Background
Liver cancer is the sixth most common cancer in incidence and the fourth leading cause of cancer-related death worldwide [
1,
2]. Hepatocellular carcinoma (HCC) is the major type of liver cancer, accounting for 75–85% of primary liver cancers [
3]. The main risks for HCC are hepatitis B virus (HBV), hepatitis C virus (HCV), aflatoxin contamination in food, alcohol abuse, obesity and so on [
3]. Treatments for liver cancer patients contain surgical therapies, tumor ablation, transarterial therapies and systemic therapies [
2]. However, the prognosis remains miserable, 5-years survival rate for liver cancer patients is 18%, only inferior to pancreatic cancer [
1].
TGF-β signaling pathway has been reported playing a multifaceted role in tumor development and progression. It promotes homeostasis and suppresses tumor progression by inducing cytostasis, differentiation, apoptosis, inflammation depression and stroma-derived mitogens in normal and premalignant cells. Meanwhile, in advanced cancer cells, TGF-β initiates immune evasion, growth factor production, differentiation to invasive phenotype, metastatic dissemination and metastatic colonies establishment [
4]. Eight SMAD proteins were identified participating in TGF-β signaling: the receptor-regulated SMAD (R-SMAD) (SMAD1, 2, 3, 5 and 8), the co-mediator SMAD (Co-SMAD) (SMAD4), and the inhibitory SMAD (I-SMAD) (SMAD6 and 7) [
5]. As R-SMAD, SMAD2 and SMAD3 are phosphorylated by receptors of TGF-β branch, whereas SMAD1, 5 and 8 are phosphorylated by other branches of receptors such as BMP receptors [
6]. Upon TGF-β stimulation, SMAD2 and SMAD3 are phosphorylated and activated by type I receptor kinase, and bind with SMAD4 to form activated SMAD complexes [
5]. Regardless of the similar working mechanisms and high identity of protein structures [
7], the role of SMAD2 and SMAD3 are often different, even opposite. Distinct effects of SMAD2 and SMAD3 were reported in breast cancer bone metastasis [
8], pancreatic ductal adenocarcinoma cell proliferation and migration [
9], HaCaT keratinocyte cell growth [
10], TGF-β autoinduction in clostridium butyricum-activated dendritic cells [
11] and TGF-β induced transcription [
12]. Our lab has previous reported SMAD3 promoted HCC metastasis by upregulating protein tyrosine phosphatase receptor epsilon (PTPRε) expression and high expression of SMAD3 was associated with poor prognosis in HCC [
13], but the impacts of SMAD2 on HCC remain largely unclear.
MicroRNAs (miRNAs) are short endogenous RNAs 19–25 nucleotides in length that post-transcriptionally regulate mRNA expression [
14]. Instead of directly silencing targeted mRNA, miRNAs in human and other bilaterian animals promote mRNA decay or repress mRNA translation mainly by recruiting RNA-induced silencing complex (RISC). As the heart of RISC, Argonaute2 (Ago2) recruits TNRC6, PABPC, and deadenylase complexes, i.e., the PAN2-PAN3 complex or CCR4-NOT complex to destabilize or repress mRNA [
15‐
17]. Numerous miRNAs have found dysregulated in HCC and involved in HCC diagnosis, prognosis and therapeutics, such as, miR-16, miR-92a and miR-500 [
18‐
20]. Lower miR-148a expression in HCC tissues was observed than that in adjacent noncancerous hepatic tissues and miR-148a-3p expression was correlated to clinical TNM stage, metastasis, status of capsular infiltration and numbers of tumor nodes in HCC [
21]. Xu et al. reported that hepatitis B virus X protein (HBx) repressed miR-148a expression and further upregulated the expression of hematopoietic pre–B cell leukemia transcription factor interacting protein (HPIP) to enhance tumorigenesis [
22]. Gailhouste et al. revealed that miR-148a promoted hepatospecific phenotype of mouse fetal hepatoblasts (MFHs) and suppressed the invasiveness of transformed cells [
23].
Our study revealed that SMAD2 exhibited oncogenic role in HCC and found that miR-148a was an upstream regulator of SMAD2 by decaying SMAD2 mRNA in an Ago2 dependent manner. miR-148a inhibited the mobility and proliferation of HCC cells and low expression of miR-148a in HCC was associated with shorter overall survival time. These results indicated miR-148a could be a promising diagnostic marker and therapeutic target for HCC patients.
Methods
Patients and tissue specimens
Human tumor and adjacent non-tumor tissues were collected from HCC patients underwent hepatectomy at the Hepatic Surgery Center, Tongji Hospital of Huazhong University of Science and Technology (HUST) (Wuhan, China). All procedures were approved by the Ethics Committee of Tongji Hospital, HUST and conducted according to the Declaration of Helsinki Principles. Prior written and informed consent was obtained from each patient.
Cell lines and culture
HCC cell lines MHCC-97H, HCC-LM3 were obtained from Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China. Hep3B, Huh7, PLC/PRF-5 (ALEX), HLF, Bel7402 and normal liver cell line HL7702 were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China). ALEX was purchased from cell bank of Chinese Academy of Sciences (Shanghai, China) [
24]. All cell lines were maintained in Dulbecco Modified Eagle Medium (DMEM) (Hyclone, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Gibico) at 37 °C in a 5% CO
2 cell incubator.
Cell counting kit 8 assay and EdU incorporation assay
Cell Counting Kit 8 assay (Dojindo, Kumamoto, Japan) was performed according to manufactures’ protocol. Briefly, 1500 indicated cells were seeded into 96-well plates. Culture medium was changed to 100 μl 10% CCK8 solution at the indicated time and incubated in cell incubator for 2 h. Optical density (O.D.) was measure by Universal Microplate Reader ELx 800 (BIO-TEK, USA) at 450 nm wave length. For each group, the absorbance values were measured by five replicates. After extracting blank value, an average of gross O.D. values was used for data analysis.
HCC cells (4000 cells/ well) were seeded into 96-well plates and cultured overnight for EdU incorporation assay by using Cell-Light™ EdU Apollo567 In Vitro Imaging Kit (Ribobio, Guangzhou, China) according to the manufacturer’s instructions. Briefly, 100 μl 50 μM EdU solutions were added into cells and cultured for 2 h. Cells were rinsed with PBS, fixed with 4% paraformaldehyde and incubated with 0.5% TritonX-100. Then, cells were stained with 100 μl 1X Apollo solution for 30 min, nucleus were stained with 1X Hoechst33342 solution. Representative images were captured with EVOS FL auto imaging system (life technologies, USA) and positive cells were counted by Image Pro. Plus version 6.0.
Dual luciferase assay
The sequence of 8284–8390, containing predicted binding site with miR-148a, in the 3′-untranslated region (3’UTR) of SMAD2 or 3′UTR-SMAD2-mutant was cloned into the psiCHECK™-2-vector (Promega, Madison, WI, United States). About 1 × 105 cells/well were seeded into 24-well plates. After 24 h, the recombinant plasmid pSicheck-2-3′UTR-SMAD2 or pSicheck-2-3′UTR-SMAD2-mutant were co-transfected into cells with miR-148a mimic, miR-148a inhibitor or their respective negative control (nc) using Lipofectamine 3000 (Invitrogen). Forty-eight h later, total protein from cells were extracted by Passive Lysis Buffer (Promega) and the luciferase activity was determined using Dual-Luciferase® Reporter 1000 Assay System (Promega) by GloMax 20/20. Firefly luciferase values were normalized against Renilla luciferase activity, and the ratio of firefly/Renilla luciferase activity was presented.
Anti-Ago2 RNA binding protein immuno-precipitation (RIP) assay
Rabbit anti-Ago2 IgG was purchased from Abcam (ab32381). Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Darmstadt, Germany) was used to enrich Ago2 binding RNA. The enriched RNA was subjected to qRT-PCR. 2-ΔCT was calculated and normalized to the 2-ΔCT of 10% input.
Animal experiments
4–5 weeks old male BALB/cA-nude mice were purchased from Beijing HFK Bioscience Co. Ltd. and maintained at SPF conditions. All animal experiments were approved by the Ethics Committee of Tongji Hospital, HUST. The whole procedure was in accordance with the “Guide for the Care and Use of Laboratory Animals” (NIH publication 86–23 revised 1985). For xenograft tumor model, 1× 106 indicated tumor cells were suspended in 100 μl serum free DMEM and inoculated subcutaneously into the flanks of nude mice. Thirty days later, all mice were sacrificed to compare the volume and weight of tumors. For lung metastasis model, 1 × 106 cells were suspended in 100 μl serum free DMEM. Then cells were injected via the tail vein of nude mice. After 2 months of injection, mice were sacrificed and lungs were resected to calculate metastatic nodules.
In situ hybridization (ISH) and immunohistochemistry analysis (IHC)
ISH was performed using the ISH Kit (Boster, Bio-Engineering Company, Wuhan, China). All procedures followed the manufacturer’s instructions. Samples were stained with hematoxylin, dehydrated with alcohol, washed with xylene, sealed with flavor sealing tablets. Oligo (5’Digoxin-ACAAAGTTCTGTAGTGCACTGA) was used as ISH probe for miR-148a. IHC staining was performed as described previously [
24]. Primary antibody for SMAD2 (1:100, 12,570–1-AP) and Ki67 (1:100, 27,309–1-AP) were purchased from Proteintech. The representative images of ISH and IHC were captured and processed using DM2300 microscope and ScopeImage 9.0 software (Nanjing Jiangnan Novel Optics Co., Ltd., China). ISH and IHC staining scores were independently determined by 3 pathologists without prior knowledge of patient information. The overall score defined by multiplying the percentage of positive cells by the staining intensity score as described previously [
24].
In silico analysis and prediction websites
Overall survival rates of HCC patients with different SMAD2 or SMAD3 level were analyzed by GEPIA [
25] and Kaplan Meier-plotter [
26] websites. TargetScan [
27], miRTarBase [
28] and miRcode [
29] websites were used to predicted miRNAs targeting SMAD2 mRNA. StarBase [
30] website was used to analyzed correlations between expression of miRNAs and SMAD2 mRNA, or overall survival rate of liver hepatocellular carcinoma (LIHC) patients with different miRNAs expression.
Statistical analyses
We used Prism 7.0 (GraphPad Software, La Jolla, CA, USA) or SPSS 13.0 (SPSS, Chicago, IL, USA) to analyze the data. Quantitative data were analyzed by Student’s t test (unpaired two-tailed comparison) or Pearson’s correlation test. Kaplan-Meier and log-rank analysis were applied to evaluate survival between two groups. Categorical data were analyzed by chi-square test. All values were presented as mean ± SEM. P values less than 0.05 were considered statistically significant.
Supplementary materials and methods were provided in additional information.
Discussion
As R-SMAD proteins, the impacts of SMAD2 and SMAD3 on cancer initiation and progression were widely investigated. Interestingly, SMAD2 and SMAD3 were found play distinct, even opposite roles under certain context [
10‐
12]. In non-small-cell lung carcinoma (NSCLC), cancer metastasis was associated with inactivation of SMAD2-mediated and activation of SMAD3-mediated transcriptional programs [
31]. During breast cancer bone metastasis progression, the TGF-β induced bone metastatic genes expression were found depend on SMAD3 but not SMAD2, and knockdown of SMAD3 in MDA-MB-231 cells inhibited bone metastasis, while SMAD3 knockdown led to a more aggressive phenotype [
8]. In HCC, SMAD3 was reported suppressing carcinogenesis in chemically inducing animal models [
32] and sustained SMAD3 activation promoted cancer metastasis [
13]. However, the role of SMAD2 in HCC was poorly focused and remained obscure. Our results showed that the expression of SMAD2 was elevated in HCC specimens and high expression of SMAD2 in HCC associated with poor prognosis. SMAD2 promoted proliferation, migration and invasion of HCC cells.
miRNAs can affect TGF-β signaling process by directly targeting canonical members in the signaling pathway or targeting its effector genes [
33]. In human, instead of directly slicing targeting mRNA by associated with an Ago protein that retained the catalytic ability of endonucleolytic cleavage, miRNAs mainly partially paired with targeted mRNA, and recruited RISC complexes to enhance mRNA decay or translational repression [
17]. In our study, we predicted the mRNA of SMAD2 can be targeted by serials of miRNAs. Among these miRNAs, we identified miR-148a as an upstream regulator of SMAD2 by analyzing the expression relevance between miRNAs and SMAD2, and the association between miRNAs expression and OS in HCC. miR-148a was found downregulated in HCC tissue and low expression of miR-148a was associated with shorter OS time, which is consistent with previous study [
21]. We further found that miR-148a downregulating SMAD2 expression through binding with Ago2, the core protein of RISC complexes.
Endeavors have been paid on exploring the effects of miR-148a on the HCC formation and progression. Researchers revealed that miR-148a promoted mouse fetal hepatoblasts (MFHs) differentiating to mature hepatocytes by directly targeting DNA methyltransferase 1 (DNMT1) [
23] and induced hepatocytic differentiation to HCC by inhibiting IKKα/NUMB/NOTCH signaling [
34]. While, Xiaojie Xu et al. found that Hepatitis B virus X protein (HBx) repressed miRNA-148a to enhance tumorigenesis in mouse animal model of HCC [
22]. The role of miRNA-148a as an HCC metastasis repressor seems consistent across different studies, miR-148a-3p suppressed the invasiveness of HCC cell by regulating c-Met [
23], Wnt1 [
35] or activin A receptor type 1 (ACVR1) [
36]. Our results showed that miR-148a inhibited proliferation and migration of HCC cells in vivo and in vitro. And the impacts of miR-148a in HCC cells were mediated by SMAD2.
Conclusions
This study demonstrated that SMAD2 was highly expressed in HCC specimens, elevated SMAD2 expression was associated with shorter overall survival time for HCC patients. SMAD2 played its tumor promoter role by enhancing migration, invasion and proliferation abilities of HCC cells. Besides, the expression of miR-148a was found negatively related with SMAD2 in HCC. Low expression of miR-148a was associated with more aggressive clinical features and predicted poorer prognosis. miR-148a inhibited HCC cells migration, invasion and proliferation in vitro, suppress tumorigenesis and metastasis in vivo. In mechanism, miR-148a recruited Ago2, bound with and decayed SMAD2 mRNA to inhibit SMAD2 expression. In conclusion, miR-148a was identified as a regulator of oncogenic SMAD2 and may serve as a promising prognostic marker or therapeutic target for HCC patients.
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