Effects Of RNA Interference Targeting HTERT Gene On The Proliferation Of The Human Malignant Melanoma Cell Line A375

Objective To establish the hTERT gene silencing model,and to analyse the effects of hTERT gene targeted silencing on the proliferation of the human malignant melanoma cell line A375.Methods A375 cells were cultured in vitro and identificated with cell STR typing.Specific siRNAs with green fluorescent labeling an different concentrations of 0,5.0,15.0 and 35.0nmol/L were transfected into A375 cells by using trans-fection reagent(Hiperfect)at different volumes of 1.5,3 and 4.5?l.The transfection efficiency of the cells was determined by fluorescen microscop,and the concentration of siRNA and volume of Hipetfect were selected.Four hTERT-siRNAs including hTERT-siRNA-203,hTERTsiRNA-210,hTERT-siRNA-217 and hTERT-siRNA-224 were constructed and transfected into A375 cells separately at the most optimal transfection condition,and the extraction of total mRNA after 24-hour treatment.Real-time fluorescence-based quantitative PCRwas conducted to select the most specific hTERT-siRNA,MTT assay to evaluate cell proliferation.Results 0,5.0,15.0 and 35.0nmol/L specific siRNAs with green fluorescent labeling were transfected into A375 cells by using 1.5?l Hiperfect,the transfection efficiency in each group were1.44%±0.49%,37.19%±0.09%,23.46%±0.17%,16.63%±0.03%;the 3.0?l Hiperfect were 3.07%±0.97%,73.46%±0.03%,34.41%±0.07%,29.16%±0.05%;the 4.5?l Hiperfect were 1.59% 0.91%,21.74%±0.04%,29.04%±0.01%,?31.15%±0.26%,there were significant differents in transfection efficiency of different concentrations of siRNA and volumes of Hiperfect(F=57.21,P<0.05).Additionally,the transfection efficiency of 5nmol/L siRNA and 3?l Hiperfect group were significantly higher than other groups(all P<0.05).The mRNA expression of hTERT differed in the each hTERT-siRNAs groups,negitive control group and blank control group(0.36 0.03,?0.79 0.07,0.57 0.01,0.64 0.03,0.93 0.05 and 1.00 0.07 respectively;F=22.78,P? ? ? ? ?<0.05),and the hTERT mRNA expression of hTERT-siRNA-203 group showed the lowest(all P<0.05),and the transfection efficiency was the best.The proliferation inhibition rates of cell at 24,48 and 72 hours were 20.45% 2.09%,31.53% 2.44% and ? ?45.40% 2.91% respectively in hTERT-siRNA-203 group,satistically different from ?those in the negative control group(2.75% 0.54%,5.05% 1.18%,8.45% 1.42%,? ? ?respectively,all P<0.01)and blank control group(3.93% 1.09%,6.84% 2.46%,12.33 ? ?% 2.84%,respectively,all P<0.01).Conclusion The siRNA-mediated hTERT gene sile-?ncing model is established successfully,and the hTERT-siRNA-203 can specifically downregulate the expressions of hTERT mRNA,and can inhibit celluler proliferation of A375 cells.