The SiRNA Sequences Of Hypoxia Inducible Factor-1α(HIF-1α)Take A Inhibitioy Role On The Hepatocarcinoma Cell Lines Gel-7402

Primary hepatocellular carcinoma is one of the commonest malignant tumors in China. Since most clinical patients are in the Later stage because of its hidden onset, HCC 's treatment is limited at present and it has a lower surgical resection rate and a higher recurrence rate.Gene therapy, as a new cancer treatment, has become the direction of medical research. RNA interference (RNAi) as a new technology has a good prospect in gene therapy . However, RNAi is still in the basic research phase, the molecular mechanism is not yet completely clear, nor the formation of a specific and efficient treatment system .This experiment found by experimental methods for a specific siRNA sequence of hypoxia-inducible factor-1α(HIF-1α) in liver cancer cell and a set of appropriate and effective RNA interference strategy. We want to provide a theoretical basis of animal experiments and clinical trials and gene therapy in the future. Part I Construct the recombinant vectors of HIF-1αsiRNA OBJECTIVE Construction of four groups siRNA recombinant expression vector targeting HIF-1αby recombinant DNA technology , to provide experimental basis for the follow-upstudy.METHODS four purpose siRNAs designed as requiredby using of network resources, and inserted into the plasmid pGenesil-1.1, named respectively: S1, S2, SN, SB. After the bacteria filter and amplified ,those plasmids was been tested whether recombinant sequence as designed by using digestion and gene sequencing .RESULTS The size of the two segments of plasmid digested are in line with design requirements; all of gene sequences of four groups are correct by tested with gene sequencing.CONCLUSION This study constructed four recombination carriers successfully.Part II: the siRNA plasmid of HIF-1αinhibited the growth of liver cancerOBJECTIVE Selected the siRNA sequence taking interference on HCC HIF-1α, as the follow-up animal tests and basis of genetic drug research.METHODS those recombination carriers will be transfected into hepatocarcinoma cell strain bel-7402 by Lipofectamine 2000. Reverse transcription polymerase chain reaction ( RT-PCR) will be used to detect the effectiveness that small Interfering RNA (siRNA) suppress the transcription of HIF-1α.Cell Count will be used to detect the growth information of those transfected Cell.RESULTS S2 group had suppress transcription of target gene HIF-1αdramatically Among the total,but S1,SN group didn't suppress the transcription of target gene efficiently . Hepatoma carcinoma cell proliferation had been suppressed by the S2 group recombination carrier. Compared to SB group, S1 and SN hadn't suppress the proliferation of Hepatoma carcinoma cell.CONSLUSION This study had sieved a specific siRNA sequence S2. The S2 group recombination carrier show a great transfection efficiency .Its transcript had suppressed transcription of target gene HIF-1αdramatically and also inhibited the proliferation of hepatoma carcinoma cell.