MiR-1180 promotes apoptotic resistance to human hepatocellular carcinoma via activation of NF-κB signaling pathway

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.

Tissue specimens. Fresh HCC tissues and normal hepatic tissues were collected from curative resection and diagnosed histopathologically at the Department of Interventional Radiology, the First Affiliated Hospital of Sun Yat-Sen University. The samples were immediately frozen and stored in liquid nitrogen (-80 °C) after surgery prior to analysis. Informed consent was obtained from all participants, and the study was approved by the Institutional Research Ethics Committee.
Colony formation assay. Cells were plated into a 6-well plate (1 × 10 4 cells per well) and transfected with miR-1180 mimic, miR-1180 inhibitor, or NC. After 24 h, the cells were treated with cisplatin (0.4 μg/ml) for another 24 h, and then cultured for 10 days. The colonies were stained with 1.0% crystal violet for 5 min after fixation with 10% formaldehyde for 15 min. All experiments were performed in triplicate.

Flow cytometry analysis. For cell cycle analysis, the cells were harvested and fixed in a solution of 70%
ice-cold ethanol with phosphate buffered saline (PBS). Propidium iodide (PI, 50 μg/ml; Sigma) and a fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody specific for Annexin V (Sigma) were incubated with the cells at 4 °C for 30 min before analysis using the FACS Calibur system (BD Biosciences, CA, USA). The data were analysed by the ModFit LT software package. Results are representative of three independent experiments with triplicate samples for each cell line.
Xenografted tumour model and treatment. BALB/c-nu mice (5-6 weeks old, 18-20 g) were purchased from the Experimental Animal Center of the Guangzhou University of Chinese Medicine and housed in barrier facilities on a 12 h light/dark cycle. The Institutional Animal Care and Use Committee of Sun Yat-Sen University approved all experimental procedures. The mice were randomly assigned to groups (n = 5/group). All mice in groups were inoculated subcutaneously with HepG2 cells (1 × 10 7 cells per mouse) in the left dorsal flank. A week later, mice in group A were injected intratumourally with 100 μl of a miR-1180 antagonist (diluted in PBS at 2 mg/ ml), and mice in group B were injected with a antagomiR control (diluted in PBS at 2 mg/ml), three times per week. After two weeks, all the mice were injected intratumourally with cisplatin (0.4 μg/ml) three times per week. Tumours were examined twice weekly; length, width, and thickness were measured with callipers, and tumour volumes were calculated. Tumour volume was calculated using the equation (L × W 2 )/2. On day 40, the animals were euthanised, and the tumours were excised and weighed.
Western blotting assay. Total protein was extracted from whole cells. The samples were heated at 90 °C for 10 min with the 1× sample buffer and separated by SDS-PAGE. Subsequently, the sample was electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membranes were probed with polyclonal rabbit antibodies against anti-Bcl-2, anti-Bax, anti-cleaved-caspase-3, anti-OTUD7B, anti-TNIP2, and anti-BAD (Abcam, Cambridge, MA, USA). For loading control, the membranes were stripped and re-probed with anti-alpha-Tubulin (Abcam).
Scientific RepoRts | 6:22328 | DOI: 10.1038/srep22328 Luciferase reporter assay. Cells were seeded in triplicate into a 24-well plate (3 × 10 4 per well) and were allowed to settle for 12 h. The pNF-κ B-luciferase plasmid (100 ng) or the control-luciferase plasmid, and an additional 10 ng of pRL-TK Renilla plasmid (Promega, Madison, WI, USA), were transfected into HCC cells using the Lipofectamine 2000 reagent (Invitrogen). Luciferase and Renilla signals were measured at 48 h, post-transfection using the Dual Luciferase Reporter Assay Kit (Promega), according to a protocol provided by the manufacturer.

RNA extraction and quantitative reverse-transcription PCR (qRT-PCR). Total cellular RNA
was extracted using Trizol reagent (Invitrogen) according to a protocol provided by the manufacturer. The SYBR Green I (Molecular Probes, Invitrogen) dye was used according to the manufacturer's instructions, and qRT-PCR was performed and analysed using a 7500 Fast Real-Time Sequence detection system software (Applied Biosystems, Foster City, CA, USA). The qRT-PCR reactions for miRNAs were performed at 95 °C for 3 min, followed by 40 cycles (each 30 s in length) at 95 °C, 58 °C, and 72 °C. The expression levels of miR-1180 were normalized with reference to the expression levels of U6 small nuclear RNA (snRNA). The qRT-PCR conditions for genes were set at 95 °C for 10 min, followed by 40 cycles at 95 °C for 20 s, 60 °C for 30 s and 72 °C for 1 min. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference gene that acts as an internal standard to normalize the mRNA expression. The fold changes were calculated by relative quantification (2 −ΔΔCt ). The primers used for stem-loop reverse-transcription PCR of miR-1180 and U6, were purchased from RiboBio (RiboBio Co. Ltd, Guangzhou, Guangdong, China). Statistical analyses. All quantitative data were analysed using a two-tailed paired Student's t-test. The data were expressed as the mean ± standard deviation (SD) for three independent experiments. Survival curves were plotted by the Kaplan-Meier method and compared by the log-rank test. P < 0.05 was considered to indicate a statistically significant difference.

Results
MiR-1180 is upregulated in HCC. By analysing a published micro-array-based high-throughput assessment (NCBI/GEO/GSE36915), we found that miR-1180 was markedly upregulated in HCC tissues (T, n = 68), compared with that in non-tumour tissues (N, n = 21; P < 0.01; Fig. 1a). To confirm the result of the published assessment, we examined the expression level of miR-1180 in our freshly collected HCC tissues using qRT-PCR. We found miR-1180 was upregulated in eight samples of HCC tissues (T) compared with their adjacent noncancerous hepatic tissues (ANT; Fig. 1b). The expression of miR-1180 was also significantly increased (P < 0.05) across a panel of HCC cell lines (i.e., Hep3B, HepG2, BEL-7402, HCCC-9810, MHCC97H, MHCC97L, Huh7, and QGY-7703) compared to normal liver epithelial cells (THLE3; Fig. 1c). As miR-1180 expression was elevated in HCC tissues and cell lines, miR-1180 appears to have a positive function on HCC progression. In addition, we found that HCC patients with higher miR-1180 expression had a shorter survival time, whereas patients with lower miR-1180 expression had a longer survival time (n = 75; P = 0.001; Fig. 1d), suggesting a potential correlation between expression of miR-1180 and the progression of HCC.

Ectopic miR-1180 promotes proliferation and inhibits cisplatin-induced apoptosis of HCC cells.
To further explore the role of miR-1180 upregulation in HCC progression, we chose to examine the HepG2 and Huh7 cell lines (Supplementary Figure 1a). We first monitored the impact of ectopic overexpression of miR-1180 on cell viability using the MTS assay. Ectopic miR-1180 significantly increased cell viability compared to the control cells (P < 0.05; Fig. 2a). Our colony formation assays showed that overexpression of miR-1180 led to more and larger-sized colonies compared to control cells (P < 0.05; Fig. 2b). To further explore the function of miR-1180 on cytotoxic reagent-induced cell apoptosis, HCC cells were exposed to an anti-tumour agent, cisplatin. Flow cytometry experiments demonstrated that, after treatment with cisplatin (0.4 μg/ml, 24 h), the percentage of Annexin-V-FITC-positive HCC cells in cells that were overexpressing miR-1180 decreased significantly compared to the control cells (Fig. 2c). We also examined the expression of the anti-apoptotic protein, Bcl-2, and the pro-apoptotic proteins, Bax and caspase-3. Overexpression of miR-1180 resulted in an increase of Bcl-2 expression, and a decrease of Bax and cleaved-caspase-3 expression (Fig. 2d, Supplementary Figure 2a). Together, these results indicate that overexpression of miR-1180 in HepG2 and Huh7 cell lines can promote cell proliferation and provide resistance to cisplatin-induced cell death.

Inhibition of miR-1180 attenuates cell viability and promotes cisplatin-induced apoptosis of HCC cells in vitro.
Suppression of miR-1180 (loss-of-function) studies using an inhibitor was performed to confirm the function of miR-1180 in HCC cells (Supplementary Figure 1b). The suppression of miR-1180 caused a significantly decreased cell viability compared to the control cells (P < 0.05; Fig. 3a). Colony formation assays showed that cells overexpressing miR-1180 showed fewer and smaller-sized colonies than control cells (Fig. 3b). Concurrently, apoptosis analysis using flow cytometry showed an increase in the percentage of Annexin-V-FITC-positive in cisplatin-treated and miR-1180-inhibited HCC cells compared to the control cells (Fig. 3c). Furthermore, inhibition of miR-1180 led to a decrease in Bcl-2 expression and an increase in Bax and cleaved-caspase-3 expression (Fig. 3d, Supplementary Figure 2b). Moreover, the ratio of Bcl-2/Bax was significantly increased by miR-1180 overexpression and decreased by miR-1180 suppression, suggesting that miR-1180 increases the anti-apoptotic ability of HCC (Supplementary Figure 2c). These results strongly suggest that inhibition of miR-1180 attenuates cell viability and promotes cisplatin-induced apoptosis of HCC cells.

Inhibition of miR-1180 suppresses HCC cells proliferation and promotes cisplatin-induced apoptosis in vivo.
We then examined the tumour suppressive role of a miR-1180 antagonist in HCC progression using an in vivo tumour model. Importantly, intratumoural injection with an miR-1180 antagonist dramatically inhibited tumour growth, while injecting a antago-miR control had no effect on tumour development  (Fig. 4a). Moreover, after the tumours injected with cisplatin, tumours treated with the miR-1180 antagonist were significantly decreased in both size and weight compared to those tumours injected with the antago-miR control (Fig. 4a-c). Collectively, these results suggest that the inhibition of miR-1180 suppresses HCC cells proliferation and promotes cisplatin-induced apoptosis in vivo. miR-1180 modulates HCC cells survival through downregulation of its target genes, OTUD7B, TNIP2 and BAD. To explore the molecular mechanism of miR-1180 in HCC cells, the publicly available algorithms (TargetScan) were used to predict the target(s) of miR-1180 in humans. The results showed that OTUD7B, TNIP2 and BAD, which are closely correlated with cell survival and apoptosis progression in tumours, were three putative targets of miR-1180 (Fig. 5a). As predicted, western blotting revealed that the expression of OTUD7B, TNIP2 and BAD decreased in HepG2 and Huh7 cells overexpressing miR-1180, and expression increased in cells transfected with the miR-1180 inhibitor compared to negative controls (Fig. 5b). To further confirm the direct correlation between miR-1180 and these putative target genes, the OTUD7B-, TNIP2-and BAD-3′ -UTR fragments, containing the miR-1180 binding site, were subcloned into a pGL3 luciferase reporter vector. Ectopic overexpression of miR-1180 decreased the activity of OTUD7B-, TNIP2-or BAD-3′ -UTR-luciferase reporters and miR-1180 suppression increased their activity (Fig. 5c-e). However, when the OTUD7B-, TNIP2-or BAD-3′ -UTR's contained a mutated binding site (in the seed sequence), the luciferase activity was not affected by miR-1180 overexpression or suppression (Fig. 5c-e). Collectively, the results revealed that OTUD7B, TNIP2 and BAD are direct targets of miR-1180, and are subsequently downregulated in HCC cells overexpressing miR-1180.

Activation of NF-κB signaling pathway is essential for the apoptosis resistant function of miR-1180 in HCC cells.
As OTUD7B and TNIP2 act as inhibitors of the NF-κ B signaling pathway [19][20][21] , we further examined the activity of NF-κ B using a luciferase reporter assay. Ectopic miR-1180 overexpression promoted NF-κ B transcriptional activity compared to controls (Fig. 6a). Moreover, the abundance of nuclear p65 significantly increased in the miR-1180-overexpressing cells, and was reduced when miR-1180 was suppressed (Fig. 6b, Supplementary Figure 3a). Meanwhile, the effect of miR-1180 on p50 nuclear translocation is the similar to that on p65 (Supplementary Figure 3b). Our result was consistent with previous reports that activation of NF-κ B leads to nuclear translocation of NF-κ B (p65/p50) heterodimer to initiate target genes' transcription [23][24][25][26][27] . The expression of NF-κ B-targeted genes, including CCND1, IL6, MYC, BcL-XL, TNFA and  (Fig. 6c). To further confirm miR-1180's function on NF-κ B signaling, cells transfected with miR-1180 were treated with an NF-κ B inhibitor, JSH23, for 24 h. The MTS assay and colony formation assay both revealed that inhibition of NF-κ B blocked the apoptosis resistant function of miR-1180 (Fig. 6d,e). Moreover, Overexpression of OTUD7B or TNIP2 indeed antagonizes miR-1180-induced NF-κ B activation, which further confirmed our conclusion that miR-1180-regulating OTUD7B (or TNIP2)-NF-κ B-signaling regulation mediates HCC resistance ( Fig. 6f and Supplementary Figure 4). These experiments indicate that miR-1180 promotes the resistance to cisplatin-induce apoptosis in HCC cells through activation of the NF-κ B signaling pathway.

Clinical relevance of miR-1180-mediated OTUD7B and TNIP2 inhibition and NF-κB activation in HCC.
Finally, we examined whether miR-1180-mediated OTUD7B and TNIP2 inhibition, and NF-κ B signaling activation, in HCC cells was clinically relevant. As shown in Fig. 7a,b, and Supplementary Figure 5, miR-1180 expression in seven freshly collected HCC samples was inversely correlated with expression of OTUD7B (r = − 0.673, P = 0.047) and TNIP2 (r = − 0.709, P = 0.033); but was positively correlated with NF-κ B activation (r = 0.761, P = 0.017). Collectively, our results demonstrate that upregulation of miR-1180 activates NF-κ B signaling via downregulation of OTUD7B and TNIP2, and thereby promotes the anti-apoptotic ability of HCC cells.

Discussion
In the present study, we showed miR-1180 is upregulated in both HCC tissues and cell lines. Ectopic overexpression of miR-1180 was capable of increasing the HCC cell growth and resistance to cisplatin-induced cell apoptosis both in vitro and in vivo. On the other hand, miR-1180 suppression enhanced cisplatin-induced cell apoptosis. Moreover, we found that the direct targets of miR-1180 are OTUD7B, TNIP2 and BAD, and their expression is downregulated by the microRNA in HCC cells. As OTUD7B and TNIP2 are common inhibitors of NF-κ B signaling, we further examined this process and found that the NF-κ B pathway was significantly activated by miR-1180, indicating its crucial role for apoptosis regulation might be through activation of NF-κ B signaling in HCC cells.
Anticancer agents aimed to induce apoptosis are considered an effective therapeutic strategy to treat tumours. Importantly, the normal regulatory network of apoptosis is essential for apoptosis-induced therapy. Defects in apoptotic pathways (including mutations, downregulation, epigenetic silencing of death receptors, overexpression of FLIP, epigenetic silencing of Caspase, and alterations in the mitochondrial pathway) can result in apoptosis resistance and therapy failure 28 . Apoptosis resistance becomes more complicated as the tumour evolves, and multiple apoptotic genes and their regulators may play a part in the resistance progression. MiRNAs contribute to the cellular proliferation, differentiation and metastasis of various human malignancies, and have recently attracted considerable attention for their effects on apoptosis and drug resistance [29][30][31][32][33][34][35] . In particular, miR-21 has been reported to protect glioblastoma cells from the chemotherapeutic drug temozolomide-induced apoptosis by decreasing the Bax/Bcl-2 ratio and caspase-3 activity 16 . In addition, Li et al. found that the miR-106a was involved in the development of drug resistance of human ovarian cancer cells by targeting PDCD4, primarily through the death receptor-mediated pathway 36 . Similarly, in our study, we discover that miR-1180 is able to inhibit cisplatin-induced apoptosis of HCC cells. We also indicate a mechanism for miR-1180 induced drug resistance by downregulation of OTUD7B, TNIP2 and BAD, the direct targets of miR-1180 in HCC cells.
OTUD7B and TNIP2 are known inhibitors of the NF-κ B signaling pathway; a pathway that has multiple functions during tumourigenicity and development, and targets genes involved in cell proliferation, anti-apoptosis, cell migration or invasion, and angiogenesis [37][38][39][40][41] . Previous studies by Hu et al. have reported that OTUD7B negatively regulates TRAF3 degradation by affecting its ubiquitination, thereby preventing aberrant activation of non-canonical NF-κ B signaling 19 . Meanwhile, TNIP2 binds to the COOH-terminal domain of the zinc-finger protein A20 to inhibit NF-κ B activation 20 . Additionally, miR-486 can disrupt multiple NF-κ B-negative feedback loops, by downregulating CYLD, Cezanne, and multiple A20 regulators, including ITCH, TNIP-1, TNIP-2 and TNIP-3 21 . In the present study, we observe that miR-1180 is also capable of augmenting the activity of NF-κ B signaling to increase the apoptosis resistance of HCC cells. This increased activity of NF-κ B signaling by miR-1180 is suggested to occur via downregulation of OTUD7B and TNIP2. In addition, our study shows that miR-1180 downregulates the BAD protein, which selectively binds to anti-apoptotic molecules of the Bcl-2 family to mediates its pro-apoptotic functions 42 . On the other hand, survival signals lead to BAD phosphorylation, which results in their inactive localization in the cytoplasm 43 . As BAD is also downregulated by miR-1180, the mechanism may also contribute to cell survival in HCC. Collectively, our results are consistent with the expected outcomes of an activated NF-κ B signaling pathway, and provide evidence that miR-1180 contributes to cisplatin-induced resistance in HCC by targeting this pathway.

Conclusions
In summary, the present study demonstrates that the upregulation of miR-1180 contributes to the proliferation and cisplatin-resistance of HCC cells both in vitro and in vivo, by targeting and suppressing OTUD7B, TNIP2 and BAD. Inhibition of OTUD7B and TNIP2 results in activation of NF-κ B, suggesting the anti-apoptotic function of miR-1180 may occur through the NF-κ B signaling pathway. (a-c) qRT-PCR analysis of miR-1180 (a), western blotting analysis (b) of OTUD7B and TNIP2 expression and EMSA analysis of NF-κ B 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-κ B activity in HCC tissues. Error bars represent the mean ± SD from three independent experiments. *P < 0.05.