Background
Colorectal cancer (CRC) is the third most common form of gastrointestinal cancers, with more than one million newly diagnosed cases each year in the world [
1]. The incidence of CRC in women and men is 9.2% and 10%, respectively, which makes CRC the second most common among women, and the third most common cancer among men [
2‐
4]. Although continued efforts have made to improve the understanding of tumorigenesis, and numerous oncogenes and tumor suppressor genes involved in CRC tumorigenesis have been identified, the biological and molecular mechanisms of CRC are still far from being fully understood.
Signal transducer and activator of transcription 3 (STAT3), a transcription factor, normally resides in the cytoplasm. When its tyrosine-705 residue is phosphorylated in response to cytokine signaling and tyrosine kinase oncoproteins, STAT3 translocates to the nucleus and controls the transcription of downstream genes that are involved in cell cycle transition and cell survival [
5,
6]. Activated phospho-STAT3 (p-STAT3) is found to be increased in CRC samples compared to normal mucosae [
7‐
9]. The Janus family kinases (JAK) are known to mediate the activation of STAT3 [
10]. AG490, a pharmacological inhibitor of JAK, and STAT3 small interfering RNA (siRNA) could suppress CRC cell growth and invasion, and induce CRC cell apoptosis [
9].
The tripartite motif (TRIM) family protein is characterized by its tripartite motif, which contains a RING domain, one or two B-box domains and a coiled-coil domain [
11]. More than 80 members have been identified in human until now. Substantial evidence has accumulated and supported the roles of TRIM proteins in innate immune response, cell proliferation, apoptosis and autophagy [
11,
12]. The dysfunction of TRIM family proteins is known to be associated with the pathogenesis of several diseases, including various human cancers [
12‐
14]. TRIM52 is a novel member of the TRIM protein family [
15]. TRIM52 can degrade the viral nonstructural protein 2A (NS2A), thus exerting antiviral activity against Japanese encephalitis virus (JEV) infection [
16]. TRIM52 is able to active the NF-κB signaling pathway [
17]. Recently, the oncogenic role of TRIM52 has been described in hepatocellular carcinoma (HCC) [
18,
19]. Hepatitis B virus X protein (HBx) may regulate TRIM52 expression [
19]. TRIM52 can facilitate cell proliferation, migration and invasion of HCC cells [
18].
Previous studies have revealed the significance of TRIM52 in HCC, but no investigation has focused on the effects of TRIM52 on CRC. In the current study, we examined the expression and function of TRIM52 in CRC. We found that the STAT3 signaling was involved in the abilities of TRIM52 to induce the proliferation and inhibit apoptosis in CRC cells.
Materials and methods
Tissue collection
This study included 80 patients with CRC who underwent surgery at the Department of Gastrointestinal Surgery, Shanghai Eighth People’s Hospital from January 2008 to December 2011. Clinical pathology features, including gender, age, tumor stage, tumor size, and histological type, were retrieved from the medical records (Table
1). The mean age of the enrolled patients ranged from 58 years (34–75 years), including 43 males and 37 females. Collected CRC tissues (n = 80) and adjacent normal colonic mucosa were formalin-fixed paraffin-embedded. This study was approved by the Ethics Review Committee of Shanghai Eighth People’s Hospital (Shanghai, China). All participants received written informed consent.
Table 1
Clinicopathological characteristics and TRIM52 expression (n = 80)
Gender
|
Male | 43 | 53.8 |
Female | 37 | 46.2 |
Age (years)
|
≥ 65 | 47 | 58.8 |
< 65 | 33 | 41.2 |
Tumor size (cm)
|
≥ 5.0 | 45 | 56.3 |
< 5.0 | 35 | 43.7 |
Clinical stage
|
I/II | 37 | 46.3 |
III | 43 | 53.7 |
Histological types
|
Non-mucinous adenocarcinoma | 60 | 75.0 |
Mucinous adenocarcinoma | 20 | 25.0 |
TRIM52 expression
|
Low | 32 | 40.0 |
High | 48 | 60.0 |
Vital status (at followed-up)
|
Alive | 20 | 25.0 |
Dead | 60 | 75.0 |
Immunohistochemistry (IHC) analysis
For IHC analyses, formalin-fixed paraffin-embedded tissue samples were cut into 5 μm-thick section. Antigen retrieval was performed in 0.01 M citrate sodium buffer solution (pH = 6.0) at 100 °C for 10 min. Endogenous peroxidase was blocked in 0.3% H2O2 at room temperature for 15 min. Non-specific binding was blocked by incubation in 10% bovine serum albumin 10% for 30 min. Then, a rabbit primary antibody against TRIM52 (diluted in 1:50; Novus Biologicals, Inc., Littleton, CO, USA; NBP2-31651) was added for overnight incubation at 4 °C. After washed three times in phosphate-buffered saline, anti-rabbit IgG was applied for 1 h incubation at room temperature. The sections were developed with DAB substrate and counterstained with hematoxylin. The stained sections were evaluated by two investigators, and classified into the TRIM52-high expression group and the TRIM52-low expression group with 20% of tumor cell positive stained as cut-off.
Cell culture
Five human CRC cell lines (SW480, LoVo, SW620, HT29 and RKO) and normal human intestinal crypt cells (HIEC) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All the cells were grown in DMEM medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Invitrogen) and 1% penicillin/streptomycin in a humidified air with 5% CO2 at 37 °C.
Lentiviral construction
Four TRIM52-specific target sequences were designed using Dharmacon siDESIGN Center (Dharmacon, Waltham, USA). Short hairpin RNAs (shRNAs) targeting these sequences (RNAi#1, RNAi#2, RNAi#3 and RNAi#4) and a negative control sequence (NC) were synthesized (Additional file
1: Table S1) by Genechem (Shanghai, China), and constructed into pLKO.1 vector (Addgene, Cambridge, MA, USA). The coding sequence of TRIM52 was amplified by reverse transcription PCR with the following primers (forward 5′-CGGAATTCATGGCTGGTTATGCCACTACTC-3′ and reverse 5′-CGGGATCCTTACTGATTATAGGCCTTGCTG-3′) and constructed into pLVX-puro vector (Clontech, Palo Alto, CA, USA). TRIM52 shRNAs lentiviruses, TRIM52 overexpression lentivirus (TRIM52OE), and control (NC and Vector) lentiviruses were produced by transient transfection of lentiviral constructs and packaging vectors into 293T cells with Lipofectamine 2000 (Invitrogen).
Immunoblot assay
Total protein was extracted by radio-immunoprecipitation assay (RIPA) buffer containing phosphatase and protease inhibitors (Beyotime, Shanghai, China). Equal amounts of protein from each sample were separated by 10% or 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and then electro-transferred onto nitrocellulose membranes (Millipore, Bredford, MA, USA). Following blocking with 5% skim milk in Tris-buffered saline Tween-20 (TBST) for 30 min at room temperature, the membranes were probed with the following primary antibodies: TRIM52 antibody from San Cruz (Santa Cruz, CA, USA), Bcl-2, Bax, p-STAT3, STAT3, PTP1B, TCPTP, SHP1, SHP2 and ubiquitin (Ub) antibodies from Abcam (Cambridge, MA, USA), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody from Cell Signaling Technology (Danvers, MA, USA) according to the manufacturers’ protocols. After being rinsed by TBST, the membranes were incubated with incubated with peroxidase labeled secondary antibody at room temperature for 1 h. Signal was detected with enhanced chemiluminescence substrate (ECL, BioRad, Richmond, CA, USA).
Immunoprecipitation
Total protein was incubated with protein A/G Plus agarose beads (Santa Cruz Biotech., Santa Cruz, CA, USA) together with specific antibodies. After incubation for 4 h at 4 °C, protein A/G Plus beads were washed three times with RIPA buffer. Immunoprecipitates were mixed with SDS-PAGE loading buffer and boiled at 95 °C for 5 min. The immunoprecipitates were analyzed by western blotting with antibodies against TRIM52, SHP2 or ubiquitin (Ub).
Cell Counting Kit-8 (CCK-8) assay
Cells incubated in 96-well plates were treated as indicated and cell proliferation was assessed by CCK-8 assay (SAB biotech. College Park, MD, USA) at 0, 24, 48 and 72 h post treatment following the manufacturer’s instruction. Optical density (OD) was recorded at 450 nm.
Flow cytometry
At 48 h post treatment, cell apoptosis was assessed by the Annexin V-FITC/PI apoptosis detection kit (Beyotime) and BD Biosciences Accuri C6 flow cytometer (Franklin Lakes, NJ, USA) following the manual procedure.
Tumor xenograft implantation in nude mice
The animal study followed the Guidelines for the Animal Care and Use approved by Shanghai Eighth’s People Hospital. Nude mice, aged 5–6 weeks old, were housed in a specific pathogen free (SPF) grade laboratory with a constant temperature (22–25 °C) and humidity (55 ± 5%). A total of 12 mice were allocated into RNAi#1 group and NC group with 6 mice in each group. LoVo cells stably expressing RNAi#1 or NC were established by puromycin selection. The stable cells were resuspended in serum-free DMEM were injected into each mouse (106 cells per mouse). Tumor volume was calculated with the following formula: volume = 1/2 × (largest diameter) × (smallest diameter)2 every 3 days after inoculation. After 33 days, the nude mice were sacrificed, and the xenografts were collected, weighed and processed for TUNEL (Terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridine triphosphate (dUTP)-nick end labeling) staining (Roche, Indianapolis, IN, USA).
Statistical analysis
Data analysis was performed with GraphPad Prism software (GraphPad Software, La Jolla, CA, USA). Fisher’s exact test was conducted to analyze the relationship between TRIM52 expression and clinical features. Kaplan–Meier survival curves followed by log-rank test were used to compare overall survival of different groups. The in vitro experiments were repeated at least three times. Student’s t test and one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison were carried out for comparison of two groups and for comparison of three or more groups, respectively. p < 0.05 was considered significant.
Discussion
The dysfunction of TRIM family proteins implicates in the pathogenesis of various human cancers [
12‐
14]. Recently, studies have investigated the functions of TRIM proteins in CRC. TRIM24 [
28] and TRIM29 [
29] are up-regulated in CRC tissues and significantly correlated with poor prognosis. Studies have demonstrated the oncogenic roles of TRIM24 [
30], TRIM27 [
31], TRIM29 [
29,
32] and TRIM59 [
28] in CRC. TRIM52, a novel member of TRIM family proteins, has been reported to be up-regulated in HCC and to promote cell proliferation, migration and invasion of HCC cells [
18,
19]. Nevertheless, the roles and mechanisms of TRIM52 in colorectal carcinogenesis have not been investigated. In present study, we provided new evidence that TRIM52 expression was elevated in CRC tissues (Fig.
1a) and cell lines (Fig.
2a), and that TRIM52 expression was significantly correlated with tumor size, tumor stage and overall survival of CRC patients (Fig.
1b and Table
2). Furthermore, knockdown of TRIM52 expression in CRC cells significantly suppress cell proliferation due to the induction of apoptosis in vitro (Figs.
2,
3) and in nude mice (Fig.
4). Evidence suggested that cellular senescence is another key event contributing to the anticancer response [
33]. The possible effect of TRIM52 knockdown on cell senescence could not be ruled out and further work will be necessary. Nevertheless, all of these findings indicate that TRIM52 may serve as an oncogene in CRC, which was consistent with its role in HCC [
18,
19].
Activated STAT3 is found to be increased in CRC samples compared to normal mucosae [
7‐
9]. AG490 and STAT3 knockdown could suppress CRC cell growth and invasion, and induce CRC cell apoptosis [
9]. A recent study reported that TRIM8 interacts with protein inhibitor of activated STAT3 (PIAS3), thus enhancing the STAT3-dependent signaling [
34]. TRIM29 knockdown in CRC cells led to a notable reduce in the phosphorylation levels of STAT3 [
32]. Here, we tried to explore the association of TRIM52 and the STAT3 signaling in CRC. TRIM52 knockdown reduced the phosphorylation of STAT3 at Tyr705 in both CRC cells and xenograft tumors (Fig.
5). SHP2, a tyrosine phosphatase, has been shown to negatively regulate the STAT3 signaling [
27]. TRIM52 interacted with SHP2 and promotes its ubiquitination, whereas the ubiquitination site on SHP2 is to be identified. Further, we found that JAK2 inhibitor AG490 can block the promotional effects of TRIM52 overexpression on CRC cell proliferation (Fig.
6). These data suggest that TRIM52 may promote SHP2 ubiquitination, thus inactive the STAT3 signaling and serve as an oncogene in CRC.
Bcl-2 family proteins, including anti-apoptotic factor Bcl-2 and proapoptotic factor Bax, are important for the regulation of apoptosis. It seems that abnormal activation of the bcl-2 gene is an early event in colorectal tumorigenesis [
35]. The protein levels of Bcl-2 [
36] and Bax [
37] may be potential prognostic indicator for CRC although controversial results exist. Inhibition of the STAT3 pathway could decrease the Bcl-2 expression and increase Bax expression in CRC cells [
9,
38]. Here, TRIM52 knockdown decreased Bcl-2 expression and increase Bax expression (Fig.
3), which may due to the decreased phosphorylation of STAT3. Complementary data were obtained in TRIM52 overexpressed cells (Fig.
6c). The increased apoptosis rate of CRC cells with TRIM52 knockdown may ascribed to the decreased ratio of Bcl2/Bax.
Conclusion
Our data also revealed that TRIM52 could promote CRC cell proliferation by inhibiting cell apoptosis through the STAT3 signaling pathway. Collectively, these findings provide new insight into the role of TRIM52 in CRC, which might serve as a prognostic indicator and a novel therapeutic target for CRC treatment.
Authors’ contributions
SLP and XJQ designed the study; SLP, YYD, JF, YHZ and ZJZ performed the experiments and prepared the figures; SLP, XKR and XJQ contributed to drafting the manuscript. All authors read and approved the final manuscript.