Mutual Regulation Of PI3K/AKT/mTOR Signaling Pathway And LSD1 In Esophageal Squamous Cell Carcinoma

Aim 1.To investigate the regulation effects of PI3 K / AKT / m TOR signaling pathway on LSD1 in esophageal squamous cell carcinoma(ESCC).2.To investigate the regulation effects of LSD1 on mTOR signaling pathway.Methods 1.After ESCC cell line ECa109 cells were treated with PI3 K / AKT / m TOR signaling pathway inhibitor(LY294002,RAD001,PP242),the proliferations of ECa109 cells were detected by CCK-8 method.2.After ECa109 cells were treated with PI3 K / AKT / mTOR signaling pathway inhibitor(LY294002,RAD001,PP242),the protein expression levels of Raptor,Rictor,p-AKT(Ser473),p-p70S6 K,LSD1 and histone H3K4me2 were detected by Western blots,.3.After ECa109 cells were treated with LY294002 combined with different concentrations of RAD001 and PP242,respectively,the proliferations of cells were detected by CCK-8,andthe combined index CI of them was calculated.4.ECa109 cells with Rictor-sh RNA vector filtered by puromycinwere treated with the PI3 K / AKT / m TOR signaling pathway inhibitor(LY294002,RAD001,PP242)in different concentrations,the proliferations of cells were detected by CCK-8.5.After ECa109 cells were transfected with Rictor-shRNA,the expression of Rictor and LSD1 was detected by Western blot.6.After ECa109 cells were treated withLSD1-shRNA,LSD1-siRNA,LSD1 inhibitor 244 and LSD1 positive control inhibitor TCP,respectively,the expressions of LSD1,Raptor,Rictor and p-p70S6 K were detected by Western blots.Results 1.The results of CCK-8 showed that LY294002,RAD001 and PP242 inhibited the proliferation of ESCC cells on a dose-dependent manner.2.The results of Western blots showed that LY294002 down-regulated the expression of p-p70S6 K and promoted the expression of Rictor,Raptor and p-AKT(Ser473),and promoted the expression of histone H3K4me2 on a dose-dependent and time-dependent manner,but had no effects on theexpression of LSD1;RAD001 inhibited the expression of Rictor,Raptor,p-p70S6 K and up-regulated the expression of p-AKT(Ser473),and inhibited theexpression of LSD1 while promoted the expression of histone H3K4me2 on a dose-and time-dependent manner;PP242 reduced the expression of Rictor and increased the expression of Raptor,p-AKT(Ser473)and p-p70S6 K,while had no effects on the expression levels of histone H3K4me2 and LSD1.3.LY294002 combined with RAD001 and PP242,respectively,had stronger inhibitory effect on the proliferation of ECa109 cells than that of cells treated with them alone.The combined index CI of LY294002 and RAD001 were less than 1,suggesting that the combination of them has synergistic effect;the combined index CI of LY294002 and PP242 were less than 1 at low concentration butmore than 1 at maximum concentration,indicating that they hadsynergistic effect on low concentration but had antagonistic effect on high concentration.4.The results of CCK-8 showed that compared to untransfected cells and cells with control-sh RNA,LY294002,RAD001 and PP242 had stronger inhibition effects on cells with Rictor-sh RNA.5.The result of Western blot showed that after ECa109 cells weretransfected with Rictor-sh RNA,compared to untransfected group,the expression of Rictor was significantly decreased and the expression of LSD1 was significantly increased.6.After ECa109 cell was treated with LSD1-sh RNA or LSD1-siRNA and LSD1 inhibitor TCP and 244,the expression level of Rictor wassignificantlyinhibited and Raptor and p-p70S6 K werepromoted,and the protein level of LSD1 was significantly down-regulated in the first three ways.Conclusions 1.The PI3 K / AKT / m TOR signaling pathway inhibitor(LY294002,RAD001,PP242)can inhibit the proliferation of ESCC cells.Down-regulated the expression of Rictor with Rictor-sh RNA can increase the sensitivity of ESCC cells tothe three inhibitors.2.There is a mutual regulation effects between PI3 K / AKT / mTORsignaling pathway and LSD1 in ESCC.The expression of LSD1 was down-regulated and thedemethylation function of LSD1 was inhibited by the suppression of m TOR.On the other hand,when LSD1 was inhibited,the expression of Raptor and Rictor and the target gene p-p70S6 K also can beregulated.