| STAT3 is an important member of Stats(signal transducer and activator of transcription),and its signal transduction pathways are closely related to cell proliferation, differentiation and apoptosis. Sustained activation of this pathway can lead to abnormal cell proliferation and malignant transformation. It has been confirmed that excessive activation and expression of STAT3 in many malignant tumors in mice and human. Now STAT3 has been defined as oncogenes.In recent years, RNAi (RNA interference) technology provides a helpful tool in studying the endogenous gene function and signal transduction pathways. DNA-based siRNA vector can be imported into cells, but expression of siRNA is transient and transfection is not stable. Using liposomes or physical methods such as transfection direct with electroporation have low efficiency and the activity of cells would be lost. Lentiviral vector can stably express siRNA with high transfection efficiency, so it is expected to become an ideal gene transfer vector. We have successfully transfected siRNA to Lewis lung cancer cells with technology In early experiments. It has been proved that silence STAT3 gene in application of RNAi technology can inhibit growth and induce apoptosis of tumor cells in various experiments.The purposes of the present study are (i) to constructing RNAi lentiviral vector of STAT3 gene, (ii) stably inhibit STAT3 gene expression in human lung adenocarcinoma cells (A549), (iii)to test changes of gene expression in transfected cells with genechip technology.The main process is as follows: according to the result of previous experiment, we select the best effective RNAi gene target against STAT3, as the Oligo DNA target sequences, annealing form double-stranded DNA, connecting with pSIH1-H1-copGFP-vector, judging the linked pSIH1-siRNA, transfent 293T cell with pSIH1-siRNA plasmid and lentiviral. Infect human lung adenocarcinoma cells with Produced SiRNA-STAT3 and select stably expression cells. Analyse mRNA expression level with Real Time-PCR, then hybridize extracted cRNA and genechip, check stably transfected cell gene expression changes.We collected lentiviral vector which can stably express siRNA with high transfection efficiency(higher than 90%) and sustained effect, also got cells which can stably inhibit STAT3 gene expression. in these cells, mRNA expression has markedly declined. Level of 29 genes was increased and 10 were declined.The results show following lentiviral vector stably inhibited STAT3 gene expression, Notch and PIK/Akt signaling pathway have more than two changes in gene expression. oncogenes of promoting cell proliferation were significantly increased and genes of inhibiting cell proliferation were significantly reduced. Some growth factor and cytokine related genes which related to interactions between cells had obviously changed. It needs further study of relationship between STAT3 and these genes.In early experiments, we silenced STAT3 genes of Lewis cancer cells with RNAi technology. It induced apoptosis only in lung cancer cells. STAT3 have high level of expression in some tumor cells and plays important role in tumor processing, growth and apoptosis. When we filter expression patterns in stably transfected A549 cells by siRNA-STAT3, we found that a mass of genes, signal transduction, protein kinase, receptors are closely related to STAT3 gene. The relationships of these genes can become representative of potential target genes of counter-resistance in treatment of tumor.So far, the major therapeutic motalities of cancer are surgery, radioth- erapy and chemotherapy, but the tumor chemotherapy and radiotherapy inevitably harm to normal cells and resistance to chemotherapeutic drugs and reduce the effect of chemotherapy. As STAT3 does not induce apoptosis in normal cells, we speculate that siRNA treatment of cancer is expected to becoming effective and have the least impact on normal cells.
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