| Objective: The incidence of gastric cancer has been high all over the world,and the fatality rate of gastric cancer in China is 40% of that in the world.Although the radical surgical treatment and adjuvant chemoradiotherapy for gastric cancer have made good progress,the therapeutic effect and prognosis of patients with gastric cancer are still not ideal due to the complexity of the pathogenesis of gastric cancer.Therefore,to explore the molecular mechanism of the occurrence and development of gastric cancer so as to determine the ideal therapeutic target is the key to the treatment of gastric cancer,and has been a hot spot in the process of cancer research.SLC12A5,a potassium chloride transporter belonging to the solute carrier family,was originally known as an important protein in maintaining chloride ion balance in and outside nerve cells.With the deepening of research,a number of studies have shown that SLC12A5 plays a carcinogenic role in a variety of cancers,but the expression and role of SLC12A5 in gastric cancer remain unclear.The purpose of this study was to clarify the expression of SLC12A5 in gastric cancer and its relationship with the prognosis of patients with gastric cancer,and to explore its mechanism in the occurrence and development of gastric cancer.Methods: 1.Pathological numbers and wax samples of patients who underwent radical gastrectomy during 2015 and 2016 were collected from the Department of Gastrointestinal Oncology,the First Affiliated Hospital of China Medical University.A total of 108 subjects meeting the criteria were included.Wax samples of gastric cancer and paired adjacent normal tissue were collected from 108 patients and their clinicopathologic data(such as sex,age,degree of differentiation,serous infiltration,lymph node metastasis,TNM stage,tumor size,and 5-year survival rate after surgery)were collected.Immunohistochemical test was used to detect the expression level of SLC12A5 in the above paired tissues,and the relationship between the immunohistochemical results and clinicopathologic data of patients with gastric cancer was analyzed.Kaplan Meier function and Kaplan Meier Plotter website(https://kmplot.com/analysis)is used to analyze SLC12A5 immunohistochemical results of the relationship with the prognosis of patients.2.The effect of SLC12A5 on the function of gastric cancer cells and its mechanism of action: Fresh cancer tissues and paired paracancer tissues of gastric cancer patients after surgery were collected,and the expression of SLC12A5 protein was detected by Western Blotting experiment.Star Base using bioinformatics analysis site(http://starbase.sysu.edu.cn/starbase2/index.ph)to predict SLC12A5 regulation of micrornas,Double luciferase reporter gene assay was used to verify the direct regulatory relationship between SLC12A5 and miR-133a-3p.The expression of miR-133a-3p in gastric cancer cells was detected by real-time quantitative PCR.Transient plasmid was used to improve the expression of SLC12A5 and miR-133a-3p in gastric cancer cells,and then Transwell assay and CCK-8 assay were used to verify the effects of SLC12A5 on the proliferation,migration and invasion of gastric cancer cells.Western Blotting experiments verified the effect of SLC12A5 overexpression on downstream pathway-related proteins.Results: 1.Immunohistochemical and Western Blotting experiments showed that the expression of SLC12A5 in gastric cancer tissues was significantly higher than that in paracancer tissues.It was positively correlated with the degree of tumor differentiation(P=0.027),serous infiltration(P < 0.001),TNM stage(P < 0.001)and lymph node metastasis(P < 0.001),but not with gender(P=0.836),tumor size(P=0.700)and age(P=0.717).Kaplan-Meier survival analysis showed that high expression of SLC12A5 was associated with poor prognosis in patients with gastric cancer.2.After SLC12A5 was successfully overexpressed by transient plasmid,CCK-8 experiment showed no significant change in the proliferation ability of gastric cancer cells,while Transwell experiment showed that SLC12A5 overexpression could significantly enhance the migration and invasion ability of gastric cancer cells.Studies on the downstream pathway showed that SLC12A5 overexpression could promote the expression of p65 phosphorylated MMP-7.When SLC12A5 overexpressed gastric cancer cells were treated with Helenalin,an inhibitor of NF-κB pathway,the migration and invasion ability of gastric cancer cells were decreased,and the phosphorylation of p65 and the expression of MMP-7 were decreased.Double luciferase reporter gene assay confirmed that miR-133a-3p could target bind to the 3’UTR region of SLC12A5 to inhibit the expression of SLC12A5.Real-time quantitative PCR experiments of all gastric cancer cell lines showed that the expression of miR-133a-3p in normal gastric mucosa GES-1was significantly higher than that of other gastric cancer cell lines.After overexpression of miR-133a-3p,the expression of SLC12A5 was successfully inhibited,and the migration and invasion ability of gastric cancer cells were decreased,without significant changes in proliferation ability.Western Blotting experiments showed that the phosphorylation of p65 and the expression of MMP-7 were decreased.Conclusion: 1.SLC12A5 is highly expressed in gastric cancer,and its expression level is positively correlated with patients’ serous infiltration,degree of tumor differentiation,TNM stage,lymph node metastasis and poor prognosis,suggesting that SLC12A5 plays a promoting role in the occurrence and development of gastric cancer and is an important marker gene for gastric cancer.2.miR-133a-3p can directly bind to the 3’UTR region of SLC12A5 and inhibit the expression level of SLC12A5 in gastric cancer cells.3.Overexpression of SLC12A5 in gastric cancer can increase the expression level of MMP-7 by activating the NF-κB signaling pathway and promote the migration and invasion of gastric cancer.SLC12A5 may be an oncogene in gastric cancer and can be used as a biomarker in the diagnosis of gastric cancer. |
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