title: Expression of P2X7R in breast cancer tissue and the induction of apoptosis by the gene-specific shRNA in MCF-7 cells.
abstract: The aim of the present study was to investigate the effects of P2X7R short hairpin (sh)RNA on the proliferation and apoptosis of MCF-7 cells, and to detect the expression of P2X7R in breast cancer and MCF-7 cells. In order to detect the expression of P2X7R in normal breast and breast cancer tissues, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blot analysis and immunohistochemistry were performed. A P2X7-targeted shRNA sequence and a scrambled sequence were inserted into the pLKO.1 expression vector, and MCF-7 cells with stable transfection of P2X7R-shRNA and P2X7R-scrambled shRNA (control) were selected. qRT-PCR was used to detect the mRNA expression levels of P2X7R in the MCF-7 cells transfected with P2X7R-shRNA and scrambled shRNA. In addition, protein expression levels of P2X7R in the fresh tumor tissues were detected by western blot analysis. An MTT assay was used to detect the proliferation rate at different time points, while flow cytometry was used to detect the growth inhibition and apoptosis rate of the stably transfected MCF-7 cells. P2X7R expression levels in the breast cancer tissues were higher when compared with the normal breast tissue, and a positive correlation was observed with the estrogen receptor (ER+), as shown by qRT-PCR, western blot analysis and immunohistochemistry. Plasmids expressing P2X7 gene-specific shRNA and scrambled shRNA were constructed and transfected into MCF-7 cells. The qRT-PCR results revealed lower mRNA expression levels of P2X7 in the P2X7R-shRNA cells when compared with the scrambled shRNA cells. Furthermore, western blot analysis demonstrated that P2X7 protein was highly expressed in the MCF-7 cells transfected with scrambled shRNA, while low expression was observed in the P2X7R-shRNA-transfected cells. Following transfection of the recombinant plasmids into the MCF-7 cells, the proliferation rate in each group was analyzed. The P2X7R-shRNA and KN-62 groups were shown to have significantly reduced rates of proliferation when compared with the normal control group. In addition, flow cytometry revealed that the P2X7R-shRNA and KN-62 groups exhibited a reduced level of cell proliferation and a higher rate of apoptosis. In conclusion, P2X7R was shown to be overexpressed in breast cancer tissues and positively associated with ER expression. A P2X7R-shRNA expression vector effectively inhibited P2X7R expression in MCF-7 breast cancer cells, which subsequently induced cell apoptosis and reduced the levels of cell proliferation. These results indicated that P2X7R may serve as a potential target for breast cancer treatment and prevention.
authors.affiliation: Institute of Molecular Biology, Medical College, China Three Gorges University, Yichang, Hubei 443002, P.R. China ; First Affiliated Hospital of China Three Gorges University, Yichang, Hubei 443000, P.R. China.
authors.forename: Chao
authors.initials: C
authors.lastname: Tan
authors.affiliation: Institute of Molecular Biology, Medical College, China Three Gorges University, Yichang, Hubei 443002, P.R. China.
authors.forename: L I
authors.initials: LI
authors.lastname: Han
authors.affiliation: Institute of Molecular Biology, Medical College, China Three Gorges University, Yichang, Hubei 443002, P.R. China.
authors.forename: Lili
authors.initials: L
authors.lastname: Zou
authors.affiliation: First Affiliated Hospital of China Three Gorges University, Yichang, Hubei 443000, P.R. China.
authors.forename: Chunhua
authors.initials: C
authors.lastname: Luo
authors.affiliation: Institute of Molecular Biology, Medical College, China Three Gorges University, Yichang, Hubei 443002, P.R. China.
authors.forename: Aihua
authors.initials: A
authors.lastname: Liu
authors.affiliation: Institute of Molecular Biology, Medical College, China Three Gorges University, Yichang, Hubei 443002, P.R. China.
authors.forename: Xiejing
authors.initials: X
authors.lastname: Sheng
authors.affiliation: Institute of Molecular Biology, Medical College, China Three Gorges University, Yichang, Hubei 443002, P.R. China.
authors.forename: Dee
authors.initials: D
authors.lastname: Xi
keywords: P2X7R; breast cancer; short hairpin RNA