The Proliferation And Apoptosis Biological Function And Mechanism Of Tumor Suppressor Gene Fragile Histidine Traid Gene (fhit) In Colon Cancer Cells And Hepatoma Cancer Cells

Fragile histidine triad gene (Fhit) is a new tumor suppressor gene, which was found in 1996. It belongs to histidine triad family. The gene encode 1.1kb mRNA, which is translated into a 16.8KD protein. Researches show that lack of Fhit expression is related to the occurrence and development of malignant tumors. The transcrips and protein expression of Fhit were changed in breast cancer, lung cancer, gastric cancer, prostate cancer, gastrointestinal cancer, head and neck cancer and other malignant tumors. Fhit increased the cell apoptosis and inhibited the proliferation of tumor cells in certain tumors, such as gastric cancer, lung cancer, head and neck cancer, esophageal cancer, when it was used for gene thrapy with the use of adenovirus or adeno-associated virus vector. However, there is little study associated with treatment of colon cancer and liver cancer. Although the role of tumor suppressor has been very clear, the mechanism of Fhit is still uncertain. It is not only further clarify the tumor suppressor function of Fhit and supply the expreiment evidence of using it into gene thrapy with the study in colon cancer cells and hepatoma cancer cells, but also provide foundation for the study of the machanism of Fhit.Firstly, we constructed a recombinant adenovirus carrying Fhit gene, and observed its biological function on the proliferation of colon cancer cells. Secondly, we constructed the gene of TAT-Fhit fusion protein containing TAT protein transduction domain and Fhit gene,and studied the internalization function and biological effect of the fusion protein after expression and purification in E coli. Finally, we used the yeast two-hybrid assay to search proteins which can interact with Fhit protein, and analysed the antitumor mechanism of Fhit.In expriment one, Fhit gene was cloned from the fetal liver cDNA library using the PCR method. The PCR product was inserted into the T vector to construct the plasmid pMD18T-Fhit. The Fhit fragment from the pMD18T-Fhit was inserted into the vector pTrack-CMV to construct a shuttle plasmid pTrack-CMV-Fhit. After PmeI digested and linearized process, pTrack-CMV-Fhit was co-transformed into Escherichia coli strain BJ5183 together with the adenovirus backbone vector pAdEasy-1 to generate a recombinant adenovirus plasmid by homologous recombination. The adenovirus plasmid was identified by PacI digestion and transfected into 293A cells to package a recombinant adenovirus which expressed the Fhit protein. Furthermore, the adenovirus rAd-Fhit was infected into colon cancer cells,and the expression of the ectogenic protein was detected by Western blotting. Finally, the proliferation of colon cancer cells was observed in adenovirus-infected cells by the MTT assay. Rusult shows that we constructed the recombinant adenovirus encoding Fhit gene and expressed it in colon cancer cells successfully. We detected that the proliferation of colon cancer cells was inhibited obviously in rAd-Fhit-infected cells with comparison to the control groups.In expriment two, we designed primers to amplify the Fhit gene, and connected it with synthesized coding sequence TAT peptide. Then the recombined fusion protein gene TAT-Fhit was cloned into expression vector pET32a. We detected the expression of the protein and purified the protein by Ni-NTA chelating agarose. The internalization function was confirmed by indirect immunofluorescence staining. The apoptosis of hepatoma cancer cells was detected by flow cytometry. The proliferation of hepatoma cancer cells was measured by MTT assay. We observed that the proliferation of the cells was inhabited and the apoptotic rate was increasing after TAT-Fhit fusion protein internalized into hepatoma carcinoma cells.In expriment three, we used the yeast two-hybrid assay to find the proteins which could interact with Fhit. Firstly, we co-transformed the baid portein Fhit with the liver cDNA library into AH109 yeast cell line. Secondly, we chose the positive clone at the nutrient-dificient medium with the high stringent screening method. Thirdly, we extracted the plasmids from the positive clones, amplified and digested them with restriction enzyme. Finally, we sequenced and analysed the different types of plasmid with bioinformatics. We found that DYNLT1 (Dynein light chain 1) may interacted with Fhit.In summery, the Fhit may act as a tumor suppressor in colon cancer cells, and the adenovirus-mediated Fhit can be a novel strategy for the colon cancer therapeutics. The TAT protein transduction domain is able to mediate Fhit protein across the plasma membrane into hepatoma cancer cells. The Fhit plays the role of anti-tumor gene in hepatoma cancer cells. The TAT transduction peptide may become a new cancer therapy method. Fhit might affect the cell condition by influencing intracellular material transport when interacted with DYNLT1. Our study provides some experiment evidences for finding the anticancer machanism of tumor Fhit.