Knockdown Of COL1A1 Expression Using ShRNA In MCF-7 Cell Lines

The diagnosis and treatment of tumors in recent years has been focused on the the newly developed technology of RNA interference, which could to efficiently silence target-specific gene expression.Gene function has become an important tool for research. Breast cancer is the most common malignancy among women and incidence rate in China kept increasing year by year. At present, it has been developed to be one of the main research methods to research gene function in breast cancer, applying RNA interference technology in breast cancer cell lines in vitro to silence related genes. COL1A1 gene encodes the pro-type I collagen fibers, whci has been reported to have specific expression in breast cancer and may be associated with the occurrence and metastasis of breast cancer. Till now, nobody has reported the exact function of COL1A1 gene in occurrence and development of the breast cancer using RNA interference method. In this study ,we use COL1A1 gene interfering shRNA recobininant plasmidto to transfect MCF-7 breast cancer cells to study its function in breast cancer.Purpose: To determine the role of COL1A1 in MCF-7 breast cancer cell lines using recombined shRNA plasmid to optimize the best reaction conditions of transfection, positive selection and testing method.Using fluorescence microscope and fluorescence quantitative PCR method to test transfection efficiency and inhibition efficiency.Methode: Transfect MCF-7 breast cancer cell lines with lipofectamin 2000 using in vitro designed and synthesized COL1A1 interferencing plasmid. Using fluorescence microscope to observe and evalue the transfect efficiency 3 days after trasfection. Using QT-PCR to test the inhibition efficiency of COL1A1 mRNA.Result: We succeeded in transfecting the MCF-7 breast cancer cell lines using recombined COL1A1 shRNA with the transfection efficiency of 35%。We use QT-PCR to show mRNA has been interfered and has reduced by 96.83%。Conclusion: 1. In our research, we have succeeded in transfecting the MCF-7 cells with Recombinant COL1A1 shRNA interferencing plasmid using liposome 2000.2.In our research, we used fluorescence microscope to test the transfection efficiency of transfected MCF-7 cells and the transfection efficiency is 35%.3. In our research, we succeeded in selecting the positive cells after transfection using G418 and the concentration of G418 is 700μg/ml.4. In this experiment we succeeded in testing the transfected cells using fluorescence quantitative PCR method. We have built the standard curve of fluorescence quantitative PCR for both pMD18-T-β-actin and pMD18-T-COL1A1.Using 2-△△Ct method we found the inhibition efficiency is 96.83%.5. In this experiment,we have optimized the best reaction conditions of transfection, positive selection and testing method, and they would be valuable in the following experiment in which the funciton of COL1A1 will be tested.