Mechanism Investigation On The Antiproliferation Of Cardamonin Mediated By Raptor On SKOV3 Cells

Aim1 To shed light on the key role that Raptor plays in the regulation of m TORC1’s activity.2 To further research in the possible regulatory mechanism through which CAR regulating m TORC1 by Raptor in inhibition of the proliferation of SKOV3 cells.Methods1 Cell cultureHuman ovarian cancer SKOV3 cell lines that transfected Raptor si RNA or not were cultured in Mc Coy’s 5A medium with 10 % FBS, 100 μg·m L-1 streptomycin and 100 U·m L-1 penicillin at 37 ℃with 5 % CO2 and then passaged when the adherent ones grew to 70 %- 80 %. Cells for cryopreservation were cultured in the frozen stock solution with 10 % DMSO and 90 % BSA, stored at 4 ℃ for 40 min, then-20 ℃ for 1 h, and-80 ℃ for 3-5 months.2 Groups and drugs interventionControl group, CAR group(5, 20 μM), RAP group(0.1 μM) and AZD8055 group(0.1 μM) were set.3 Measurements and methodology3.1 Inverted microscope was used to observe the cells’ morphology and growth;3.2 MTT method was used to measure the cells’ proliferation;3.3 PCR and real time PCR were used to measure the transfection efficiency of SKOV3 cells;3.4 Western blotting was used to detect the expression quantity, phosphorylation level of m TOR, Raptor, PRAS40 and S6K1.Results1 CAR significantly inhibited the phosphorylation of m TOR, Raptor, PRAS40 and S6K1 respectively, and inhibited the total protein expression of Raptor in a concentration-dependent way(P ? 0.05);2 Raptor si RNA was well transfected into SKOV3 cells as the expression of Raptor m RNA and protein were decreased notably;3 The activity of m TOR, Raptor, PRAS40 and S6K1 and the total protein expression of Raptor in Raptor si RNA transfected SKOV3 cells were lower than that in untransfected cells, and which got much lower when cells were incubated with CAR(P ? 0.05);4 CAR strikingly inhibited the proliferation of Raptor siRNA transfected or not SKOV3 cells, with a higher inhibition ratio in former one(P ? 0.05).ConclusionsThe expression and phosphorylation of Raptor play an important role in regulating the activity of m TORC1, while down-regulating Raptor may be a mechanism that CAR inhibited the SKOV3 cells’ proliferation.