Background
As one of the most common malignant tumors, colorectal cancer (CRC) remains the third most incident cancer, with the incidence of colon cancer rising rapidly worldwide [
1]. Previous data showed that among colorectal cancer patients, 25% were often accompanied by distant metastases, and about 40–50% who had not been found with primary colorectal cancer metastasis eventually developed distant metastasis. Moreover, the median survival of untreated colorectal cancer metastases in advanced patients was only 5–6 months [
2,
3]. The occurrence and development of colorectal cancer is a process of multi-gene participation and multi-stage evolution. A current research indicates that colorectal cancer is a sequence evolutionary process in which adenomatous polyps eventually become cancerous and metastasize to the primary cancer nest [
4,
5]. The inactivation of tumor suppressor genes (e.g., APC, TP53 and TGFBR2), and the mutation of oncogenes (e.g., RAS, BRAF and PI3KCA), which can inhibit or activate downstream-related signaling pathways and ultimately lead to the occurrence of adenomatous polyps and malignant tumors, are involved in the process [
6]. Surgical treatment, chemotherapy, radiotherapy and other common clinical and technical methods are difficult to effectively treat late metastatic tumors, which eventually cause 90% cancer patients death [
7]. Therefore, finding new biomarkers and further understanding the molecular mechanism may help prevent and treat colorectal cancer.
Epithelial-mesenchymal transformation (EMT) refers to the transformation of epithelial cells into cells with mesenchymal characteristics [
8]. The process includes the loss of epithelial markers such as E-cadherin and cytokeratin, accompanied by increased expression of interstitial markers such as N-cadherin, vimentin and fibronectin [
9]. Previous studies suggested that EMT could cause a variety of changes in cells, such as enhanced ability of cell migration and invasion, and enhanced resistance to apoptosis and senescence, which play a very important role in the formation of tumor metastases [
10].
Snail1 is considered a key factor in the aggressive expression of tumors for its critical role in the EMT pathway associated with tumor metastasis [
11]. Studies showed that Snail1 expression was significantly higher in non-small cell lung cancer tissues than in normal non-cancer tissues, which suggested a possible reaction of Snail1 in tumor [
12]. Moreover, in normal tissues Snail1 gene was silent, but in tumor tissues its expression was up-regulated, and exerted a functional role by controlling the expression of related proteins [
13]. Therefore, it’s still worth exploring the detailed molecular mechanism of Snaill in CRC.
Specification peptidase 18 (USP18) is a depolymerase of the ubiquitin-like modified enzyme system that can reduce the modification effect of ISG15 on the target protein by removing ISG15 from the bound target protein, which is called “deubiquitination” and has a regulatory effect on the body’s multiple signaling pathways and homeostatic maintainance [
14,
15]. Recent studies showed that USP18 could also affect tumorigenesis by regulating interferon production and immune cell function, and recent researches found its expression in various tumors [
16,
17]. The deletion of the USP18 gene induced the expression of exogenous apoptosis-related genes such as TRAIL by activating the I-IFN-related signaling pathway [
18]. The knockdown also inhibited the EGFR expression by up-regulating miR-7, which further inhibited the growth of tumor cells and increased apoptosis [
19]. Moreover, the lack of USP18 gene could inhibit the formation of leukemia induced by BCR-ABL virus through up-regulating the I-IFN signaling pathway [
20]. However, the roles of USP18 and Snaill in CRC are still poorly studied. In this study, we explored whether USP18 affects CRC cells through regulating Snail1 ubiquitination.
Methods
Tissue samples and cells
Sixty colorectal cancer samples and their paired normal tissues were collected in the department of pathology of the First Affiliated Hospital of Fujian Medical University between Jan 2019 and Dec 2019. The ethics committee of The First Affiliated Hospital of Fujian Medical University had reviewed and approved all experimental protocols. All patients had read and signed the informed consent. The detached tissues were quickly frozen with fluid nitrogen and stored at − 80 °C. FHC, HCT116, SW480, DLD1, and LOVO cells were purchased from ATCC (Virginia, USA). Cells were cultured with RPMI 1640 with 10% FBS (Invitrogen, Carlsbad, CA) in a humidified chamber at 5% CO2, at 37 °C. SW480 cells were plated on six-well plates (5 × 105 cells per well). OPTI-MEM serum-free medium (M5650, Sigma Aldrich) and Lipofectamine 2000 reagent (Thermo Fisher Scientific, USA) were used in transfection tests. The final concentration of 100 nM siRNA was introduced in this study. Meanwhile, pEZ-Lv201 Vector (Biovector, China) was employed to construct the USP18 overexpression system in the DLD1 cells. Lentiviral particles generated with a standardized protocol were used to produce the highly purified plasmids. Endo Fectin-Lenti™ and Titer Boost™ reagents (CWBio, China) were used to co-transfect DLD1 cells. The supernatant was collected after 48 h transfection and stored at − 80 °C.
Effect of USP18 on chemotherapy sensitivity of CRC cells
Three common chemotherapy drugs (fluorouracial, doxorubicin, and cisplatin) were used. Overexpression or knockdown of USP18 in CRC cells were established as described above. Then, CRC cells were treated with different concentrations of fluorouracial (0, 20, 40, 60, and 80 g/mL), doxorubicin (0, 0.5, 2.5, 5, and 10 µM), or cisplatin (0, 10, 20, 30, and 40 µM) for 24 h. Then, the cell survival was measured using CCK-8 assay.
qRT-PCR analysis
Total RNA was extracted with M5 SuperPure Total RNA Extraction Reagent (SuperTRIgent) (mei5bio, China). The mRNA expression was examined with the Q225 system (Kubotechnology, China). The PCR reaction contained 10μL GoldStar Probe Mixture (CWBio, China), 1μL sense primer (10 nM), 1μL anti-sense primer (10 nM), 2μL cDNA template (10 ng), and 6μL H
2O. The program for qRT-PCR was set as follows: 95 °C, 30 s, 40 cycles (95 °C, 5 s, and 60 °C, 10 s). 2
−ΔΔCt cycle method was used to calculate the relative expression level of mRNAs. GAPDH was employed as the internal control. Primer sequences used were listed in Additional file
1: Table S1.
Western blot analysis
Cellular protein in different groups was extracted with 1% PMSF a RIPA Lysis and Extraction Buffer (Beyotime, China). Sodium dodecy lsulfate–polyacrylamide gel electrophoresis was used to perform further examination. In this step, the proteins were transfered onto a polyvinylidene difluoride layer (Novus, USA). After blocking for 1 h at room temperature, the layer was brooded with anti-Rabbit USP18 (1:1000) (#4813, CST, USA), E-cadherin (1:1000) (# 3195S, CST, USA), Vimentin (1:1000) (# 5741S, CST, USA), N-cadherin (1:1000) (#13116S, CST, USA), CD133 (1:1000) (#64326, CST, USA), CD44(1:1000) (# 37259S, CST, USA), Snail1 (1:1000) (#3879, CST, USA), and GAPDH (1:1000) (#2118, CST, USA), overnight. Proteins were hatched with the corresponding secondary antibodies for 1 h at room temperature after being treated with ECL Chemiluminescence Detection Kit (PromoCell, German). The bands were observed with Chemiluminescence Imaging (Clinx Ltd., China).
Immunohistochemical staining analysis
The immunohistochemical SP method was used to stain cancer tissue sections. Tissue sections were baked in a 60 °C incubator for 1 h, and then were subjected to multiple treatments, including immersion in xylene to dewax, gradient alcohol hydration, microwave antigen repair, and 3% hydrogen peroxide treatment. After blocking using goat serum, the sections were added in an anti-rabbit USP18 monoclonal antibody Snail1 (1:1000) (#3879, CST, USA) and incubated at 4 °C overnight. An optical microscope was used for observation.
Migration and invasion assay
EZCell™ Cell Migration/Chemotaxis Assay Kit (24-well) (K911-12, Biovision, USA) and EZCell™ Cell Invasion Assay (Basement Membrane) (96-well Kit) (K912-100, Biovision, USA) were used to perform cell migration and invasion, respectively.
CCK8
The differently-treated cells were digested, centrifuged and resuspended. The cells were diluted with complete medium. The cells were counted using a cell glass counting plate, and then diluted to 2000 cells/ml. 100 μL cell suspension (2000 cells/mL) was added to each well in a 96-well plate. There were 5 replicate wells in each group and the five replicates were set and observed at five-time points. Subsequently, we incubated the cells in a 5% CO2, 37 °C incubator overnight. Next day, 10 μL CCK-8 solution (Beyotime, China) was added to the medium. Then, the plate was incubated in a 37 °C incubator for 2 h. The absorbance at OD450 was measured.
Immunofluorescence analysis
Cancer cells in the logarithmic growth phase were inoculated into 24-well plates with cell slides and cultured for 48 h. We discarded the medium, removed the cell slides, and washed 3 times with PBS. Sections were fixed using 4% paraformaldehyde at 4° C for 30 min. After washing 3 times with PBS (5 min/time), 0.1% Triton was used to treat sections for 10 min. Then, PBS was used to wash sections for 5 min, and goat serum was used for blocking. After washing 3 times with PBS (5 min/time), first antibody was used to cultivate sections at 4 °C overnight. Subsequently, secondary antibody was used to culture sections for 1 h at room temperature in a wet box. After washing 3 times with PBS (10 min/time), an inverted fluorescence microscope was used to observe results.
Co-IP detection
Cancer cells in the logarithmic growth phase was used in this step. Total protein was extracted using the RIPA Lysis and Extraction Buffer (89900, ThermolFisher Scientific, USA). The beads were washed with 100 μL ice-cold buffer. 100 μL antibody binding buffer was added to spin the antibody and magnetic beads for 30 min. The beads were washed 3 times with 200 μL buffer. Cell lysate and antibody-conjugated magnetic beads were used to incubate for 1 h at room temperature and then washed 3 times with 200 μL buffer. 20 μL elution buffer was used to wash the beads once and the supernatant was collected.
Scratch test
Differently Snail1 knocked-down cells were resuspended and counted. The scratch test insert after alcohol disinfection was carefully placed in a 12-well plate (3 replicates per group). The complete medium was used to dilute the cells to 500 cells/μL. 70 μL cell suspension was added to each well. Twenty-four hours later, the cells were gently washed twice with PBS and then, 1 ml 1% FBS medium was added. Cell status was observed under the microscope at 0 h and 24 h.
Statistical methods
SPSS16.0 statistical software was used and data were expressed as χ ± s. Two groups were compared using the t test. One-way analysis of variance was used for comparison between groups. P < 0.05 was considered to be significant difference.
Discussion
In China, colorectal cancer remains as one of the most common malignant tumors in the digestive system, with a third incidence rate among all tumors and being increasing over the past decades due to environmental and dietary factors [
22,
23]. Studies have found the relationship between its incidence and intestinal polyposis, chronic stress and inflammation, and a family history of cancer [
24]. Previous studies suggested that the main cause of death was the invasion and migration of advanced colorectal cancer [
25]. Improving diagnostic techniques, including surgery, chemotherapy and radiotherapy treatments, could help detect tumors early and improve patient survival. Better understanding the pathogenesis progression may provide new therapeutic strategies for the prevention and treatment of CRC.
USP18 is an effective regulator of epidermal growth factor receptors (EGFR) [
19], and the low expression of it could lead to a down-regulated expression of carcinogenic targets thus decreasing cell proliferation and increasing cell apoptosis [
26]. In MCF-7 cells and glioblastoma, knocking out USP18 can induce apoptosis of tumor cells [
27,
28]. The low expression of USP18 can significantly reduce the metastasis and invasion of lung cancer cells [
29]. However, what role USP18 plays in the progression of CRC has not been reported.
Ubiquitination plays an important role in several biological processes including metabolism, protein degradation, cellular localization, inflammatory immunity, transcription regulation, and cell cycle [
30]. Meanwhile, ubiquitination is closely linked with the regulation of tumors. It was reported that over-expressed DUSP4 could promote chemotherapy-induced apoptosis. In addition, silence of DUSP4 could activate the Ras-ERK signaling pathway and further promote the proliferation and migration of tumor cells [
31]. However, the role of USP18 in the regulation of tumor cells is poorly understanded. In this study, we demonstrated that the change of USP18 expression was closely related to the invasion, migration, and proliferation of CRC cells. Therefore, USP18 may provide a potential target for the treatment of CRC. Recent reports indicated that abnormal expression of USP18 in CRC tissues was associated with a poorer prognosis [
17]. Those results are consistent with our findings, which showed that the expression of USP18 was higher in CRC tissues than in adjacent tissues, and overexpression or knockout of USP18 could affect the proliferation, and migration of CRC cells. Therefore, USP18 might be a biomarker for the diagnosis of CRC.
Snail1 is a nuclear transcription factor that can control the transcription efficiency of DNA to messenger RNA [
32]. Previous study suggested that Snail1 can inhibit the expression of the downstream gene Cyclin D2 and promote cell survival [
33]. Meanwhile, it was suggested that Snail1 can upregulate the expression of myosin that could promote the migration of tumor cells [
34]. However, whether USP18 could affect the proliferation, migration and invasion of CRC cells through targeting Snail1 remains unclear.
EMT process has been believed to act a key role affecting tumor metastasis. EMT is characterized by decreased expression of epithelial proteins such as E-Cadherin, and increased level of mesenchymal protein such as vimentin. Low E-Cadherin expression means decreased of epithelial connexin, and further facilitate tumor metastasis. In this study, we found that E-Cadherin, Neadherin, and Vimentin were remarkably influenced in the overexpression and knockdown models of USP18. Protein stability is mainly affected by proteasome degradation pathways and autophagolysosomal degradation pathways.
Snail1 is an important transcription factor of EMT, and the expression level of it is linked with the invasion, migration, and apoptosis of tumor cells. Snail1 is believed to be an important factor affecting the neural tube and development of mesoderm, but also plays an important role in tumor metastasis. Snail1 is the most important E-cadherin transcriptional repressor, and it could down-regulate the expression of claudins and occludins protein. Our results revealed that DUSP18 and Snail1 could regulate EMT of CRC through E-caderin, N-caderin, and Vitmentin. Snail1 can directly interact with USP18 in cellular.
In this study, it was notable that Snail1 expression was significantly affected in USP18 over-expressed or knocked-down cells. Snail1 could directly interact with USP18 in cells. Moreover, USP18 could reduce Snail1 protein expression without affecting its transcription. Moreover, our results suggested that USP18 affected the protein degradation pathway of Snail1 through ubiquitination modification. Meanwhile, knockdown and overexpression of Snail1 could affect cell migration and invasion, and EMT-related molecules including E-cadherin, N-cadherin, and Vimentin. We proved that Snail1 could effectively reverse the influence of USP18 on cell proliferation, migration, invasion, and EMT of CRC cells.
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