Experimental Study On The Therapy Of Cholangioc-arcinoma By RNAi-Rac1

Cholangiocarcinoma is a malignant tumor of high mortality, morbidity, accounting for 2% of the total number of malignant tumors. With the development of the clinical pathology, the cholangiocarcinoma will be diagnosed more and more. Racl is a Rho family GTPase Ras superfamily of small molecular G proteins, as molecular switches to regulate cell signaling pathways. Racl plays key element in many important cellular processes work, such as in gene transcription, the process of cell cycle regulation and cell apoptosis, tumor invasion. Racl in human normal tissues was low expression or no expression, but in many tumor tissues and cell lines was high expression.By detecting the expression of Racl in cholangiocarcinoma, the Racl role was investigated. After targeting Racl closed, QBC939 cell proliferation and apoptosis was observed by the miRNA technology. The miRNA gene targeting Racl closed cholangiocarcinoma cells, cyclophosphamide, fluorouracil and the combination of any two groups were compared with QBC939 cholangiocarcinoma cell proliferation and apoptosis. For the gene therapy of cholangiocarcinoma, we try to find a new target and open up a new idea.1. A total of 74 cases which were from in Inner Mongolia from 2007-2009 Affiliated Hospital of cholangiocarcinoma,17 patients in the normal patients, and the expression of Racl protein were detected by immunohistochemical SP method. Within the 74 ECC cases analyzed,17 (22.97%) were highly differentiated adenocarcinomas,29 (39.19%) moderately differentiated adenocarcinomas, and 28 (37.84%) slightly differentiated adenocarcinomas. Racl in the incidence of cholangiocarcinoma play an important role in the development process, with the invasion and metastasis of cancer. Its detection can help predict the biological behavior of laryngeal squamous cell carcinoma and the prognosis.2. Molecular biology techniques were used to simulate the endogenous miRNA, miRNA genes in the design targeting Racl. Then the plasmid miRNA was transfected into cholangiocarcinoma cell QBC939 by the Lipofectamine 2000, transfection efficiency was observed by inverted fluorescence microscope, to obtain stable transfected cell lines, the G418 was used to select the positive cells. After transfection, Racl mRNA and protein levels were detected by RT-PCR and Western-blot. Through blank RNAi transfection,24 hours later cells and blank Racl cells to see significant green fluorescence, while in the blank group without. RT-PCR and western blot proofed the Racl's expression in cells were decreased in Racl interference. It was indicating the successful cell screening.By lipofection, and with the G418 selection, identified by PCR and Western blot, it was found that Racl interference cells were obtained successfully.3. By fluorescence quantitative PCR and Western blot, the Racl in the expression of mRNA and protein in cholangiocarcinoma cell lines QBC939 were detected. With MTT, the growth curve was drawn. Cell cycle was detected by flow cytometry. The invasion of the QBC939 was detected by Transwell. In Racl interference group, the Racl expression level and the inhibition rate of cell growth was higher than the other groups (P<0.05). After flow cytometry Racl blocking cells in the S group was significantly lower (P<0.05), while in G2 /M higher (P<0.05). The Racl blocking group also had the highest rate of apoptosis. Through Transwell experiments, showing that Racl blocking group was significantly lower than the number of penetrating cells in the other two groups (P<0.05). Racl in the cell growth process QBC939 play an important role to activities the role of molecular switch. And it was closely related to apoptosis and proliferation.4. Cholangiocarcinoma of the transfected cells detected by real-time quantitative PCR QBC939 and Western blot, fluorouracil, and blank control group significantly decreased expression level. In the cyclophosphamide+fluorouracil, fluorouracil+Racl blocking group, cyclophosphamide+Racl blocking group, the expression of Racl was lower in the latter two groups (P<0.05). MTT assay by inhibition of QBC939 cells proliferation, after 72 hours, Racl blocking inhibitory effect was significantly lower than the other two chemotherapy drugs groups, and in the cyclophosphamide+fluorouracil, cyclophosphamide+Racl blocking group and fluorouracil+Racl blocking group, the effect of phosphorus amide+ fluorouracil was lowest, the same as flow cytometry result. If the single drug, cyclophosphamide, and fluorouracil on cell growth inhibition QBC939 better and, if the combination therapy, RNAi is involved in the group are all good, indicating that the effect of combination therapy may be stronger than the single drug.We find that Racl in growth, proliferation, apoptosis, invasion and metastasis of QBC939 cell plays an important role in regulating function. Silenced Racl by RNAi technology can be effective in promoting cholangiocarcinoma cell proliferation and death, Inhibiting the cell invasion and metastasis. We found that the gene therapy is less than the role of strong chemotherapy drugs, but RNAi-Racl therapy combined with chemotherapy drugs can be a better therapeutic effect. Therefore, gene therapy or in combination with chemotherapy drugs can inhibit the expression of Rac1. This may be new ideas for the treatment of cholangiocarcinoma.