Effect of shRNA targeting PAX6 on proliferation, apoptosis and epithelial mesenchymal transition of MCF-7 human breast cancer cells

TANG Xin; WANG Jungu; HE Xinbin

Abstract：Objective To investigate the effect of short hairpin RNA( shRNA) targeting gene paired?box 6 ( PAX6) on the proliferation, apoptosis and epithelial mesenchymal transition ( EMT) of breast cancer MCF?7 cells. Methods According to the Gen?Bank sequence, the PAX6 sequence was designed and the shRNA sequence of PAX6 was synthesized, and then cloned into the vector of Puro pSUPER. plasmid. The MCF?7 cells were randomly transfected with pSuper. puro PAX6 shRNA plasmid ( transfection group) or negative control plasmid pSuper. puro luciferase shRNA ( negative control group) as to establish the MCF?7 cell line with stable low expression of PAX6. The MCF?7 cells without transfection were used as blank control group. After the verification by qPCR, thiazolyl blue( MTT) method was used to detect the proliferation inhibition rate with the plasmid for 24, 48, 72 and 96 h. The flow cytometry was employed to evaluate the apoptosis and cell cycle. Western blotting was used to detect the levels of EMT related markers( E?cadher?in, Fibronectin and Vimentin) after transfection for 96 h. Results The level of PAX6 in the transfection group was lower than that in the negative control group and blank control group ( P<0?05) . Compared with other two groups, the proliferation inhibition rate, apop?tosis rate and mRNA level of Bax and Bad were increased, while the Bcl?2 level was decreased in transfection group with statistical sig?nificant difference ( P<0?05) . The transfection with pSuper. puro PAX6 shRNA plasmid increased the proportion of G0/G1 phase but decreased the proportion of S phase and G2/M phase (P<0?05). The levels of E?cadherin in the transfection group were increased, while the levels of Fibronectin and Vimentin were decreased with significant differences ( P<0?05) . Conclusion The shRNA targeting PAX6 inhibits proliferation, induces apoptosis and cell cycle arrest and has a certain inhibition effect on EMT process, thus providing a basis for the gene silencing target of breast cancer.