NCX1 Promotes Proliferation Of Gastric Cancer Through Ca²⁺/AKT/β-Catenin Signaling Pathway

Part 1 Comparison of expression of NCX1 in human primary gastric cancer tissues and gastric cancer cell lines.Objectives:To investigate the protein expression changes of NCX1 in human gastric cancer tissues and adjacent tissues,gastric cancer cell lines and gastric mucosal epithelial cels.Methods:Immunohistochemistry was applied for examining the protein expression of NCX1 in human gastric cancer tissues and adjacent tissues and the correlation was analyzed with clinicopathological features and survival prognosis.Western blot was used to detect the expression of NCX1 in human gastric cancer tissues and adjacent tissues,gastric cancer cell lines and gastric mucosal epithelial cel s GES-1.Results:Immunohistochemistry showed that the protein expression of NCX1 in human gastric cancer tissues was higher than that in adjacent tissues.The gastric cancer patients with high expression of NCX1 had larger tumors,shorter survival time and poor prognosis.Western blot results also confir med the above conclusion.It also manifested that the protein expression of NCX1 in human gastric cancer cell lines was higher than that in gastric mucosal epithelial cel s GES-1.Conclusions:NCX1 was highly expressed in human gastric cancer tissues and gastric cancer cell lines,suggesting that it may be a cancer-promoting molecule in the development of gastric cancer.Part 2 The effects of NCX1 in the proliferation of gastric cancer cel s.Objectives:To study the effects of NCX1 in the proliferation of gastric cancer cel s.Methods:CCK-8 assay was carried out to detect the effects of Ca²⁺and the reverse inhibitor KB-R7943 of NCX1 in the proliferation of gastric cancer cells MKN-45,SGC-7901,AGS and GES-1 cells.Western blot was used to detect the effects of Ca²⁺and KB-R7943 stimulation on NCX1 protein expression in gastric cancer cells.After the knockdown of NCX1,verified the above experiment.Results:CCK-8 results showed that Ca²⁺promoted the proliferation of gastric cancer cells and it was inhibited by KB-R7943,while Ca²⁺had no significant effects in the proliferation of GES-1 cells.Western blot manifested that Ca²⁺promoted the protein expression of NCX1 in gastric cancer cells and it was also inhibited by KB-R7943.Knockdown of NCX1 confirmed the above conclusion.Conclusions:Ca²⁺promoted the proliferation of gastric cancer cells by enhancing the protein expression of NCX1.Part 3 Molecular mechanism of NCX1 for promoting the proliferation of gastric cancer cels.Objectives:To investigate the role of NCX1 in regulating calcium concentration in gastric cancer cells and the molecular mechanism that promoted the proliferation of gastric cancer cels.Methods:Digital Ca²⁺imaging system was used to detect the regulatory effects of NCX1 on the concentration of Ca²⁺in gastric cancer cells.Western blot was carried out to examine effects of NCX1 on the phosphorylation of AKT（Ser473）and-catenin（Ser675）.Results:Extracellular Na⁺-free solution activated the reverse mode of NCX1 in gastric cancer cells,thereby detecting an increase of intracellular Ca²⁺signal and it could be inhibited by KB-R7943.Ca²⁺promoted the phosphorylation of AKT（Ser473）and-catenin（Ser675）and they could be inhibited by KB-R7943 and the knockdown of NCX1.Conclusions:NCX1 worked in a reverse mode to transfer extracellular calcium into the cytoplasm,thereby activating AKT（Ser473）,enhancing the expression ofβ-catenin（Ser675）,and finally promoting the proliferation of gastric cancer cels.