The Research Of Molecular Mechanism Of Inducing Malignant Transformation Of3T3Cells By Piwil2

BackgroundPiwil2which is needed for jellyfish, mice, and human biological stem cellself-renewal belongs Argonaute family protein. Piwil2only in the testis andhematopoietic stem cells in normal human expression, but were also expressed in manytumors and tumor cell lines.Chen et al [1] confirmed that cancer stem cells (CSC) andthe cancer stem cell precursor cells (pCSC) specific express Piwil2protein that are alsoimportant regulatory genes in cell division and play. a major role in the formation anddevelopment of stem cell differentiation and tumor, Positive expression in tumor tissueand tumor cell lines Piwil2sake pCSC/CSC.Our studies also show, Cervical cancerstem cell-specific expression Piwil2[2], and cervical cancer tissues and high expressionPiwil2.A small amount of piwil2expressed in CIN Ⅱ/Ⅲ rather than CINⅠandnormal cervical epithelium.piwil2as a cancer-testis antigen(CTA) provides a highlyspecific target for the treatment of tumor-induced differentiation. In order to furtherdetermine Piwil2gene expression in tumor. The study applications a non endogenousPiwil2gene commonly used in the study gene malignant transformation of the cell lines-mouse fibroblasts3T3. Genes stably expressing Piwil2the3T3-Piwil2cell lines aswell as the empty vector control are established to research the regulatory biologicalcharacteristics of Piwil2in3T3cells...ObjectiveThe study found that Piwil2expression only in the testis and the hematopoieticstem cells in normal human, but are expressed in many tumor tissues and tumor cell lines。Piwil2is a CTA (cancer-testis antigen, CTA) specifically expressed in CSCprecursor cell (pCSC) and the CSC， is a key molecular regulation of stem celldifferentiation, but the molecular mechanism has not yet been fully elucidated. Thisstudy established3T3cells stably expressing exogenous Piwil2gene， preliminarystudy regulation of the biological characteristics of3T3cells.Methods（1）Constructed the eukaryotic expression recombinant plasmid pIRES2-EGFP-Piwil2.（2）By using gene transfection technology through liposome-mediated method andG418selection, establish3T3cell lines the stable expression Piwil2gene.（3）By using the colony formation experiment, observe3T3cells of high expressionPiwil2gene colony forming ability changes, compared with the control group.（4）By using the cell proliferation experiment, observe3T3cells of high expressionPiwil2gene growth ability changes, compared with the control group.（5）By using cell cycle analysis, observe3T3cells of high expression Piwil2gene cellcycle changes, compared with the control group.（6）By using nude mice experiments, observe3T3cells of high expression Piwil2genetumorigenic ability changes, compared with the control group.Results(1) Successfully constructed the eukaryotic expression recombinant plasmidpIRES2-EGFP-Piwil2.（2）Successfully established the stable expression Piwil2gene in3T3cell lines.（3）Compared with the control group,3T3cells of high expression Piwil2gene colonyforming ability enhanced, the difference between the groups was statistically significant(p <0.05).（4）Compared with the control group,3T3cells of high expression Piwil2gene proliferation ability enhanced, the difference between the groups was statisticallysignificant (p <0.05).（5）Compared with the control group, G0/G1phase was decreased and S phase wassignificantly increased, the difference between the groups was statistically significant (p<0.05).（6）Nude mice experiment results show that the tumor volume was significantlygreater than the control group, the difference between the groups was statisticallysignificant (p <0.05).Conclusion（1）High expression Piwil2gene in3T3cell can change colony formation, growth rate,cell cycle and tumorigenicity.（2）Through the activation of Stat3signaling pathway, accelerate cell division byenhanced cyclin D1and inhibit apoptosis by the BCL-2gene affect p53inactivation.