Study On The Relationship Between Cardamonin Targeted Of MTOR And FKBP12

Background and ObjectivemTOR, a serine/threonine kinase, plays an important role on regulating the cell growth and proliferation, and is the drug targets on treating various diseases. Our previous works suggested that cardamonin (Cardamonin, CAR), a kind of flavonoids, inhibited the expression and activity of mTOR and proliferation of VSMCs and A549cells. Overexpressed of FKBP12and mTOR, CAR only inhibited the activity of mTOR but did not combine with FKBP12. This study is to investigate the mode of action of cardamonin on mTOR activity by silencing the expression of FKBP12. For further observed the effect of CAR on mTORC2, the protein expression of AKT was analyzed. It is aimed to provide an experimental basis for a novel mTOR inhibitors.Method1Screening the best sequence of transfected FKBP12-siRNA1.1Determination of the transfection efficiencyHela cells of Lipofectamin transfection marked with FAM (NC-FAM) served as negative control was incubated in a CO2incubator for12h, then the number of cells with fluorescence was counted under the fluorescence microscope.1.2Determination of the transfection conditionsThe designed siRNA which contraposed the positive control GAPDH mRNA sequence was transfected into Hela cells by different concentration. RNA was extracted at24h and48h, respectively, then the expression of GAPDH gene was analyzed by PCR and RT-PCR.1.3Confirmation of the best FKBP12-siRNA sequenceThe Hela cell was transfected by interference sequence which contraposed FKBP12mRNA included FKBP12-264, the FKBP12-296and FKBP12-397, the total RNA and protein were extracted at24h and72h later, respectively. Then the expression of FKBP12mRNA and protein expression of FKBP12were determined by PCR, RT-PCR and Western blotting method.1.4Confirmation of the best time of protein detection by transfectionThe Hela cell was transfected by the best interference FKBP12-397, total protein was extracted at24h,48h,72h, and96h later, respectively, then the expression of target protein was detected by Western blotting.2Analysis of the cell proliferation by MTTThe MTT was added in the cultured cells, then OD value was readed at the spectrophotometry (Thermo Labsystems) at490nm by cells transfected in24h,48h,72h and96h, respectively.3Determination of protein expression by Western blottingThe effect of CAR on the protein expression of mTOR, AKT, p70S6K1, FKBP12and phosphorylation, FKBP12, IL-2in normal Hela cells and transfection Hela cells was preformed.4Confirmation of the binding site of CAR on FKBP12in Hela cellsExperimental groups on cell proliferation:Control group of Hela cells(CG), Control group of Transfection Hela cells (TCG), Control group of solvent (0.1%DMSO)(SG), CAR group (CARG1:3×10-5mol·-1, CARG2:10-5mol·L-1,CARG3:3×10-6mol·L-1, CRAG4:10-6mol·L-1, CRAG5:10-7mol·L-1), AZD8055group (AZDG1:10-7mol·L-1, AZDG2:10-6mol·L-1), FK506group (FKG1:3×10-6mol·L-1, FKG2:10-5mol·L-1), RAP group (RAPG:10-7mol·L-1). Each group set up6repeated holes.Experimental groups on protein expression:Control group of Hela cells (CG), Control group of transfection Hela cells (TCG), CAR group (CARG2:10-5mol·L-1, CARG3:3×10-6mol·L-1, CRAG4:10-6mol·L-1), AZD group (AZDG1:10-7mol·L-1), FK506group (FKG2:10-5mol·L-1), RAP group (RAPG:10-7mol·L-1).Results1Transfection efficiencyThree representative vision was selected in each hole from there repeated hole,100cells were observed for fluorescent cell in each vision, the rate of transfection was85%.2Transfection condition The GAPDH-siRNA expression reduced in end concentration of50,100,150nmoL conditions for positive control GAPDH-siRNA (P<0.05, P<0.01), the best effect that mRNA expression can be reduced by85.3%in the condition of24h,100nmol compared with negative control (NC).3The best interference sequence of FKBP12-siRNAThe interference efficiency of FKBP12-264, FKBP12-296and FKBP12-397were33.08%,7.25%and74.8%. The gray ratio of FKBP12protein expression was0.86±0.08,0.99±0.1,0.54±0.08vs1.12±0.04(P<0.05, P>0.05, P<0.01), the best effect of interference was siRNA-397.4The best time of FKBP12-siRNA interferenceThe protein expression of FKBP12was strengthened with the extension of time after FKBP12-397transfected Hela cells at24h,48h,72h and96h later, respectively. The gray ratio of FKBP12protein expression were0.89±0.07,0.71±0.04,0.44±0.06,0.61±0.04vs0.93±0.08(P>0.05, P<0.05, P<0.01, P<0.01). The best time was72h after transfection.5The effect of transfected FKBP12-siRNA on the protein expression of mTOR, p70S6K1and IL-2in Hela cells.The protein expression of IL-2was significantly reduced, and that of p70S6K1and mTOR had no obviously changed in the condition of transfected FKBP12-siRNA.6The effect of transfection FKBP12-siRNA on cell proliferationThe Hela cells with transfected FKBP12-397grew slowly. The OD value were0.262±0.017vs0.261±0.004,0.469±0.019vs0.549±0.019,0.486±0.006vs0.839±0.009,0.48±0.012vs0.946±0.016(P>0.05, P<0.01, P<0.01, P<0.01) at24h,48h,72h and96h, respectively.7The effect of Cardamonin on the proliferation of Hela cells with transfection FKBP12-siRNAThe inhibition ratio of cell proliferation has no significant difference between normal Hela cells and Hela cells transfected with FKBP12-siRNA, which were treated with CAR, AZD and FK506. The inhibition effect on FKBP12-siRNA transfected Hela cells was significantly weakened by RAP.8The effect of Cardamonin on the protein expression of FKBP12-siRNA transfected Hela cells CAR and RAP can significantly inhibite the protein expression of p-mTOR and p-p70S6K1in the normal and FKBP12-siRNA transfected Hela cells group, and had no obvious effect on the total protein of p-Akt and Akt, mTOR, p70S6K1. CAR and AZD had a similar effect on the above protein, the protein expression was no obvious difference between normal group and transfected group, but the protein expression of p-mTOR and p-p70S6K1can be significantly weakened by RAP. FK506and RAP can inhibit the protein expression of FKBP12and IL-2in normal Hela cells, but FK506can not effect the protein expression of p-Akt, p-mTOR, p-p70S6K1and its total protein in normal group and transfected group.Conclusions1The FKBP12-siRNA sequence can be transfected successfully with the liposome method and it significantly reduced the mRNA and protein expression of FKBP12in Hela cells.2CAR obviously inhibited the proliferation of Hela cells in a concentration-dependent way.3The inhibitory effect of CAR on mTOR has no correlation with the expression of FKBP12.4CAR did not affect Akt and has no effectory on the phosphorylated proteins expression of...