Expression And Significance Of STAT3/NANOG Signaling Pathway In Gastric Carcinoma Cells

In this study,we observed the activation of STAT3/NANOG signaling pathway in gastric cancer cells and normal gastric mucosa epithelial cells,and compared the expression of key signaling molecules of STAT3/NANOG signaling pathway.When the STAT3/NANOG signaling pathway inhibitors NSC74859 and Napabucasin act on cells,we compared and analyzed the differences among NANOG,PIAS3,SOX2,OCT4,STAT3 and p-STAT3 in each cells from the level of cell transcription,translation,and cell biology.To explore the molecular pathology mechanism of STAT3 and p-STAT3,and to provide scientific basis for target therapy of gastric cancer.At the same time,to explore the isolation and extraction of gastric cancer stem cells,and to lay a foundation for studying the expression of STAT3/NANOG signaling pathway in gastric cancer stem cells.Objectives: 1.Compare and analyze the expression differences of STAT3,NANOG,PIAS3,SOX2,OCT4 and other genes in poorly differentiated gastric cancer cell lines BGC-823,MGC-803,moderately differentiated gastric cancer cell line SGC-7901 and normal gastric mucosal epithelial cell GES-1.Verify the activation of STAT3/NANOG signaling pathway in gastric cancer.2.Observe the effects of STAT3 inhibitor NSC74859 on poorly differentiated gastric cancer cell lines BGC-823,MGC-803,moderately differentiated gastric cancer cell line SGC-7901 and normal gastric mucosa epithelial cells GES-1,and explore the possible molecular mechanism of STAT3/NANOG signaling pathway regulating gastric cancer cells to provide more experimental evidence for the application of this pathway inhibitor to the clinic.3.Investigate the effect of Napabucasin,a inhibitor of STAT3,on signal transduction of poorly differentiated gastric cancer cell lines BGC-823,MGC-803 and moderately differentiated gastric cancer cell line SGC-7901 in order to provide more experimental evidence basic research and clinical treatment for gastric cancer.4.Explore the extraction of gastric cancer stem cells,initially.This study lays a foundation for exploring whether STAT3/NANOG signaling pathway exists in gastric cancer stem cells.Provide academic basis for the prevention and treatment of the genetic level of gastric cancer,and for the clinical research of new targeted therapeutic drugs and the search for new combined treatment options.Methods: 1.Culture human poorly differentiated gastric cancer cell lines BGC-823,MGC-803,moderately differentiated gastric cancer cell line SGC-7901 and normal gastric mucosal epithelial cell GES-1.Real-time fluorescence quantitative PCR was used to determine the signal molecules STAT3,NANOG and PIAS3 of STAT3/NANOG signaling pathway and the mRNA expression of,stem cell-specific transcription factors SOX2,OCT4 and other related genes.The expression of total protein STAT3,NANOG,PIAS3,SOX2,OCT4 and phosphoprotein p-STAT3 was detected by Western Blot.2.The STAT3 targeting inhibitor NSC74859 was used to intervene in poorly differentiated gastric cancer cell lines BGC-823,MGC-803,moderately differentiated gastric cancer cell line SGC-7901 and normal gastric mucosal epithelial cell GES-1.CCK8 assay was used to detect the effect of NSC74859 on cell proliferation;RT-qPCR was used to measure the mRNA expression of STAT3,NANOG,PIAS3,SOX2,OCT4 and other genes;Western blot was used to detect expression of total protein STAT3,NANOG,PIAS3,SOX2,OCT4 and p-STAT3;changes in cell cycle distribution and apoptotic rate were measured by flow cytometry.3.STAT3 stem inhibitor Napabucasin was used to intervene in poorly differentiated gastric cancer cell lines BGC-823,MGC-803,and moderately differentiated gastric cancer cell line SGC-7901.The effect of Napabucasin on cell proliferation was detected by CCK-8,mRNA expressions of STAT3,NANOG,PIAS3,SOX2,OCT4 and so on were detected by RT-qPCR.The quantitative expression of total protein STAT3,NANOG,PIAS3,SOX2,OCT4 and p-STAT3 were detected by Western Blot.4.Explore the isolation and extraction of gastric cancer stem cells.Isolate and extract the Cancer stem cells were isolated and extracted from cell lines and identifyied them.Results: 1、Transcriptional and translational expression of key signal molecules of STAT3/NANOG pathway in poorly differentiated gastric cancer cell lines BGC-823,MGC-803,moderately differentiated gastric cancer cell line SGC-7901 and normal gastric mucosal epithelial cell GES-1.Compared with control group GES-1,the expression levels of NANOG,PIAS3 and stem cell-specific genes SOX2,OCT4 mRNA in SCC-7901 cells were significantly higher than those in GES-1,with significant differences(P<0.01).The mRNA expression of NANOG,PIAS3 and stem cell-specific genes SOX2 and OCT4 in BGC-823 cells was lower than that of GES-1(P<0.01).The mRNA expression of STAT3,PIAS3 and stem cell-specific genes SOX2 and OCT4 in MGC-803 cells was lower than that of GES-1(P<0.01).The expression of p-STAT3 protein in STAT3/NANOG signaling pathway was significantly lower in GES-1 cells than in BGC-823,MGC-803,and SGC-7901(P<0.05).The expression of NANOG protein in normal stomach The mucosal epithelial cells in GES-1 were significantly higher than those in BGC-823,MGC-803,and SGC-7901 cells(P<0.05).The expression of NANOG in moderately differentiated cell line SGC-7901 was significantly higher than that in poorly-differentiated BGC-823,MGC-803(P<0.05).2、Effects of STAT3 inhibitor NSC74859 on the biological function of poorly differentiated gastric cancer cell lines BGC-823,MGC-803,moderately differentiated gastric cancer cell line SGC-7901 and normal gastric epithelial cells GES-1 by inhibiting STAT3/NANOG signaling pathwayAfter 24 hours treatment of MGC-803,BGC-823,SGC-7901,and GES-1 cells with different concentrations of NSC74859,it was found that within this concentration range,MGC-803,BGC-,compared with the control group,increased with the concentration of NSC74859.The proliferation of 823,SGC-7901 and GES-1 cells gradually decreased(P<0.05).When the concentration of MGC-803 and BGC-823 cells in the experimental group was 25 μmol/L,the cell proliferation rate was 110.6%,which was higher than that in the control group.At this time,the inhibitors had a promoting effect on cells at low concentrations,but promoted cell growth instead.Flow cytometry analysis of the effect of NSC74859 on the cell cycle of SGC-7901 revealed that the proportion of S phase cells gradually increased,the proportion of G1 phase cells gradually decreased,and the proportion of G2 phase cells also gradually decreased(P<0.05).At the same time,apoptosis detection showed that NSC74859 had pro-apoptotic effects on MGC-803,BGC-823,and SGC-7901 cells in a dose-dependent manner.In GES-1 cells,as the concentration of NSC74859 increased,the expression level of STAT3 mRNA increased and the expression levels of NANOG,SOX2,and OCT4 first decreased and then increased,and the expression level of PIAS3 mRNA increased.After the high reduction,there is a significant difference(P<0.05).Compared with the control group,there was no significant difference in total STAT3 protein expression(P>0.05).With the increase of NSC74859 concentration,the expression levels of p-STA3,NANOG and PIAS3 protein gradually decreased,and the difference was statistically significant(P<0.05).In BGC-823 cells,as the concentration of NSC74859 increased gradually,the expression levels of STAT3,NANOG,SOX2,and OCT4,the key genes in the STAT3/NANOG pathway,decreased continuously with the concentration of 50μmol/L and 100μmol/L.The expression of PIAS3 was continuously increased,with significant differences(P<0.05).Compared with the control group,there was no significant difference in the expression of p-STAT3 protein(P>0.05).The expression levels of STAT3,NANOG and PIAS3 protein were lower than those in the control group.With the increase of the concentration,the protein expression gradually decreased.Statistically significant(P<0.01).In MGC-803 cells,mRNA expression levels of STAT3 and OCT4 increased gradually with the increasing concentration of NSC74859,and the mRNA expression levels of NANOG,PIAS3,and SOX2 gradually decreased compared with the control group,with significant differences(P< 0.05).There was no significant difference in total STAT3 protein expression(P>0.05).With the increase of NSC74859 concentration,the expression of PIAS3 protein gradually decreased,and the difference was statistically significant.In SGC-7901 cells,as the concentration of NSC74859 increased,the mRNA expression of STAT3 increased continuously,and the mRNA expression levels of NANOG,PIAS3,SOX2,and OCT4 decreased gradually compared with the control group(P< 0.05).There was no significant difference in total STAT3 protein expression(P>0.05).The expression levels of p-STA3,PIAS3 and OCT4 protein were lower than those in the control group(P<0.05).The expression level of NANOG protein increased with the concentration.High and increased(P<0.05),the difference was statistically significant.3、Effects of STAT3 inhibitor napinbucasin on the biological function of poorly differentiated gastric cancer cell lines BGC-823,MGC-803 and moderately differentiated gastric cancer cell line SGC-7901 by inhibiting STAT3/NANOG signaling pathway.After 72 hours of Napabucasin treatment,the concentrations of MGC-803,BGC-823,and SGC-7901 cells in the treated group were increased with the concentration of Napabucasin at concentrations of 12.5,25,50,100,and 150 nmol/L.The cell proliferation ability gradually decreased.In BGC-823 cells,the mRNA expression levels of STAT3,NANOG,PIAS3,SOX2,and OCT4 decreased with the increase in the concentration of napabucasin compared with the control group,with significant differences(P<0.05).There was no significant difference in the total STAT3 protein expression(P>0.05).The expression of p-STA3 protein increased with the increasing concentration of Napabucasin,and its expression gradually decreased.The difference was statistically significant(P<0.01).The protein expressions of PIAS3,NANOG and OCT4 increased with the increase of Napabucasin concentration,and the difference was statistically significant(P<0.01).In MGC-803 cells,the expression of STAT3/NANOG pathway key genes STAT3,NANOG,PIAS3 and stem cell-specific genes SOX2,OCT4 mRNA was gradually increased with the increase of napabucasin concentration compared with the control group,with a significant difference(P<0.05).Compared with the control group,there was no significant difference in mRNA expression levels of SOX2 and OCT4 genes in each dose group.There was no significant difference in total STAT3 protein expression(P>0.05).The expression of p-STA3 protein increased with the increase of Napabucasin concentration,and its expression gradually decreased.The difference was statistically significant.The protein expression of PIAS3 and NANOG increased with the increase of Napabucasin concentration,and the difference was statistically significant(P<0.01).In SGC-7901 cells,the expression of STAT3/NANOG pathway key genes STAT3,NANOG,PIAS3 and stem cell-specific genes SOX2,OCT4 mRNA was gradually increased with the concentration of napabucasin compared with the control group,and the expression level was significantly different(P <0.01).There was no significant difference in the total STAT3 protein expression(P>0.05).The protein expression of p-STA3,PIAS3,and NANOG increased with the increase of Napabucasin concentration.The expression of STAT3 protein gradually decreased and the difference was statistically significant(P<0.01).4.The successful isolation of gastric cancer stem cells has been characterized by the characteristics of cancer stem cells.Conclusions: 1、The STAT3/NANOG signaling pathway is active in human poorly differentiated gastric cancer cell lines BGC-823,MGC-803,and moderately differentiated gastric cancer cell line SGC-7901.This abnormal signal transduction pathway is related to the occurrence and development of tumors.2、STAT3 inhibitor NSC74859 can inhibit the transcription and translation of key genes involved in STAT3/NANOG signaling pathway,which can inhibit the proliferation of gastric cancer cells,arrest cells and induce apoptosis,thus inhibiting the growth of gastric cancer cells and revealing STAT3 Correlation between STAT3/ NANOG signaling pathway and the effect of chemotherapy in gastric cancer.3 、 The STAT3 stem inhibitor napabucasin can inhibit the biological activity of cells by inhibiting STAT3/NANOG signaling pathway,which can inhibit gastric cancer cell proliferation,and inhibit the growth of gastric cancer cells.The STAT3/NANOG signaling pathway inhibitor napabucasin may be used as an effective anticancer drug for clinical use.4、It can successfully isolate gastric tumor stem cells and lay a foundation for subsequent study of the expression of STAT3/NANOG signaling pathway in gastric cancer stem cells.