RNAi Induced Down Regulation Of MARCH6Expression Inhibits Proliferation Of Breast Cancer Cell Lines In Vitro

Objective:Silence MARCH6gene expression in breast cancer MCF-7cells through lentiviral RNA interference, and investigate its effects on MCF-7cell proliferation and cell cycle.Methods:1. transfect MARCH6targeting shRNA lentivirus gene into293T cells and use fluorescence microscopy to observe the amount of fluorescence to verify the transfection efficiency; To knock down the expression of MARCH6gene expression in MCF-7cells through lentiviral infection, and use fluorescence microscope to observe the amount of fluorescence to determining the initial effect of silencing;2. Real-time PCR and Western blot is used to detect the expression of MARCH6in MCF-7cells which is infected by lentivirus;3.After knocking down MARCH6in MCF-7cell, MTT and BrdU assay detect changes of cell proliferation and PI staining flow cytometry detect cycle distribution of MCF-7cells.Results:1. MARCH6shRNA lentivirus successfully transfected293T cells, observating under the fluorescence microscope showing that about80%of the cells with green fluorescent by contrast with the bright field; lentiviral was successfully infected MCF-7cells, observating under the fluorescence microscope showing that about90%of the cells with green fluorescent by contrast with the bright field; 2. Western Blot and Real-Time PCR verifies the level of MARCH6transcription and protein was significantly decreased After MARCH6shRNA lentivirus infect MCF-7cells;3. By MTT assay, BrdU method validate MCF-7cell proliferation significantly decrease after infected by MARCH6shRNA lentivirus (p <0.01), flow cytometry shows that after knock down MARCH6in MCF-7cells, the cell cycles are inhibited in the G1phase cells and proliferation index significantly reduced.Conclusions:Silencing MARCH6gene by RNA interference can inhibit the proliferation of MCF-7cells, this result suggests that the expression of MARCH6promotes proliferation of breast cancer cells.