Study On The Mechanism For TGF-β1-induced Epithelial-to-mesenchymal Transition Via PKM2 Relative-mTOR/p70s6k Signaling Pathway In Cervical Carcinoma

BACKGROUND: Cervical cancer was in the second place in cancer ranks,which of incidence was still the highest in the developing countries,and seriously threaten women’ health.Now cervical cancer was still increased,and had a younger trend.The mTOR signaling pathway upstream was phosphorylated by mTOR to produce a response effect to regulate its downstream molecular ribosomal S6 protein kinase(S6K),which regulated the specific subgroup of m RNAs.Ribosomal S6 protein kinase(S6 kinase)was an important target protein for the downstream of mTOR,which was a main regulator of protein translation in S6 K.P70S6K was a kind of serine / threonine protein kinase,which could promote the synthesis of protein.mTOR had an important effect to effect the transition of G1 to S phase.If mTOR was specifically inhibited,G1 phase was blocked in the cells,thus induced apoptosis.mTOR inhibitor was used to inhibit the activity of mTOR pathway,thereby blocking the phosphorylation of P70S6 K and protein synthesis,G1 phase was blocked to regulate the role of cell proliferation and growth.The main feature of the tumor was that the cell cycle regulation disorder of normal cells,and the balance between cell proliferation and death clearance was broken.The mTOR signaling could regulate cell cycle,cell proliferation,cell migration,energymetabolism and protein synthesis,and so on.Warburg stated the metabolic tumors was the results of a large amount of sugar and lactic acid production.Pyruvate kinase showed that it was a key enzyme in the abnormal glucose metabolism of tumor cells.There were two main states of PKM2,the dimer active form,and the two polymer weak form.The state of the two mer was present in the tumor cells and named it Tumor-M2-PK.PKM2 was also known as tumor PKM2.Due to the necrosis and metastasis of tumor cells,the tumor PKM2 was released into the blood.The functions of PKM2 showed that it was diverse,including anabolism and regulation of glycolysis.Recent reports suggested that mTOR induce Warburg effects by upregulation of PKM2 expression.Epithelial-to-mesenchymal transition(EMT)increased the invasion and metastasis of tumor.Some studies reported that TGF-beta 1 induced the establishment of a EMT model which affected mTOR involvement in the regulation of non Smad independent signaling pathways.Metformin was an oral hypoglycemic agent that reduced insulin resistance and fasting plasma insulin levels.A retrospective study found that patients withⅡtype diabetes who were treated with metformin were significantly less to suffer cancers than those who used other antidiabetic drugs.A large number of cohort studies and epidemiological investigation showed that the risk of using metformin could reduce the prevalence of a variety of tumors.Studies showed that the long term use of metformin improved cancer patients survival.Metformin inhibited tumor growth and mTOR pathway,which was closely linked to effect cervical cancer cells,through inhibition of the AMPK dependent on mTOR pathway: metformin activated AMPK sigaling to induce phosphorylation of TSC2,and the TSC1/TSC2 complex could directly inhibit mTOR and ultimately inhibited downstream signal factor of ribosomal protein kinase activity,which affected on the transcription and synthesis of proteins.OBJECTIVES: To identify the mechanism of epithelial mesenchymal transition in cervical cancer,TGF-beta 1 induced the EMT state,and assisted with or without rapamycin to blockmTOR pathway,to certify the key EMT proteins and prove the existence of mTOR/p70s6k/PKM2 pathways,and used metformin to verify the mTOR/p70s6k/PKM2 pathway in the EMT state.METHODS: In this experiment,we used different doses of rapamycin to block mTOR pathway and detected the effect of rapamycin on biological characteristics and its mechanism.Next,we indeced EMT state: TGF-beta 1 induced cervical cancer cells in 48-96 hours.The morphological and biological characteristics were observed.The relationship between EMT and PKM2 was identified.Then,after the establishment of EMT state,cells were treated with or without rapamycin and TGF-beta 1.Under the EMT state,the mTOR inhibitors influenced the biological characteristics and their mechanisms.Further,cervical cancer cells were treated with or without metformin and TGF-beta 1 to verify the mTOR/p70s6k/PKm2 pathway.Specific experimental methods were following: CCK-8 detected the cell proliferation,scratch experiments tested cells migration,flow cytometry detected cells apoptosis.E-cadherin,STATA3,Snail2,vimentin,p-p70s6 k and PKM2 protein expression were detected by Western blot.RESULTS:(1)Rapamycin was able to inhibit the proliferation of Siha and Hela cells in a dose-and time-dependent manner.At 72 h,25 n M,50 n M,100 n M rapamycin treatment group had significant statistical significance.In Hela cell phase G0/G1,the number of cells increased(50.8% to 64%),and the number of cells in stage S decreased(40.2% to 30.7%).In Siha cells,rapamycin resulted in a significant increased in the number of cells in the G0/G1 phase(55.2% to 70.7%),whereas the number of cells in phase S decreased from 38.3% to 24%.With the increase of rapamycin dose,the percentage of total apoptotic cells in Siha cells(early apoptosis + late apoptosis)increased significantly(0.8% to 53.5%).Similarly,apoptotic cells in Hela cells increased(0.1% to 77%).After treatment with the mTOR inhibitor(rapamycin),the downstream levelsof phosphorylated p70S6 K were reduced,which downregulated PKM2 expression.(2)After 48 h treatment with TGF-beta 1 for cervical cell,the majority of cervical cancer cells underwent epithelial mesenchymal degeneration,which were characterized to disappearance epithelial cell polarity and intercellular space enlargement.The cell viability showed that the growth inhibition rate was not reduced at all concentrations of TGF-beta 1.And 10ng/ml TGF-beta 1 significantly promoted cell proliferation.The scratch test showed that the scratch distance of the 10ng/ml dose group were smaller than the control groups.After induction the EMT by TGF-beta 1,apoptosis was significantly reduced.In hela cells,0.1% of the early apoptosis decreased to 0%,and the total apoptosis decreased from 1.2% to 0.1%.Similarly,early apoptosis in Siha cells induced by TGF-beta 1 decreased by 0.6% to 0.4%,and total apoptosis decreased from 1.4% to 1.3%.After treatment with TGF--beta 1 in 48 h,E-cadherin obviously decreased,while vimentin was significantly increased.We verified that the EMT model was successfully established.PKM2 significantly increased,after induction of the EMT state.(3)TGF-beta 1 induced the epithelium mesenchymal changes in cervical cancer cells and it promoted cells proliferation and migration.Rapamycin reversed cell proliferation and reversed by TGF-beta 1 induced EMT in cervical cancer epithelial mesenchymal changes.After cells treated with TGF-beta 1,the total apoptosis(early apoptosis + late apoptosis)significantly decreased compared to the control groups.The apoptosis rate of 50 n M rapamycin treatment group significantly increased.In addition,the addition of rapamycin significantly reversed the anti apoptotic effect induced by TGF-beta 1 in Hela and Siha cells.Western blot results showed that: after the TGF-beta 1 was given,the expression of E-cadherin was decreased and vimentin was increased,while the relative expression of E-cadherin in rapamycin group was increased,and vimentin was decreased.In addition,the mTOR pathway was activated under TGF-beta 1,and its downstream phosphorylation of P70S6 K was increased,while rapamycin inhibited themTOR pathway and reduced the phosphorylation of P70S6 K expression.In addition,inhibition of the mTOR pathway also reduced PKM2 expression in EMT state.(4)Metformin inhibited the proliferation of Hela cells in a dose-dependent manner.TGFbeta 1 treatment significantly increased cells in 72 h and metformin could reduce the proliferation in Hela cells.Siha cells had the similar effects.TGF-beta 1 significantly increased the migration ability of Hela cells,and metformin reversed the migration ability induced by TGF-beta 1.Similarly,metformin reduced the migration of Hela cells.In addition,Siha cells had the same cell migration.In TGF-beta 1 treated cells,Hela and Siha cells,the total apoptosis cells(early apoptosis + late apoptosis)significantly decreased compared to untreated cells.Apoptosis rates were significantly increased in the cells treated with 10 m M metformin.In addition,metformin significantly reversed the anti apoptotic effects of Hela and Siha cells.In addition,the antitumor effect of metformin was similar to that of mTOR inhibitors,which inhibited tumor cells.Metformin partially reversed the epithelial mesenchymal change established by TGF-beta 1.In Hela and Si Ha cells,TGF-beta 1 significantly induced phosphorylation of P70S6 K.Western blot showed that TGF-beta 1 significantly increased PKM2 expression.Metformin reduces the expression of p-p70s6 k and PKM2.Metformin reversed TGF-beta 1 induction and decreased the expression of p-p70s6 k and PKM2.CONCLUSION:(1)It is indicated rapamycin result in cell apoptosis and cell cycle arrest,which related with mTOR/p70s6k/PKM2 signaling.(2)TGF-1 could successfully induce cervical cancer cells to establish the EMT situation.PKM2 expression was closely related to the EMT situation.(3)TGF-β1 built the EMT state,by which mTOR pathway was activated.mTOR pathwayinhibitors inhibited the induction of EMT and mTOR/p70s6 k signal reduction,reduced PKM2 expression.(4)Metformin reversed the EMT state,which inhibit mTOR/p70S6K/PKM2 pathway.