Overexpression And Inhibition Of JTV1 Gene And Its Influence On The Proliferation And Apoptosis Of K562 Cells

PARTⅠCONSTRUCTION AND FUNCTIONAL IDENTIFICATION OF HUMAN JTV1 GENE SIRNA RECOMBINANT VECTORObjective: To construct the recombinant vector siRNA targeting JTV1 gene and to identify its effect on the expression of JTV1 gene.Methods: The siRNA sequence targeting JTV1 gene was synthesized and cloned into the plasmid pGeneSil-1 to form the recombinant vector pGeneSil-1-JTV1 siRNA. Then the recombinant vector was transfected into K562 cells. The mRNA and protein levels of target JTV1 gene were detected by RT-PCR and Western blotting.Results: The recombinant vector containing siRNA targeting JTV1 gene was successfully constructed. The transcription and translation levels of JTV1 gene in K562 cells were significantly inhibited by JTV1 siRNA recombinant. Conclusions: The recombinant vector pGeneSil-1-JTV1 siRNA significantly inhibited the transcription and translation levels of JTV1 gene, and the K562 cell clones in which JTV1 gene's expression was stably inhibited were obtained by G418 screening.PARTⅡINHIBITION OF JTV1 GENE PLAYS A PART IN PROLIFERATION AND APOPTOSIS OF K562 CELLS AND ITS MECHANISMObjective:To investigate the tumor-suppressing function of JTV1 gene on the proliferation and apoptosis of K562 cells, the effect on the apoptosis related factor Bcl-2, c-Myc and Bax genes in human chronic myeloid 1eukemia (CML) cell line K562, and to search the mechanism.Methods:The recombinated vector pGeneSil-1-JTV1-1.1 siRNA, negative vector pGeneSil-1-N.1 and the empty vector pGeneSil-1 were transfected into K562 cells. The cell proliferation ability of K562 cells was evaluated by colony formation experiment; the mRNA levels of apoptosis related genes Bax, Bcl-2 and c-Myc were detected by RT-PCR; the protein levels of Bax, Bcl-2 and c-Myc were detected by Western Blotting.Results: The colony formation experiment results showed that the proliferation ability of K562 cells was increased when the expression of JTV1 gene was inhibited; compared with the negative group and the empty vector group, the transcription and the translation levels of Bax gene of the pGeneSil-1-JTV1-1.1 siRNA group were significantly decreased; and the transcription and the translation levels of Bcl-2 and c-Myc genes were significantly increased.Conclusions: The expression of Bcl-2 gene and c-Myc gene were upregulated when the gene JTV1 was inhibited, however, the expression of Bax gene was downregulated, which may promote the proliferation ability of K562 cells and inhibit the apoptosis effect of K562 cells. Inhibited the expression of JTV1 gene clould promote proliferation ability of K562 cells and inhibit the apoptosis effect of K562 cells via up-regulating the expression of Bcl-2 gene and down-regulating the expression of c-Myc and Bax genes. PARTШCONSTRUCTION OF PCDNA3.1-JTV1 VECTOR AND ITS EXPRESSION IN K562 CELLSObjective: To construct the pcDNA3.1-JTV1 vector and obtain the positive K562 cell clones expressing JTV1 gene stably, and to establish a cell model for studying the effect of JTV1 gene on cell proliferation.Methods: The cDNA of human JTV1 gene was amplified by RT-PCR from the total RNA isolated from the mononuclear cells and was inserted into pcDNA3.1 vector to construct the recombinant vector pcDNA3.1-JTV1. The recombinant plasmid was then transfected into K562 cells by liposome. 0verexpression of JTV1 gene in the transfected K562 cells was confirmed with RT-PCR and Western blotting.Results: By the use of RT-PCR, a 1200 bp DNA fragment was successfully amplified from the mononuclear cells, which matched the length of cDNA encoding JTV1 gene. The results of Enzyme digestion analysis and DNA sequencing analysis showed that the target gene was cloned into recombinant vector successfully. Western blotting results revealed that the JTV1 protein could be expressed stably in K562 cells pcDNA3.1-JTV1 vector transfected.Conclusions: The pcDNA3.1-JTV1 vector was successfully constructed. The positive K562 cell clones expressing JTV1 stably were obtained, which has provided solid foundation and a cell model for further research on the function of JTV1 gene and its relationship with cell proliferation ability.PARTⅣEFFECT OF JTV1 ON PROLIFERATION AND APOPTOSIS OF K562 CELLS AND ITS MECHANISMEObjective: To investigate the effect of tumor-suppressing gene JTV1 on proliferation and apoptosis ability of K562 cells and the influence on apoptosis related genes Bcl-2, c-Myc and Bax in K562 cell line. Methods: The recombinated vector pcDNA3.1-JTV1 and the empty vector pcDNA3.1 were transfected into K562 cells. The proliferation ability of K562 cells was evaluated by colony formation experiment; the cell cycle and apoptosis rate were assessed by flow cytometry (FCM); the mRNA levels of Bax, Bcl-2 and c-Myc apoptosis related genes were detected by RT-PCR; the protein levels of Bax,Bcl-2 and c-Myc were detected by Western blotting.Results: The colony formation results showed that the proliferation ability of K562 cells was decreased when the expression of JTV1 gene was upregulated; the FCM results showed that the cells of G phase pcDNA3.1-JTV1 positive transfected were increased compared with the negative control group and the pcDNA3.1 empty vector transfected, the differences were statistically significant; compared with the negative control group and the empty vector group, the mRNA level and the protein levels of Bax gene were significantly increased; the mRNA level and the protein levels of Bcl-2 and c-Myc gene were significantly reduced.Conclusions: The expression of Bcl-2 and c-Myc genes were inhibited when the gene JTV1 was upregulated, but the expression of Bax gene was increased, which may inhibit the proliferation of K562 cells and promote apoptosis of tumor cells. Over expression of JTV1 gene could inhibit the proliferation ability of K562 cells and promote the cell apoptosis probability via inhibiting the expressions of Bcl-2 and c-Myc genes and up-regulating the expression of Bax gene. PARTⅤVERIFICATION OF INTERACTION BETWEEN GLUTAMATE-AMMONIA LIGASE AND NUCLEAR LOCALIZATION SIGNAL-RETINOIC ACID RECEPTORαObjective: To verify the interaction between glutamate-ammonia ligase (GLUL) and nuclear localization signal-retinoic acid receptorα(NLS-RARα) protein by yeast two-hybrid and co-immunoprecipitation method.Methods: The two plasmids expressing NLS- RARαbait-protein and GLUL protein were co-transformed into yeast AH109 to investigate the interaction in vivo. Tagged fusion protein eukaryotic expression vectors were co-transformed into HEK 293 cells, then HA- NLS- RARαprotein was immunoprecipitated by anti-HA polyclonal antibody, and GLUL-cMyc protein expression was confirmed by Western blotting analysis using anti c-Myc monoclonal antibody.Results: Positive blue clones were found in the QDO/X-α-gal plate. Eukaryotic expression vectors were co transfected into HEK 293 cells, then HA-NLS-RARαprotein was immunoprecipitated by anti-HA polyclonal antibody, and GLUL-cMyc protein expression was confirmed by Western blotting analysis using anti c-Myc monoclonal antibody.Conclusions: The interraction between NLS-RARαand GLUL has been verified by both yeast two-hybrid and co-immunoprecipitation.