Study On The Biological Function And Mechanism Of CLDN1 Gene On Anoikis Of Esophageal Squamous Carcinoma Cell

Objective: Our research is aimed at investigating that CLDN1 regulate anoikis and its molecular mechanism,in esophageal squamous cancer cells.Methods: We culture HEEC,Eca109,Eca9706,TE1,TE10,TE11,Kyse150 cells,in vitro and Screening the high and lower expression of CLDN1 mRNA esophageal squamous cancer cells by qRT-PCR.Construct and transfect virus vector harboring sequences of GPF-Puro-shR-CLDN1 and GFP-Pruo-CLDN1 for CLDN1 respectively into TE11 and TE10 cells.Puromycin screen stable low expression and high expression of cell line after 72h;and then we determine the expression of CLDN1 of stable expression cell line,as well as the cell transfection ratio though fluorescence microscopy and FCM;cell suspension culture,determine each cells of anoikis by FCM.Transfect positive cells with Lv-GFP-RFP-LC3B-adenovirus to observe the formation of autophagosome under laser scanning confocal microscope.In addition,we detect the expression of LC3 B,p62 and some others proteins related with AMPK ?-ULK1 pathways of autophagy by western blot.Western blot detect the expression of CLDN1,phosphorylation AMPK alpha and ULK1 and LC3BII/LC3 BI ratio,which could be reversed by MET,an activator of AMPK signaling.in vivo we validation the effection of CLDN1 on anoikis through the nude mice subcutaneous tumor-burdened.Results: In vitro,we successfully screen the CLDN1 knock down cells and cells of over expression CLDN1.Compared with NC group,the expression of CLDN1 were significantly lowered in sh-CLDN1 group which disturb the expression of CLDN1.(sh-CLDN1 group:0.235 ±0.020 VS NC group:0.706±0.023,P<0.05).Compared with NC group,the expression of CLDN1 were significantly higher in CLDN1 group which CLDN1 protein isover-expression(CLDN1 group:0.796±0.019 VS NC group 0.135±0.051,P<0.05).The rate of anoikis in sh-CLDN1 group is higher than NC group(38.901±2.541VS19.882±3.568%,P<0.05).The rate of anoikis in CLDN1 group is lower than NC group(17.170±2.122 VS 39.121±3.281%,P<0.05).Results show that disturb the expression of CLDN1 can promote esophageal squamous cancer cells anoikis,over-expression of CLDN1 inhibits esophageal squamous cancer cells anoikis,The confocal laser scanning microscope had observed that inhibiti of CLDN1 decrease the number of autophagosome between sh-CLDN1 group and NC group(5.671±1.267 VS.11.50±1.50,P<0.05),but CLDN1 promote the number of autophagosome in CLDN1 group compared with NC group(22.670±2.510 VS.5.671± 0.58,0P<0.05).3-MA,a inhibitor of autophagy can promote the rate of anoikis in CLDN1-group cells(CLDN1group:6.505±2.104% VS.CLDN1+3MA group:16.684± 2.337%,P<0.05).on the contrary,rapamycin(RAPA)a agonist of autophagy can restrain the number of autophagosome in sh-CLDN1 group(sh-CLDN1:42.035±3.092% VS.sh-CLDN1+RAPA group:25.984±3.356%,P<0.05).In vivo and vitro,disturbing the expression of CLDN1 down regulate the expression of phosphorylation AMPK ? and ULK1 and LC3BII/LC3 BI ratio(0.590± 0.036,0.59± 0.05,0.627± 0.150,P<0.05),over-expression of CLDN1 up regulate the expression of phosphorylation AMPK ? and ULK1 and LC3BII/LC3 BI ratio.In vitro,which could be reversed by CLDN1 and metformin,an activator of AMPK signaling.Conclusion: Our study show that up regulation of CLDN1 mediated by AMPK ? /ULK1 pathway to promote autophagy and contributes to anoikis resistance of esophageal squamous cancer cells.