Autophage Activated By ASIC1 Protein Related With Poor Prognosis In Gastric Carcinoma And Its Mechanism

Part Ⅰ The expression of ASIC1a and its clinical significance in gastric carcinomaObjective:We focused on the expression of ASIC1a in cancer and peri-cancer tissues and the relations between ASIC1a expression and gastric carcinoma.Methods:We investigated the expression of ASIC1a in gastric carcinoma by RT-PCR,IHC and westernblot.Performed in cancer and peri-cancer tissue of 32 patients who underwent operation for gastric cancer.The clinical significance of expression of ASIC1a was analyzed.Result:We found that:By RT-PCR,the ASICla mRNA level was significantly higher in gastric carcinoma than those in their adjacent nontumorous tissues(P<0.05).ASIC1a protein level detected by westernblot was significantly higher in HCC than those in their adjacent nontumorous tissues.Overexpression ASICla was found to correlate significantly with the tumor size,tumor infiltration depth,lymph node metastasis,TNM stage,nerve invasion,serum CEA,serum CA724.Conclusion:ASIC1a is highly expressed in gastric carcinoma.It played a certain role in promoting cancer。Part Ⅱ The expression and its control of ASICs in AGS cell lineObjective:To investigate the expression and mechanisms of ASICs in AGS cell line.Methods:To investigate the expression and function of ASICs in AGS cell line,Real-time PCR,immunofluorescence staining and western blotting were used in examining the mRNA and protein levels of ASICs.The electrophysiological recording was applied in the determination of ASICs function.Both Amiloride(a well-known blocker of ASICs)and PcTx1(a specific antagonist of homomeric ASIC1a channel)were used here as positive controls on proton-gated currents in AGS cell line.Result:Different methods were performed to verify the presence of ASICs of various isoforms including ASICla,ASIC1b,ASIC2a,ASIC2b,ASIC3,and ASIC4 in AGS cell line.Furthermore,the proton-gated currents were reversibly inhibited by amiloride and PcTxl,a specific blocker of ASIC1a,in a dose-dependent manner.Conclusion:Our results identified the existence of six subunits of ASICs including ASICla,ASIC1b,ASIC2a,ASIC2b,ASIC3 and ASIC4 in AGS cell line.The proton gated currents are mediated by ASICs.ASIC protein expression and its current can be regulated by Ami and PcTxl.Part Ⅲ The impact on gastric cancer biological characteristics by regulation of ASIC la expressionObjective:Explore the impact of ASIC1a on tumor growth,invasion,migration,apoptosisand metastasis in vivo and in vitro experiments.Methods:Using lentivirus vector,we transfected si ASIC la knock-down in AGS cell line,to constructed stable cell lines of AGS-siASIC1a.the expression of ASIC1a was determined by qRT-PCR,western blot.The ability of proliferation and invasion and migration was tested by MTT and transwell in the stable transfection cell.The human gastric cancer mode was established with AGS cell lines in athymic nude mouse.the gross tumor volumes were measured dynamically.Result:In vitro experiments,our results showed that:Knocking down ASIC1a inhibited the proliferation,migration and invasion of gastrc cancer cells,it could be affected by PH in cell environment.In vivo,the tumor mass in the control group higher than the ASIC1a silencing group.ASIC1a has a carcinogenic effect.Conclusion:Knocking down ASIC1a can inhibit the proliferation,migration and invasion of the gastric cancer cells.Part Ⅳ Autophage activated by ASIC1 protein related with gastric carcinoma and its mechanismObjective:To investigate the invasion and metastasis of gastric cancer cells mediated by ASIC1a through activating autophagy signaling pathway.Methods:In vitro,taking human AGS gastric cancer cells as the research object,cell damage induced by pH6.0 acid broth,Single cell patch clamp technique test current,Immunoprecipitation detect autophagy marker protein LC3-Ⅱ/LC3-Ⅰ,ASIC1a protein expression.The expression of autophagy marker LC3-Ⅱ and its co-localization with ASIC1a protein were detected by immunoprecipitation.Result:Single-cell patch clamp technique detected the generation of functional current on AGS cells,which could be blocked by the blocking agent of ASIC1a.Immunoprecipitation showed that ASIC1a protein was co-located with LC3-Ⅱ.Conclusion:Carcinogenic factors such as tissue acidification led to abnormal activation of ASIC1a on AGS cells and mediated invasion and metastasis of gastric cancer cells by regulating the activity of autophagy signaling pathway.