摘要
目的:研究生长抑制和DNA损伤诱导45G蛋白(growtharrest and DNA-damage-inducible 45 gamma,Gadd45G)对人结肠癌细胞增殖的影响,并初步探讨可能的作用机制。方法:构建携带有四环素可调控(tetracycline-on,Tet-on)基因表达系统的真核表达载体pTRIPZ-Gadd45G,采用慢病毒感染的方法转入人结肠癌细胞系HCT116和SW480,并在强力霉素(doxycycline,Dox)的诱导下过表达Gadd45G。分别采用MTT法、FCM和衰老相关半乳糖苷酶染色法检测Gadd45G过表达对结肠癌细胞增殖、凋亡、细胞周期和衰老的影响,实时荧光定量-PCR(real-time fluorogenic quantitative-PCR,RFQ-PCR)和蛋白质印迹法检测Gadd45G过表达对衰老相关细胞因子白细胞介素-8(interleukin-8,IL-8)及细胞周期相关蛋白表达的影响。结果:在Dox诱导后,含Tet-on基因表达系统的结肠癌HCT116和SW480细胞可稳定表达Gadd45G蛋白。过表达的Gadd45G能显著抑制结肠癌HCT116和SW480细胞的增殖(P<0.001),且使细胞周期被阻滞在G2/M期。Gadd45G的表达可造成结肠癌细胞衰老(P<0.001),但不能诱导细胞凋亡。在Gadd45G过表达抑制细胞增殖的过程中,IL-8mRNA的表达水平明显提高,衰老标志物γ-H2A蛋白的表达水平明显升高,p53、p21/CDKN1a、p16/INK4a和Rb蛋白的表达水平并未发生明显变化。结论:应激蛋白Gadd45G过表达可通过诱导细胞衰老而抑制结肠癌细胞的增殖。
Objective： To investigate the effect of Gadd45G （growth arrest and DNA-damage-inducible 45 gamma） overexpression on cell proliferation of colon cancer cells and the possible related mechanism.Methods： To explore whether Gadd45G functions in regulating cell proliferation of colon cancer cells, a lentiviral Tet （tetracycline）-inducible expression system was used for ectopic Gadd45G expression in colon cancer HCT116 and SW480 cells. The effects of Gadd45G over-expression on cell proliferation, cell cycle distribution, apoptosis and cellular senescence were analyzed by MTT method, FCM （flow cytometry）and senescence-associated beta-galactosidase assay, respectively. The expression of senescenceassociated secrete phenotype IL-8 （interleukin 8） and cell cycle-regulators were measured by RFQ-PCR （real-time fluorogenic quantitative-PCR） and Western blotting, respectively. Results： After doxycycline induction, Gadd45G expression was validated in HCT116 and SW480 cells. Gadd45G overexpression significantly inhibited the proliferation of HCT116 and SW480 cells （P 〈 0.001）, and resulted in cell cycle arrest at G2/M phage. Intriguingly, Gadd45G over-expression efficiently elicited cellular senescence （P 〈 0.001） but not apoptosis. Moreover, the protein levels of cell cycle regulators p53, p21/CDKN1a, p161NK4a and Rb were not significantly altered in the colon cancer cells upon Gadd45G induction,while the expressions of IL-8 mRNA and senescence marker ~＇-H2A protein were significantly increased. Conclusion： The over-expression of stress sensor Gadd45G inhibits the proliferation of colon cancer cells through inducing cellular senescence.
出处
《肿瘤》
CAS
CSCD
北大核心
2013年第3期207-213,共7页
Tumor
基金
国家自然科学基金资助项目(编号:81201542)
