高表达PRMT2剪接体对人甲状腺癌TT细胞E2F1基因表达的影响-内分泌专业论文.docxVIP

PAGE PAGE 10 达 PRMT2 剪接体能下调 TT 细胞 E2F1 蛋白的表达。 关键词：PRMT2 剪接体，甲状腺癌，基因转染，E2F1 The effection of E2F1 gene in HumanThyroid Cancer Cell Line TT by over-expressing Exogenous PRMT2 Splice Variants Abstract Objective: To investigate the impact of exogenous of PRMT2 Splice variants on human thyroid cancer cell growth, and to test the effects on E2F1 expression in human thyroid cancer cells over-expressed exogenous PRMT2 splice variants. Methods: Human thyroid cancer cell line, TT cells were transfected with expression plasmid pcDNA3.2/V5-PRMT2α, pcDNA3.2/V5-PRMT2β, pcDNA3.2/V 5-PRMT2γ or pcDNA3.2/V5 (empty plasmid as control) by using Lipofectamine 2000 to establish cell lines, which stably expressed PRMT2 splice variants. mRNA and proteins for PRMT2 splice variants in transfected TT cells were detected by RT-PCR and Western blot after G418 selection. The localization of the exogenous PRMT2 splice variants in transfected TT cells was then measured by Immunofluoresence. Furthermore, stable transfected TT cell lines with highly expressed PRMT2 splice variants were used to detect cell proliferation by MTT assay and malignant transformation of cells by colony formation assay. Finally, we test the expression of E2F1 in stable transfected TT Cell lines by Western-blot. Results: PRMT2 splice variants mRNA and protein levels significantly increased in stable transfected TT cells by RT-PCR and Western blot. Immunefluorescence further indicated that the PRMT2 proteins highly expressed in the cytoplasm of TT cells transfected PRMT2 splice variants compared to control cells transfected with empty plasmid. Cell proliferation were significantly inhibited in those TT cells over-expressed with PRMT2 splice variants (PRMT2α, PRMT2β and PRMT2γ) by 33.75%, 36.38% and 37.21% respectively compared to control group (P0.05) by using MTT assay. Colony formation assay showed that PRMT2 splice variants also significantly attenuated the anchorage dependent growth in PRMT2 over-expressed TT