The Effect Of RAPA On Human RetinoBlastoma HXO-RB₄₄ Cells Line And MTOR Expression

Objective:Retinoblastoma cell lines was treated with the mTOR inhibitor rapamycin,in order to explore the effect of rapamycin on the HXO-RB44cells lines in vitro anti-proliferative and cell cycle,and to explore its molecular mechanism.Methods:Human Retinoblastoma HXO-RB44was treated with different concentrations (0,0.625,1.25,2.5,5,10,20nmol/L) of rapamycin.In the situation of pre-and post-treatment of rapamycin,Cell proliferation of the HXO-RB44Cell lines in response to rapamycin was assessed by MTT;Cell cycle and apoptosis analyses were assayed by flow cytometry;To detect different RNA’s quantity of mTOR in experimental groups cell using QRT-PCR;To detect different protein’s quantity of change of mTOR、p-mTOR in experimental groups cell.Result:(1) The cell growth was inhibited by rapamycin,and the result indicated that the group with rapamycin concentration-dependent manner which followed concentration gradient increased with proliferation rate reduce; compared with the control group,0.625nM have no significant differernce (P>0.05), the other results have significant difference(P <0.01).(2) The result of FCM technology indicated that the group with rapamycin which followed concentration gradient increased with apoptosis rate increase; compare the group with rapamycin in all concentration with the control group have significant differernce (P <0.01);Treatment of rapamycin was shown to increase in the proportion of cells in the G1phase and decrease in the proportion of cells in the S phase in0.625nM cell lines compared to untreated cells,the result has significant differernce (P<0.01).(3) the result of QRT-PCR indicated that compared mRNA’s quantity of mTOR in group with rapa with the control group have no significant difference(P>0.05).(4)The result of Western blot indicated that compared the protein’s quantity of change of mTOR in group with rapamycin concentration in0.625,1.25,2.5,5nM with the control group have no significant difference(P>0.05),compared with the control group the protein’s quantity of change of P-mTOR in group with rapamycin in all concentration was shown to reduce.Conclusion:(1) Rapamycin can inhibit growth and proliferation of HXO-RB44cell lines。(2)Rapamycin inhibit cell growth and proliferation mainly through the mechanism of effectively arrests HXO-RB44cells in the G1phase of the cell cycle,and meanwhile can induce apoptosis.(3)Compared the mRNA’s and protein’s quantity of change of mTOR in group with rapamycin with the control group have no significant difference, which demonstrated that rapamycin didn’t play a role directly influence mRNA’s and protein’s quantity of mTOR.(4) Compared with the control group,the protein’s quantity of change of P-mTOR in group with rapamycin in all concentration was shown to reduce.(5)Therefore,our finding provides evidences that rapamycin,a specific inhibitor of mTOR can be thought of as a potential target for RB targeted therapy.