Histone Deacethylases, Caspases, Polypyrimidine Tract Binding Protein And RUNX3 In The Control Of Colorectal Carcinoma

Part 1 The constitution of hunman PTB and RUNX3 lentivirus vectors and check their efficiencyAim To constitute RNA interference lentiviruses of both PTB and RUNX3, and check their efficiency in HEK 293T cell lines.Methods Design two pairs of primers according to the gene sequences of PTB and RUNX3, then design and synthesize the DNA oligo of si RNA which is then anneal to the pSUPER vector which is digested with the enzymes of BgⅢHindⅢ, transformed to competent cells. PCR is used to check the positive colonies. Choose the positive colonies and mini preparation plasmids. Digest the plasmids with ClaI and EcoRI, then do gel extraction and DNA purification. At the same time digest pLVTHM vector with the enzymes of Clal and EcoRI, do the ligation with T4 ligase and then transformed to competent cells. PCR is used to check the positive colonies by using primers of multiple cloning sites at both ends of the two genes. Positive clones are mini prepared and sequenced. Select the clone of correct sequence, and use lipofecting 2000 to transfect HEK 293T cell lines with the plasmids of pLVTHM-PTB/ pLVTHM-RUNX3, packaging plasmid and envelop plasmid. Transfect the lentivirus to 293T cell lines at different concentration to the wells, negative control is also prepared. RT-PCR and western-blot is used to check the efficiency of the two lentiviruses.Results The sequencing results showed that the plasmids of pLVTHM-PTB and pLVTHM-RUNX3 are correct. Maxi of the two plasmids are prepared for the lentivirus packaging. HEK 293T cells are seed in six well plate and transfected with lentiviruses at different concentration. There are three groups with the concentration of Oμl/ml,1μl/ml and 2μl/ml respectively. Cells are ovserved with immunofluorescence microscope three days after transfect. The results show that both of the two lentiviruses have the best effect at the concentration of 2μl/ml, which is then used in the following experiments. Then the two lentiviruses are transfected in to hepatocellular carcinoma cell line SMMC-7721, RNA and protein are extracted at 3 days and 4 days after transfect. Then RT-PCR and western-blot are performed to check the efficiency of gene suppression. The results show that PTB is effectively decreased on the day of 3 and 4 after transfect, RUNX3 decrease is more obvious at day 4 than day 3.Conclusion pLV-PTB and pLV-RUNX3 lentivirus are successfully prepared by using lipofectamine 2000. These two lentiviruses are transfected to HEK293T cells and HT-29 cells. We used PCR and western-blot to check the effectivity of transfect and it proved to be good enough.Part 2 Histone Deacethylases, Caspases, Polypyrimidine Tract Binding Protein and RUNX3 in the control of Colorectal CarcinomaObjective:To investigate the process of colorectal tumorigenesis induced apoptosis of colorectal cancer related genes and signaling pathways.Methods:RT-PCR, western-blot were used to detect related genes the expression levels of transcription and translation in vivo and in vitro, and use real-time PCR is used for quantitative analysis.Results:HT-29 cells were transfected with different concentrations of pLVTHM-PTB lentivirus after the first 3,4 days, cell protein were extracted, detected by western-blot results of the level of RUNX3 expression increased at translational leval. HT-29 cells were transfected with different concentrations of pLVTHM-RUNX3 lentivirus after the first 3,4 days, cell protein were extracted, detected by western-blot results of the expression of PTB in translation is on a increasing trend. HT-29 cells successfully transfected pLVTHM-PTB lentivirus days after the first 3,4,5,6, cell protein were extracted, detected by western-blot results of HDAC 1,2,5 expression levels decreased at translational leval. In the HT-29 cells were incubated with histidine deacetylase inhibitors (HDACi) to increase the fracture of the PTB, this process is caspase dependent signaling pathway activated by external causes.Conclusion RUNX3 and PTB have an effect of downregulation to each other in colorectal carcinoma; HDAC can upregulate PTB expression in colorectal carcinoma. In the process of colorectal cancer cell regulation, caspase-dependent external signal pathway is involved in PTB protein fraction caused by HDAC defect.