Helicobacter Pylori Infection Promotes The Growth And Invasion Of Gastric Cancer Cells Through C-JUN-DKK1-PI3K/AKT/mTOR Signaling

Objective:To explore Helicobacter pylori(H.pylori)infection caused by the proto-oncogene c-JUN up-regulated DKK1 expression,as well as the role and molecular mechanism of DKK1 in the growth and invasion of gastric cancer cells.Methods:(1)Gastric cancer cells AGS and SGC-7901 were transfected with c-JUN shRNA 48h,and H.pylori 1004 was added to infect the cells for 6h.The cells were collected,total protein were extracted,and the c-JUN and DKK1 protein expression levels each group was detected by Western blot.(2)Gastric cancer cell lines NCI-N87and SNU-16 were transfected with the over-expression vector of c-JUN plasmid gene for 48h,and then cells were collected,and total RNA and protein were extracted,as well as the c-JUN and DKK1 mRNA and protein expression levels were detected by RT-qPCR and WB,respectively.(3)Dual luciferase experiment:(1)DKK1 promoter sequences were obtained,and a dual luciferase expression vector(DKK1 P)containing the DKK1 promoter sequences was construced and identified.(2)AGS cells were co-transfect with over-expression vectors of FOSL1,c-JUN and DKK1genes separately or simultaneously,and then cells were lysed at 48h after transfection,and the activities of firefly luciferase(Fluc)and renilla luciferase(Rluc)were detected,to calculate the ratio of Fluc to Rluc.(4)The stable cell lines overexpressing or silencing DKK1 gene were screened to extract total RNA and total protein of cells.RT-qPCR and WB were used to detect the expression levels of DKK1 mRNA and protein,respectively.The experiments were divided into blank control group(Control),negative control group(shNC/NC),silencing/overexpressing DKK1 group.CCK8 assay was used to detect the cell growth at 0,24,48,72,96,120 and 144h after cell culture,and and crystal violet staining was used to count the clone number in each group.Transwell test was used to detect the cell migration and invasion ability.Flow cytometry was used to detect the cell cycle and apoptosis after silencing DKK1 gene.(5)AGS and BGC823 cells for over-expresson of DKK1 gene were treated with a PI3K inhibitor LY294002 and divided into DKK1 overexpression group,DKK1overexpression+0.5%dimethyl sulfoxide group(DKK1+0.5%DMSO)and DKK1overexpression+LY294002 group(DKK1+LY29400.CCK8 asssy was used to detect the cell growth in each group after culture for 0,24,48,72,96,120 and 144h.Crystal violet staining was used to count the clone number of cells after 2 weeks of culture.Transwell test was used to detect the cell migration and invasion ability.(6)Stable transfected cell lines silencing or overexpressing DKK1 gene were established.RT-qPCR was used to detect the expression of PI3K,AKT and mTOR mRNA,WB was used to detect the expression of PI3K,AKT,and mTOR proterins and their phosphorylated proteins.In the silence experiment,H.pylori 1004 was used to carry out a rescue experiment.(7)Nude mouse tumorigenesis experiment:Nude mice(3-4weeks)were inoculated subcutaneously with stably DKK1-transfected BGC823 cells at 3.5×10~6 cells and sacrificed after 18 days of inoculation.Then xenografts in nude mouse were harvested,and the tumors volume and weight were measured.The xenograft tissues were fixed to detect the expression of DKK1 and Ki67 by WB and identified by HE staining.Results(1)Compared with the negative control group,the expression of DKK1 protein in the c-JUN gene-silencing group was reduced.After adding H.pylori 1004,the expression of DKK1 protein was restored,and the difference was statistically significant(P<0.05).(2)Compared with the negative control group,the expression of DKK1 mRNA and protein in c-JUN overexpression group was up-regulated,and the differences were statistically significant(P<0.05).(3)The dual luciferase experiment results show that:FOSL1 and c-JUN can combine to DKK1 promoter to promote the expression of luciferase gene,and c-JUN and FOLS1can coordinately enhance the expression of luciferase genes.The differences was statistically significant(P<0.05).(4)The results showed that the stable cell lines silencing or overexpressing DKK1 gene was successfully constructed and compared with the shNC group,the cell growth,proliferation,migration and invasion were inhibited,cell apoptosis was increased,and cell arrest was at G2/M phase in the DKK1-silencing group.The differences were statistically significant(P<0.05).Further,compared with the NC group,overexpression of DKK1 gene can promote cell growth,proliferation,migration and invasion,and the differences were statistically significant(P<0.05).(5)After PI3K signaling pathway inhibitor LY2940002 was used to treat the DKK1-overexpressed cells at a concentration of 50?mol/L for 48h,the results showed that that the cell growth,clone number and the number of migrating and invading cells were decreased significantly compared to control cells(P<0.05).(6)After silencing the DKK1 gene,the expression of PI3K signaling pathway-related proteins was down-regulated,and after adding H.pylori 1004 into cells,the expression levels of these proteins were all recovered.The differences were statistically significant(P<0.05).Contrarily,after overexpression of DKK1 gene,the expression of PI3K signaling pathway-related proteins was up-regulated,and the differences were statistically significant(P<0.05).(7)The results of in vivo tumor formation in nude mice showed that compared with the control group(NC),the tumor volume and weight in nude mice in the DKK1-overexpressed group were increased and there were the hemorrhage and necrosis on the surface of tumor,as well as the expression of DKK1 and Ki67 in the tumor tissue was increased.Conclusion:(1)Helicobacter pylori infection promotes DKK1 expression by up-regulating c-JUN.(2)c-JUN alone or combined with FOSL1 to form AP-1 promotes the activity of DKK1promoter,and c-JUN and FOSL1 collectively play the strongest.(3)DKK1 regulates the malignant biological phenotype of gastric cancer cells through the PI3K/AKT/mTOR signaling pathway.