The Mechanism Of Action Of TKTL1 On Proliferation Of Human Colon Cancer Cell Line(LoVo)

Cloning and sequence analyzing of the recombinant plasmid affecting gene TKTL1 translation by RNA interferingObjective: To clone the recombinant plasmid affecting gene TKTL1 translation by RNA interfering and analyze the nucleic acid sequence of the recombinant for further searching new gene therapy method of tumor.Methods: Two DNA sequences containing small hairpin structure were designed and synthesized. The complement form was obtained by annealing and inserted into vector pEGFP-C1-U6, and the recombinant plasmid was transformed into DH5аstrain. Finally the plasmid identified by restriction enzyme was used for sequence analysis. Results: The recombinant was cloned and the aim sequence was obtained.Conclusion: Successful cloning of the recombinant helps to search new gene therapy method of tumor. The effect of TKTL1 gene on proliferation of human colon cancer cell lineObjective: The aim of this study was to investigate the effect of TKTL1 on proliferation of LoVo cell in vitro.Methods: A plasmid, which carries the DNA to be transcribed into a siRNA against TKTL1 mRNA, was transfected human colon cancer cell. Real-time PCR were used to determine the mRNA expression of TKT gene family in the human colon cell line. Continuous monitoring assay were used to determine total transketolase activity. Flow cytometry and MTT were used for detecting the effect of anti-TKTL1 siRNA on cell proliferation and cell cycle in the LoVo cell.Results: There was no significant difference in the expression level of TKT and TKTL2 gene between the LoVo cells transfected with siRNA TKTL1 construct and the cells transfected with control vector or untransfected LoVo cells (P>0.05). However, the expression level of TKTL1 gene was significantly down regulated in the LoVo cells transfected with siRNA TKTL1 construct compare with the cell transfected with control vector. The total transketolase activity significant decrease, cancer cells proliferation was significantly inhibit, and cancer cells were arrested in G0/G1 phase cyclein the LoVo cells transfected with siRNA TKTL1 construct.Conclusion: TKTL1 plays an important role in the total transketolase activity and cell proliferation of human colon cancer. It also indicates that TKTL1 can be seen as a potential target for novel anti-transketolase cancer therapies.