Background
Lung cancer is the most frequently diagnosed malignancy. It is the leading cause of cancer death with over 1 million death annually in the world [
1]. Non-small cell lung carcinoma (NSCLC) is the most frequently occurring of lung cancer and accounts for approximately 85 % of lung cancer [
1]. NSCLC includes adenocarcinoma (ADC), squamous cell carcinoma (SCC), large cell carcinoma (LCC) and others [
2]. Despite recent advances in diagnosis and treatment, the prognosis of lung cancer is still poor [
3]. Therefore, a better understanding of which pathways or proteins are active in lung tumor progression will contribute to the development of early detection and targeted therapy for lung cancer [
4‐
6].
Tripartite Motif Containing (TRIM) proteins are characterized by the presence of tripartite motif, which is composed of a RING domain, 1 or 2 B-box motifs and a coiled-coil region (RBCC) [
7]. Most members of TRIM proteins, including TRIM11 could be defined as E3 ubiquitin ligases. Besides RBCC domain, TRIM11 contains a PRY domain and a SPRY domain. TRIM11 is thought to destabilize Humanin (24-amino-acid neuroprotective peptide) [
8], activator-recruited cofactor 105-kDa component (ARC105) [
9], PAX6 (a member of the paired-box family of transcription factors) [
10] and PHOX2B (a paired box homeodomain transcription factor) [
11]. Earlier studies on TRIM11 have revealed its roles in nervous system function and development [
8,
10,
11]. Recently, TRIM11 expression was found elevated in high-grade gliomas and it may exert an oncogenic function in glioma biology [
12]. Other members of TRIM proteins, such as TRIM25 [
13] and TRIM59 [
14], have been reported to be upregulated in lung cancer, while TRIM16 [
15] and TRIM31 [
16] were found decreased in NSCLC. However, few investigation has been performed to test the expression and functions of TRIM11 in lung cancer.
In this study, TRIM11 expression was frequently higher in lung cancer tissues than corresponding adjacent non-neoplastic tissues. We investigated whether TRIM11 regulated cell proliferation and metastasis of lung cancer. Our study showed that TRIM11 promoted cell migration and invasion by activating the PI3K/AKT signal pathway. Our findings suggest that TRIM11 is a new potential target in lung cancer.
Methods
Samples
120 patients with lung cancer undergoing surgical resection at Department of Thoracic Surgery, Northern Jiangsu People’s Hospital (Yangzhou, China) were enrolled in this study. The age of enrolled patients was between 34 and 72 (median 56) years. 63 participants (52.5 %) were male and 57 (47.5 %) were female. Primary lung cancer tissues were collected from all enrolled patients, while adjacent non-cancerous tissues were obtained from 35 patients of the enrolled patients. The follow-up lasted 5 years. The study was reviewed and approved by Research Ethic Committee in Northern Jiangsu People’s Hospital (Yangzhou, China). Written informed consent was obtained from all patients.
RNA isolation and real-time PCR
Total RNA from tissues and cells was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The residual DNA was removed by treating with DNase I (Roche, Indianapolis, IN, USA). Real-time PCR was used to evaluate TRIM11 mRNA levels. Total RNA (2 μg) was reverse-transcribed to cDNA with M-MLV Reverse Transcriptase Kit (Thermo Fisher, Rockford, IL, USA). The resulted cDNA was used for real-time PCR with SYBR Green qPCR Master Mix (Thermo Fisher) on ABI 7300 system (Applied Biosystem, Foster City, CA, USA) following manufacturer’s instruction. The relative TRIM11 mRNA levels were calculated by normalization to GAPDH mRNA levels. The PCR primers were as follows: TRIM11 forward, 5’- CACCTAAGCTGCACAGTTCC-3’; TRIM11 reverse, 5’- GGCTGCCTCCTAATTCTTCC -3’; GAPDH forward, 5’-CACCCACTCCTCCACCTTTG-3’; GAPDH reverse, 5’- CCACCACCCTGTTGCTGTAG -3’.
Western blotting
Frozen tissue sample (about 0.1 g) was ground into powder using liquid nitrogen. Protein was extracted from frozen tissue powder and cultured cells by using RIPA lysis buffer (JRDUN Biotech., Shanghai, China) with fresh-added proteinase inhibitor cocktail (Sigma, St. Louis, MO, USA) on ice for 15 min and centrifuged at 12,000 rpm for 20 min. Protein concentrations were determined using a bicinchoninic acid kit (Thermo Fisher). For Western blotting analysis, lysates (30 μg/well) were subjected to SDS-PAGE and transferred onto nitrocellulose filter membranes. After blocking in 5 % skim milk at room temperature for 30 min, the membranes were incubated with primary antibodies overnight at 4 °C. Horseradish peroxidase conjugated secondary antibodies were subsequently used. Signals were detected using chemiluminescenct substrate (ECL; Bio-Rad, Richmond, CA, USA) and exposed to X-ray film. The results were scanned and analyzed using Image J software (
http://rsb.info.nih.gov/ij/, Bethesda, MD, USA). Antibodies against anti-TRIM11, proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, MMP-9, Twist1, PI3K and p-PI3K were from Abcam (Cambridge, MA, USA). Antibodies against CyclinD1, Snail, E-cadherin (CDH1), AKT, p-AKT, ERK, p-ERK and GAPDH Cell Signaling Technology (Danvers, MA, USA).
Cell culture
Human lung adenocarcinoma cell lines A549, NCI-H1975 and PC-9, human large cell lung carcinoma cell lines NCI-H460, small cell lung cancer cell line NCI-H446 and human embryo lung fibroblasts MRC-5 were obtained from cell bank of Shanghai Biology Institute, Chinese Academy of Science (Shanghai, China). The lung cancer cell lines were cultured in RPMI 1640 medium (Invitrogen; Carlsbad, CA, USA). MRC-5 cells were maintained in Eagle’s Minimum Essential Medium (EMEM). Both medium were supplemented with 10 % fetal bovine serum (FBS; Gibco, Los Angeles, CA, USA), 1 % penicillin/streptomycin and 2 mM L-glutamine. All cell lines were cultured in at 37 °C, 5 % CO2.
Knockdown of TRIM11 expression by small interference RNA (siRNA) transfection
Three siRNA targeting human TRIM11 mRNA (siRNA1, 5’- CAGAAGUUGUGCCUAUGGA -3’; siRNA2, 5’- GCUAUUACAAUUCCUCGGA -3’; and siRNA3, 5’- CUAUUCAUCUUUCCCGAGA -3’) and a non-specific control siRNA sequence (NC) were synthesized by Genepharma (Shanghai, China) and transfected into A549 and NCI-H446 cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) per the manufacture’s instruction. Real-time PCR and Western blot analysis were performed at 48 h after transfection to assess knockdown efficiency.
Construction of TRIM11 lentivirus
Full-length human TRIM11 was cloned into the pLVX-AcGFP1-C1 (Clontech, Palo Alto, CA, USA). Lentiviral constructs of pLVX-AcGFP1-C1 empty vector or pLVX-AcGFP1-C1-TRIM11 were cotransfected with viral packaging plasmids (psPAX2 and pMD2.G) into 293 T cells with Lipofectamine 2000. At 48 h post transfection, viral supernatant was collected to infected NCI-H460 and NCI-1975 cells.
Cell proliferation assay
A proliferation assay was carried out using Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan) according to the manufacturer’s protocol [
17]. Briefly, 2000 cells/well were seeded into 96-well plates and transfected with indicated siRNA. The cells were cultured for 0, 24, 48 and 72 h, then 10 μL CCK-8 reagent was added to each well and the cells were incubated at 37 °C for 1 h. The absorbance was recorded at 450 nm with a microplate reader (Bio-Rad).
Transwell migration and invasion assays
Cell migration was assayed using Transwell with 8-μm-pore filters (Corning; New York, NY, USA) as previously described [
5]. Cells were treated with the desired siRNA or expression virus. At 24 h after treatment, cells were trypsinized, resuspended in serum-free RPMI1640 medium and placed in the upper chamber (5 × 10
4 cells/well) and treated with MK-2206 (Merck, Germany) or DMSO (Sigma). Then RPMI 1640 medium supplemented with 10 % FBS was added to the lower chamber. Non-migrating cells in the upper chamber were completely removed with a cotton swab 24 h later. Migrated cells were stained with 0.5 % crystal violet and counted in five random fields.
Cell invasion assays were also performed in the same condition expect that the upper chamber was pre-coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA).
Statistical analysis
All values are expressed as the mean ± SD. Statistical analysis was tested using Graphpad Prism (Graphpad Software, San Diego, CA, USA). For comparison between groups, statistical differences were tested with one-way analysis of variance (ANOVA), followed by a Sidak’s test for multiple comparisons. Fisher’s exact test was carried out to evaluate the relationship between TRIM11 mRNA expression and clinicopathological features. Kaplan-Meier survival curves were generated and compared by log-rank analysis. P < 0.05 was considered significantly different.
Discussion
The altered expression of TRIM proteins has been reported in a variety of human cancers, including lung cancer [
13‐
16]. Di, K. et al. demostrated diagnostic and prognostic value of TRIM11 in gliomas [
12]. However, the expression and function of TRIM11 in lung cancer has been poorly characterized. Here, by analyzing online available dataset (Fig.
1a), we found that TRIM11 mRNA was elevated in lung cancer tissues, which was confirmed by real-time PCR analysis on 120 patients admitted at Department of Thoracic Surgery, Northern Jiangsu People’s Hospital (Fig.
1b). Fisher’s exact tests (Table
1) and Kaplan-Meier survival curves (Fig.
1c) demonstrated the strong association between TRIM11 expression and tumor size, TNM stage, lymph node metastasis, as well as overall survival of lung cancer patients. Thus, TRIM11 may serve as a useful diagnosis and prognosis marker for lung cancer although further in-depth clinical study is needed.
Then we investigated the functions of TRIM11 in lung cancer. The in vitro experiments demonstrated that inhibition of TRIM11 expression in lung cancer cells with higher expression of TRIM11 (A549 and NCI-H446 cells) suppressed cell growth (Fig.
3), migration (Fig.
4a) and invasion (Fig.
4b). On the contrary, ectopic expression of TRIM11 in in lung cells with lower expression of TRIM11 (NCI-H460 and NCI-H1975 cells) had inverse effects (Additional file
1: Figure S2 and Fig.
6). These data suggest that TRIM11 may play an oncogenic role by promoting the proliferation, migration and invasion of lung cancer cells. Our results of in vitro experiments were consistent to our clinical observation that high expression of TRIM11 associated with lymph node metastasis and tumor size. Furthermore, cancer cells gain migratory and invasive properties through the process of EMT [
22]. Functional loss of E-cadherin has been considered as a key event during the process of EMT [
23]. Twist1 and Snail can repress the expression of E-cadherin, and has been regarded as EMT-inducers [
24]. VEGF [
25] and MMP-2/9 [
26] are well-known to play an important role in tumor metastasis. Here, knockdown of TRIM11 decreased the expression of metastasis (VEGF, MMP-2 and MMP-9) and EMT-related proteins (Twist1 and Snail), while increased the expression of E-cadherin. Our data suggested that TRIM11 siRNA may inhibit lung cell invasion via suppressing EMT.
Further, in addition to cellular phenotype, we tried to investigate the molecular mechanism through which TRIM11 works as an oncogene in lung cancer. ERK and PI3K/AKT pathways can promote cell growth and metastasis [
27,
28]. Both ERK [
20] and PI3K/AKT [
21] pathways are frequently over-activated in various tumor types. Di, K. et al. found that knockdown of TRIM11 in glioblastoma multiforme cells had no effects on PI3K/AKT activity, but suppressed ERK activity [
12]. In the current study, we found a notable decrease of ERK and PI3K/AKT activation (Fig.
5) in TRIM11 knocked down lung cancer cells. The discrepancy of data and the previous study [
12] could be due to the different cell types used. Accumulating evidence points to the important role of cell motility and invasion in cancer progression and metastasis [
29]. Phosphorylation levels of AKT was found to be associated with the invasion and metastasis of NSCLC [
30]. In the present study, TRIM11 overexpression can promote the PI3K/AKT pathway (Additional file
1: Figure S2). The promotion effects of TRIM11 overexpression on cell migration and invasion were significantly weakened by the exposure of AKT inhibitor, MK-2206 (Fig.
6), which suggested that TRIM11 might promote cell migration and invasion partially by activating AKT pathway. Thus, we speculated that elevated TRIM11 will facilitate the development and invasive property of lung cancer through AKT pathways. However, the mechanisms are needed to be further elucidated in detail.
Abbreviations
ARC105, activator-recruited cofactor 105-kDa component; CCK-8, Cell Counting Kit-8; EMEM, Eagle’s Minimum Essential Medium; EMT, epithelial-mesenchymal transformation; ERKs, extracellular signal-regulated kinases; FBS, fetal bovine serum; MMP, matrix metalloproteinase; NSCLC, non-small cell lung carcinoma; PCNA, proliferating cell nuclear antigen; TCGA, The Cancer Genome Atlas project; TIRM, Tripartite Motif Containing; VEGF, vascular endothelial growth factor.
Acknowledgments
Not applicable.