| Gastric cancer is one of the most common malignancies in humans.According to reports published by the World Health Organization,the worldwide incidence of gastric cancer is 13.86 / 100,000 per year,and the mortality rate of gastric cancer ranks second among all tumor mortality.China is a country with a high incidence of gastric cancer.The annual morbidity and mortality of gastric cancer are more than twice the world average.Therefore,studying the pathogenesis of gastric cancer and the target of drug action has become a global research hotspot.Atrial natriuretic peptide(ANP)has normal physiological effects such as relaxing vascular smooth muscle,increasing vascular permeability,reducing blood pressure,diuretic and regulating electrolyte balance.Early studies by Japanese scholars have shown that ANP can inhibit the invasion and metastasis of lung cancer cells.However,the effect of ANP on gastric cancer has not been reported.The occurrence and development of gastric cancer is a very complicated process,which is believed to be related to abnormal changes in signaling pathways.Current research shows that the occurrence,development,metastasis and poor prognosis of gastric cancer are closely related to the excessive activation of PI3 K / Akt signaling pathway.Studies have reported that inhibitors of the PI3 K / Akt signaling pathway can inhibit the invasion and metastasis of pancreatic cancer cells.The cytoskeleton Nestin protein is related to the aggressive growth and metastasis of tumors.However,the effect of cytoskeleton Nestin protein on gastric cancer cell,s growth,invasion and metastasis is almost unknown.Whether the effect of cytoskeleton Nestin protein on invasion and metastasis of gastric cancer cells is affected by ANP and PI3 K / Akt signaling pathway has not been reported at home and abroad.In this experiment,human gastric cancer cell line MGC-803 was used as the research object.ANP and PI3K-specific inhibitor LY294002 were applied to MGC-803 to investigate the effect of ANP on the proliferation and migration of gastric cancer cells,and further study whether the effect of ANP on MGC-803 cells is related to the regulation of the PI3 K / Akt signaling pathway and the cytoskeleton Nestin protein,thereby inhibiting the proliferation and migration of gastric cancer cells.The purpose of this study is to provide theoretical basises for ANP as an inhibitor of gastric cancer cell proliferation and metastasis and Nestin protein as a potential drug target for drug action,and provide new ideas for studying the mechanism of gastric cancer.This research is conducted in two parts: Part I Effect of ANP on Proliferation and Migration of Human Gastric Cancer MGC-803 CellObjective:In this study,we use human gastric cancer MGC-803 cells as the research object,to explore the effects of ANP and PI3 K specific inhibitor LY294002 on the proliferation and migration of human gastric cancer MGC-803 cells.Methods:1.MGC-803 cells were treated with different concentrations of ANP for 12 h,24h and 48 h,and the effect of ANP on the proliferation of MGC-803 cells was detected by CCK-8 assay.2.MGC-803 cells were treated with different concentrations of LY2940 02 for 24 h,48h and 72 h,and the effect of LY294002 on the proliferation of MGC-803 cells was detected by CCK-8 assay.3.The proliferation activity of MGC-803 cells was detected with contr-ol,ANP,LY294002,ANP+LY294002 treatment for 48 h by CCK-8 assay.4.The cell number and morphology differences of MGC-803 cells were observed with control,ANP,LY294002,(ANP+LY294002)treatment for 48 h by inverted microscope.5.The migration capacity of MGC-803 cells was detected with control,ANP,LY294002,ANP+LY294002 treatment for 24 h by scratch test.Results:1.The results of CCK-8 assay showed that higher concentrations of ANP could significantly inhibit the proliferation of MGC-803 cells with time-meter dependent.2.The results of CCK-8 assay showed that LY294002 could significantly inhibit the proliferation of MGC-803 cells with time-meter dependent.3.The results of CCK-8 assay showed that the ANP+LY294002 group had a more significant inhibitory effect on the proliferation of MGC-803 cells than when they were used alone.4.Under the inverted microscope,it was found that the MGC-803 cells in the group control were well attached.Compared with the control group,the number of cells in the ANP group decreased and the state was slightly worse.Compared with the control group,the cells in the LY294002 group had severely restricted growth,and some of the cells retracted and even showed a state of rupture and suspension and the cells in the ANP+LY294002 group had the worst growth,showing that some of the cells retracted or even ruptured in abnormal cell morphology.5.The scratch test showed that both ANP and LY294002 could significantly inhibit the migration of MGC-803 cells.The scratch test showed that ANP+LY294002 group had a more significant inhibitory effect on the migration of MGC-803 cells than when they were used alone.Conclusion:1.ANP can inhibit the proliferation of human gastric cancer cell MGC-803,and the inhibitory effect of ANP on human gastric cancer cell MGC-803 shows a dose-time dependent relationship,and ANP can also inhibit the migration of human gastric cancer cell MGC-803 effect.2.ANP combined with LY294002 was applied to human gastric cancer cell line MGC-803,which had stronger inhibitory effect on the growth and migration of MGC-803 than when they were applied alone.Part II Relationship between the effect of ANP on inhibiting the proliferation and migration of gastric cancer cell MGC-803 and the PI3 K / Akt signaling pathway and cytoskeleton Nestin proteinObjective:According to the results of the first part,we further analyzed whether the inhibitory effect of ANP on human gastric cancer MGC-803 cells was related to the regulation of PI3K/Akt signaling pathway and nestin protein.Methods:1.The expression levels of Nestin,t-Akt,and p-Akt-Ser473 protein was detected with control,ANP,LY294002,ANP+LY294002 treatment for 48 h by Immunocytochemistry(ICC).2.The expression levels of Nestin,t-Akt,and p-Akt-Ser473 protein was detected with control,ANP,LY294002,ANP+LY294002 treatment for 48 h by Western Blot.3.Experimental data were statistically analyzed by SPSS 19.0 softwareResults:1.Immunocytochemistry and Western Blot results showed that Nestin protein,t-Akt protein,and p-Akt-Ser473 protein were all expressed in human gastric cancer MGC-8032.Immunocytochemistry and Western Blot results showed that compared with the control group both LY294002 and ANP groups could significantly down-regulate the expression of Nestin protein and p-Akt-Ser473 protein.Compared with ANP group and LY294002 group,the expression levels of Nestin and p-Akt-Ser473 in ANP + LY294002 group were the lowest.3.Immunocytochemistry and Western Blot results showed that there was no difference in the expression of t-Akt protein among the control group,ANP group,LY294002 group,and ANP + LY294002 group.Conclusion:1.ANP may affect the activity of the PI3K/Akt signaling pathway by inhibiting Akt phosphorylation,thereby reducing the expression of Nestin,and then affecting the proliferation and migration of human gastric MGC-803 cells.2.ANP combined with LY294002 synergistically inhibit the PI3 K / Akt signaling pathway and down-regulated Nestin protein in the cytoskeleton to further inhibit the proliferation and migration of gastric cancer cells MGC-803.Conclusion1.ANP can inhibit the proliferation and migration of human gastric cancer cell MGC-803,so ANP can be used as an inhibitor of gastric cancer cell proliferation and migration.2.ANP can inhibit the proliferation and migration of gastric cancer cell line MGC-803 by inhibiting the PI3 K / Akt signaling pathway to down-regulate cytoskeletal Nestin protein.Therefore,cytoskeleton Nestin protein can be used as a potential drug target for the treatment of gastric cancer.3.The study that ANP inhibited the proliferation and migration of gastric cancer cell line MGC-803 by inhibiting the PI3 K / Akt signaling pathway to down-regulate the cytoskeleton Nestin protein provides a new idea for studying the pathogenesis of gastric cancer.4.The study that ANP combined with LY294002 synergistically inhibited the PI3 K / Akt pathway to down-regulate the cytoskeleton Nestin protein and thereby inhibiting the proliferation and migration of MGC-803,provides a new idea for combined target inhibitors to treat gastric cancer. |
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