Effects Of Silencing ADAM17Gene On Proliferation Of Human Breast Cancer MCF-7Cells Under Hypoxia

Objective To investigate the effects of shRNA (short hairpin RNA, shRNA) targetingADAM17(a disintegrin and metalloprotease17, ADAM17) gene on the proliferation ofhuman breast cancer MCF-7cells under hypoxia. The relationship between ADAM17and breast cancer under hypoxic conditions will provide a new direction for thedevelopment of targeted therapy for breast cancer research.Methods The research object is the human breast cancer MCF-7cells in the experiment.The specific pGPU6/GFP/Neo-ADAM17shRNA expression vector, which could expressgreen fluorescent protein, was designed for ADAM17mRNA. Four kindsGPU6/GFP/Neo-ADAM17shRNA expression vectors were shRNA297、shRNA1134、shRNA1219and shRNA1508. They were transfected into human breast carcinoma cellline MCF-7cells by lipofectamineTM2000or electroporation. Fluorescence microscopywas used to observe the efficiency of transfection. Real time PCR (Real time polymerasechain reaction, Real time PCR) was used to screen the most effective silencing shRNAexpression vectors. Experiment groups were divided into normoxic and hypoxic groups.Normoxic groups made up with normoxic control group, normoxic shNC transfectedgroup and normoxic shRNA transfected group. Hypoxic groups were made up withhypoxic control group, hypoxic shNC transfected group and hypoxic shRNA transfectedgroup. So there were a total of six groups. Hypoxic environment was acquired by a threegas incubator flushed with an atmosphere saturated with1%O2,5%CO2and94%N2. Realtime PCR was used to study the expression levels of ADAM17mRNA. The proliferativeability and cell growth curve of MCF-7cells were detected by iCELLigence. Cell cycledistribution of MCF-7cells was analyzed by FCM (flow cytometry, FCM).Results1MCF-7cells were transfected transiently by ADAM17shRNA expressionvector with an average efficiency of85%. The expression level of ADAM17mRNA wassignificantly lower in MCF-7cells transefected by shRNA297, shRNA1134, shRNA1219and shRNA1508, compared with the blank control group and negative control group(P<0.05). Among them, shRNA1219had the highest inhibitory rate (P<0.01).2MCF-7cells were transfected transiently by ADAM17shRNA1219expression vector. And thenthey were cultured under normoxic or hypoxic environment. Real time PCR showed that ADAM17mRNA relative expression of MCF-7cells under hypoxic conditions wassignificantly lower than the ones cultured under normoxic conditions. The relativeexpression of ADAM17mRNA in MCF-7cells transfected by shRNA1219wassignificantly lower than control groups and shNC transfected groups (P<0.05), with noother significant differences between control groups and shNC transfected groups.3Human breast cancer MCF-7cells were cultured under hypoxia. ICELLigence systemtests showed that MCF-7cells produced a rapid proliferation on the hypoxic stressinitially. With the extension of time for about24h under hypoxia, MCF-7cellsproliferation and proliferation activity were significantly inhibited.4ADAM17wassilenced successfully and cultured under hypoxia. The iCELLigence system displaysshowed the proliferation of MCF-7cells transfected with shRNA1219under hypoxia wassignificantly reduced than other groups.5Human breast cancer MCF-7cells werecultured under hypoxia. FCM showed that the proportion of S phase cells decressed andG0/G1phase stopped with the extension of time (0h,12h,24h and48h). Hypoxia for along time could lead to inhibite the proliferation of MCF-7markedly and prevent cellsinto proliferating cycle.6FCM showed that MCF-7cells growth inhibition all appearedin hypoxia group, hypoxia shNC transfection group and hypoxia shRNA transfectiongroup. The results in hypoxia shRNA transfection group were more significant (P<0.05).The proportion of MCF-7cells into the S and G2/M phase is lowered, with the vastmajority of cells remaining in the G0/G1phase. Compared with normoxic groups, cellproliferation was inhibited. So the difference was statistically significant (P <0.05).Conclusions The RNA interference technology could effectively silence the expressionof ADAM17in human breast cancer MCF-7cells by ADAM17shRNA expression vector.ADAM17shRNA may be an effective method of inhibiting the proliferation of humanbreast cancer cells. The proliferation of MCF-7cells was inhibited and the cell cycle wasblocked by shRNA transfection or hypoxia incubation. It still could not suggest that thesynergistic effect of ADAM17and hypoxia synergistically inhibits the proliferation oftumor cells.