Knockdown Of Smac Expression By RNAi Enhances Growth And Cisplatin Resistance Of Human Lung Cancer Cells

Part I Preparation of the Vector Expressing SMAC RNAi, Screening of the Target Genes and Package of LentivirusPurpose:The aim was to construct RNAi Plasmid Vector containing siRNA sequences targeting Smac, and to make package lentivirus and preparations of stable cell strains infected with lentivirus.Materials and Methods:1. multiple RNA interference target sequences were designed aiming target gene sequences and according to the design principles of RNA interference from public websites;and then they were assessed by professional software and some targets with the best kinetic parameters were chosen for subsequent experiments. The double strand DNA oligo with cohesive ends of restriction site at both ends containing interference constructed by Genechem Co., Ltd was directly connected to vector containing siRNA. The binding products were transformed to the prepared competent cells. The cell clones were chosen as those with successfully constructed plasmid after PCR analysis and comparisons in sequence analysis.2. A549 cells were infected with lentivirus gene transfer vector (pGC-FU vector) containing siRNA sequences targeting Smac. The efficiency of gene silencing of shRNA-lentivirus for the target SMAC was verified not only under light microscope, but also from mRNA and protein levels by RT-PCR and Western Blot method respectively.3. The recombinant virus particles encoding the lentivirus and the two auxiliary packed vectors were extracted with endotoxin-free large number of high-purity plasmid method. According to the instructions of lipofectamine2000 kit, T293 cells were cotransfected, cultured and then cell supernatant full of lentiviral particles was collected and concentrated. The virus titers were tested and demarcated.Results:1.The psc-1 clones containing vshRNA fragments were the successfully constructed vectors with target siRNA.2. The efficiency of gene silencing for the target SMAC was verified not only under light microscope, but also from mRNA and protein levels after the induction of shRNA-lentivirus into the target A549 cells, which indicated the useful target for RNA interference was successfully identified.3. The cells used for tools and virus vectors were successfully packed and stable cell strains infected with lentivirus were prepared successfully.Conclusions:The RNAi Plasmid Vector containing siRNA sequences targeting Smac were successfully constructed and the useful target for RNA interference was successfully identified. The titers of virus were tested after package and the stable cell strains infected with lentivirus were prepared successfully, which laid the foundation of our subsequent study on the functions of SMAC. Partâ…¡Construction of a Lentivirus Vector over-expressing SMACPurpose:The aim was to a Lentivirus Vector over-expressing SMAC as a tool for our subsequent study on the gene functions.Materials and Methods:1. The target gene was obtained from the plasmid containing the gene or from the cDNA library. Genechem Co., Ltd carried out the followings:the primer synthesis and identification of the target gene, and its PCR amplification, purification and enzyme digestion, and direct connection to recombinant clone, confections to the competent cells, and PCR identification of recombinant transformants. The company performed the sequencing and alignment of the positive clones and so the correct clones were chosen as the successfully constructed plasmid.2. A549 cells were infected with lentivirus gene transfer vector (pGC-FU vector) containing full-length coding region of human Smac (for overexpression). pGC-FU vector containing siRNA with a green fluorescent protein (GFP) sequence at the C-termination and the control vector were also transfected. The expressions of the vectors were verified from protein levels by western blot method.3. according to the instructions of lipofectamine2000 kit, T293 cells were cotransfected, cultured and then cell supernatant full of lentiviral particles was collected and concentrated. The virus titers were tested and demarcated.Results:SMAC was over expressed in the pGC-FU vector containing full-length coding region of human Smac, which was verified from protein levels.Conclusions:The Lentivirus Vector over-expressing SMAC was successfully constructed providing a reliable tool for our subsequent study on the gene functions. Partâ…¢Study of the effects of SMAC on the growth and cisplatin resistance of human lung cancer cellsPurpose:We investigated the effects of the Smac knockdown through lentivirus-mediated Smac RNAi on growth and drug resistance to cisplatin in lung cancer cells, while the effects of the Smac on growth and drug resistance to cisplatin in lung cancer cells are further proved by Smac over-expression through transfection with pOE vector containing complete encoding sequence of Smac. Thus the potential mechanism of Smac is verified pro and con, which laid the foundation of our further study on the mechanisms on the effects of the Smac on growth and drug resistance to cisplatin of lung cancer cells.Materials and Methods:1. the knockdown of Smac expression was established by knockdown of Smac gene. The changes of proliferation ability of A549 and 95D cells were tested after and before the Smac knockdown by the method of MTT, colony-formation assay, and amphochromophil Annexinâ…¤-PI flow cytometry after the Smac knockdown mediated by RNAi.2. the changes of growth and apoptosis of A549 and 95D cells were tested after and before the intervention of cisplatin and the Smac knockdown by the method of MTT and amphochromophil Annexinâ…¤-PI flow cytometry so as to test the effect of cisplatin-resistance.3. Smac over-expression was achieved through transfection with pOE vector containing complete encoding sequence of Smac, and the changes of cellular growing ability were checked by the method of MTT, Brducell proliferation assay so as to further test the effect of cisplatin-resistance.Results:1. To assess the effect of Smac knock-down on proliferation and growth of human lung cancer cell lines A549 and 95D, MTT assay and colony-forming assay were performed. We found that pKD-transfected cells had a significantly increase in proliferation as compared with that of pNC-transfected cells in both cell lines, which indicates that knockdown of Smac promotes the proliferation ability of human lung cancer cells. In addition, the results from colony-forming assay showed that the colony amounts and size of pKD-transfected cells had an obvious increase by comparing with pNC-transfected cells, providing evidence for that Smac knockdown significantly promoted colony-formation of lung cancer cell A459.2. flow cytometry showed that the apoptosis induced by cisplatin was also inhibited by Smac knockdown. In both lung cancer cells lines, the proportion of cells at early and late stages of apoptosis (LR and UR) was largely reduced when Smac was down-regulated, while at the same the proportion of living cells was dramatically increased.3. Protein content of Smac was greatly increased in pOE-transfected A549 cells which validated the effectiveness of lentivirus mediated Smac overexpression. In contrast to Smac knockdown, overexpression of Smac significantly inhibited cell growth and increased cisplatin sensitivity of A549 cells. These data demonstrated the important roles of Smac in cell growth and drug sensitivity of lung cancer cells from another aspect.Conclusions:In our study, Smac significantly stimulated cell proliferation and enhanced susceptibility to cisplatin. Smac may be a useful target to predict the cisplatin sensitivity in lung cancer cells. Our results provided robust evidence for the important roles of Smac in suppressing lung cancer cells growth and sensitizing them to chemotherapeutic drugs. Therefore, Smac is also a promising target for the development of effective drugs for lung cancer treatment, and small molecule mimics of Smac will also function in the treatment of human lung cancer.