| Introduction:Gastric cancer is the14th most common cancer and cause of cancer death in the United States, with an estimated21,000new cases/year and over10,000deaths. The disease affects men disproportionately, with more than60%of new cases occurring in men. It is a disease of older individuals, with peak incidence in the seventh decade of life. Among racial groups, the disease is more common and has a higher mortality in African Americans, Asian Americans, and Hispanics compared with whites. Worldwide, gastric cancer is the fourth most common cancer and the second leading cause of cancer death. It is especially prevalent in East Asia and South America and has been increasing in developing countries, which now have almost two thirds of all distal gastric cancer cases. In contrast, rates have been decreasing in the United States. Among developed countries, Japan and Korea have the highest rates of the disease. Gastric cancer is the most common cancer in Japan. As a result, gastric cancer screening in Japan was started in the1970s and the mortality rate has dropped by50%since that time. Although there has been an increase in proximal tumors in Japan, most are distal gastric cancers. The major risk factors for gastric cancer include environmental and genetic factors. In1994, the international agency for research on cancer labeled H. pyloria definite carcinogen. A number of longitudinal prospective studies have demonstrated an association with the development of gastric cancer. The primary mechanism is thought to be the presence of chronic inflammation. Long-term infection with the bacteria leads to gastritis, primarily within the gastric body, with ventral gastric atrophy. In some patients, this progresses to intestinal metaplasia, dysplasia, and ultimately intestinal-type adenocarcinoma. A wide range of molecular alterations in intestinal metaplasia have been described and may affect the transformation into gastric cancer. These include overexpression of cyclooxygenase-2and cyclin D2, p53mutations, microsatellite instability, decreased p27expression, and alterations in transcription factors such as CDX1and CDX2. It is clear that intestinal metaplasia is a risk factor for the development of gastric carcinoma; however, not every patient with intestinal metaplasia develops invasive cancer. Host inflammatory responses play an important role in this process. Specifically, individuals with high levels of interleukin-1expression are at increased risk of gastric cancer development. High-salt foods, particularly those with salted or smoked meats that contain high levels of nitrate, along with low fruit and vegetable intake, are linked to an increased risk of gastric cancer. The mechanism is thought to bethe conversion of nitrates in the food to N-nitroso compounds by bacteria in the stomach. Fresh fruits and vegetables contain ascorbic acid, which can remove the carcinogenic N-nitroso compounds and oxygen free radicals. At present, although surgical resection combined with chemotherapy or radiotherapy is utilized in gastric cancer treatment, the outcomes for patients with metastasis are relatively poor. Therefore, it remains an extremely urgent matter to identify novel factors for early diagnostic and prognostic assessment.Most studies focus on intracellular signal molecules, such as oncogenes or tumor suppressor genes. As biomarkers, p53, E-cadherin, and v-erb-b2avian erythroblastic leukemia viral oncogene homolog2(HER-2) predict poor prognosis in gastric cancer. It is worth noting that various proteolytic enzymes, including matrix metalloproteinases (MMPs) and serine proteases, also play crucial roles in gastric cancer progression due to the degradation of extracellular matrix components. Serine protease inhibitor (serpin) peptidase inhibitor clade E member2(SERPINE2, also known as prostate nexin-1[PN-1]), a member of the serpin protein family, mainly modulates thrombin, urokinase, plalsmin, and trypsin activity. SERPINE2was first discovered as a susceptibility factor for chronic obstructive pulmonary disease and panlobular type emphysema. Recent studies have shown that SERPINE2is closely associated with tumorigenesis. Altered SERPINE2expression has been reported in several types of tumors, such as breast cancer, colon cancer, and oral squamous cell carcinoma. SERPINE2is also a tumor suppressor gene that decreases cell proliferation and vessel formation in prostate cancer. In contrast, another report demonstrated that SERPINE2promoted lymph node metastasis (LNM) in a xenograft model of testicular cancer, suggesting its function as an oncogene. However, SERPINE2expression and its relationship with gastric cancer, as well as its biological function on gastric cancer cells, remain unclear.In the present study, the expression levels of the SERPINE2gene in243gastric cancer tissues and matched adjacent normal mucosa were measured by quantitative PCR. We aimed to establish whether SERPINE2expression levels correlate with clinicopathological parameters and prognosis for patients with gastric cancer. Moreover, the biological function of SERPINE2was investigated in the gastric cancer cell line SGC7901.Methods:1. The levels of SERPINE2mRNA in243gastric cancer tissues and paired non-cancerous mucosa were determined using quantitative PCR.2. Inhibition of SERPINE2expression by small interfering RNA (siRNA) was detected by western blotting. Tetrazolium, soft agar, and Transwell assays were performed to evaluate the proliferation, anchorage-independent growth, and motility of gastric cancer SGC7901cells transfected with SERPINE2siRNA.3. Fisher’s exact test was used to analyze the association between SERPINE2expression and clinicopathological variables. OS or DFS curves were plotted using the Kaplan-Meier method and the differences were compared with the log-rank test. Independent risk factors were determined using a Cox proportional hazard model.Results:1. Real-time PCR:The data revealed a stepwise upregulation of SERPINE2mRNA in normal mucosa, tumor, and tumor with LNM. SERPINE2mRNA was upregulated 5.09-fold in gastric cancer tissues and4.18-fold in SGC7901cells compared with the normal mucosa (P=0.0089,0.0015, respectively). Compared with the normal mucosa, SERPINE2mRNA in the tumor with LNM group was upregulated6.79-fold (P=0.004). SERPINE2mRNA in the tumor without LNM group was2.24-fold higher than that in the normal mucosa, although no statistical difference was found between the two groups (P=0.0682). It is worth noting that compared to the tumor without LNM group, SERPINE2mRNA was increased3.02-fold in the tumor with LNM group (P=0.023).2. Association between SERPINE2mRNA expression and clinicopathologic factors in gastric cancer:of the243paired specimens, we considered a>1.25-fold increase as the cutoff for SERPINE2overexpression in gastric cancer:157cases (64.4%) had high SERPINE2expression; expression was low in the remaining86cases (35.6%). To determine the potential clinical implications of SERPINE2, the associations between clinicopathological factors and SERPINE2expression level were analyzed. High SERPINE2mRNA expression was significantly correlated with LNM (N classification, P=0.01),distant metastasis (M classification, P=0.018), and clinical stage (International Union Against Cancer [UICC], P=0.001). However, no correlations were found between SERPINE2mRNA level and age, sex, tumor size, tumor location, differentiation, or Lauren classification (P>0.05).3. Association of SERPINE2mRNA expression with prognosis of patients with gastric cancer:To further explore the prognostic value of high SERPINE2, the correlation between SERPINE2expression and OS or DFS was analyzed using Kaplan-Meier survival curves. Patients with high SERPINE2expression had significantly worse OS and DFS than patients with low SERPINE2expression (both P<0.001). Univariate analysis revealed that both decreased OS and DFS were associated with T classification, LNM, distant metastasis, clinical stage, poor differentiation, and SERPINE2expression. Multivariate analysis further demonstrated that high SERPINE2expression was an independent prognostic factor for patients with gastric cancer (P=0.000, hazard ratio [HR]=3.05,95%confidence interval [95%CI]=1.78-4.96; P=0.002, HR=2.30,95%CI=1.57-3.55). 4. SERPINE2siRNA had no effect on cell proliferation in vitro:To test the effect of increased SERPINE2on cell biological behavior in vitro, the proliferation ability of SGC7901cells treated with SERPINE2siRNA or negative control was detected by MTS assay. After transfection, the interference efficiency of SERPINE2was first confirmed by western blotting. Normalized to the negative control, SERPINE2expression was downregulated56.83±11.80%in the SERPINE2siRNA group (P=0.0039). Then, the cell proliferation ability was monitored at24h,48h, and72h. However, as illustrated in Figure3B, no difference in cell proliferation rate was observed between the two groups (P>0.05).5. SERPINE2siRNA decreased cell migration and invasion and inhibited soft agar colony formation in vitro:As SERPINE2expression was associated with LNM and distant metastasis, we were interested in determining whether SERPINE2affected cell migration and invasion ability in vitro. The migration and invasion activity of SGC7901cells transfected with SERPINE2siRNA was assessed by Transwell assay. In contrast to the negative control group, a significantly lower number of cells in the SERPINE2siRNA group migrated to the lower surface of the membrane in the migration and invasion assays (Figure3C, P=0.0158,0.008, respectively). Furthermore, to verify whether decreased SERPINE2expression would alter colony formation ability, a soft agar assay was performed after SERPINE2siRNA transfection. As expected, there was a significant reduction in colony formation by SERPINE2siRNA-transfected cells on soft agar compared to the negative control group (Figure3D, P=0.001). These data imply that SERPINE2controls the migration, invasion, and anchorage-independent growth capability of SGC7901cells.Conclusion: SERPINE2is overexpressed in gastric cancer tissues and correlates with poor survival, which suggest that SERPINE2might be involved in gastric cancer progression. SERPINE2is involved in gastric carcinogenesis by promoting cell migration, invasion, and anchorage-independent growth. We propose that SERPINE2is a potential target of gene therapy for gastric cancer. |
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