MCM7 Promotes The Proliferation Of The Acid-adapted HepG2,UMUC3 Cells

BackgroundCancer cells proliferate rapidly and mainly utilize aerobic glycolysis to produce energy.Aerobic glycolysis produces excessive lactic acid,coupled with the higly expression of NHE1 increases acid excretion and the vascular function of the tumor is insufficient,resulting in an acidic tumor microenvironment.Previous studies show that acidic microenvironment could promote malignant transformation,make cancer cells resistant to radiotherapy.However,the current researches on this aspect are mostly limited to phenotypic observation,and are mostly based on short-term acid stress.long-term acid stress effects on tumor cells prolifertive charicteritics as well as intracellular pH(pHi)and NHE1 expression need to be further determined.Epithelial mesenchymal transition(EMT)is one of the main mechanisms of malignant transformation of tumor cells.EMT is often reported to be positively related with high metastatic and invasive ability of tumor cells.Many studies reported that acidic environment can induce epithelial to mesenchymal transition.MET is a reverse process of EMT,in which mesenchymal cells transforming into epithelial cells,and MET is connected with rapid proliferation and facilitate metastatic growth.However,the relationship between EMT,MET and tumor cell proliferation in acidic environment is still unclear.Cdc6 and Mcm7 are important members of DNA pre-replication complex(pre-RC).Their main function is to initiate DNA replication.Recent studies found that besides initiating DNA replication,Cdc6 and Mcm7 also participate in epigenetic regulation to promote cancer cell malignant transformation.However,the role of CDC6 and MCM7 in promoting malignant transformation of tumor cells in acidic environment 1s still unclear.Objective1.To determine the EMT phenotype,pHi and proliferation characteristics of cancer cells after long-term acidic stress;2.To clarify the role of pre-RC proteins CDC6 and MCM7 in the proliferation oft cancer cells under long-term acid stress.Methods and Results1.Acid stress induces EMT in cancer cells:After cultured in acidic medium(Panel,HepG2:pH 6.8;UMUC3:pH6.5)for 24h,E-cadherin mRNA was downregulated,while vimentin mRNA was upregulated,and cancer cells showed enhanced tolerance to cytotoxic drug cisplatin in the acidic medium;After cultured in acidic medium for 7 days,cancer cells showed increased expression of the mesenchymal maker vimentin,moreover,stelnness genes mRNA were increased in these acid-cultured cells2.Acidic extracellular incubation for 7days makes cells in quiescent state with acidic pHi:After cultured in acidic medium for 7days,cancer cells enter into non-cycling quiescent state,and NHEI was down-regulated in quiescent cells;Results of BCECF一AM fluorescent probe showed that pHi of quiescent cells reduce.3.Long-term acidity stress induces MET and obtains acid-adapted cancer cells:HepG2 and UMUC3 cells were adapted to acidic environment with proliferation capability recovered after cultured in acid medium for 2 months,and acid-adapted cells undergo a MET transition with N-cadherin and vimentin proteins were significantly downregulated;In additonal,NHE1 of acid-adapted cells was increased and pHi returned to a normal level.4.MCM7 promote acid-adapted cells proliferation and help maintain MET phenotype:Acid-adapted cancer cells showed an unique proliferation characteristics with MCM7 recovered rather than E2F1(G1-S important transcription factor),which was consistent with the cell proliferation state;Knock down of MCM7 inhibit acid-adapted cancer cell proliferation,decreased protein levels of Cyclin D1 and E1,and resulted in apparent increased expression of vimentin;Simvastatin(MCM7 inhibitor)can also significantly inhibit acid adaptation to cancer cell proliferation.On the contrary,the stable overexpression of MCM7 promotes the proliferation of HepG2 cells in acidic environment and inhibited the EMT process induced by acidic environment.Conclusions1.Cancer cells display a phenotypic transition between EMT and MET under long-term acidic condition2.The EMT/MET phenotype is related to proliferation state,cells with quiescent state mainly are mesenchymal phenotype,and cells in proliferation state mainly are epithelial phenotype.3.Acid-adapted cancer cells show distinct proliferative mechanism,in which MCM7 can promote the cancer cells proliferation in acidic environment and is association with the phenotypic transition between EMT and MET.