Background
Hepatocellular carcinoma (HCC) is one of the most common malignant primary liver tumors and is the third leading cause of global cancer death, particularly in East Asia and South Africa [
1]. Like other cancers, HCC is involved in a complex and multistep process which is related to a variety of genetic and epigenetic changes. Although many efforts have been made, one of the major problems with HCC is that there are no obvious symptoms, so most patients with HCC are only diagnosed at an advanced stage, and the patients’ survival rates are therefore discouraging [
2,
3]. Cirrhosis caused by hepatitis B virus (HBV) or hepatitis C virus (HCV) is reported to be associated with HCC [
4]. At present, nonalcoholic fatty liver disease is becoming the leading cause of HCC, and there are no ideal targeted therapies [
5]. Additionally, it was also found that oncogenes and/or loss of tumor suppressor genes could be potential biomarkers, as the dysregulated expression level was associated with clinicopathologic characteristics, even prognosis [
6,
7]. However, little is known about the molecular and cellular mechanisms. Understanding the mechanisms underlying key genes is essential for further clarifying the pathogenesis of HCC and can provide opportunities for the development of novel therapeutic strategies.
Protein phosphatase, Mg
2+/Mn
2+ dependent 1A (PPM1A) is a member of the protein phosphatase 2C (PP2C) family of serine/threonine protein phosphatases, which negatively regulate the cellular stress response pathway [
8]. Overexpression of PPM1A can activate the expression of p53 and its downstream p21, which are tumor suppressor genes in cancer development, leading to G2-M phase cell cycle arrest as well as apoptosis, and may be involved in the regulation of tumor pathogenesis [
9]. PPM1A can dephosphorylate Smad1, thereby regulating the signaling pathway of bone morphogenetic protein (BMP) in transforming growth factor-β (TGF-β) superfamily [
10]. PPM1A can also specifically terminate the TGF-β signaling pathway by dephosphorylation of Smad2/3, which has expanded the substrate type of PPM1A and its signaling pathways [
11]. It has been found that PPM1A was downregulated in the HCC tissues compared with the corresponding pericarcinous tissues and that down-regulation of PPM1A through increasing its proteasomal degradation and ubiquitination resulted in the increased ability of HCC cell migration and invasion [
12,
13].
Ubiquitination is an important post-translational modification and E3 ubiquitin ligase plays an important role in ubiquitination [
14]. Tripartite motif (TRIM) family proteins are considered as E3 ubiquitin ligase with more than 80 members and are associated with developmental disorders, neurodegenerative diseases and viral infections as well as cancer cell growth and differentiation through regulating cell proliferation and apoptosis [
15‐
17]. Tripartite motif containing 52 (TRIM52) is a novel TRIM family protein. A previous study demonstrated that TRIM52 inhibited Japanese encephalitis virus (JEV) replication by degrading the viral nonstructural protein 2A (NS2A) [
18]. Additionally, TRIM52 promotes HCC cell proliferation in HBV-associated HCC and HBV X protein (HBx) may regulate TRIM52 expression via the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway [
19]. However, the function of TRIM52 in regulating cell cycle and motility of HCC is still largely not understood.
In this study, we have identified that TRIM52 up-regulation in HCC and promote HCC cell proliferation, migration and invasion. Our data suggest that ubiquitination of PPM1A by TRIM52 may be a novel mechanism underlying HCC carcinogenesis.
Methods
Study subjects
The human HCC tissue microarrays used in this study were prepared by Shanghai Outdo Biotech Co., Ltd. (Shanghai, China). The clinicopathologic characteristics of the 87 patients enrolled in the study are summarized in Table
1. This study was approved by the Ethics Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital.
Table 1
Clinicopathologic characteristics of the patients with HCC
Age (years) |
≤53 | 47 (54.0%) |
> 53 | 40 (46.0%) |
Sex |
Female | 8 (9.2%) |
Male | 79 (90.8%) |
Tumor size (cm) |
≤4 | 25 (28.7%) |
> 4 | 62 (71.3%) |
Recurrence (2 years) |
Yes | 63 (72.4%) |
No | 24 (27.6%) |
Differentiation |
Low | 39 (44.8%) |
Moderate | 38 (43.7%) |
High | 10 (11.5%) |
Tumor number |
Single | 64 (73.6%) |
Multiple | 23 (26.4%) |
TNM stages |
I | 15 (17.2%) |
II | 22 (25.3%) |
III | 20 (23.0%) |
IV | 30 (34.5%) |
Pathologic stages |
I | 6 (6.9%) |
II | 34 (39.1%) |
III | 40 (46.0%) |
IV | 7 (8.0%) |
Lung metastasis |
Yes No | 16 (18.4%) 71 (81.6%) |
HBsAg |
Negative | 7 (8.0%) |
Positive | 80 (92.0%) |
HBV infection |
Absent | 7 (8.0%) |
Present | 80 (92.0%) |
Immunohistochemistry (IHC) analysis
After deparaffinization, rehydration and antigen-retrieval, hepatic tissue slides (4–7 μm) were blocked by 3% H2O2 for 10 min and incubated with anti-Ki67, anti-p-Smad2/3 and anti-MMP2 antibody (Abcam, Cambridge, MA, USA) at 4 °C overnight. The slides were then stained with horseradish peroxidase (HRP)-labeled IgG (Shanghai Long Island Biotec, Shanghai, China) at 25 °C. Subsequently, the sections were stained with diaminobenzidine (DAB), counterstained with hematoxylin and washed in water. The immunoreactive cells were counted in five visual fields of each section under a 200 × light microscope.
Cell culture and transfection
Human high metastatic HCC cell line MHCC-97H and low metastatic HCC cell line MHCC-97L, and immortalized normal human liver cell line LO2 were obtained from ATCC (Manassas, VA, USA) and cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (HyClone, Logan, UT, USA) complemented with 10% fetal bovine serum (FBS) (Gibco, Detroit, MI, USA) along with 1% Penicillin-Streptomycin solution separately incubated in 37 °C with 5% CO2 and 95% air.
Oligonucleotides encoding short hairpin RNA (shRNA) targeting human TRIM52 (point 670–692, 5’-GGGCATGTGCTTTAAACAC-3′) and scramble shRNA were cloned into the pLKO.1 lentiviral vector. The cDNA encoding TRIM52 and PPM1A was obtained by reverse transcription PCR (RT-PCR) and cloned into pLVX-Puro for constructing pLVX-Puro-TRIM52 and pLVX-Puro-PPM1A expressing vector, respectively. pLKO.1-scramble shRNA (NC) and blank pLVX-Puro (Vector) were used as the negative controls. 293T cells were plated in 6-well plates and transfected with constructs for 4–6 h using Lipofectamine Reagent (Invitrogen, Carlsbad, CA, USA) according to the instructions of the manufacturer. After incubation in a CO2 incubator at 37 °C, recombined lentivirus was collected 48 h after transfection and used for MHCC-97H and MHCC-97L cells infection.
Cell proliferation
After indicated transfection, MHCC-97H and MHCC-97L cells (4 × 103 cells/well) were plated in 96-well plates and cultured for 0, 24, 48 and 72 h, and the cells were added with 10 μL Cell Counting Kit-8 (CCK-8) solution (Signalway Antibody, College Park, MD, USA) with 5% CO2 at 37 °C for 1 h, after which the absorbance readings were obtained at 450 nm.
Cell cycle
Propidium iodide (PI) staining was performed in MHCC-97H and MHCC-97L cells. Briefly, the cells were trypsinized, resuspended in phosphate buffer solution (PBS) containing 10% FBS and fixed in 70% chilled ethanol before cultured in 10 mmol/L RNase at 37 °C for 10 min. After 5 min centrifugation at 1000×g, the cells were resuspended with RNase A (1 mg/mL) for 30 min. Then PI (50 μg/mL) was added in the cells for 10 min at 37 °C under dark. The fluorescence intensities were assessed by flow cytometer (BD Biosciences, San Jose, CA, USA).
Transwell assay
The invasive ability of MHCC-97H and MHCC-97L cells to pass through filters was measured using a Transwell insert coated with Matrigel. Briefly, MHCC-97H and MHCC-97L cells transfected with indicated lentivirus vectors were serum-starved for 24 h, following which 5 × 103 cells/well in 300 μL serum-free DMEM were placed in the upper chamber at 37 °C. The DMEM medium containing 10% FBS (700 μL) was added into the lower chamber. After 48 h incubation, the cells migrated through to the bottom surface of the membrane were washed with PBS, fixed, stained with 0.5% crystal violet and counted. The invasive cells found on the bottom site of each inserts were then photographed and counted under a microscope (× 200; Olympus, Tokyo, Japan). The migratory ability of MHCC-97H and MHCC-97L cells was measured using Transwell without Matrigel coated.
Quantitative real-time PCR (qRT-PCR)
Whole RNA was extracted from the HCC cell lines by using TRIzol reagent (Invitrogen) and stored at − 80 °C in RNA secure RNase Inactivation Reagent (Thermo Fisher, St. Louis, MO, USA), and PrimeScript reverse-transcription reagent Kit (Thermo Fisher) was used to carry out reverse transcription reaction on RNA in accordance with the protocols of the manufacturer. Quantitative analysis on the change in expression level was conducted by SYBR Green qPCR Master Mixes (Thermo Fisher) and performed using the GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA, USA). The primer sequences were shown subsequently:
-
TRIM52 forward: 5’-GCCATCTGCTTGGATTACTTC-3′;
-
TRIM52 reverse: 5’-TTCATCTTCCTCCTCGTTCTG-3′;
-
PPM1A forward: 5’-CCCTTGTTTCCTCTACTTTC-3′;
-
PPM1A reverse: 5’-TAATCCTTCCCTACCTATCC-3′;
-
GAPDH forward: 5’-AATCCCATCACCATCTTC-3′;
-
GAPDH reverse: 5’-AGGCTGTTGTCATACTTC-3′.
The change in the expression of mRNA was assessed by the 2-ΔΔCt approach.
Western blot assay
Total protein extraction was performed with RIPA Lysis Buffer (Solarbio, Beijing, China) and then centrifuged at 12,000×g for 10 min. Proteins in cell lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA), then incubated into TRIM52, p21, matrix metalloproteinase 2 (MMP2), PPM1A, p-Smad2/3, Smad2/3 or GAPDH antibodies overnight at 4 °C. The next day, the membranes were washed and subsequently incubated with HRP-conjugated secondary antibodies (1:1000; Beyotime, Nanjing, Jiangsu, China) for 1 h at 37 °C. Then, the membranes were developed using an enhanced chemiluminescence (ECL) detection Kit (Pierce Biotechnology, Rockford, IL, USA).
Co-immunoprecipitation (Co-IP) and ubiquitination in vitro
Co-IP was performed as described previously [
20,
21]. Briefly, cold PBS was used to wash the cells for three times, and the cells were scraped into lysis buffer containing complete protease inhibitors and centrifuged at 14,000×g for 20 min at 4 °C. The supernatants were incubated with normal IgG or anti-HA tag antibody, and the immunocomplexes were then associated with Protein A-Sepharose. Anti-TRIM52 (1:500), anti-PPM1A (1:500), anti-HA-tag (1:500) and normal rabbit/mouse IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used for Western blot analysis. And the blots were immunoblotted by using anti-HA for ubiquitin.
Nude mouse xenograft experiment
Twelve nude mice (4–5 week-old BALB/c; Shanghai SLAC laboratory animal Co., Ltd., Shanghai, China) were housed in the animal facility at 25 °C with humidity of 60–70%. MHCC-97H cells (4 × 106) transfected with pLKO.1-shRNA-TRIM52 or pLKO.1-scramble shRNA (NC) were subcutaneously injected into each of the six nude mice, respectively. Tumor volume (mm3) was calculated using the following standard formula: (the longest diameter) × 0.5 × (the shortest diameter)2. After 33 days, the mice were sacrificed and the tumors were weighted. Care of the laboratory animals and animal experimentation were performed in accordance with the animal ethics guidelines and approved protocols of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital.
Statistical analysis
Data are presented as mean ± standard deviation (SD), and each test was repeated at least three times. The independent-samples t test was applied to two-group analyses, while one-way ANOVA and post hoc Bonferroni test was used when analyzing more than two groups. All statistical analyses were carried out with the GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA). Two-tailed P < 0.05, the difference between groups was considered to be statistically significant.
Discussion
Cellular carcinogenesis is a multistep process involving multiple factors and genes, which is accompanied by changes in a variety of gene expression patterns and which in turn affects the proliferation, apoptosis and differentiation modulated by these genes. The occurrence and development of HCC is also a complex process with multiple genes and steps [
22,
23], so it is of great theoretical and practical significance to elucidate the abnormal expression of genes in the process of HCC carcinogenesis. In the present study, we found that TRIM52 was up-regulated in HCC tissues and cell lines. TRIM52 expression was correlated with tumor size, TNM stages and tumor number. Up-regulation of TRIM52 promoted HCC cell proliferation, migration and invasion in vitro and cell growth in vivo through the ubiquitination of PPM1A. Moreover, PPM1A up-regulation inhibited TRIM52-mediated enhancement of HCC cell proliferation, migration and invasion.
A number of recent studies have focused on the function of TRIM proteins in HCC. TRIM3, TRIM16 and TRIM26 down-regulation contributes to poor prognosis in patients with HCC, suggesting the tumor suppressor function in HCC [
24‐
26]. On the contrary, TRIM11 and TRIM31 function as oncogenes of HCC, showing up-regulation in HCC tissues and contributing to HCC cell proliferation and invasion [
27,
28]. Except for up-regulation in HCC tissues and cell lines as well as enhancement of HCC cell proliferation reported in our recent study, no other effect has been reported in HCC carcinogenesis involving TRIM52 [
19]. In line with the recent findings, our results also demonstrated TRIM52 up-regulation in the HCC tissues compared with the adjacent non-tumor hepatic tissues, and in HCC cell lines, including high metastatic MHCC-97H and low metastatic MHCC-97L, compared with normal human liver cell line LO2. TRIM52 up-regulation promoted HCC cell proliferation, migration and invasion in vitro, and down-regulation of TRIM52 inhibited HCC cell invasion, migration and proliferation, induced G0-G1 phase cell cycle arrest in vitro, and inhibited cell growth and Ki67, p-Smad2/3 and MMP2 expression in vivo.
Next, we studied the mechanism by which TRIM52 regulated HCC carcinogenesis. p21 is considered as a regulator of cell cycle progression at G1 through binding to and inhibiting the activity of cyclin D-CDK2 or cyclin D-CDK4 complexes [
29]. Previous studies have indicated that MMP2 activation can enhance invasion, migration and metastasis of HCC [
30,
31]. PPM1A down-regulation increased epithelial-to-mesenchymal transition (EMT) progress and invasion through increasing the activity of Smad2/3 signaling pathway in bladder cancer [
32]. Simultaneous Smad2/3 phosphorylation was significantly associated with poor outcome of HCC patients after surgery [
33] and increased HCC cell growth, invasion and migration [
34,
35]. In this study, our results demonstrated that TRIM52 up-regulation inhibited p21 and PPM1A expression, increased MMP2 expression and induced Smad2/3 phosphorylation in HCC cells, which were reversed by TRIM52 down-regulation. These results suggest that these proteins may associate with TRIM52-mediated HCC cell proliferation, invasion and migration.
PPM1A inhibited the invasion and migration of HCC cells and induced the dephosphorylation of Smad2/3 [
11‐
13], suggesting an opposite effect between PPM1A and TRIM52 in HCC. It therefore provides a hypothesis that TRIM52 might involve in HCC carcinogenesis through down-regulating PPM1A. Co-IP and ubiquitination assay confirmed that TRIM52 interacted with PPM1A and TRIM52 down-regulation inhibits the ubiquitination of PPM1A, which was similar to the regulatory mechanism of PPM1A in HBV-related HCC [
13]. Moreover, down-regulation of PPM1A via promotion of proteasomal degradation and ubiquitination may result in HCC cell invasion and migration [
12]. However, conclusive molecular mechanism by which TRIM52 enhances the ubiquitination of PPM1A need to be further studied. Our experiments demonstrated that PPM1A up-regulation inhibited TRIM52-mediated enhancement of the proliferation, migration and invasion of HCC cells.