Hepatocellular carcinoma (HCC) is a prevalent malignancy that ranks the third leading cause of cancer mortality worldwide [
]. High frequencies of recurrence and metastasis are the major causes for the poor clinical outcomes of HCC patients. Increasing evidences have supported that miRNA deregulation is in correlation with HCC progression [
]. By targeting the 3′-untranslated region (UTR) of the mRNA, miRNA leads to inhibition of targeted mRNA depending on total or partial complementarity [
]. miR-182-5p, a member of the miR-183/96/182 cluster, emerged as a high-priority miRNA in HCC and has been proven to be related to various cancers. However, the function of miR-182-5p is complicated because it can be an oncogene or a tumor suppressor in the context of different cancers. miR-182-5p is identified as onco-miR in ovarian cancer [
], breast cancer [
], and melanoma [
] and acts as tumor suppressor in RCC [
] and glioblastoma [
]. In HCC, miR-182-5p contributes to HCC metastasis by targeting metastasis suppressor 1 (MTSS1) [
], and upregulated miR-182-5p increases drug resistance in cisplatin-treated HCC cells by regulating tumor protein 53-induced nuclear protein 1 (TP53INP1) [
]. In addition, increased miR-182-5p can be of diagnostic and prognostic value in HCC patients [
]. However, whether miR-182-5p was involved in early recurrence of HCC remained unknown. We here investigated the relationship between miR-182-5p and early recurrence of HCC patients underwent curative surgery and further explored the underlying mechanisms of miR-182-5p in promoting HCC progression.
FOXO3a, a member of Forkhead box O (FOXO) transcription factor family, mediates many genes through its transcriptional activity, with important roles in cell fate decisions and is also suggested to play a pivotal role as a tumor suppressor in a wide range of cancers [
]. FOXO3a is an important target of PI3K/Akt pathway. Activated AKT phosphorylates FOXO3a and leads to its cytoplasmic translocation and subsequently degradation [
]. In addition to AKT, there are other negative regulators of FOXO3a, such as serum and glucocorticoid-regulated kinase (SGK) [
]. It has been reported that activation of the Wnt signaling pathway induced expression of SGK1 and lead to nuclear exclusion of FOXO3a [
], indicating that FOXO3a was under the regulation of Wnt/β-catenin signaling pathway. In contrast, FOXO3a was reported to inhibit the expression β-catenin by transactivating miR-34b/c in prostate cancer [
], and FOXO3a can directly bind to β-catenin and compete with T cell factor (TCF) for the interaction to β-catenin, thereby inhibiting β-catenin/TCF transcriptional activity [
In the present study, we demonstrated that miR-182-5p could be a potential predictor for early recurrence of HCC patients underwent curative surgery, and miR-182-5p acted as a promoter of HCC growth both in vitro and in vivo
. Notably, we found that miR-182-5p activated Wnt signaling pathway by inhibiting the degradation of β-catenin and enhancing the interaction between β-catenin and TCF4, which was mediated by repressed FOXO3a. These results provide new insight into the mechanism of miR-182-5p in promoting HCC progression.
Cell culture and transfection
HEK293T was originally obtained from the American Type Culture Collection (ATCC). MHCC-97H and MHCC-97L are human hepatocellular carcinoma cell lines with high metastatic potential [
], obtained from Liver Cancer Institute. All cell lines were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, 10 U/mL penicillin, and 10 mg/mL streptomycin. Cells were grown in a humidified atmosphere at 37 °C at gas tensions of 20% O
for normoxic incubation.
Plasmids used in the experiment:
miRNA inhibitor scrambled control: CmiR-AN0001-AM03, anti-miR-182-5p: HmiR-AN0239-AM03, miR-182-5p: HmiR0115-MR03, miR-NC: CmiR0001-MR03, and Lenti-Pac™ HIV Expression Packaging Kit (Cat.No. HPK-LvTR-20) were all purchased from the GeneCopoeia.
Cellular proliferation assay and transwell assay
For the cell proliferation assay, 5 × 10
3 cells were plated in 96-well plates. Cell growth was determined by using CCK8 assay. After transfection, MHCC-97H and MHCC-97L cells were suspended in 100 μL serum-free medium and placed in the upper chambers of the transwell and incubated at 37 °C for 72 h for the invasion assay. The cells that penetrated the matrigel-coated filters were counted at a magnification of × 200 in eight randomly selected fields, and the mean number of cells per field was recorded.
Whole-cell extracts were prepared in immunoprecipitation (IP) lysis buffer. The extracts were incubated overnight at 4 °C with 4 μg antibody and for an additional 2 h with protein A/G agarose beads. Beads were then washed three times with the lysis buffer, and the beads with the immunoprecipitates were resuspended in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer and heated at 95 °C for 10 min. Subsequently, the supernatants were analyzed by SDS-PAGE followed by western blot.
Western blot analyses were performed as previously described [
AKT #4685 CST; p-AKT Ser473 #4060 CST; Bcl-2 #15071; Bcl-xl #2764 CST; Cyclin D1 #2922 CST; SGK #12103 CST; β-actin #3700 CST; Wnt3a #2721 CST; Wnt/β-catenin Activated targets antibody Sampler Kit #8655 CST; Forkhead Signaling Antibody Sampler Kit #9946 CST.
RNA preparation and real time-PCR (RT-PCR)
Total RNA including miRNAs was extracted from cells using TRIzol reagent (Invitrogen) following the manufacturer’s protocol. For analysis of miR-182-5p expression, reverse transcription and stem-loop RT-PCR were performed using the TaqMan MicroRNA assays (Applied Biosystems, Foster City, CA, USA) and amplified by TaqMan Universal PCR Master Mix (Applied Biosystems). U6 snRNA was probed as a loading control.
In situ hybridization (ISH)
ISH was used to detect miR-182-5p in tissue microarrays using digoxigenin-labeled sense and antisense miR-182-5p probes (Exiqon, 610341-360, Denmark). The slides were de-paraffined and rehydrated before incubation with Proteinase K at 37 °C for 40 min, then wash the slides three times with PBS for 15 min. After incubation with 5× SSC solution at room temperature for 15 min, miR-182-5p probes were added for hybridization at 50 °C for 1 h. Next, the slides were washed with graded-diluted SSC solutions at 50 °C for 30 min, followed by incubation with an antibody against digoxigenin (1:1000, Roche, Mannheim, Germany) at 4 °C overnight. Finally, hybridization signals were visualized by NBT/BCIP (Sigma), and the reaction was stopped by washing with water for 5 min. At last, slides were counterstained with nuclear fast red for 1 min and then mounted using an aqueous solution.
Dual-luciferase reporter assay
The 3′-UTR segments of FOXO3a including the wild type or the mutant type of miR-182-5p binding sites were cloned into the downstream of the luciferase reporter, the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, USA), between the SacI and SalI sites and verified by sequencing. HEK 293 T cells were plated into 24-well plates and transfected with 50 nM miR-182-5p or NC and 100 ng of the luciferase vector (pmirGLO). Cells were harvested 48 h after the transfection. The relative luciferase activity was measured by the Dual-Glo luciferase assay kit (Promega).
This study was approved by the clinical research ethics committee of the Shanghai Zhongshan Hospital of Fudan University. The tumor specimens for tissue microarrays were obtained from 119 patients who underwent surgery during July 2014 to May 2015 at the Shanghai Zhongshan Hospital of Fudan University. The disease-free survival was calculated from the date of resection to the date of tumor recurrence.
Tumor xenografts in nude mice
The orthotopic HCC implant tumor model in nude mice was performed as previously described [
]. All surgical procedures and care administered to the animals were in accordance with the institutional ethics guidelines.
Immunohistochemistry (IHC) data were analyzed using a χ
2 test. A two-tailed
t test was used to compare the means between two sets, and a one-way analysis of variance was used to compare the means among three groups. By the Kaplan-Meier method and the log-rank test, survival curve analysis was performed to study the role of miR-182-5p in HCC progression. The data were analyzed with SPSS software version 19.0 (SPSS Inc., Chicago, IL, USA).
P < 0.05 (two-sided) was considered statistically significant.
Metastasis and recurrence are the main problems that limiting the 5 years’ survival rate of HCC. In HCC patients, high-serum VEGF has been shown to associate with tumor recurrence, metastasis, and poor survival [
]. In addition to targeted drugs for VEGF in HCC, non-invasive detection of hydrodynamic changes in the field of industry has made a lot of progress [
], which might be applied in the near future in the field of medicine.
Our results showed that miR-182-5p was overexpressed in both HCC cell lines and HCC tissues. Moreover, miR-182-5p was related to poor prognosis and early recurrence in HCC. By univariate and multivariate analyses, we found that the expression of miR-182-5p could be regarded a potential predictor for early recurrence of HCC patients under curative surgery.
miR-183, miR-182, and miR-96 act as regulators of FOXO expression in various cancer types [
]. MiR-182-5p has been reported to promote melanoma metastasis by repressing FOXO3 [
]. However, the specific binding target site in the 3′-UTR was not showed. Here, we exhibited that miR-182-5p suppressed FOXO3a expression by binding to the 72–79 site, but not 914–921 site in the 3′-UTR of FOXO3a.
Overexpression of miR-182-5p induces G1-phase arrest via inhibition of AKT/FOXO3a signaling in RCC [
]. However, we proved that miR-182-5p promoted AKT phosphorylation resulting in inactivation of FOXO3a in HCC. Moreover, knockdown of FOXO3a could further activate AKT, indicating a positive feedback loop between AKT and FOXO3a. The apoptosis inhibiting protein Bcl-2 and Bcl-xl, downstream target of AKT, were upregulated by overexpression of miR-182-5p. Taken together, miR-182-5p promoted cell proliferation by repressing FOXO3a, subsequently activating AKT/FOXO3a pathway.
It has been shown that Wnt/β-catenin activated miR-183/96/182 expression in hepatocellular carcinoma and promoting cell invasion [
]. Furthermore, β-catenin enhances expression of primary and mature miR-96, miR-182, and miR-183 [
]. We proved that miR-182-5p activated Wnt signaling by inhibiting β-catenin degradation and promoting β-catenin/TCF4 interaction, forming a positive feedback loop between Wnt signaling and miR-182-5p.
Wnt/β-catenin signaling pathway plays a critical role in the proliferation and cell cycle regulation of HCC cells [
]. When Wnt signaling is activated, β-catenin accumulates in the nucleus interacting with the T cell factor/lymphoid enhancer-binding factor (TCF/LEF) to transcriptionally activate downstream gene expression, such as cyclin D and c-Myc which exert promoting role in cell cycle [
]. β-catenin directly binds to FOXO and enhances FOXO transcriptional activity which is particularly important under condition of oxidative stress [
]. FOXO3a competes with TCF for interaction with β-catenin, thereby inhibiting TCF transcriptional activity [
]. Thus, β-catenin appears function as a switch that determining whether a cell chooses TCF or FOXO3a in response to stimuli.
We showed that miR-182-5p activated Wnt signaling by inhibiting β-catenin degradation, and miR-182-5p negatively regulate FOXO3a, resulting in the enhancement of β-catenin/TCF interaction, increasing the expression of β-catenin targeted genes. As a downstream factor of Wnt signaling, SGK1 preferentially phosphorylates Ser315, and AKT mediates the phosphorylation of Ser253 of FOXO3a [
]. SGK1 negatively regulates the transcription factor FOXO3a via phosphorylation and exclusion from the nucleus, leading to FOXO3a degradation [
]. When Wnt signal was activated by miR-182-5p, SGK was upregulated and lead to the phosphorylation of FOXO3a. As a result, the inactivation of FOXO3a might further promote Wnt signal, forming a positive feedback loop.