| Background and objective:Gastric cancer is the fifth most common cancer in the world.It is mostly diagnosed at an advanced stage,having a high rate of mortality,which makes it the third most severe cancer in the world.Helicobacter pylori(H.pylori,HP)is listed by the World Health Organization as a class I carcinogen,which is the most important factor in the occurrence of gastric cancer and an important initiator of the development of gastric mucosal Correa cascade(chronic non-atrophic gastritis –intestinal metaplasia – dysplasia – gastric cancer.About 89% of non-cardia adenocarcinomas are related to HP infection.In recent years,several studies have shown that HP infection could induce gastric epithelial-μlmesenchymal transition(EMT)process and contributes significantly to the migration and invasion of gastric tumor cells.The CD44 protein family could be roughly divided into CD44 standard isoforms(CD44s)and CD44 variant isoforms(CD44v),which are a class of transmembrane glycoproteins.At present,more and more studies focus on the role of CD44 s and various CD44 v in tumorigenesis and progression.CD44v9 is a member of CD44 v.It has been reported that CD44v9 is highly expressed in tumors and is associated with malignant phenotypes such as tumor prognosis,invasion,lymph node metastasis and EMT.Individual studies have shown that HP infection status is closely related to the appearance of CD44v9 positive cells in the gastric mucosa.Hypoxia-inducible factor 1α(HIF-1α)is a major regulator of cells adapting to hypoxia,and is involved in the invasion,migration and EMT process of gastric cancer cells.The expression of HIF-1α in gastric mucosa is also affected by HP infection.Studies have shown that CD44 s can regulate the expression of HIF-1α.The high expression of CD44v9 is related to the poor prognosis of gastric cancer patients.The current research on CD44v9 is mostly limited to its correlation in tumor prognosis and clinicopathological characteristics,and lack of studies on the biological processes and mechanisms involved.The biological process which CD44v9 involved in the progression of gastric cancer,especially the malignant phenotypes such as migration and invasion,has not been studied,as well as the regulatory mechanism.What is the role of CD44v9 in the pathogenesis of HP infection and its molecular mechanism? Therefore,based on the above research background,this study expounds the role of CD44v9 in malignant phenotypes such as migration,invasion and EMT of gastric cancer cells from human tissue specimens and cytology.We also explore the role of CD44v9 in the pathogenesis of HP infection and its regulatory mechanism,and whether CD44v9 regulates the migration and invasion of gastric cancer cells induced by HP infection by activating the HIF-1α signaling pathway.Materials and methods:1.CD44v9 is over expressed in gastric cancer tissues and is related to poor prognosis and clinicopathological features of gastric cancerPurchased gastric cancer tissue microarray chip,and detected the expression of CD44v9 in cancer tissue and adjacent tissue by immunohistochemical staining;In combination with gastric cancer patients’ clinical data,CD44v9 was analyzed in relation to gastric cancer patients’ prognosesThe correlation between CD44v9 and clinicopathological features was analyzed;2.CD44v9 expression correlated with HP infection status(1)CD44v9 expression was detected by immunohistochemical staining in different stages of gastric mucosal lesions(CNAG,IM,DYS,GC)and different HP infection states;(2)HP strains with different multiplicity of infection(MOI)were co-cultured with AGS cells for 12 hours,and the expression of CD44v9 in AGS cells was detected by Western blot;3.High expression of CD44v9 promoted gastric cancer cells migration,invasion and EMT process(1)AGS cells with knockdown of CD44v9 and BGC-823 cells with overexpression of CD44v9 were constructed by lentiviral infection,and the knockdown and overexpression effects were verified by Western-blot method;(2)The abilities of cell migration and invasion were assessed using the transwell migration and invasion Assay,and Western blot was used to detect E-cadherin,Vimentin,ZEB1 and other EMT markers;4.The role of CD44v9 in HP-induced gastric cancer cells migration,invasion and EMT process(1)After knocking down the expression of CD44v9 in AGS cells,they were co-cultured with HP strains for 48 hours,and the abilities of cell migration and invasion were assessed using the transwell migration and invasion Assay,(2)HP strains were co-cultured with AGS cells for 12 hours,and the expressions of EMT-related indicators were detected by Western-blot: E-cadherin and N-cadherin;After knocking down the expression of CD44v9 in AGS cells,they were co-cultured with HP strains for 12 hours,and the expressions of EMT-related indicators were detected by Western-blot: E-cadherin and Vimentin;5.The role of CD44v9/HIF-1α axis in HP infection promoting gastric cancer cells migration and invasion.(1)HIF-1α expression in CD44v9 knockdown AGS cells and CD44v9 overexpressed BGC-823 cells was detected by Western-blot;The correlation between the expression of CD44v9 and HIF-1α gastric mucosa was analyzed by Pearson correlation analysis according to the results of immunohistochemical staining;(2)The interaction between CD44v9 and HIF-1α in 293 T,BGC-823 and AGS cells infected with Flag-CD44v9 lentivirus was detected by co-immunoprecipitation(COIP);(3)HP was co-cultured with gastric cancer cells with different MOI for 12 h,and the HIF-1α expression was detected by Western-blot;(4)After transfected with HIF-1α small interfering RNA(Si-HIF-1α)or treated with HIF-1α inhibitor(YC-1),AGS cells were co-cultured with HP for 48 hours,and the migration and invasion abilities of the cells were detected by transwell migration and invasion assay;Results:1.CD44v9 was highly expressed in gastric cancer tissue and was associated with poor prognosis and clinicopathological characteristics of gastric cancer patients(1)Gastric cancer tissues showed significantly higher CD44v9 level than adjacent tissues(P<0.0001);(2)Compared with gastric cancer patients in the low expression group of CD44v9,the patients in the high expression group of CD44v9 had shorter overall survival(P=0.0007);(3)The high expression of CD44v9 was significantly correlated with gastric cancer TNM stage(P=0.001),vascular invasion(P=0.018),nerve invasion(P=0.001)and lymph node metastasis(P=0.013).2.The expression of CD44v9 was related to HP infection status(1)In comparison with the CNAG group,the expression of CD44v9 increased gradually in the IM,DYS,and GC groups(P<0.001).In the IM,DYS and GC groups,the expression of CD44v9 in the HP positive group was higher than that in the HP negative group(P<0.05);(2)HP infection could up-regulate the expression of CD44v9 in gastric cancer cells compared to the control group(P<0.05);3.High expression of CD44v9 promoted the migration,invasion and EMT process of gastric cancer cells(1)The CD44v9 knockdown cell line AGS(the knockdown effects of targets #2and #3 were more obvious)(P<0.05)and the CD44v9 overexpression cell line BGC-823(P<0.001)were constructed successfully;(2)Comparing to the negative control group,knockdown of CD44v9 significantly attenuated the migration(P<0.001)and invasion ability(P<0.01)of AGS cells,and over expression of CD44v9 significantly improved the migration ability of BGC-823 cells(P<0.01).And in comparison with the negative control group,knockdown of CD44v9 protein significantly inhibited the EMT process of AGS cells(the negative EMT indicator E-cadherin was up-regulated,and the EMT positive indicators ZEB1 and Vimentin were down-regulated)(P<0.05),while overexpression of CD44v9 protein significantly promoted the EMT process of BGC-823 cells(the negative EMT indicator E-cadherin was down-regulated,and the EMT positive indicators ZEB1 and Vimentin were up-regulated)(P<0.05);4.The role of CD44v9 in HP-induced migration,invasion and EMT of gastric cancer cells(1)In comparison with the HP-uninfected group,the migration and invasion abilities of AGS cells in the HP-infected group were significantly improved,while knockdown of CD44v9 expression significantly reduced HP-induced AGS cell migration and invasion.(Fig.P<0.05);(2)As compared to the HP uninfected group,HP infection significantly decreased E-cadherin expression and significantly increased N-cadherin expression.HP infection promoted the EMT process of AGS cells(P<0.001).Contrary to the negative control group,the CD44v9 knockdown group showed a significant down-regulation of the EMT positive indicator Vimentin,and an up-regulation of the EMT negative indicator E-cadherin(P<0.01);5.The role of CD44v9/HIF-1α axis in HP infection-induced migration and invasion of gastric cancer cells.(1)After CD44v9 was knocked down,HIF-1* expression in AGS cells was significantly decreased(P0.01);and a significant increase in HIF-1α expression was also observed after CD44v9 overexpression(P<0.001);The Pearson correlation analysis of the expressions of CD44v9 and HIF-1α in different stages of gastric mucosal lesions showed that there was a significant correlation between them(r =0.610,P<0.0001).In conclusion,CD44v9 regulates the expression of HIF-1α;(2)COIP experiment showed that: Ig G group did not detect HIF-1α band,only Flag group detected HIF-1α band,which indicated that there was an interaction between CD44v9 and HIF-1α protein;(3)In comparison with the control group,a significant increase in HIF-1αexpression has been observed in gastric cancer cells in the HP infection group(P<0.05);(4)In comparison the Si-NC group,the migration and invasion abilities of gastric cancer cells in the Si-HIF-α group were significantly attenuated.In comparison with the HP group,the migration and invasion abilities of cells which were tread with the si RNA of HIF-1α first and co-cultured with HP subsequently were significantly weakened.(P<0.05)The same results as above were obtained after the treatment of HIF-α inhibitor YC-1(P<0.05);Conclusions:1.CD44v9 was highly expressed in gastric cancer tissue,which promoted the invasion and migration of gastric cancer cells,and HP infection could up-regulate the expression of CD44v9;2.HP infection promotesd the migration and invasion of gastric cancer cells through the regulation of HIF-1α expression by CD44v9. |
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