Effective Interference Fragment Screening And Construction Of Lentiviral Mediated RNA Interference Of Mammalian Target Of Rapamycin

Lung cancer is the tumor with the highest mortality rate in the world, and has become a global problem which threats to human health. The incidence of lung cancer continues to rise in the trend in China still. The Male lung cancer accounts for the first of a variety of tumor incidence, and the female lung cancer accounts for the second. The majority of patients with lung cancer treatment is already in locally advanced or distant metastasis, generally, the conventional treatment is surgery, radiotherapy, chemotherapy, biological cell therapy and comprehensive treatment, but the results are far from ideal, so, searching new methods for the treatment of lung cancer is the current research focus.mTOR, mammalian target of rapamycin, is a atypical serine/threonine kinase and it is also an important signal transduction molecule. The protein encoded by mTOR gene which is located in Ip36.2consists of2549ami no acids, the molecular weight is289KD. It is paid close attention for the location in the central link of the signal transduction pathway. It plays a central regulator in the process of cell growth, differentiation, proliferation, and other basic functions, meanwhile, it also plays important physiological functions in regulating the cell cycle, protein synthesis and degradation, cellular energy metabolism and the other pathways. So, mTOR has become a hot topic of the present tumor-targeting gene therapy.RNAi, RNA interference, is a kind of the small21nt-23nt of double-stranded RNA or short hairpin RNAs molecules, which can broken the expression of body-specific gene sequence efficiently and specifically at the mRNA level, and then lead to the gene deletion process of the reduced phenotype. RNAi has been widely used in the study of gene function and disease gene therapy because of its speediness, economic, efficiency, simple operation and other characteristics, moreover, the theory and related technologies about RNAi have also been penetrated into various fields of life sciences.The key factors of gene therapy lie in choosing the best of transgenic carrier, the most appropriate purpose gene, and realizing controllability of purpose gene. Lentiviral mediated can infect most of cell, not only including the cells of split period, but also the cells of non-split period, particularly, adapt to RNAi experiment of various cells. The cells can be infected efficiently by lentiviral mediated, and be used to mediate the express of exogenous gene in the cells. Lentiviral mediated can be studied growth controlling of the cells, and the integration of the specific gene expression. In addition, the cells do not produced lesion after effecting by lentiviral mediated, and we can establish the cell lines of long-term continuous expression exogenous gene.ObjectiveThis research uses RNAi technology, and chemistry to integrate siRNA to select the high efficiently target of mTOR gene in lung adenocarcinoma A549cells, and constructing restructuring lentiviral expression mediated, and explore new methods for researching gene therapy of lung cancer.Methods1. To design four interference targets and Negative control (FAM) sequence for mTOR gene in strict accordance with principle of RNA interference design, and chemistry compounds effective siRNA fragment. To use the reage Lipofectamine2000to transfecte the lung adenocarcinoma A549cells, after24hours, and detecte the express of FAM with help of fluorescence microscope everyday. To detecte mTOR gene’s express in the level of mRNA after24hours and protein after48hours by RT-PCR and Western-Blot, choosing the high efficiently target for establishing lentiviral mediated.2. To use those effective disturb target spot of mTOR gene compounds double stranded DNA of target sequence, inserting pGCL-GFP mediated, transfecting293T cells with plasmid pHelper1.0and pHelper2.0, producing lentiviral. The express level of GFP protein in293T cells is used to detected and identified virus titer.Results1. Choose the high efficiently target of mTOR gene successfully.2. mTOR siRNA infecte of the human lung adenocarcinoma A549cells, and result mTOR gene expression significantly reduced in the level of mRNA and protein.3. Constructe lentiviral mediated of siRNA about mTOR gene successfully, receiving serum of virus and identificating virus titer of1x10~8UT/ml.Conclutions1. mTOR siRNA infected of the human lung adenocarcinoma A549cells and lead to downward in expressing of mTOR gene.2. Constructe lentiviral siRNA mediated of the mTOR gene successfully.