The Mechanism And Effect Of JAZF1 Overexpression On Proliferation And Apoptosis Of BCPAP Cells In Papillary Thyroid Carcinoma

PART ONE Overexpression and verification of JAZF1 in BCPAP cellsObjective:JAZF1 was overexpressed in BCPAP cells using recombinant adenovirus vector(Adv-JAZF1-GFP).Methods:BCPAP cells were infected with recombinant adenovirus,which were divided into three groups: negative control group(NC group),un-loaded group(Adv-GFP group)and experimental group(Adv-JAZF1-GFP group).48 hours after transfection,total RNA,was extracted and synthesized into c DNA,by SYBR-Green I method,ABI7500 and q RT-PCR two-step method to detect the expression level of JAZF1 m RNA.GAPDH was used as an internal reference,and 2-△△CT value was used to represent the relative expression of gene.The expression of JAZF1 m RN was detected by q RT-PCR.The total cell protein was extracted from each group,then the relative expression level of JAZF1 protein in each group was detected and analyzed by Western blot.Results:48 hours after recombinant adenovirus transfection into BCPAP cells,there was no significant difference between the negative control group and the un-loaded group,but the relative expression levels of JAZF1 m RNA and protein in the experimental group were significantly higher than those in the negative control group and the un-loaded group(P<0.001).Conclusion:The BCPAP cell line with overexpression of JAZF1 was successfully constructed,which lays a solid foundation for further study of the effect of JAZF1 gene on the biological behavior of thyroid papillary carcinoma and its related mechanism.PART TWO Effects of overexpression of JAZF1 on proliferation and apoptosis of BCPAP cellsObjective:To observe the effect of overexpression of JAZF1 on proliferation,colony forming ability and apoptosis of thyroid papillary carcinoma BCPAP cells.Methods:After the completion of cell culture and transfection,the cells growth and proliferation abilities were detecting for MTT assay,colony formation assay,and flow cytometry Annexin V-FITC/PI double staining to detect cell apoptosis in each group.The total protein of each group was extracted and the protein concentration was quantified.The relative expression levels of Bcl-2 and Bax apoptotic proteins in each group were detected by Western blot.Results:The results of MTT assay showed that compared with the negative control group and un-loaded group,the proliferation of BCPAP cells in the experimental group was significantly inhibited at 24 h,48 h and 72 h(all P<0.001).Colony formation assay showed that the efficiency of clone formation in experimental group was significantly lower than that in negative control group and un-loaded group(all P<0.001).The results of flow Annexin V-FITC/PI double staining showed that the apoptosis rate of the experimental group(23.02 ±0.35%)was significantly higher than that of the NC group(8.63 ±0.40%)and the un-loaded group(7.95 ±0.32%)(all P < 0.001).The results of Western blot showed that the expression of Bcl-2 in the experimental group was significantly down-regulated by 66.0% compared with the control group and the un-loaded group(P <0.001);and the expression of Bax was significantly up-regulated by 64.8%(P <0.001).Conclusion:Overexpression of JAZF1 can effectively inhibit the colony formation and proliferation of BCPAP cells,and promote apoptosis.PART THREE The regulatory effect of overexpressed JAZF1 on PI3K/AKT/ GSK-3β signaling pathwaysObjective:To explore the possible mechanism of overexpression of JAZF1 on proliferation and apoptosis of thyroid papillary carcinoma.Methods:48 hours after transfection,the total protein was extracted from each group,and the protein relative expression levels of PI3 K,Phosphorylated-PI3 K,AKT,Phosphorylated-AKT and GSK-3β were detected by Western blot.Results:The results of Western blot showed that overexpression of JAZF1 could inhibit the relative expression of p-PI3 K and p-AKT protein in BCPAP cells(all P <0.001),significantly down-regulate the ratio of p-PI3K/t-PI3 K and p-AKT/t-AKT(all P < 0.001);at the same time,inhibit the relative expression level of p-GSK-3β protein(P <0.001)and down-regulate the ratio of p-GSK-3β / t-GSK-3β(P <0.001).Conclusion : JAZF1 inhibits the proliferation of BCPAP cells by regulating PI3K/AKT/GSK-3β signaling pathway,which may play an important role in the development of PTC.