| Objective:Gastric cancer is the most common malignant tumor in digestive system,which has the second highest incidence rate and mortality rate among human tumors.Therefor it is of great importance to clarify the etiology and pathogenesis of this disease,as well as get early diagnosis.Slc26a9 is a newly found chloride channel protein,with high expression in normal gastric tissue of both human and mouse.Using Slc26a9 gene knockout mice researchers found that Slc26a9 is necessary in the maintanance of parietal cell function and viability,and normal morphology of stomach.Our previous work found that Slc26a9 gene deletion led to spontaneous precancerous lesions in mouse stomach,and eventually the carcinogenesis of gastric cancer.Besides,as Slc26a9 expression in human stomach is similar to that of mouse,it can be assumed that Slc26a9 gene deletion or downregulation may contritute to the initiation and progression of gastric cancer in human.There are no reports about the role of Slc26a9 in any tumor by far.Our research focuses on the effect of Slc26a9 on the initiation and progression of gastric cancer and underlying molecular mechanism,to provide theoretical basis for the etiology and pathogenesis of this disease,and guideline for the prevention of it.Methods: 1.IHC was performed to detect Slc26a9 expression in gastric cancer tissues and normal gastric mucosa tissues;2.qRT-PCR and western blot techniques were used to compare Slc26a9 mRNA and protein expression in Human normal gastric mucosal cell line GES-1 and human gastric cancer cell line AGS,BGC-823,MKN-28,MKN45,SGC-7901,HGC-27 and human gastric signet-ring cell carcinoma tissues;3.Overexpression of Slc26a9 in AGS cell line was achieved by lentiviral transfection,and qRT-PCR and western blot were used to confirm that;4.Proliferation of gastric cancer cells were detected by CCK-8 experiment and growth curve assay in three groups-normal control group,transfection control group,and Slc26a9 overexpression group;5.Ki-67 expression was detected by IHC in three groups;6.Flow cytometry were performed to analyze early stage apoptotic cells and late stage apoptotic cells in three groups,and TUNEL method to analyze late stage apoptotic cells;7.Migration of cells in three groups were investigated by Transwell experiment;8.At last,qRT-PCR and western blot were performed to learn about the mRNA and protein expression of p53-related gene.Result: 1.IHC revealed that there was a obviously lower expression of Slc26a9 in gastric cancer tissues than in normal gastric mucosa tissues(p<0.001),and the expression is decreased in different types of gastric cancer,and the expression level was significantly correlated with age,depth of tumor invasion,lymph node metastasis,and TNM stage of gastric cancer(p<0.05),it was not related to gender and distant metastasis.2.qRT-PCR and Western blot confirmed that Slc26a9 expressed in all of the cell lines detected,with highest expression in Human normal gastric mucosal cell line GES-1 and lowest expression in human gastric cancer cell line AGS;3.As demonstrated by qRT-PCR and western blot,Slc26a9 was overexpressed in AGS cell line(p<0.01);4.CCK-8 experiment and growth curve assay indicated cell proliferation was suppressed in Slc26a9 overexpression group,compared with the other two groups;5.Ki-67 expression was reduced in Slc26a9 overexpression group,indicating supressed proliferation(p<0.001);6.Flowcytometry combined with TUNEL method established that Slc26a9 overexpression could induce early stage and late stage apoptosis(p<0.01);7.Transwell experiment indicated that migration was inhibited when Slc26a9 overexpressed(p<0.001);8.By perfroming qRT-PCR and Western blot,we found that p53 signaling pathway was activated when Slc26a9 overexpressed,and proteins in p53 signaling pathway were activated,too.Compared with the other two groups,Slc26a9 overexpression group had higher p53 expression and lower P-AKT and Bcl-2 expression(p<0.05,p<0.01,p<0.001).Conclusion:Slc26a9 may be a novel tumor suppressor gene,whose down-regulation may contribute to the occurance of gastric cancer.Maybe it is through the AKT/p53/Bcl-2 signaling pathway that Slc26a9 overexpression inhibits proliferation and migration of gastric cancer cell AGS,and induces apoptosis... |
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