【作者】 陈军；

【导师】 苗里宇；

【作者基本信息】 吉林大学 ， 内科学， 2012， 博士

【摘要】 目的：对人第10号染色体缺失的磷酸酶及张力蛋白同源的基因（phosphatase andtensin homologue deleted chromatosome10，PTEN）低表达在肾透明细胞癌（clear-cellrenal-cell carcinoma，CCRCC）耐药性中所起的作用及其可能机制进行了探讨，以期为临床CCRCC的有效治疗提供新的有效靶位点。方法：采用免疫组化和免疫蛋白印迹的方法检测了抑癌基因PTEN在CCRCC中的表达情况；采用基因沉默的方法结合流式细胞分析或者CCK8分别研究了PTEN沉默对CCRCC细胞凋亡和细胞增殖能力的影响；采用基因沉默，免疫共沉淀结合免疫印迹的方法研究了PTEN对etoposide诱导的细胞凋亡的影响及其作用机制。结果：CCRCC中PTEN mRNA和蛋白表达显著降低。转染PTEN shRNA后CCRCC细胞系ACHN中PTEN mRNA和蛋白的表达被有效抑制，etoposide诱导的细胞凋亡明显减少，细胞增殖能力增强，说明PTEN抑制能够抵制凋亡，促进细胞增长。Etoposide诱导后，细胞内PTEN的表达量没有明显改变，说明etoposide对PTEN并没有直接的作用。Etoposide诱导引起p-Akt、p-HDM2、HDM2的表达量减少，而p53、PUMA和Cleaved PARP的表达量增加，提示etoposide通过抑制Akt信号通路，并可能通过p53依赖的细胞凋亡发挥作用；与之相反，PTEN沉默后，p-Akt、p-HDM2、HDM2的表达量增加，而p53、PUMA和Cleaved PARP的表达量减少，说明etoposide诱导的Akt信号通路抑制被PTEN抑制所解除，同时其诱导的p53依赖的凋亡受到抑制。Western blotting结果显示，在CCRCC组织中同样发现p-Akt、p-HDM2、HDM2的表达量增加，p53的表达量减少；组织免疫化学实验得出了同样的结果，进一步证实了体外实验的结果。细胞免疫共沉淀显示，etoposide诱导后， ACHN中与HDM2相互作用的p53的量减少，PTEN沉默后与HDM2相互作用的p53的量增加；CCRCC组织免疫共沉淀结果也显示，与正常肾脏组织相比，与HDM2相互作用的p53的量也明显增多。结合前面的实验结果说明，etoposide处理抑制了HDM2与p53的相互作用，阻滞了HDM2导致的p53降解，从而促进了p53依赖的细胞凋亡；而PTEN抑制则逆转了etoposide对HDM2与p53的相互作用的抑制，阻止了p53依赖的细胞凋亡。结论：CCRCC中Akt/HDM2信号通路被激活，同时p53的表达被抑制。PTEN的沉默引起etoposide诱导的细胞凋亡受到抑制，促进了细胞增殖。同时Akt/HDM2信号通路被激活，HDM2与p53之间的相互作用增强，引起p53的抑制。说明CCRCC中PTEN的表达降低引起Akt/HDM2信号通路的激活，阻滞了etoposide诱导的p53依赖的细胞凋亡，从而引起CCRCC对etoposide产生耐药性。更多还原

【Abstract】 Objective: To investigate roles of low expression of PTEN in CCRCC chemoresistance,and to elucidate its possible mechanisms.Methods:Western blotting and immunochemistry were used to determine theexpression of tumor suppressor gene PTEN in CCRCC; PTEN knockdown, together withflow cytometry analysis or CCK8analysis, was used to study the effects of PTEN silence onCCRCC cell apoptosis and cell proliferation ability, respectively; PTEN knockdown,together with immunoprecipitation and western blotting, was used to investigate the effectsof PTEN on etoposide induced cell apoptosis and the related molecular mechanisms.Results: PTEN mRNA and protein expression levels were significantly decreased inCCRCC tissues compared with the adjacent non-neoplastic renal samples. After transfectionwith PTEN shRNA, PTEN mRNA and preotein expression levels were inhibited efficientlyin ACHN cells, apoptosis induced by etoposide was reduced significantly, and cellproliferation ability was enhanced, which indicated inhibition of PTEN led to apoptosisresistance, and promotion of cell proliferation; After treating with etoposide, PTEN proteinexpression levels did not show any obvious changes, which illustrated etoposide did noteffect on PTEN directly. P-Akt, p-HDM2and HDM2erpression levels in ACHN cellstreated with etoposide were reduced; Meanwhile, p53, PUMA and Cleaved PARP erpressionlevels were increased, all this pointed out that etoposide inhibited Akt signaling pathway, andmight induced cell apoptosis through a p53-dependent pathway; On the opposite, afterknocking down of PTEN in ACHN cells, p-Akt, p-HDM2and HDM2erpression levels wereincreased, accompanying with decreased expression of p53, PUMA and Cleaved PARP, thisshowed us that inhibition of Akt signaling induced by etoposide was released by PTENinhibition, and simultaneously p53-dependent apoptosis was restrained. The results ofwestern blotting revealed that p-Akt, p-HDM2and HDM2erpression levels were increasedin CCRCC tissue with decreased p53expression, and immunochemistry experiments furtherconfirmed the results; Immunoprecitation experiments results showed that amount of p53which interacts with HDM2was decreased, and it was increased after PTEN knocking out.In CCRCC tissue, the amount of p53which interacts with HDM2was also increaseddefinitely. Together with the above results, we can conclude that the interaction between HDM2and p53was inhibited by etoposide, which abolished the degradation of p53inducedby HDM2; And this results was reversed by PTEN inhibition.Conclusion: Akt/HDM2signaling pathway was constitutively activated in CCRCCtissues, which was concomitant with low p53expression level. Silence of PTEN in CCRCCcells resulted inthe inhibition of apoptosis induced by etoposide, as well as the enhancedproliferation ability. Meanwhile, Akt/HDM2signaling pathway was activated, interactionbetween HDM2and p53was increased and the level of p53protein was lowered, whichinhibited the activity of p53. All in all, low expression of PTEN activates Akt/HDM2signaling pathway in CCRCC, and contributes to chemoresistance through inhibitingp53-dependent apoptosis.更多还原