【作者】 周乔丹；

【导师】 米粲；

【作者基本信息】 重庆医科大学 ， 组织工程与细胞工程， 2010， 博士

【摘要】 10号染色体缺失的磷酸酶及张力蛋白同源物(phosphatase and tensin homology deleted on chromosome ten,PTEN),是第一个被发现具有磷酸酶功能的抑癌基因,在多种肿瘤中存在突变或缺失。我们前期以小鼠胚胎成纤维细胞系PTEN+/+ MEFs为基础,成功建立了体外条件敲除PTEN基因具有恶性表型的小鼠胚胎成纤维细胞系PTEN-/- MEFs。运用蛋白质组学技术,研究PTEN+/+ MEFs和PTEN-/- MEFs细胞蛋白表达谱的差异。我们发现在PTEN缺失MEFs细胞中同属抗氧化防御系统的铜锌-超氧化物歧化酶(Cu/Zn-SOD)以及过氧化物还原酶(Prx)5, 6均表达下调。进一步运用Northern blot和Western blot对这一结果进行验证,显示PTEN缺失MEFs细胞中Cu/Zn-SOD和Prx1, 2, 5, 6 mRNA及蛋白表达均下调。抗氧化防御系统包括超氧化物歧化酶(Superoxide Dismutase,SOD)、过氧化物还原酶家族(Peroxiredoxins, Prxs)、过氧化氢酶(Catalase, CAT)以及谷胱苷肽过氧化物酶(Glutathione Peroxidase, GPxs)等,是体内活性氧(ROS)的清除系统。过量的ROS会形成氧化压力(oxidative stress),引发脂质过氧化反应,导致DNA分子的损伤,从而引起基因组不稳定性。正常生理情况下,人体主要依靠抗氧化防御系统来维持ROS的生成与清除之间的动态平衡状态。前期研究结果显示:PTEN-/- MEFs细胞抗氧化防御能力降低;细胞内基础ROS水平升高;细胞脂质过氧化损伤增强;细胞DNA氧化损伤增强。到目前为止,没有任何关于PTEN对Cu/Zn-SOD以及Prxs表达调控机制的报道。对PTEN的功能研究显示:PTEN具有脂磷酸酶的活性,对脂类底物3,4,5-三磷酸磷脂酰肌醇(PIP3)有强去磷酸化作用,而PIP3是PI3K/AKT信号转导通路的主要信号分子。PTEN的缺失可引起PI3K/AKT信号级联的活化,从而刺激细胞增殖与迁移。已知AKT可通过下游的Forkhead家族转录因子FoxO(s包括FoxO1, FoxO3a和FoxO4)对同属于抗氧化防御系统的CAT和Mn-SOD基因转录进行调控。在此调控过程中,AKT对FoxOs的磷酸化可促进FoxOs从核内排出,继而发生降解失活,减弱其在核内的转录调控活性,使CAT和Mn-SOD基因转录水平降低。结合国内外研究进展以及本实验室前期研究发现,我们提出以下假设:PTEN可通过拮抗PI3K/AKT通路对FoxOs转录因子的磷酸化降解途径,进而影响Prx1, 2, 5, 6以及Cu/Zn-SOD的调控和转录。基于上述假设,本研究分三部分进行:1.PTEN是否通过PI3K/AKT/FoxOs通路调控抗氧化防御蛋白的蛋白表达:(1)Western Blot比较PTEN+/+ MEFs和PTEN-/- MEFs细胞基础FoxO1、FoxO3a和FoxO4磷酸化水平以初步判断可能相关的转录因子。(2)Western Blot检测PTEN+/+ MEFs瞬时和稳定转染AKT-WT、AKT-AC质粒以及PTEN-/- MEFs瞬时和稳定转染PTEN-WT、AKT-DN质粒,P-FoxOs和抗氧化防御蛋白的蛋白表达水平差异,确定PTEN是否通过PI3K/AKT通路影响FoxOs在核内的转录调控活性从而调控抗氧化防御蛋白的表达。为进一步明确PTEN通过FoxOs亚家族的哪一个转录因子参与对Prx1, 2, 5, 6和Cu/Zn-SOD表达的调控提供线索。2.检测PTEN是否通过PI3K/AKT通路对细胞内活性氧和抗氧化防御能力产生影响:(1)DCFH-DA(2’7’-二氯荧光素双乙酸盐)和DHE(二氢乙啶)荧光探针分别标记细胞内H2O2及O2·ˉ,结合流式细胞学检测稳定转染AKT-WT、AKT-AC质粒的PTEN+/+ MEFs以及稳定转染PTEN-WT、AKT-DN质粒的PTEN-/- MEFs细胞中DCF和Eth荧光强度,以明确PTEN是否通过AKT通路影响细胞内基础H2O2及O2·ˉ的水平。(2)中性彗星电泳检测PTEN-/- MEFs和稳定转染PTEN-WT质粒的PTEN-/- MEFs细胞内不同浓度H2O2诱发的DNA DSBs水平变化,从而明确重建PTEN表达对MEFs细胞内抗氧化防御能力的影响。3.检测PTEN是否通过PI3K/AKT通路对细胞内DNA双链断裂产生影响:(1)中性彗星电泳检测PTEN-/- MEFs和稳定转染PTEN-WT质粒的PTEN-/- MEFs细胞内DNA DSBs水平;(2)免疫荧光染色检测0.01umol/L H2O2处理15min后,稳定转染AKT-WT、AKT-AC质粒PTEN+/+ MEFs以及稳定转染PTEN-WT、AKT-DN质粒PTEN-/- MEFs细胞按不同时间进行培养,胞核中γH2AX位点数(其直接反映DNA DSBs位点数),从而明确PTEN是否通过AKT通路对细胞内DNA双链断裂水平产生影响。主要结果如下:1. PTEN通过PI3K/AKT通路的活化抑制调控Prx1, 2, 5, 6及Cu/Zn-SOD的蛋白表达:Western Blot结果显示瞬时和稳定转染AKT-WT、AKT-AC质粒的PTEN+/+ MEFs细胞中Prx1,2,5,6和Cu/Zn-SOD蛋白水平明显下调(p<0.01),说明在PI3K/AKT通路失活的PTEN+/+ MEFs中,活化AKT可下调抗氧化防御蛋白的表达。瞬时和稳定转染PTEN-WT/AKT-DN质粒的PTEN-/- MEFs中Prx1,2,5,6和Cu/Zn-SOD蛋白水平上调(p<0.05),说明在PTEN缺失细胞中抑制AKT可使抗氧化防御蛋白的表达增强。以上结果表示PTEN可通过拮抗PI3K/AKT通路调控Prx1, 2, 5, 6及Cu/Zn-SOD蛋白表达。2. PTEN通过拮抗PI3K/AKT通路抑制转录因子FoxO1磷酸化:Western blot结果显示PTEN缺失细胞中转录因子FoxO1和FoxO3a磷酸化水平升高(p<0.01),提示FoxO1和FoxO3a可能参与PTEN对抗氧化防御蛋白的调控;Western blot检测稳定转染AKT-WT/AKT-AC质粒的PTEN+/+ MEFs和稳定转染PTEN-WT/AKT-DN质粒的PTEN-/- MEFs细胞中P-FoxO1蛋白表达,结果表明AKT活化后的PTEN+/+ MEFs细胞P- FoxO1蛋白表达增高(p<0.05),AKT抑制后的PTEN-/- MEFs细胞P- FoxO1蛋白表达显著降低(p<0.01),说明PTEN可通过拮抗PI3K/AKT通路抑制下游转录因子FoxO1磷酸化,维持FoxO1在核内的转录调控活性。3. PTEN通过抑制PI3K/AKT通路降低细胞内基础ROS水平:运用流式细胞仪检测DCFH-DA和DHE探针标记稳定转染AKT-WT/AKT-AC质粒的PTEN+/+ MEFs以及稳定转染PTEN-WT/AKT-DN质粒的PTEN-/- MEFs细胞内DCF和Eth荧光强度,结果显示AKT活化后的PTEN+/+MEFs细胞内DCF和Eth荧光强度明显增高(p<0.05),AKT抑制后的PTEN-/- MEFs细胞内DCF和Eth荧光强度则显著降低(p<0.01)。说明PTEN可通过抑制PI3K/AKT通路降低细胞内基础ROS水平。4. PTEN再表达引起细胞抗氧化防御能力增强:运用中性单细胞凝胶电泳检测发现0.01 mmol/L H2O2作用PTEN-/- MEFs细胞就出现具有统计学意义的平均尾力矩增长(p<0.01),而稳定转染PTEN-WT质粒的PTEN-/- MEFs细胞则在0.05 mmol/L H2O2作用时才出现有统计学意义的平均尾力矩增长(p<0.01)。说明PTEN再表达抑制PI3K/AKT通路会引起MEFs细胞的抗氧化防御能力明显增强。5. PTEN通过拮抗PI3K/AKT通路减少细胞内DNA DSBs水平:中性彗星电泳检测PTEN-/- MEFs和稳定转染PTEN-WT质粒的PTEN-/- MEFs细胞,显示稳定转染PTEN-WT质粒的PTEN-/- MEFs细胞的平均尾力矩(20.34)明显低于PTEN-/- MEFs细胞(27.23)(p<0.01)。说明PTEN再表达可显著降低细胞内DNA DSBs水平。0.01umol/L H2O2作用15min后按不同时间培养,免疫荧光染色显示PTEN+/+ MEFs细胞中γH2AX位点数在30min时即出现了有统计学意义的差异(p<0.05),稳定转染AKT-WT/AKT-AC质粒的PTEN+/+ MEFs细胞则在4h才出现有统计学意义的降低(p<0.05)。PTEN-/- MEFs细胞中γH2AX位点数在4h出现有统计学意义的降低(p<0.05),而稳定转染PTEN-WT/AKT-DN质粒的PTEN-/- MEFs细胞在4h时出现显著的降低(p<0.01)。说明PTEN通过拮抗PI3K/AKT通路促进DNA DSBs损伤的修复,并由此维持基因组稳定性。结论如下:1. PTEN通过PI3K/AKT/FoxOs通路调控Prx1, 2, 5, 6及Cu/Zn-SOD蛋白表达;2.PTEN通过拮抗PI3K/AKT通路降低细胞内基础ROS水平,增强细胞的抗氧化防御能力,减少细胞DNA DSBs损伤水平。说明PTEN在保护细胞免于氧化损伤、维持细胞内氧化还原环境和基因组稳定性方面有着重要作用。更多还原

【Abstract】 PTEN (phosphatase and tensin homologue deleted on chromosome 10) is the first phosphatase identified as a tumor suppressor. Reduced expression or loss-of-function mutations of the PTEN gene are found at high frequency in a wide variety of human tumors. In early studies, using two-dimensional gel electrophoresis, combined with Western and Northern blot analyses, we observed that PTEN deficiency in mouse embryonic fibroblasts (MEFs) showed deregulated expression of several antioxidant enzymes, including peroxiredoxins 1, 2, 5, and 6 and Cu, Zn superoxide dismutase.The present study aimed to demonstrate the mechanism of PTEN for regulation the protein expression of peroxiredoxin(Prx)1, 2, 5, 6 and copper/zinc superoxide dismutase (Cu/Zn-SOD) and the effect of the deregulated expression of Prx1, 2, 5, 6 and Cu/Zn-SOD on the basal levels of reactive oxygen species (ROS), antioxidant defense ability and DNA DSBs damage in PTEN+/+ MEFs transfected stably with plxsp-HAGFP -AKT-WT/AKT-AC and PTEN-/- MEFs transfected stably with plxsp-HAGFP - PTEN-WT/AKT-DN. In this study, we show for the first time that mechanism of PTEN for regulation the protein expression of Prx1,2,5,6 and Cu/Zn-SOD.Methods:1. To explore whether PTEN can regulate the expression of antioxidant enzymes through inhibiting PI3K/AKT/FoxOs signaling pathway, the protein expression of Prx1, 2, 5, 6 and Cu/Zn-SOD was measured with Western blot in PTEN+/+MEFs transfected transiently with pIRES2-EGFP-AKT-WT /AKT-AC and stably with plxsp-HAGFP-AKT-WT/AKT-AC and PTEN-/- MEFs transfected transiently with pIRES2-EGFP-PTEN-WT/AKT-DN and stably with plxsp-HAGFP-PTEN-WT/AKT-DN. Western blot was further applied to detect the basal expression of FoxOs in PTEN+/+ MEFs and PTEN-/- MEFs and the expression levels of FoxO1 in PTEN+/+ MEFs transfected stably with plxsp-HAGFP-AKT-WT/AKT-AC and PTEN-/- MEFs transfected stably with plxsp-HAGFP-PTEN-WT/AKT-DN to identify if PTEN regulate the phosphorylation of the transcription factor FoxO1 through inhibiting PI3K/AKT signaling pathway.2. To detect the effect of PTEN on the basal levels of ROS and antioxidant defense ability through inhibiting PI3K/AKT signaling pathway. Using 2’,7’-dichlorofluorescein diacetate (DCHF-DA) and dihydroethidium (DHE), the intracellular generation of H2O2 and superoxide anion(O2·ˉ) was determined by flow cytometry in PTEN+/+ MEFs transfected stably with plxsp-HAGFP-AKT-WT/AKT-AC and PTEN-/- MEFs transfected stably with plxsp-HAGFP-PTEN-WT/AKT-DN. The comet assay was used for the evaluation of various concentrations of H2O2-induced DNA double-strand breaks (DSBs) in PTEN-/- MEFs and PTEN-/- MEFs transfected stably with plxsp-HAGFP-PTEN-WT.3. To determine the effect of PTEN on DNA DSBs through inhibiting PI3K/AKT signaling pathway. The comet assay was applied to detect the levels of DNA DSBs. The expression ofγH2AX was further detected by immunofluorescence in PTEN+/+ MEFs transfected stably with plxsp-HAGFP- AKT-WT/AKT-AC and PTEN-/- MEFs transfected stably with plxsp-HAGFP- PTEN-WT/AKT-DN that were cultured for different hours after being treated with 0.01u mol/L H2O2 for 15 minutes.Results:1. PTEN can increase the protein expression of Prx1, 2, 5, 6 and Cu/Zn-SOD through inhibiting PI3K/AKT signaling pathway: western blot displayed the protein expression of Prx1, 2, 5, 6 and Cu/Zn-SOD down-regulated in PTEN+/+MEFs transfected transiently and stably with AKT-WT/AKT-AC and up-regulated in PTEN-/- MEFs transfected transiently and stably with PTEN-WT/AKT-DN, indicating that PTEN can increase the expression of Prx1, 2, 5, 6 and Cu/Zn-SOD through inhibiting PI3K/AKT signaling pathway.2. PTEN can decrease the phosphorylation level of FoxO1 through inhibiting PI3K/AKT signaling pathway: western blot displayed that the protein expression of P-FoxO1 and P-FoxO3a in PTEN-/- MEFs was higher than that in PTEN+/+ MEFs(p<0.01). Western blot further showed that the protein expression of P-FoxO1 was up-regulated in PTEN+/+ MEFs transfected stably with plxsp-HAGFP-AKT-WT/AKT-AC (p<0.05) and down-regulated in PTEN-/- MEFs transfected stably with plxsp-HAGFP-PTEN-WT/AKT-DN(p<0.01),suggesting that PTEN can decrease the phosphorylation level of FoxO1 through inhibiting PI3K/AKT signaling pathway.3. PTEN can decrease ROS levels in MEFs through inhibiting PI3K/AKT signaling pathway: A flow-cytometry-based DCHF-DA and DHE analysis indicated that the levels of both DCF and Eth fluorescence were higher in PTEN+/+ MEFs transfected stably with plxsp-HAGFP-AKT-WT/AKT-AC (p<0.05) and lower in PTEN-/- MEFs transfected stably with plxsp-HAGFP- PTEN-WT/AKT-DN(p<0.01).4. Increased antioxidant defense ability in PTEN-reexpression MEFs: The comet assay displayed that exposure to each concentration of H2O2 resulted in a highly statistically significant increase in the frequency of DNA DSBs in PTEN-/- MEFs (p<0.01) compared with H2O2-untreated control at each concentration, whereas this increase was seen only in the 0.05 mmol/L H2O2-treated PTEN-reexpression MEFs(p<0.01 compared with H2O2- untreated control), indicating the increased antioxidant defense ability in PTEN-/- MEFs transfected stably with plxsp-HAGFP-PTEN-WT.5. PTEN can decrease DNA DSBs damage in MEFs through inhibiting PI3K/AKT signaling pathway: The comet assay showed that mean tail moment in PTEN-/- MEFs transfected stably with plxsp-HAGFP-PTEN-WT was significantly lower than that in PTEN-/- MEFs(20.34 vs 27.23, p<0.01). The frequency of DNA DSBs was measured by immunofluorescence assay forγH2AX. The result displayed that a statistically significant decrease in the frequency of DNA DSBs in PTEN+/+ MEFs at 30 minuates was detected (p<0.05), whereas this decrease was found only at 4h in PTEN+/+ MEFs transfected stably with plxsp-HAGFP-AKT-WT/AKT-AC (p<0.05). And this decrease in the frequency of DNA DSBs was seen at 4h in PTEN-/- MEFs(p<0.05), whereas a higly statistically significant decrease was showed at 4h in PTEN-/- MEFs transfected stably with plxsp-HAGFP-PTEN-WT /AKT-DN(p<0.01). These results suggest that PTEN can promote DNA damage repair through inhibiting PI3K/AKT signaling pathway.Conclusion:1. PTEN regulates the protein expression of Prx1, 2, 5, 6 and Cu/Zn-SOD through PI3K/AKT/FoxOs signaling pathway.2. PTEN can reduce ROS levels, increase antioxidant defense ability and decrease DNA DSBs damage in MEFs through inhibiting PI3K/AKT signaling pathway. These findings show that PTEN plays an essential role in promoting DNA damage repair and maintaining genomic stability against oxidative damage.更多还原