泛素交联酶UBE2D3在调节人乳腺癌MCF-7细胞放射敏感性中的作用

Effect of UBE2D3 in regulation of radiosensitivity in human breast cancer MCF-7 cell

目的研究泛素交联酶UBE2D3(ubiquitin-conjugating enzyme E2D3)在端粒酶介导的放射敏感性调节机制中的作用。方法针对UBE2D3基因序列,设计3条干扰序列,通过筛选选取1条干扰效率最优的序列并合成pshRNA-UBE2D3;在MCF-7细胞中,转染过表达质粒p EGFP-UBE2D3以及干扰质粒pshRNA-UBE2D3,Western blot检测UBE2D3对h TERT蛋白表达的影响;通过转染p EGFP-h TERT,Western blot法检测h TERT蛋白表达对UBE2D3的影响;通过转染pshRNA-UBE2D3抑制UBE2D3的表达,PCR-ELISA法检测端粒酶活性,MTT法检测MCF-7细胞周期的变化,CCK-8法检测MCF-7细胞增殖情况,克隆形成法检测MCF-7细胞放射敏感性的变化。结果成功筛选出一条针对UBE2D3的最优干扰序列,干扰UBE2D3能显著降低MCF-7细胞的放射敏感性;UBE2D3能参与h TERT调控放射敏感性的调节,抑制UBE2D3的表达可能通过上调h TERT和Cyclin D1的表达、增加h TERT的活性、加速G1期向S期转变以及提高MCF-7细胞的增殖从而降低MCF-7细胞的放射敏感性。然而,过表达UBE2D3未能检测到h TERT的表达差异。结论抑制UBE2D3可能通过上调h TERT以及Cyclin D1的表达参与h TERT调节的放射敏感性的过程。

Objective To explore the role of UBE2D3 in h TERT-mediated radiosensitivity in MCF-7 cells. Methods Based on UBE2D3 gene sequences,three interference sequence were designed and a optimal jamming efficiency sequence was selected to synthesize pshRNA-UBE2D3. In MCF-7 cells,p EGFP-UBE2D3 and pshRNA-UBE2D3 were transfected respectively,and the influence of UBE2D3 on h TERT protein expression was detected by Western blot. Through transfection of p EGFP-h TERT,the influence of h TERT protein expression on UBE2D3 was detected by Western blot.UBE2D3 expression was dampened by pshRNA-UBE2D3 transfection,telomerase activity was detected by PCR-ELISA,the change of MCF-7 cell cycle was detected by MTT,MCF-7 cell proliferation was detected by CCK-8 and the change of MCF-7 cellular radiosensitivity was detected by colony-forming assay. Results An optimal jamming efficiency sequence was selected,which could significantly reduce the radiosensitivity of MCF-7 cell; UBE2D3 could participate in the regulation of h TERT radiosensitivity,inhibit UBE2D3 expression,up-regulate h TERT and Cyclin D1 expression,increase the activity of h TERT,accelerate G1 turn to S phase,and improve the MCF-7 cell proliferation to reduce the MCF-7 cell radiosensitivity. However,overexpression of UBE2D3 failed to detect the expression differences of h TERT. Conclusion UBE2D3 is inhibited by up-regulating h TERT and Cyclin D1 expression to participate in the regulation of h TERT radiosensitivity.