together, our findings suggest that the HDAC1/4/Sp1/miR-200b/E2F3 pathway is responsible for chemoresistance of docetaxel-resistant LAD cells.

Figure 1. Histone deacetylase inhibitors (Trichostatin A, TSA, and valproic acid, VPA) elevate the expression of miR-200b and reverse chemoresistance of docetaxel-resistant LAD cells

(A) qRT-PCR detection of relative miR-200b expression in docetaxel-resistant LAD cells (SPC-A1/DTX and H1299/DTX) and their parental LAD cells (SPC-A1 and H1299). Data were normalized to U6 RNA. (B) qRT-PCR detection of relative miR-200b expression in SPCA-1/DTX and H1299/DTX cells after treatment with DNA methyltransferase inhibitor (5-aza-2'-deoxycytidine, 5-aza-dC). Data were normalized to U6 RNA and determined relative to 0 µmol/L group. (C) qRT-PCR detection of relative miR-200b expression in SPCA-1/DTX and H1299/DTX cells after treatment with histone deacetylase inhibitor (TSA or VPA). Data were normalized to U6 RNA and determined relative to 0 µmol/L group. (D) MTT analysis of the IC50 values for docetaxel or paclitaxel in SPC-A1/DTX and H1299/DTX cells after treatment with TSA or VPA. (E) Western blot analysis of acetyl-histone H3 protein expression in SPC-A1/DTX and H1299/DTX cells after treatment with TSA or VPA. Data were the means ± standard error of at least three independent experiments. *P < 0.05, **P < 0.01; ^#P < 0.05, ^##P < 0.01 vs 0 nmol/L or 0 mmol/L group.

Figure 2. Effects of histone deacetylases (HDACs) on miR-200b expression

(A) Relative levels of miR-200b as measured by qRT-PCR after transfection of the indicated siRNA-HDACs or siRNA-NC into H1299/DTX or SPC-A1/DTX cells. Data were normalized to U6 RNA. (B) Relative levels of HDAC1 or HDAC4 mRNA as measured by qRT-PCR in parental or docetaxel-resistant LAD cells. Data were normalized to GAPDH. (C) Western blot detection of HDAC1 or HDAC4 protein expression in parental or docetaxel-resistant LAD cells. β-actin was used as an internal control. Data were the means ± standard error of at least three independent experiments. ^##P < 0.01; *P < 0.05, **P < 0.01 vs control.

Figure 3. HDAC1/4 decreases miR-200b promoter activities

(A) Schematic representation of the human miR-200b promoters with the putative Sp1-binding sites and the sequence of the point mutation. (B) Luciferase activity of docetaxel-resistant LAD cells cotransfected with Renilla and either wild-type miR-200b promoter or empty vector firefly reporter construct. Data were normalized to Renilla luciferase activity and determined relative to empty vector promoter activity. (C) Luciferase activity after transfection of sh-HDAC1#2, sh-HDAC4#3 or shRNA-control vector into SPC-A1/DTX cells co-transfected with Renilla and either wild-type miR-200b promoter, Sp1-binding mutant or empty vector firefly promoter reporter construct. Data were normalized to Renilla luciferase activity and determined relative to empty vector promoter activity. (D) qRT-PCR detection of relative miR-200b expression in H1299/DTX or SPC-A1/DTX cells transfected with siRNA-HDAC1, siRNA-HDAC4 or siRNA-NC or co-transfected with siRNA-HDAC1 or HDAC4 and siRNA-Sp1. Data were normalized to U6 RNA and determined relative to control group. Data were the means ± standard error of at least three independent experiments. ^#P < 0.05, ^##P < 0.01, *P < 0.05, **P < 0.01 vs control.

Figure 4. HDAC1/4 repression upregulates histone-H3 acetylation level at miR-200b promoters partially through the Sp1-dependent pathway

(A) HDAC1/4 co-immunoprecipitates with Sp1 in vivo in SPC-A1/DTX cells. The levels of HDAC1/4 and Sp1 proteins were evaluated by western blot. (B) Sp1 and (C) HDAC1/4 binds to the miR-200b promoters in vivo. ChIP assays were performed in SPC-A1/DTX cells with antibodies against Sp1, HDAC1/4 or IgG control. Immunoprecipitated DNA was amplified by qRT-PCR with primers designed to amplify the sequences containing the putative Sp1-binding sites. Data are shown relative to qRT-PCR products amplified with input DNA before immunoprecipitation. p21 was used as a positive control. (D) HDAC1/4 decreases the histone-H3 acetylation level at the miR-200b promoters. ChIP assays were performed with antibody against acetyl-histone H3 in SPC-A1/DTX cells transfected with sh-control, sh-HDAC1#2 or sh-HDAC4#3. Immunoprecipitated DNA was amplified by qRT-PCR with primers designed to amplify the sequences containing the putative Sp1-binding sites. Data was normalized to qRT-PCR products amplified with input DNA before immunoprecipitation. Data were the means ± standard error of at least three independent experiments. *P < 0.05, **P < 0.01.

Figure 5. Inhibition of HDAC1/4 reverses chemoresistance of docetaxel-resistant LAD cells partially in a miR-200b-dependent manner

(A) The IC50 values for docetaxel or paclitaxel as determined by MTT assay after transfection of sh-control, sh-HDAC1#2 or sh-HDAC4#3 vectors into docetaxel-resistant LAD cells without (or with) previous transfection with miR-200b inhibitor. Upregulation of HDAC1 and HDAC4 reversed the chemoresistance of docetaxel-resistant LAD cells. (B) The colony formation assay was performed as described in Methods. The number of colonies was counted and compared. (C) Flow cytometric analysis of apoptosis after transfection of sh-control, sh-HDAC1#2 or sh-HDAC4#3 vector into H1299/DTX or SPC-A1/DTX cells, without (or with) previous transfection of miR-200b inhibitor. (D) Protein levels of cleaved caspase-3 as measured by western blot after transfection of sh-control, sh-HDAC1 or sh-HDAC4 vectors into H1299/DTX or SPC-A1/DTX cells, without (or with) previous transfection of miR-200b inhibitor. Total caspase-3 was used as an internal control. (E) Flow cytometry analysis of cell cycle in H1299/DTX or SPC-A1/DTX cells transfection with sh-control, sh-HDAC4#3 or combined with DTX treatment or co-transfected with miR-200b inhibitor. The data were the means ± standard error of at least three independent experiments. *P < 0.05, **P < 0.01; ^#P < 0.05 vs control group.

Figure 6. Downregulation of HDAC1/4 suppresses expression of E2F3, survivin and Aurora-A partially through the miR-200b-dependent pathway

(A) Relative mRNA expression of E2F3, survivin and Aurora-A as measured by qRT-PCR after transfection of sh-control, sh-HDAC1#2 or sh-HDAC4#3 vectors into H1299/DTX and SPC-A1/DTX cells, without (or with) previous transfection of miR-200b inhibitor. Data were normalized by GAPDH and determined relative to sh-control vector group. (B) Protein levels of E2F3, survivin and Aurora-A as measured by western blot after transfection of shRNA-control, shRNA-HDAC1#2 or shRNA-HDAC4#3 vectors into H1299/DTX or SPC-A1/DTX cells, without (or with) previous transfection of miR-200b inhibitor. β-actin was used as an internal control. Data were the means ± standard error of at least three independent experiments. *P < 0.05, **P < 0.01.

Figure 7. HDAC1/4 involvement in the regulation of survivin or Aurora-A through the miR-200b/E2F3-dependent pathway (SPC-A1/DTX cells)

(A) Luciferase activity assays revealed that downregulation of HDAC1/4 significantly elevated Aurora-A (left) or survivin (right) promoter activity partially in a miR-200b/E2F3-dependent manner. Data were normalized by Renilla luciferase activity and determined relative to empty vector group. (B) Downregulation of HDAC1 and HDAC4 decreases the amount of E2F3 binding to the survivin promoter, while suppression of HDAC4 reduces the amount of E2F3 binding to the Aurora-A promoter. ChIP assays were performed with E2F3 antibody in SPC-A1/DTX cells transfected with sh-control and either sh-HDAC1#2 or sh-HDAC4#3. Immunoprecipitated DNA was amplified by qRT-PCR with primers designed to amplify the sequences containing the putative E2F3-binding sites. Data were normalized to qRT-PCR products amplified with input DNA before immunoprecipitation. Data were the means ± standard error of at least three independent experiments. *P < 0.05, **P < 0.01.

Figure 8. Suppression of HDAC1/4 or upregulation of miR-200b reverses in vivo chemoresistance of docetaxel-resistant LAD cells (H1299/DTX cells)

(A) Growth of tumors in nude mice subcutaneously transplanted with H1299/DTX cells stably transfected with sh-control, sh-HDAC1#2, sh-HDAC4#3 or miR-200b vectors (six mice in each group). Representative photographs of tumors were obtained 6 weeks after inoculation. Data were the means ± standard error. *P < 0.05. (B) The relative mRNA level of miR-200b as measured by qRT-PCR in tumors 6 weeks after inoculation. Data were normalized to U6 RNA and determined relative to control group Data were the means ± standard error. ^##P < 0.01 vs. control group. (C) HDAC1, HDAC4, E2F3, survivin and Aurora-A protein levels as measured by western blot in tumors 6 weeks after inoculation. GAPDH was used as an internal control.

Figure 9. HDAC1/4, inversely correlated with miR-200b, is upregulated in docetaxel-insensitive human LAD cases compared with docetaxel-sensitive LAD cases

qRT-PCR was performed to detect the relative mRNA level of HDAC1 (A) and HDAC4 (B) in docetaxel-sensitive (n=31) and insensitive (n=37) LAD tissues. HDAC1 (C) or HDAC4 (D) and miR-200b mRNA levels were inversely correlated in 68 LAD tissues as determined by linear regression analysis. Spearman rank test rho and P values (2-tailed) were shown. The HDAC1/4 level was normalized to GAPDH and miR-200b was normalized to U6. **P < 0.01.