A Study On The Function And Mechanism Of MiR-129in Docetaxol Resistance Of Breast Cancer Cells

The incidence of breast cancer has been showing an upward trend in our country.Though the effect of chemotherapy for breast cancer has been acknowledged, it can’t bedenied that the multi-drug resistance of tumor cells is a critical cause for the treatmentfailure. With the deepening study on the microRNA in the aspects of tumor formation anddrug resistance, we hope to predict the related microRNA molecules which may beinvolved in the multidrug resistance process by applying the bioinformatics technology.Moreover, we also hope to build a lentiviral vector by using the related molecular cloningtechnology, and sort out the microRNA molecules which are most likely involved in themultidrug resistance process based on the combination of dual luciferase reporter genesystem, Western Blot experiments and the clear biological phenomena after the infectionof microRNA virus. Thus, we can make a further study on the biological function of acertain microRNA molecule and its acting mechanisms, which will provide an effectiveway to reverse the multidrug resistance of breast cancer and improve the clinical effect of the chemotherapy. The whole experiment is consisted of three parts:1. Establishing multidrug resistant breast cancer cell lines and the stable transfectedcell lines of multidrug resistance (MDR1) gene. One of the main reasons for the multidrugresistance phenomenon in the treatment of breast cancer is the over-expression of the geneMDR1. Using the molecular cloning techniques, we successfully established the stabletransfection cell lines of multidrug resistance MDR1gene in the MDA-MB-231andMCF-7cells, which laid a good experimental basis for the in-depth study of the drugresistance mechanisms. First, we selected the lentiviral vector as the gene transductioninstrument because it can integrate exogenous genes into the chromosomes of the hostcells, so as to realize the long-term stable expression. Compared to a retroviral vector, thebiggest advantage of a lentiviral vector is that it can infect both dividing and non-dividingcells. Besides, with a wider range of infection, successful cases have been reported ininfecting neuronal cells, lymphocytes, macrophages and other types of cells by using alentiviral vector. These advantages make it promising both in basic research and the genetherapy field. We then restructured the entry vector, containing the complete sequence ofthe MDR1gene, into the backbone vector pLenti6.3, which was achieved by adopting theInvitrogen Corporation’s core technology-Getway. In order to realize the steadyexpression of the exogenous gene MDR1in MDA-MB-231and MCF-7cells, thethree-plasmid lentiviral packaging system was applied to co-transfect the humanembryonic kidney293T cells with the high incidence up to90%in the target cells. Inorder to obtain the stable integration of MDR1cells after the infection,4weeks blasticidin(Blasticilin10ug/ml) selecting process was adopted, and after that, the MDA-MB-231and MCF-7cell lines with the stable over-expression of gene MDR1were ultimatelyestablished. Through the consistent docetaxol selection, docetaxel resistant cell lines wereestablished also. Further experimental verification of the MDR1gene and its encodedprotein may play a significant role in the drug resistance research of breast cancer, whichwill provide a new strategy to expolre the function and mechanisms of the gene MDR1.2. Constructing the microRNA lentiviral vector acting on the target gene MDR1. Wepredicted the five kinds of microRNAs targeting the MDR1gene by the use of biological information technology, and successfully established the lentviral expressing vector usingthe molecular cloning techniques. Judging by the combination of dual luciferase reportergene system, Western Blot experiments and the cell proliferation-toxicity test concerningthe direct infection of the breast cancer cells MDA-MB-231and MCF-7, it was concludedthat miR-129was most likely involved in the drug resistance process of breast cancer.3.Study on the biological funcion of miR-129in the evolvement of drug resistanceand its impact on the malignant biological behavior of breast cancer cells. In order todevelop a further study on the role of miR-129in breast cancer resistance evolvement, wesuccessfully constructed the cell lines MDA-MB-231-miR-129and MCF-7-miR-129withthe stable over-expression of miR-129. The cell proliferation-toxicity experimentconfirmed the role of miR-129in generating docetaxel resistance. In addition, the platecloning experiment and EDU staining method were also adopted to study the role ofmiR-129in the proliferation of breast cancer cells. Moreover, based on the RT-PCRanalysis, we found the significantly high expression of miR-129-3p in the stabletransfectants MDA-MB-231-miR-129and MCF-7-miR-129, and the direct correlationbetween miR-129-3p high expression and the evolvement of drug resistance, which wasthen further confirmed by the chemosynthetic transfectants miR-129-3p minics andmiR-129-3p inhibitor.