Effect Of Silencing FAK Gene Expression By RNAi On Characteristics Of Human Breast Cancer MCF-7 Cell Line

Breast cancer is one of the most common types of female malignant tumors, according to statistics, 7% to 10% of the systemic malignant tumors are breast cancer, and its morbidity and mortality occupied the first of all women malignant tumors in western countries. In china the morbidity of breast cancer is rising year by year and with the tendency in an earlier age. Breast cancer has become the biggest threat to women's health. The treatments to breast cancer are mainly by surgery, chemotherapy, radiotherapy and hormone. As the treatment technology continuously improved, the survival has greatly improved, the mortality has decreased significantly. The major cause in the lethal progression of this type cancer is the invasion and metastasis of the malignant tumer cells, which represents a major prognostic indicator and serves as a guide for therapeutic strategies. Many efforts had been devoted to make better understandings of the molecular mechanisms involved in the progression of this type of cancer in order to predict more accurate and reliable biomarkers in tumer metastases. The adhesion of the malignant tumer cells to the extracellular matrix is one of the fundamental pathways that promote invasion and metastasis. A key factor involved in singal transduction guided by integral protein is the focal adhesion kinase (FAK), an intracellular tyrosine kinase protein which is localized to cellular focal contact sites. Evidences have suggested that FAK overexpressed in the tumer and correlated with the metastasis and invasive potential, such as carcinomas of the breast, colon, thyroid, liver and prostate. This study was to evaluate FAK mRNA and protein expression in breast cancer, to study the effect of RNA interference (RNAi) on FAK gene expression and the biological characteristics of human breast cancer MCF-7 cell.Objective: To construct the siRNA expression vector of FAK gene, to study the effect of RNA interference on FAK gene expression and the biological characteristics of human breast cancer MCF-7 cell.Methods: The specific recombinant plasmids FAK-pGenesil-siRNA was designed according to the FAK sequence in GenBank. A negative control sequence FAK-pGenesil-HK was designed randomly. The sequence analysis was performed. The constructed recombinant plasmid was transfected into human breast cancer MCF-7 cell using LipofectamineTM 2000. The transfection efficiency was observed under fluorescence confocal microscopy, Expression of FAK mRNA and protein were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot respectively. Distribution of cell cycle was assessed by flow cytometry, cell penetrate matrigel capacity was determined by invitro experiment.Results:(1) According to optimizing the transfected conditions, the most highly efficiency was over 60% when the ratio of recombinant plasmids to lipofectamineTM2000 was 1:4(μg/μl).(2) RT-PCR,Western blot results: The constructed recombinant plasmid FAK-pGenesil-siRNA downregulated the expression of FAK mRNA and protein. The inhibition rate of FAK mRNA was 61.80%, with significant difference compared to the HK group (p<0.05), the inhibition rate of FAK protein was 46.55%, with significant difference compared to the HK group (p<0.05).(3) Flow cytometry (FCM) results: 48 hours after transfection of recombinant plasmids, the proliferation inhibition ratio of the FAK-pGenesil-siRNA reached to 25.89±0.26%, while the HK and control group were 37.96±0.60%,8.21±0.56% respectively, with significant difference compared to the siRNA group (p<0.05);FCM analysis showed that more cells stay at G0-G1 phase in the HK and control group than siRNA. Transwell invitro invasion experiment results: 48 hours after transfection of recombinant plasmids, the cells numbers through Matrigel membrane in the FAK-pGenesil-siRNA group were 30.33±1.53, while the HK and control group were 37.96±0.60,38.21±0.56 respectively, with significant difference compared to the siRNA group (p<0.01). Conclusion: The recombinant plasmids FAK-pGenesil-siRNA was constructed successfully. FAK-pGenesil-siRNA inhibited the expression of FAK mRNA and protein, degraded the proliferation and the invasion capability of human breast cancer cell. This result will facilitate further studies of gene therapy for tumers such as breast cancer.