MiR-1180 promotes apoptotic resistance to human hepatocellular carcinoma via activation of NF-κB signaling pathway - PubMed (original) (raw)
MiR-1180 promotes apoptotic resistance to human hepatocellular carcinoma via activation of NF-κB signaling pathway
Guosheng Tan et al. Sci Rep. 2016.
Abstract
Apoptosis resistance in human hepatocellular carcinoma (HCC) is a significant factor in carcinogenesis. Therefore, understanding the molecular mechanisms involved in apoptosis resistance is crucial for developing anticancer therapies. Importantly, small non-coding microRNAs (miRNAs) have been reported as key biomarkers for detecting tumour onset and progression. In the present study, we demonstrate that miR-1180 is upregulated in HCC. Ectopic expression of miR-1180 has an anti-apoptotic effect in HCC, while miR-1180 inhibition increases cell apoptosis, both in vitro and in vivo. Moreover, our results show that miR-1180 directly targets key inhibitors of the nuclear factor (NF)-κB signaling pathway (i.e., OTUD7B and TNIP2) and the pro-apoptotic Bcl-2 associated death promoter (BAD) protein by post-transcriptional downregulation. Therefore, the anti-apoptotic function of miR-1180 in HCC may occur through NF-κB pathway activation via downregulation of its negative regulators. In conclusion, our study reveals the critical role of miR-1180 during apoptosis resistance in HCC.
Figures
Figure 1. miR-1180 is upregulated in HCC.
(a) The expression of miR-1180 in HCC tissues (T) compared to normal hepatic tissues (N), as measured using a published micro-array-based high-throughput assessment (P < 0.05; NCBI/GEO/GSE36915). (b) qRT-PCR analysis of miR-1180 expression in eight cancerous tissues (T) paired with their adjacent noncancerous hepatic tissues (ANT); Log2 value was used to show the expression difference. (c) qRT-PCR analysis of miR-1180 expression in a panel of hepatocellular carcinoma cell lines compared to normal liver epithelial (THLE3) cells. (d) Kaplan-Meier curves of HCC patients with low- versus high-expression of miR-1180 (n = 75; P = 0.001, log-rank test). The average miR-1180 expression was normalized using U6 expression. Each bar represents the mean ± SD of three independent experiments. *P < 0.05.
Figure 2. Ectopic miR-1180 promotes cell viability and inhibits cisplatin-induced apoptosis of HCC cells.
(a) Ectopic miR-1180 promotes cell viability in HepG2 and Huh7 cell lines compared with a negative control (NC) as measured by the MTS assay. (b) Representative micrographs (left) and quantifications (right) of crystal violet stained cell colonies formed by HepG2 and Huh7 cell lines, 10 days after inoculation. (c) Flow cytometry experiments using Annexin V/PI double staining to determine the percentages of apoptotic cells in HepG2 and Huh7 cell lines. (d) Western blotting analysis of Bcl-2, Bax and cleaved-caspase-3 expression in miR-1180 overexpressing cells compared with a negative control (NC); α-Tubulin is the loading control. Each bar represents the mean ± SD of three independent experiments. *P < 0.05.
Figure 3. Inhibition of miR-1180 suppresses cell viability and promotes cisplatin-induced apoptosis of HCC cells in vitro.
(a) Inhibition of miR-1180 (with the miR-1180-in) decreases cell viability in HepG2 and Huh7 cell lines compared with a negative control (NC) as measured by the MTS assay. (b) Representative micrographs (left) and quantifications (right) of crystal violet stained cell colonies formed by HepG2 and Huh7 cell lines, 10 days after inoculation. (c) Flow cytometry experiments using Annexin V/PI double staining to determine the percentages of apoptotic cells in HepG2 and Huh7 cell lines. (d) Western blotting analysis of Bcl-2, Bax, and cleaved-caspase-3 expression in miR-1180 overexpressing cells compared to control cells; α-Tubulin is the loading control. Each bar represents the mean ± SD of three independent experiments. *P < 0.05.
Figure 4. Inhibition of miR-1180 with a miR-1180 antagonist (AntagomiR-1180) suppresses tumour growth and promotes cisplatin-induced apoptosis of HCC cells in vivo.
(a) Tumour volumes were measured on the indicated days and presented as the mean ± SD. Red arrow indicates the first time point of cisplatin treatment. (b) Images of tumours from all mice in each group. (c) Tumour weights (mg) in each group. *P < 0.05.
Figure 5. MiR-1180 directly targets the 3′-UTR of OTUD7B, TNIP2, and BAD mRNA.
(a) Schematic representation of the mature miR-1180 sequence, the miR-1180 target site in the 3′-UTR of OTUD7B, TNIP2, and BAD mRNA, and a 3′-UTR mutant of OTUD7B, TNIP2, and BAD mRNA containing three altered nucleotides in the putative target site (_OTUD7B-_3′UTR-mut, _TNIP2-_3′UTR-mut, and _BAD_-3′UTR-mut). (b) The expression levels of OTUD7B, TNIP2, and BAD protein in HCC cells overexpressing miR-1180 or transfected with the miR-1180 inhibitor, compared with control cells (NC), by western blotting 48 h post-transfection; α-Tubulin is the loading control. (c) Luciferase assay of pGL3-_OTUD7B_-3′UTR or pGL3-OTUD7B -3′UTR-mut reporter cotransfected with miR-1180 mimic or miR-1180 inhibitor in indicated cells. (d) Luciferase assay of pGL3-_TNIP2_-3′UTR or pGL3-_TNIP2_-3′UTR-mut reporter cotransfected with miR-1180 mimic or miR-1180 inhibitor in indicated cells. (e) Luciferase assay of pGL3-_BAD_-3′UTR or pGL3-_BAD_-3′UTR-mut reporter cotransfected with miR-1180 mimic or miR-1180 inhibitor in indicated cells. Each bar represents the mean ± SD of three independent experiments. *P < 0.05.
Figure 6. MiR-1180 activates the NF-κB signaling pathway.
(a) NF-κB transcriptional activity measured by the luciferase assay in HepG2 and Huh7 cell lines in the presence of the miR-1180 mimic, the miR-1180 inhibitor, and a negative control (NC). (b) Western blotting analysis of NF-κB p65 expression in the cytoplasm (C) and nucleus (N) in the indicated cells. Nuclear protein p84 is a nuclear protein marker and EF-1α is a loading control. (c) The expression of NF-κB-targeted genes, CCND1, IL6, MYC, BcL-XL, TNFA and VEGF, measured by qRT-PCR; GAPDH is the control. (d) Cell viability in miR-1180 overexpressing cells treated with NF-κB inhibitor (NF-κ B-in) as measured by the MTS assay. (e) Quantifications of crystal violet stained cell colonies formed in miR-1180 overexpressing cells treated with NF-κB inhibitor (NF-κB in). (f) The relative NF-κB transcriptional activity measured by the luciferase assay in the indicated cells. Each bar represents the mean ± SD of three independent experiments. *P < 0.05.
Figure 7. The expression of miR-1180, OTUD7B and TNIP2, and NF-κB activity in HCC tissues.
(a–c) qRT-PCR analysis of miR-1180 (a), western blotting analysis (b) of OTUD7B and TNIP2 expression and EMSA analysis of NF-κB activity (c) in HCC tissues. Log2 value was used to show qRT-PCR results. (d) The correlation between miR-1180, OTUD7B and TNIP2 expression and NF-κB activity in HCC tissues. Error bars represent the mean ± SD from three independent experiments. *P < 0.05.
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