Background
The leading cause of cancer mortality is lung cancer in the worldwide. Non–small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for more than 85% of all lung cancer cases[
1]. Despite the enormous improvements made in chemotherapy and radiotherapy over the past few decades, the outlook for patients with NSCLC was dismal, with only slightly more than 15% of patients alive 5 years after diagnosis. NSCLC can be further classified into adenocarcinomas, carcinoma, large cell carcinoma and bronchoalveolar carcinomas (BAC)[
2]. The distant metastases are responsible for the failure of lung cancer therapy and the poor prognosis of lung cancer. However, the mechanisms of metastasis have not yet been fully elucidated.
MicroRNAs (miRNAs) are small, non-coding RNA molecules that negatively regulate gene expression, mainly through direct interaction with the 3’-untranslated region (3’-UTR) of corresponding target messenger RNAs (mRNA)[
3]. After binding to target mRNAs, miRNAs form a complex with target mRNAs and decrease the levels of the encoded protein, either by degrading the mRNA or by suppressing translation of the target mRNA[
4]. It has been reported that miRNAs can post-transcriptionally regulate 30% of human genes, thereby suggesting that miRNAs may have pivotal roles in physiological and pathological processes, including human carcinogenesis[
5]. Over the past 10 years, evidence has emerged that miRNAs were crucial for the initiation, promotion, and progression of human cancers. A large number of miRNAs have recently been implicated in cancer metastasis[
6]. For example, miR-155, miR-222, miR-210, miR-107, and miR-10a have a role in pancreatic cancer[
7‐
9], miR-148a, miR-23a, and miR-193a in hepatocellular carcinoma[
10‐
12], and miR-200, miR-218 in gastric cancer[
13,
14]. Furthermore, miR-133b, miR-143 and miR-32 in colorectal cancer[
15‐
17]. However, only a few miRNAs known involved in NSCLC metastasis have been reported. A better understanding of the changes in miRNA expression during NSCLC invasion may lead to a better understanding of NSCLC development, as well as to possible improvements in the diagnosis and treatment of advanced NSCLC.
Previously, we established a highly invasive (SPC-A-1sci) cell subline and a weakly invasive (SPC-A-1) cell subline by in vivo selection in NOD/SCID mice[
18]. We compared the global miRNA profiles of SPC-A-1sci and SPC-A-1 cell, and revealed low expression levels of miR-200c had influence on the invasion and migration ability of NSCLC cell lines[
19]. In this work, miR-200c has been investigated in much greater detail, because miR-200c has been reported to be correlated with EMT[
20], and SPC-A-1sci cells display phenotypic changes consistent with EMT[
18]. Recently, miR-200c has been reported in several tumors, including breast cancer, lung cancer, esophageal cancers, colorectal cancer, and pancreatic cancer[
21‐
25]. These findings indicate that miR-200c may function importantly in human carcinogenesis. However, for miR-200c, the potential roles and related target genes in NSCLC metastasis are still not well elucidated.
Discussion
In recent years, many studies have shown that the expression of miRNAs is aberrant in human cancer[
30]. Identification of tumor-associated miRNAs and their target genes is critical for understanding the roles of miRNAs in tumorigenesis and may be important for novel therapeutic targets[
31].
In our previous work, we isolated invasive and non-invasive cell subpopulations from human NSCLC SPC-A-1 cell lines by in vivo selection in NOD/SCID mice[
18]. We identified 117 novel metastasis-related miRNAs in NSCLC based on a well-established metastasis cell model[
19]. The finding that miR-200c was downregulated in metastatic SPC-A-1sci cells was intriguing, because decreased miR-200c levels have been reported in several other types of tumor[
21,
23‐
25], thus indicating that decreased miR-200c may be a common event in the tumorigenesis. Other reports showed serum miR-200c associated with poor prognosis in patients with lung cancer[
32]. In H1299 cells miR-200c targets multiple non-small cell lung cancer prognostic markers DLC1, ATRX, and HFE[
33]. However, its precise biological role in NSCLC metastasis remains largely elusive.
We focused on the effect of miR-200c on NSCLC metastasis and showed that miR-200c acted as a tumor suppressor during NSCLC metastasis. The expression of miR-200c was negatively correlated with the invasion and migration of NSCLC cell lines in vitro. Moreover, our results suggest that decreased miR-200c levels promoted, increased miR-200c levels inhibited NSCLC cell migration and invasion in vitro and metastasis in vivo. The activity of miR-200c in relation to EMT- associated phenotypes has been extensively studied[
34]. In the current study, we also found miR-200c was associated with EMT. Together, these findings suggest that miR-200c functions as a key mediator of metastasis in NSCLC.
As part of our research on how the miR-200c affects NSCLC metastasis, several bioinformatics tools for screening putative miRNA target genes were used, including miRNAMap, PicTar and miRanda and up-regulated genes in gene chip. We demonstrated that USP25 was a critical downstream target of miR-200c. To test this assumption, we investigated whether miR-200c inhibited USP25 mRNA and protein levels, then found that up-regulation of miR-200c led to a significant decrease in USP25 mRNA and protein levels, thereby suggested that USP25 was a functional target of miR-200c. Lastly, the dual-luciferase reporter assays suggested that USP25 was one of the functional downstream targets of miR-200c. The effect of USP25 on tumor metastasis has not been studied. In the current study, we found that knockdown of USP25 expression reduced NSCLC cell metastasis similar to that of the restoration of miR-200c.
To determine the potential clinicopathological implications of altered miR-200c expression, we investigated the expression levels of miR-200c in 73 NSCLC tissues and non-tumor tissues by qRT–PCR. The results showed that the miR-200c expression level in NSCLC tissues was lower than that in non-tumor tissues, and negatively associated with advanced clinical stage, lymph node metastasis. Next, we investigated the expression levels of USP25 in these patients, the results revealed higher mRNA levels of USP25 were found in NSCLC tissues than non-tumor tissues, correlated with distant metastasis, and advancing stage. Lastly, we found a low level of miR-200c tended to express high levels of USP25, whereas tumors with a high level of miR-200c tended to express low levels of USP25. In addition, there was a significant association between USP25 protein levels positively correlated with clinical stage, histological grade, and lymphatic metastasis by Immunohistochemical staining in 256 NSCLC patients. These findings indicated that the invasion suppression effect of miR-200c was at least partly mediated through a decrease in USP25 expression.
USP25 is a member of ubiquitinating-specific proteases (USPs) family, which contain two short, conserved fragments (lysine and histidine boxes) that contain Cys, His and Asn residues, which form a catalytic triad that can remove ubiquitin molecules from large proteins[
35]. Ubiquitination is a post-translational protein modification referring to the attachment of the small protein ubiquitin to a lysine residue of a target protein. This modification is more versatile than other post-translational modifications because several ubiquitin molecules can be added to the first, creating chains of ubiquitin on the target proteins that modify the signal[
36]. The ubiquitin proteasome system regulates all processes that are involved in carcinogenesis, among them invasion and metastasis, EMT, the process that endows neoplastic cells with invasive and metastatic potential, is intimately interwoven with other neoplastic processes, being served by several common pathways, several of which are regulated by the ubiquitin proteasome system[
37]. USP28 is a key regulator of the DNA damage checkpoint, high expression levels of USP28 are found in colon and breast carcinomas, and stabilization of MYC by USP28 is essential for tumour-cell proliferation[
38]. USP22 are found significantly associated with progression and unfavorable clinical outcome in esophageal squamous cell carcinoma, NSCLC[
39,
40]. USP4 is regulated by AKT phosphorylation and directly deubiquitylates TGF-β type I receptor[
41].
Recently, USP25 was significantly frequent mutation in HCC[
42], and was involved in negative regulation of IL-17-mediated signaling and inflammation by interacting with TRAF5 and TRAF6,and regulates TLR4-dependent innate immune responses through deubiquitination of the adaptor protein TRAF3[
26,
27]. Seven members of the TRAF family have been identified (TRAF1-7), and play an important role in a variety of signaling pathways. TRAF6, a unique TRAF family member, possesses a unique receptor-binding specificity, which is important for its crucial role as the signaling mediator for not only the TNF receptor superfamily but also the IL-1R/Toll-like receptor superfamily[
43,
44]. The downstream signals activated by TRAF6 mainly include NF-κB and AP-1, while NF-κB and AP-1 play an important role in the transcription and expression of numerous genes (including carcinogenesis, invasion and metastasis etc.) in organism[
45].
Methods
Cell culture
The human lung cell lines A549, H1299, SPC-A-1sci, SPC-A-1, XL-2, H460, H358, and HEK-293T were maintained in our laboratory. XL-2 cells were established from the abdomen of a NSCLC patient. All cell lines were routinely maintained in DMEM medium (HyClone) supplemented with 10% fetal bovine serum (Biowest, South America Origin), 100U/ml penicillin sodium, and 100mg/ml streptomycin sulfate at 37°C in a humidified air atmosphere containing 5% CO2. Cells were used when they were in the logarithmic growth phase.
Cell transfections
The miR-200c mimics, and miR-200c inhibitors that were used in this study were synthesized by Ribobio (Guangzhou, China). Genepharma synthesized the USP25 siRNA. The following sequences were as follows: USP25 siRNA sense: 5’-GGGAGUACUUGAAGGUAAATT-3’; anti-sense: 5’-UUUACCUUCAAGUACUCCCTT-3’; negtive control sense: 5’-UUCUCCGAACGUGUCACGUTT-3’; anti-sense: 5’-ACGUGACACGUUCGGAGAATT-3’. Oligonucleotide transfection was performed with Lipofectamine 2000 reagents according to the manufacturer’s instructions (Invitrogen, CA). For migration, invasion, Western blotting assays and real-time quantitative RT-PCR (qRT-PCR), cells were collected 48h after transfection.
In vitro migration and invasion assays
Cell migration and invasion assays were performed in a 24-well plate with 8-mm pore size chamber inserts (Corning, New York, NY, USA). For migration assays, 5 × 104 cells expressing miR mimic, miR inhibitor, miR control and negative control were placed into the upper chamber per well with the non-coated membrane. For invasion assays, 1 × 105 cells expressing miR mimic, miR inhibitor, miR control and negative control were placed into the upper chamber per well with the Matrigel-coated membrane, which was diluted with serum-free culture medium. In both assays, cells were suspended in 200 ml of DMEM without fetal bovine serum when they were seeded into the upper chamber. In the lower chamber, 800 ml of DMEM supplemented with 10% fetal bovine serum was added.After incubation for some hours at 37°C and 5% CO2, the membrane inserts were removed from the plate, and non-invading cells were removed from the upper surface of the membrane. Cells that moved to the bottom surface of the chamber were fixed with 100% methanol for 20 min and stained with 0.1% crystal violet for 30 min. Then, the cells were imaged and counted in at least 10 random fields using a CKX41 inverted microscope (Olympus,Tokyo, Japan). The assays were conducted three independent times.
Establishing stable NSCLC cells
The miR-200c lentiviral vector, miR-200c inhibitor lentiviral vector and miR-control lentiviral vector were synthesized by Genepharma. The sh-USP25 lentiviral vector and sh-control lentiviral vector were synthesized by Openbiosystems (USA).
Real-time quantitative RT-PCR (qRT-PCR)
The expression of miRNAs was measured using the TaqMan stem-loop RT-PCR method from Applied Biosystems (Foster City, CA, USA). Approximately 10 ng of total RNA was reverse-transcribed according to the MicroRNA Reverse Transcription kit and a specific stem-loop primer according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). Samples were normalized to RNU6B. SYBR green RT-PCR (TaKaRa) was performed to detect USP25. The sequences are as follows: USP25 Sense: 5’-CGGTCCCAAACGATTCCC-3’; Antisense: 5’–CTCCCTGTTCTGTTGTGCT-3’. All RT-PCR experiments were performed on the GeneAmp® PCR System 9700 (Applied Biosystems, Foster City, CA). All RT-PCR assays were carried out using a 7300 Real-Time PCR System with SDS RQ Study software (Applied Biosystems).
For the experimental metastasis mouse xenograft model, SPC-A-1 cells transfected with miR-control,miR-200c inhibitor lentiviral vector, SPC-A-1sci cells stably expressing miR-control, miR-200c lentiviral vector, knocked-down USP25 expression, the negative control were inoculated into the tail vein of five-week-old BALB/C-nu/nu nude mice(N = 12). For spontaneous metastasis mouse model,SPC-A-1sci cells stably expressing miR-control, miR-200c lentiviral vector, knocked-down USP25 expression, the negative control were injected subcutaneously into the right upper flank region of five-week-old NOD/SCID mice(N = 10). The animals were maintained under specific pathogen free (SPF) conditions. The mice were manipulated and housed according to protocols approved by the Shanghai Medical Experimental Animal Care Commission. Each tumor cell subline was injected 2 × 106 per mouse. After some weeks, the mice were sacrificed, and the lungs were harvested at necropsy and fixed in 10% neutral PB-buffered formalin (pH 7.4). The fixed samples were then embedded in paraffin, and five non-sequential serial sections were obtained per animal. The sections were stained with H&E and analyzed for the presence of metastases.
Western blot
Cell proteins were extracted and separated in SDS-PAGE gels, and transferred to nitrocellulose filter membranes (Millipore, USA). Western blot analyses were performed according to standard procedures. Western blot loading control was β-actin (Sigma-Aldrich, 1:30000). The following antibodies were used: anti-USP25 (Abcam, 1:1000), anti-E-cadherin (CST, 1:1000), anti-N-cadherin (CST, 1:1000).
Immunofluorescence and immunohistochemical analysis
Cells were plated and grown on glass slides for 18 ~ 20 hours and fixed with 4% paraformaldehyde. The slides were then blocked and incubated with the following primary antibodies: Anti-E-cadherin was obtained from Santa Cruz (1:25), Anti-N-cadherin was obtained from Santa Cruz (1:25). Finally, the slides were incubated with fluorescence conjugated secondary antibody (Sigma 1:50) and viewed with a Fluoview FV1000 microscope (Olympus, Japan).
Luciferase assay
Total cDNA from HUVEC cells was used to amplify the 3’UTR of USP25 by PCR, using the forward primer: 5’-CCGCTCGAGACTGCACACTTTCCCTGAACACAC-3' ; the reverse primer:5’–GAATGCGGCCGCAGAATGTCATAAAATATTAAGGATACTTTCTTTAC-3'. Construction of the 3’-UTR of the USP25-mutant by PCR was performed, using the forward primer: 5’–CCTGTTGGTGATATACTTTGCTTTTATCTTTTC-3'; and the reverse primer:5’–CAAAGTATATCACCAACAGGAAGGTGGCAAAAG-3'. The XhoI and NotI restriction enzyme sites were used. HEK293T cells were plated into 96-well plates at 50% confluence 24 h before transfection. The pmiR report-control vector and Luc-USP25, Luc-USP25-mu, or Luc-control vectors were co-transfected into HEK293T cells using Lipofectamine 2000. Lysates were prepared at 48 h post-transfection. Luciferase activity was measured using a Dual-Luciferase Reporter System (Promega).
Human NSCLC tissues
Patient samples in this study were obtained following informed consent, according to an established protocol approved by the Ethic Committee of Shanghai Jiao-Tong University School. The data did not contain any information that may lead to the identification of the patients. 73 NSCLC tissues and matched adjacent noncancerous tissues for RNA extraction were provided by Shanghai chest hospital. Of these 73 NSCLC tissues, 16 were squamous cell carcinomas and 57 were non squamous cell carcinomas. Matched pairs of NSCLC tissues and matched adjacent noncancerous tissues were used for the construction of a tissue microarray (Shanghai Biochip Co., Ltd. Shanghai, China) as previously described[
46]. There were 256 NSCLC tissues and matched adjacent noncancerous tissues in the tissue microarray. Of these 256 NSCLC tissues, 134 were squamous cell carcinomas and 122 were non squamous cell carcinomas. Immunohistochemical staining was performed to detect the expression of USP25 in NSCLC tissues and matched non-cancerous tissues. The primary antibody against USP25 was obtained from Abcam (1:50). Scoring was measured by the percentage of positive cells with the following staining intensities: less than 5% scored “–”; 5–24% scored “+”; 25–49% scored “++”; 50–74% scored “+++”; and more than 74% scored “++++”.
Statistical analysis
The results are presented as mean ± SD. Comparisons of quantitative data were analyzed by Student’s t- test between two groups (two-tailed; P < 0.05 was considered significant). Fisher’s exact test was used to compare qualitative variables. Analysis was performed with SAS 9.0 for Windows.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Conceived and designed the experiments: MY, XHH, JJL Performed the experiments: JL, QT, MXY, LL, HCL, FYZ, CG, TY, FLZ. Analyzed the data: JL Contributed reagents/materials/analysis tools: GLB, HW, K Wrote the paper: JL. All authors read and approved the final manuscript.