Effect Of FANCF Gene Silence On Biological Character In Breast Cancer Cell Line MCF-7

Objiective FA/BRCA pathway participate in cell cycle and apoptosis control,regulation of detoxification,survival signal transduction and DNA repair.Disruption of this pathway results in chromosome instability and hypersensitivity to DNA cross-linking agents. Stabilization of FA complex and monoubiquitination of FANCD2 are the critical events and FANCF plays an extremely vital role as a key factor.Low expression and loss function of FANCF could interfere in the FA/BRCA pathway and participate in tumorigenesis and development of drug resistance.At present,It has been confirmed that FANCF is related to development and drug resistance of many kinds of tumors. But there are no reports about whether FANCF participate in development and drug resistance of breast cancer.We investigate effect of FANCF gene silence on biological character and drug resistance of breast cancer cell and whether FANCF gene contribute to development and drug resistance of breast cancer.Methods1.Construction of FANCF-shRNA plasmid:Design FANCF-shRNA template and ligate with p Sliencer^TM4.1-CMV neo plasmid;Tranformate it into E coli competent cells JM109 for selection and amplification;Identificate FANCF-shRNA plasmid by PCR and sequencing.2.Establishment of MCF-7-FANCF-RNAi cell model:Extract FANCF-shRNA plasmid and transfect into MCF-7 cells;Detect mRNA expression of FANCF by RT-PCR to confirm FANCF gene silence.3.After FANCF gene silence, investigate the proliferation of MCF-7 cell by MTT assays;The cell cycles and apoptosis cells are measured by flow cytometry after the ceils staining by PI only and Annexin-V and PI respectively;Investigate the mRNA expression of drug resistance associated genes by RT-PCR.Results1.Successful Construction of FANCF-shRNA plasmid:PCR product of bacteria suspension with FANCF-shRNA plasmid is about 250bp;Sequencing results demonstrate the insert is correct.2.Successful establishment of MCF-7-FANCF-RNAi cell model:Compared to negative control,mRNA expression of FANCF gene is reduced in the 2nd,3rd,5th and 7th day after transfection with FANCF-shRNA(P＜0.05).It is suggested FANCF gene silence.3.Effect of FANCF gene silence on biological character of MCF-7 cells: Compared to negative control,FANCF gene silence could inhibit the proliferation of MCF-7 cells(P＜0.05),increase the percentage of cell cycle in S-phase(P＜0.05)and apoptosis rate of MCF-7 cells(P＜0.05).4.Effect of FANCF gene silence on drug resistance of MCF-7 cells:Compared to negative control,After FANCF gene silence,mRNA expression of BCRP is obviously reduced(P＜0.05)and P-gp mRNA expression is reduced slightly(P＜0.05)and there is no obvious difference of mRNA expression of MRP and LRP.Conclusion1.FANCF gene silence could inhibit the proliferation of MCF-7 cell,increase the percentage of cell cycle in S and apoptosis rate of MCF-7 cell.2.FANCF gene silence could inhibit mRNA expression of BCRP and contribute to regulation of breast cancer.