Silencing PRSS1 suppresses the growth and proliferation of gastric carcinoma cells via the ERK pathway

Background: Gastric carcinoma (GC) is one of the most common malignant tumors and seriously threatens human life and health. Methods: In the present study, 243 differentially expressed proteins in GC were identified using laser capture microdissection (LCM) combined with isotopically labeled quantitative proteomics technology. The expression of serine protease 1 (PRSS1) protein was analyzed by immunohistochemistry and Western blot. MTT and colony formation assays were employed to determine the effect of PRSS1 expression on the growth and proliferation of GC cells. Then, we observed the expression of miR-146a-5p in GC by qRT-PCR. A dual luciferase assay was performed to determine whether PRSS1 is a target gene of miR-146a-5p. We also explored the influence of miR-146a-5p expression on PRSS1 expression and on the growth and proliferation of GC cells. Finally, Western blotting was used to analyze the effect of PRSS1 expression on the activation of the ERK signaling pathway. Results: We confirmed that PRSS1 expression was significantly increased and was positively correlated with the differentiation, tumor size and lymph node metastasis of GC. Subsequently, we found that overexpression of PRSS1 promoted the growth and proliferation of cells, whereas silencing PRSS1 expression inhibited the growth and proliferation of MGC803 cells by inhibiting activation of the ERK signaling pathway via reductions in PAR-2 activation. MiR-146a-5p targets PRSS1 and suppresses the growth and proliferation of MGC803 cells. Conclusions: miR-146a-5p targets PRSS1 and suppresses the growth and proliferation of MGC803 cells. Silencing PRSS1 expression inhibits the ERK signaling pathway by reducing PAR-2 activation, resulting in suppressed growth and proliferation of MGC803 GC cells.


Introduction
Gastric carcinoma (GC) is one of the most common malignancies, ranking fifth in global incidence among all malignancies and third in cancer-associated mortality [1,2]. However, the majority of cases of GC are usually not diagnosed until an advanced stage; therefore, the outcome is often poor with a 5-year survival rate of no more than 30%, including among patients who have undergone surgery [3]. It is key to improve the 5-year survival rate and quality of life of patients for the early Ivyspring International Publisher diagnosis and management of GC. Therefore, it is worthwhile to explore methods and biomarkers for the early diagnosis of GC. However, specific and sensitive molecular markers for the early diagnosis of GC have been poorly defined.
The development of proteomics technology has provided a new way to research molecular markers for the early diagnosis of GC [4]. Laser capture microdissection (LCM) technology is one of the best methods for separating cells from tissues and obtaining high-purity tissues [5]. Isobaric tags for relative and absolute quantitation (iTRAQ) technology is beneficial for comparative analysis of abundance in mass spectrometry and has advantages relating to quantitative and differential expression results. The quadrupole time-of-flight tandem mass spectrometer (Q-TOF MS/MS) is suitable for the analysis of trace molecules. This machine is significantly better than traditional techniques for not only its resolution, accuracy and sensitivity but also its ability to determinate proteins with very low molecular weight, extreme pH and low abundance [6].
The PRSS1 gene is located on human chromosome 7 and encodes 247 amino acids, comprising cationic trypsin 1. Recent studies have shown that the expression of PRSS1 protein is significantly increased in pancreatic cancer, colorectal cancer and cervical cancer and may be involved in the pathological process of tumor progression [7][8][9]. MicroRNAs (miRNAs) are a type of noncoding RNA widely found in eukaryotes. They mainly recognize the 3′-UTR of their target mRNA to either degrade the mRNA or inhibit its translation [10]. MiR-146a-5p is dysregulated in a variety of cancers and plays an important role in the development of cancer [11,12]. Previous studies reported that miR-146a-5p is dysregulated in GC [13]; however, the signaling mechanisms need further research. Protease-activated receptor-2 (PAR-2) is a seven-transmembrane G protein-coupled receptor consisting of 397 amino acid residues that is expressed in a variety of tumor cells. It has been reported that various proteases can exert hydrolytic activity and activate PAR-2 [14], which consequently affects tumor proliferation, metastasis and angiogenesis through the ERK pathway [15]. However, it remains unclear whether PAR-2 activation affects the proliferation of gastric cancer cells.
In this study, we identified differentially expressed proteins in LCM-purified gastric mucosal epithelial cancerous tissues using iTRAQ labeling and 2D LCMS/MS and selectively verified a subset of the differentially expressed proteins with consistent results. Among them, PRSS1 protein was significantly overexpressed, which was positively correlated with the differentiation, tumor size and lymph node metastasis of GC. Subsequently, PRSS1 is a target gene of miR-146a-5p. Finally, we found that knockdown of PRSS1 expression inhibited activation of the ERK signaling pathway by reducing PAR-2 activation, resulting in suppressed proliferation of GC MGC803 cells.

Patients
We collected 20 matched pairs of fresh normal gastric mucosa (NGM), atypical hyperplasia (AH), poorly differentiated gastric adenocarcinoma (GPDAC) and lymph node metastasis adenocarcinoma (LMGAC) tissues for LMC (Supplementary Table 1). In addition, 81 pairs of GC and adjacent normal formalin-fixed paraffin-embedded tissues were collected for immunohistochemical (IHC) staining with a PRSS1 antibody. Tissue chips made from 141 GC specimens were used for IHC staining with a PAR-2 antibody. All samples were diagnosed in the Department of Pathology of the First Affiliated Hospital of University of South China. All samples were obtained from patients with GC with approval of the medical ethics committee.

Laser capture microdissection (LCM)
Tissue samples were removed from liquid nitrogen and sliced into 8-µm-thick frozen sections using a cryostat device carrier (Leica Biosystems GmbH, Wetzlar, NJ). Frozen tissue sections were stained with methyl green and placed on an LCM (Leica LMD6, Leica Microsystems GmbH). The target tissues were outlined on the display and automatically cut from the slice with the laser. We collected the target tissues in a tube containing 2-3 µl of protease inhibitor (Roche Diagnostics, Basel, Switzerland) and stored them at -80 °C.

Protein extraction and isobaric tags for relative and absolute quantitation (iTRAQ) labeling
Total protein from LMC-treated tissues was extracted using lysis buffer (CW2333S, Cwbiotech, Beijing, China) following the manufacturer's protocol. The total proteins from NGM, AH, GPDAC and LMGAC tissue samples were labeled with different iTRAQ reagents (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Four labeled lysates were mixed and lyophilized before they were desalted. Then, the samples were dissolved in deionized water containing 0.1% formic acid (FA, Tedia Company, Fairfield, OH, USA) and eluted three times with Sep-Pak C18 1 cc Vac cartridges (Waters Corporation, Milford, MA, USA). The cleaning solution was collected and lyophilized to obtain the final samples.

2D LC-ESI-MS/MS analysis and identification of differentially expressed proteins
The samples marked with iTRAQ were dissolved in cation-exchange (SCX) buffer for SCX separation.
The four samples containing mesenchymal proteins from NGM, AH, GPDAC and LMGAC tissues were mixed and loaded into a polysulfoethyl column and segregated by an LC-20AD high-performance liquid chromatography (HPLC) system (Shimadzu Corporation, Kyoto, Japan) following the manufacturer's instructions. Next, the products were concentrated by vacuum centrifugation for reverse-phase HPLC-mass spectrometry (MS) analysis. The samples were dissolved in 50 µl of 5% ACN containing 0.1% FA and loaded into a Zorbax 300SB-C18 column (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's instructions. The data were analyzed using QSTAR-XL (Applied Biosystems, Thermo Fisher Scientific) and tandem MS (MS/MS). Finally, the IPI human database (version 3.45, URL: http://www.ebi.ac.uk/IPI) was searched for protein information, the confidence level was set as >95%, and the ion peak areas of m/z 114 and 118 were integrated.

Cell line and cell culture
GC cell lines (SGC7901, BGC823, MGC803) and the immortalized gastric epithelial cell line GES-1 were obtained from Cancer Research Institute of the University of South China for the present study. HEK293T cells were obtained from the ATCC (American Type Culture Collection, Manassas, VA). Cells were cultured in either RPMI 1640 (HyClone) medium containing 10% fetal bovine serum (HyClone) or serum-free keratinocyte medium (K-SFM, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) in a humidified 5% (v/v) CO2 atmosphere at 37 °C.

MTT assay
Cells were seeded into 96-well plates at 2×10 3 cells/well. The number of viable cells was detected by the MTT method according to the manufacturer's instructions (Molecular Probes, Eugene, OR, USA) every 24 hours in triplicate. The OD values were measured at 570 nm with a microplate reader (17260, BIO-RAD, USA).

Colony formation assay
The cells were suspended before they were seeded in a six-well plate at 1×10 3 cells/well at 37 °C for 2 weeks. The colonies were fixed with methanol for 25 min and then stained with 0.4% crystal violet for 30 minutes at room temperature (E607309, BBI, Shanghai, China). The number of cell colonies was counted under a microscope (TS100, Nikon, Japan) and analyzed.

Soft agar clone formation assay
Cells were seeded into semisolid agar K-SFM medium [base layer, 0.6% (w/v); upper layer, 0.3% (w/v)] at a density of 5×10 4 cells/well in 6-well plates. After 2-week incubation at 37 °C with 5% (v/v) CO2, the number and size of colonies (≥50 cells as one colony) were observed and analyzed.

Immunocytochemical staining
Cell slides were prepared and fixed with 4% paraformaldehyde for 30 min. The immunocytochemical staining procedure was the same as that used for IHC according to the S-P kit instructions (Maixin, Fujian, China). The cells were incubated with antibodies targeting proliferating cell nuclear antigen (PCNA) (ab137867, 1:500, Wanleibio, Shanghai, China) and PAR-2 (ab1809, 1:100, Abcam, UK) overnight at 4 °C and stained by adding DAB. Finally, the cells were observed under a microscope (BX53, Olympus, Japan).

Luciferase reporter assay
PRSS1-WT and PRSS1-MUT are vectors containing the recombinant wild-type and mutant form of PRSS1, respectively. All vectors were confirmed by sequencing (Sangon Biotech). HEK 293T cells were seeded into 24-well plates at a density of 5×10 4 cells/well and cotransfected with the PRSS1-WT or PRSS1-MUT plasmids and the miR-146a-5p mimics or NC using LipoFilter transfection reagent (HB-TRLF-1000, Hanheng, Shanghai, China). Luciferase activity was measured by a dual-luciferase reporter assay system (Lux-T020, BLT, Guangzhou, China) after 48 hours of transfection following the manufacturer's instructions. Relative luciferase activity was expressed as the fold-change after normalization to Renilla luciferase activity. Recombinant expression vectors were confirmed by sequencing (Sangon Biotech).

Statistical analysis
Statistical analyses were performed using GraphPad Prism 8 software. Data are expressed as the means±standard deviation (M±SD). The results of the immunohistochemical staining were analyzed by the Kruskal-Wallis H test, and Fisher's exact probability test was used for pairwise comparisons. One-way ANOVA was used to compare the means of samples between groups. The MTT experiment results were statistically analyzed using two-way ANOVA. Student's t-test was performed when the variance between groups was similar, and the correlation test used was Spearman's correlation analysis. *P<0.05, **P<0.01, and ***P<0.001 were considered to indicate a statistically significant difference.

Identification of differentially expressed proteins during human gastric mucosal carcinogenesis by Proteomics
To explore differentially expressed proteins related to gastric mucosa carcinogenesis, we used LCM technology to extract proteins from 20 normal gastric mucosa (NGM), atypical hyperplasia (AH), poorly differentiated gastric adenocarcinoma (GPDAC) and lymph node metastasis adenocarcinoma (LMGAC) tissues ( Figure 1A). A total of 243 differentially expressed proteins were identified by iTRAQ labeling, two-dimensional liquid chromatography separation and Q-TOF MS/MS mass spectrometry (the Life Science Research Center of Fudan University). The MS/MS map of PRSS1 peptides is shown in Figure 1B. There were significant differences in the levels of the PRSS1, TGM2, SerpinA3, P180 and HSP90α/β proteins, and their expression gradually increased in NGM, AH, GPDAC and LMGAC tissues (Table  1). We constructed a regulatory network of gastric mucosa carcinogenesis-related proteins via DAVID Online Bioinformatics Analysis Software (http://david. abcc.ncifcrf.gov) ( Figure 1C).

Expression and clinical significance of PRSS1 in GC
To further confirm the quantitative proteomics results, we detected the expression levels of some of the differentially expressed proteins in 20 NGM, AH, GPDAC, and LMGAC tissues by Western blot. The results of the Western blot analysis were consistent with those of the quantitative proteomics analysis (Figure 2A). Having demonstrated that, we evaluated the expression and clinical significance of PRSS1 in GC. Western blot and IHC staining analyses indicated that PRSS1 was highly expressed in GC cells and tissues ( Figure 2B, 2C and Table 2). Subsequently, we explored the correlation between PRSS1 expression and the clinicopathological status of GC patients. According to statistical analysis, PRSS1 overexpression was positively correlated with the differentiation, tumor size and lymph node metastasis of GC (Table  3). Then, we predicted the clinical significance of PRSS1 in patient prognosis. As shown in Figure 2D, there was poor overall survival (OS) of patients with high expression of PRSS1. Therefore, PRSS1 may be a potential marker for the early diagnosis and prognosis of GC.

Effect of PRSS1 expression on the growth and proliferation of GC cells
Subsequently, we investigated the effect of PRSS1 expression on the growth and proliferation of GC cells. A GES-1 cell line with ectopic  Figure 3A and 3E). We performed MTT, colony formation and soft agar colony formation experiments to examine the changes in the proliferation of GES-1/pcDNA3.1-PRSS1 cells and GC MGC803/miR-PRSS1 cells. The MTT assays showed that overexpression of PRSS1 significantly increased the growth of GES-1 cells, whereas downregulation of PRSS1 significantly inhibited the growth of MGC803 cells ( Figure 3B and 3F). In the colony formation and soft agar assays, the colony-forming ability of GES-1 cells was significantly increased after upregulation of PRSS1 expression; in contrast, the colony-forming ability of MGC803 cells was significantly reduced after downregulation of PRSS1 expression ( Figure 3C-D and 3G-H). The results indicated that increased expression of PRSS1 promoted cell growth and proliferation, and silencing PRSS1 expression inhibited the growth and proliferation of MGC803 cells.

MiR-146a-5p targets PRSS1 and inhibits MGC803 cell growth and proliferation
Next, we used miwalk and miRanda bioinformatics software to find whether miR-146a-5p has a potential binding site with PRSS1. We confirmed that miR-146a-5p expression was significantly decreased in GC cells ( Figure 4A) while PRSS1 mRNA was significantly increased in the same cells ( Figure  4B). The expression level of miR-146a-5p was negatively correlated with PRSS1 mRNA expression as measured by qRT-PCR ( Figure 4C). To investigate whether miR-146a-5p affects the growth and proliferation of MGC803 cells, we transfected miR-146a-5p mimic, mimic NC, inhibitor, and inhibitor NC into MGC803 cells and found that the growth and colony-forming ability of MGC803 cells were significantly reduced after miR-146a-5p expression was upregulated. Conversely, the growth and colony-forming ability of MGC803 cells were significantly increased after miR-146a-5p expression was downregulated ( Figure 4D-E). At the same time, Western blot analysis showed that miR-146a-5p mimic reduced the protein expression of proliferating cell nuclear antigen (PCNA), while the miR-146a-5p inhibitor increased PCNA expression ( Figure 4F). The immunocytochemistry (ICC) results were consistent with the Western blot results ( Figure 4G).

PRSS1 mRNA is a direct target of miR-146a-5p
Subsequently, we predicted the binding site of miR-146a-5p to PRSS1 mRNA via the TargetScan database ( Figure 5A). To confirm this finding, we performed a luciferase reporter experiment to verify whether miR-146a-5p directly targets PRSS1 mRNA. First, we constructed luciferase reporters containing the wild-type and mutant sequences of the PRSS1 3′UTR. We cotransfected miR-146a-5p mimic or NC with each luciferase reporter gene into HEK 293T cells and measured luciferase activity. The results indicated a significant reduction in luciferase activity when wild-type PRSS1 (PRSS1-WT) was cotransfected with miR-146a-5p. However, there was a significant increase in luciferase activity when mutant PRSS1 (PRSS1-MUT) was cotransfected with miR-146a-5p ( Figure 5B). In addition, we further confirmed the results of the luciferase assays by qRT-PCR and Western blot analysis. Our data demonstrated that the miR-146a-5p mimic significantly downregulated the expression of PRSS1 mRNA and protein, while the miR-146a-5p inhibitor upregulated the expression of PRSS1 mRNA and protein ( Figure 5C-E). Therefore, PRSS1 is a direct target gene of miR-146a-5p, and miR-146a-5p inhibits the growth and proliferation of GC by downregulating the expression of PRSS1.

PRSS1 affects cell proliferation via the PAR-2-activated ERK signaling pathway
In searching for the molecular mechanism downstream of PRSS1 that affects GC MGC803 cell proliferation, we found that PAR-2 was highly expressed in well-differentiated, moderately differentiated and poorly differentiated gastric adenocarcinoma and lymph node metastatic cancer tissues compared with normal gastric mucosa ( Figure  6A, Table 4) as measured by IHC. The expression of PAR-2 in GC tissues was associated with TNM stage and lymph node metastasis (Table 5). Moreover, compared with GES-1 cells, MGC803 GC cells showed high expression of PAR-2 ( Figure 6B). We transfected the miR-PRSS1 interference plasmid into MGC803 cells to silence the expression of PRSS1, but there was no significant difference in the expression of PAR-2 ( Figure 6C). Then, we treated MGC803 cells with FSLLRY-NH2 (PAR-2 inhibitor) and SLIGKV-NH2 (PAR-2 agonist) and observed cell growth and proliferation as well as measured the levels of phosphorylated ERK1/2. The results indicated that cell growth and proliferation and the levels of phosphorylated ERK1/2 in MGC803/miR-PRSS1 cells were significantly reduced compared those in with MGC803 and MGC803/miR-NC cells ( Figure 6D-F). Additionally, cell growth, proliferative ability and levels of phosphorylated ERK1/2 were reduced in MGC803 cells treated with FSLLRY-NH2 ( Figure  6D-F). Interestingly, when SLIGKV-NH2 was added to MGC803/miR-PRSS1 cells, cell growth, proliferative ability and phosphorylated ERK1/2 levels were restored ( Figure 6D-F). However, we found that the changes in PRSS1 expression and the addition of either FSLLRY-NH2 or SLIGKV-NH2 did not affect the expression level of PAR-2 protein ( Figure 6F). The results suggest that knockdown of PRSS1 expression inhibits ERK signaling pathway activation by reducing PAR-2 activation, resulting in weakened growth and proliferation of MGC803 cells.

Discussion
Improving the 5-year survival rate and quality of life of patients is key for the early diagnosis and management of GC. There is an urgent need to identify of specific and sensitive molecular markers for the early diagnosis of GC. The development of proteomics technology has provided a new way to research molecular markers for the early diagnosis of GC [4]. In this study, we identified differential PRSS1 proteins in gastric mucosal epithelial cancerous tissue via iTRAQ labeling and 2D LCMS/MS.
A previous report showed that PRSS1 may be an oncogene for pancreatic tumors [16]. Recent studies have shown that the PRSS1 protein is important in the development of pancreatic, colorectal and cervical cancer [7][8][9]. However, whether and how the PRSS1 protein is involved in the progression of GC is unknown. In the present study, we confirmed that PRSS1 was highly expressed in GC, which was positively correlated with tumor differentiation, tumor size, TNM stage and lymph node metastasis of GC and was associated with poor prognosis. Cell abnormalities and unchecked proliferation are hallmark features of malignant tumors [17]. Moreover, we found that PRSS1 overexpression promoted cell growth and proliferation, while the opposite result was obtained when PRSS1 expression was knocked down. Therefore, PRSS1 may play an oncogenic role in GC and be involved in the process of GC. However, the underlying mechanism by which PRSS1 affects GC is poorly understood.
MicroRNAs are small noncoding RNAs that regulate the expression of target genes through posttranscriptional modification and are involved in tumor progression [18]. Previous studies have reported that miR-146a-5p is involved in the occurrence and development of multiple tumors [11,19]. Other reports have demonstrated that miR-146a-5p expression levels are significantly reduced in GC, which inhibits the proliferation, migration and invasion of GC [20,21]. However, some studies have shown that miR-146a-5p is highly expressed in GC and promotes the progression of GC [22,23]. In our study, we confirmed that miR-146a-5p expression is significantly reduced in GC, and increasing the expression of miR-146a-5p inhibits the growth and proliferation of GC cells. In contrast, we observed a decrease in cell growth and proliferation in MGC803 cells lacking miR-146a-5p expression. In addition, PRSS1 is a direct target gene of miR-146a-5p. Our data indicated that miR-146a-5p targets PRSS1 and inhibits the growth and proliferation of MGC803 cells.
PAR-2, a member of the family of proteaseactivated receptors widely distributed on the surface of human cells, is mainly activated by proteases [24]. According to the PAR-2 activation mechanism, there are multiple modulators of PAR-2 activity that are commonly used: the PAR-2 agonist SLIGKV-NH2 and the PAR-2 inhibitor FSLLRY-NH2 [25]. These regulators are powerful tools for PAR-2-related research. PAR-2 is highly expressed in GC and may be associated with GC proliferation and angiogenesis [26,27]. Chen reported that PAR-2 activation plays a key role in maintaining tumor cell proliferation [28]. In our cell proliferation assays, both knockdown of PRSS1 expression and inhibition of PAR-2 activity effectively inhibited MGC803 cell growth and proliferation; however, the growth and proliferative ability of MGC803/miR-PRSS1 cells treated with the PAR-2 agonist SLIGKV-NH2 was completely restored. This suggested that low expression of PRSS1 leads to a decrease in the PAR-2 activation level in MGC803 cells, indicating that PRSS1 plays an important role in maintaining cell growth and proliferation in a PAR-2-dependent manner.
PRSS1, a T cell receptor protein, is an important molecule for tumor signal transduction and activity regulation. PRSS1 activates receptors or ligands on the cell surface and is involved in cell growth, proliferation, differentiation and migration through the MAPK/ERK signaling pathway [29][30][31]. The ERK signaling pathway is a classic pathway of the mitogen-activated protein kinase (MAPK) family [32], which is mainly responsible for transmitting extracellular signals to the nucleus and regulating cell proliferation, differentiation, metastasis and survival [33]. Activated PAR-2 is involved in ERK signaling and mediated tumor cell proliferation [34]. However, the function of the ERK signaling pathway in GC needs further study. Phosphorylated ERK1/2 is an important marker of ERK signaling pathway activation [35]. In the present study, we confirmed that knockdown of PRSS1 expression significantly inhibited the activation of the ERK signaling pathway, and the inhibitory effect was comparable to that of MGC803 cells treated with FSLLRY-NH2. Interestingly, when MGC803/miR-PRSS1 cells were incubated with SLIGKV-NH2, the level of phosphorylated ERK1/2 was completely restored. Our data indicated that knockdown of PRSS1 expression prevents the activation of the ERK signaling pathway by reducing PAR-2 activation in MGC803 cells. This is consistent with previous reports that PAR-2 activation may affect ERK signaling pathway activation [36].
Together, our data demonstrate that PRSS1 protein expression was significantly increased in GC and was positively correlated with differentiation, tumor size, and lymph node metastasis. In addition, PRSS1 is a target gene of miR-146a-5p. MiR-146a-5p targets PRSS1 and suppresses the growth and proliferation of MGC803 cells. Silencing PRSS1 expression inhibits ERK signaling pathway activation by reducing PAR-2 activation, resulting in suppressed growth and proliferation of MGC803 cells. Further research and attention on PRSS1 will help us better understand the occurrence and progression of GC and provide novel evidence for the identification of biomarkers or potential targets for the early treatment of GC.