突变型PUMA(S10A)对Hela细胞的凋亡作用及其分子机制

Mutant type PUMA can accelerate the apoptosis of Hela cells in vitro and its molecular mechanism

目的观察突变型PUMA和野生型PUMA对Hela细胞在促凋亡及抑制肿瘤细胞增殖方面的生物学功能差异,并探讨导致这些差异的分子机制。方法实验随机分为:空载体质粒组、野生型PUMA组、突变型PUMA 3个实验组,应用脂质体转染方法分别将空载质粒、野生型PUMA质粒、突变型PUMA质粒转染Hela细胞中,转染24、48 h后用实时定量PCR和Western blot法检测各组PUMA表达情况;野生型实验组和突变型实验组分别加入蛋白质合成抑制剂放线菌酮后,Western blot法检测PUMA蛋白的表达;MTT及流式细胞术检测野生型PUMA与突变型PUMA对细胞增殖抑制和促凋亡作用。结果在相同转染效率的情况下,通过Western blot法分析野生型PUMA组和突变型PUMA组的表达水平,结果表明突变型PUMA蛋白比野生型PUMA蛋白有一个更高的稳定状态。转染24 h后诱导表达野生型和突变型的PUMA细胞中均加入放线菌酮,结果表明,突变型PUMA蛋白降解延迟、半衰期延长;光密度值测定结果显示细胞转染两种质粒后,细胞存活率随着时间的延长逐渐下降,且转染突变型PUMA质粒在24、48 h的存活率均比野生型PUMA组低(P<0.05,P<0.01);流式细胞术结果显示,随着PUMA转染时间的增加,野生型和突变型两组肿瘤细胞的凋亡率均逐渐升高,并且突变型组细胞比野生型组细胞的凋亡率增加更加明显(P<0.05,P<0.01)。结论 PUMA具有抑制Hela细胞增殖、促进其凋亡的作用,而其10号位的丝氨酸被丙氨酸取代后增加了突变型PUMA的稳定性、延长了其半衰期,导致突变型比野生型PUMA对Hela细胞的抑制作用及促凋亡功能得到加强。

Objective To investigate the differences of wild type PUMA protein mutant PUMA protein in apoptosis and proliferation inhibition function and to explore the molecular mechanism of these differences, which may pave the way for further study on tumor suppressor function of PUMA and its post-translational regulation on mechanism. Methods Hela cells were divided into 3 groups：wild type PUMA transfection group, mutant PUMA transfection group, and the empty vector control group. After transfection 24 h and 48 h the expression of PUMA was detected by Western blot and qPCR. The cellular proliferation inhibition was examined by MrlT. The cellular apoptosis rates were examined by FCM. Wild type group and mutant group after joining protein synthesis inhibitors cycloheximide, degradation of PUMA protein were detected by Western blot, respectively. Results We analyzed the expression levels of wild type PUMA and mutant PUMA by Western blot. This revealed that mutant PUMA was expressed at a higher steady state level than wild type PUMA in the same under the condition of transfection efficiency. Cells ex- pressing wild type PUMA and mutant PUMA were treated with cycloheximide, and we then tested wild type PUMA and mutant PUMA protein degradation. This assay revealed that degradation of mutant PUMA protein delayed deg- radation and prolonged the half life. After mutant PUMA and WT plasmids cell transfection, the OD value results displayed certain degree proliferation inhibitory effect in a time-dependent manner Hela cells, and transfected with mutant PUMA was significantly lower than that after being transfected with WT at the 24 h and 48 h time point. There was significant difference （ P 〈 0. 05, P 〈 0. 01 ）. FCM results showed that with the increase of transfection time, wild type and mutant of two groups of tumor cell apoptosis rate increased, and the mutant type group cell ap- optosis rate increased more obviously, the difference was statistically significant（ P 〈 0. 05, P 〈 0. 01 ）. Conclusion PUMA can inhibit Hela cells proliferation and promote its apoptosis. 10 of serine was replaced by alanine can in- crease the stability of mutant PUMA, prolong its half-life, which results in inhibition and pro-apoptotic function strengthened in the mutant than wild type PUMA on Hela cells.