Foxo3a-dependent miR-633 regulates chemotherapeutic sensitivity in gastric cancer by targeting Fas-associated death domain

Abstract

The development of chemotherapeutic drugs resistance such as doxorubicin (DOX) and cisplatin (DDP) is the major barrier in gastric cancer therapy. Emerging evidences reveal that microRNAs (miRNAs) contribute to chemosensitivity. In this study, we investigated the role of miR-633, an oncogenic miRNA, in gastric cancer chemoresistance. In gastric cancer tissue and cell lines, miR-633 expression was highly increased and correlated with down regulation of Fas-associated protein with death domain (FADD). Inhibition of miR-633 significantly increased FADD protein level and enhanced DOX/DDP induced apoptosis in vitro. MiR-633 antagomir administration remarkably decreased tumor growth in combination with DOX in vivo, suggesting that miR-633 targets FADD to block gastric cancer cell death. We found that the promoter region of miR-633 contained putative binding sites for forkhead box O 3 (Foxo3a), which can directly repress miR-633 transcription. In addition, we observed that DOX-induced nuclear accumulation of Foxo3a leaded to the suppression of miR-633 transcription. Together, our study revealed that miR-633/FADD axis played a significant role in the chemoresistance and Foxo3a regulated this pathway in gastric cancer. Thus, miR-633 antagomir resensitized gastric cancer cells to chemotherapy drug and had potentially therapeutic implication.

Keywords: FADD; Foxo3a; chemosensitivity; gastric cancer; miR-633.

Figure 1.

FADD loss is associated with high expressions of miR-633 in gastric carcinoma. (a) The predicted binding sequences for the miR-633 within the human FADD 3ʹ-UTR. b and c, FADD protein expression levels in gastric carcinoma tissues (b) and gastric cell lines (c), c = cancer tissues; n = matched adjacent tissues. (d) Immunohistochemistry staining of FADD in paraffin sections of patient tissues, scale bar = 50 μm. (e) normalized miR-633 expression levels in normal and cancer tissues from 26 pairs of gastric cancer patients. (f) qRT-PCR data of miR-633 expression in the gastric cell lines. (g) the correlation of miR-633 level and FADD protein level. Error bars represent S.D. Data are expressed as the mean ± SD of 3 independent experiments. *P < 0.05

Figure 2.

FADD participates in chemotherapy drugs induced apoptosis in gastric tumor cells. (a and b), the mRNA and protein expression levels of FADD in SGC-7901 cells exposed to 2 μM DOX (a) or 30 μM DDP (b) treatment for 24 h. (c), the mRNA expression and protein expression of FADD in SGC-7901 cells after transfected with FADD siRNA (si-FADD) or scramble siRNA (FADD-sc). (d and e), inhibition of FADD by transfecting si-FADD or FADD-sc prevented apoptosis in SGC-7901 treated with 2 μM DOX for 24 h, apoptotic cells were analyses by TUNEL assay (d) and counted (e). f), inhibition of FADD by transfecting si-FADD or FADD-sc prevented apoptosis in SGC-7901 treated with 30 μM DDP for 24 h.

Figure 3.

miR-633 suppresses apoptosis and induced chemoresistance in gastric cancer cells. (a), the expression levels of miR-633 were analyzed by qRT-PCR in SGC-7901 cells exposed to 2 μM doxorubicin (DOX) or 30 μM cisplatin (DDP) treatment for 24 h. (b), real-time PCR in SGC-7901 cells to detect the expression of miR-633 after transfected with miR-633 antagomir (Anta-633) or antagomir control (Anta-NC). (c), TUNEL assay in SGC-7901 cells to detect apoptosis after transfected with Anta-633 or Anta-NC as the indicated time. (d, e, f and g), overexpression of miR-633 by transfecting miR-633 mimics or mimics control (NC) prevented apoptosis in SGC-7901 treated with 2 μM DOX for 24 h, representative images show TUNEL-positive cells (d), scale bar = 100 μm. Apoptotic cells were counted (e), miR-633 expression were detected (f) and cleaved caspase-3 was analyzed by western blot (g). (h, i and j), inhibition of endogenous miR-633 transfected with Anta-633 enhanced apoptosis in SGC-7901 after 24 h 0.2 μM DOX treatment. MiR-633 expression were detected (h), the apoptotic cells were counted after DOX treatment (i), and cleaved caspase-3 was analyzed by western blot (j). Error bars represent S.D. Data are expressed as the mean ± SD of 3 independent experiments.*P < 0.05.

Figure 4.

miR-633 regulates chemotherapy resistance through FADD in gastric tumor cells. (a), the mRNA and protein expression levels of FADD were analyzed by qRT-PCR and western blot in SGC-7901 cells after miR-633 inhibition. (b), overexpression of miR-633 prevented the FADD protein expression increase in SGC-7901 treated with 2 μM DOX for 24 h. (c), inhibition of miR-633 promoted the FADD protein expression in SGC-7901 treated with 0.2 μM DOX 24 h. (d), inhibition of miR-633 enhanced cell death in SGC-7901 in response to 0.2 μM DOX treatment for 36 h, which was reversed by FADD siRNA. (e), luciferase activity detected in HEK-293 transfected with synthesized miR-633 mimics or mimic control, along with human FADD 3ʹUTR luciferase reporter constructs as indicated. (f), detection of FADD 3ʹUTR by qRT-PCR in SGC-7901 cells transfected with Bio-633, Bio-633mut, or Bio-nc by RNA pull-down assay and results were normalized to human GAPDH (n = 3). Error bars represent S.D. Data are expressed as the mean ± SD of 3 independent experiments.*P < 0.05. Mut, mutated; Wt, wild type.

Figure 5.

miR-633 is a transcriptional target of Foxo3a. (a), the promoter region of human miR-633 contains two optimal Foxo3a binding sites (BS1 and BS2). The wild type miR-633 promotors (Wt1 and Wt2) linked to luciferase report gene. The mutations were introduced into the potential binding sites (m-BS1 and m-BS2) as indicated. (b), CHIP analysis was performed in SGC-7901 cells to identify the binding site of Foxo3a in miR-633 promoter treated with 2 μM DOX or 30 μM DDP for 12 h. (c), immunoblotting was performed to analyze the phosphorylated Foxo3a (p-Foxo3a) and total Foxo3a in the cellular fractions of nuclei or cytosol. Proliferating cell nuclear antigen (PCNA) is a nucleic marker. Tubulin is a cytosolic marker. (d), 2 μM DOX induced Foxo3a nuclear relocation from cytoplasm in SGC-7901 cells with 2 μM DOX treatment as the indicated time. Subcellular location was definitude by immunofluorescent staining, cell nuclei were stained by 4,6-diamidino-2-phenylindole (DAPI), scale bar = 10 μm. (e and f), Luciferase activity of reporter constructs harboring wild-type or mutated miR-633 promoter in HEK-293 cells infected with adenoviral Foxo3a as indicated. Foxo3a inhibits miR-633 promotor activity (e), and DOX or DDP induced miR-633 promotor activity reduction was rescued by inhibition of Foxo3a siRNA (f). (g), the miR-633 level and Foxo3a protein expression were analyzed by qRT-PCR and western blot in SGC-7901 cells after infected with adenoviral Foxo3a. Error bars represent S.D. Data are expressed as the mean ± SD of 3 independent experiments.*P < 0.05. Mut, mutated; Wt, wild type, β-gal, β-galactosidase.

Figure 6.

Foxo3a regulates miR-633-induced chemoresistance dependenting on FADD in gastric cancer cells. (a), TUNEL assay in SGC-7901 cells to detect apoptosis after infected with adenoviral Foxo3a. (b and c), inhibition of Foxo3a by transfect with Foxo3a siRNA (si-Foxo3a) or scramble siRNA (Foxo3a-sc) rescued 2 μM DOX induced cell death in SGC-7901. Real-time PCR to detecte miR-633 level, FADD protein expression were detected by western blot (b) and TUNEL assay to detect cell apoptosis in SGC-7901 cells (c). (d and e), overexpression of Foxo3a treated with 0.2 μM DOX for 24 h in SGC-7901 cells, miR-633 level, FADD protein expression (d) and TUNEL assay (e) were detected. (f), the mRNA and protein expression levels of FADD analyzed by qRT-PCR and western blot in SGC-7901 cells after infected with adenoviral Foxo3a. (g), overexpression of Foxo3a elevated FADD protein level in SGC-7901 cells in response to 0.2 μM DOX treatment for 36 h, which reversed by miR-633 mimics. (h), overexpression of Foxo3a increased cell death in SGC-7901 in response to 0.2 μM DOX treatment for 36 h, which was reversed by FADD siRNA. FADD protein level analyzed by western blot. Error bars represent S.D. Data are expressed as the mean ± SD of 3 independent experiments.*P < 0.05.

Figure 7.

Inhibition of miR-633 promotes chemotherapeutic effect in vivo. BALB/c nude mice subcutaneously transplanted with a total of 1 × 10⁷ SGC-7901 cells. When tumors reached 250–300 mm³, intratumorally injected with miR-633 antagomir or antagomir control and intraperitoneally treated with DOX (DOX; 2 mg/kg) or PBS (Control) was given every other day. (a and b), Representative images of xenografts (a, upper), tumor weight (a, bottom) and tumor volume (b) are shown. (c), immunoblot of FADD protein (upper) and miR-633 expression levels (bottom) in generated xenograft tumors were analyzed. (d), TUNEL assays were performed to detect tumor cell apoptosis in xenograft tumor tissues. n = 6 each group. Error bars represent S.D. Data are expressed as the mean ± SD of 3 independent experiments.*P < 0.05 compared with DOX (1 mg/kg) alone.