【作者】 张玲；

【导师】 沙卫红；

【作者基本信息】 南方医科大学 ， 消化病学（专业学位）， 2016， 博士

【摘要】 研究背景及目的结肠癌是一种人类高发的恶性肿瘤,在全球恶性肿瘤发病率中位居第三位,其死亡率居恶性肿瘤死因的第二位,是男性罹患的第四大恶性肿瘤,女性所患恶性肿瘤中结直肠癌居第三位,2014年国际癌症研究机构在全球范围内涉及184个国家的多种常见恶性肿瘤的调查结果显示,美国2014年结直肠癌新增病例约96,830例,死亡人数39,590例,由此可见目前发达国家的结直肠癌的治疗效果亦不尽如人意,在我国结直肠癌是威胁人民健康的第五大恶性肿瘤,近10余年,我国的结肠癌发病率、死亡率一直呈上升趋势,患者的5年生存率也徘徊不前。目前,早期结直肠癌患者可以通过根治性手术得到治疗,并获得较好的预后,但是由于非侵入性检查对于空腔脏器的不敏感性以及结肠镜无症状筛查理念的未被普及等因素的影响下,大多结直肠癌患者被检出时病变已经发展至中晚期,目前对于中晚期结直肠癌的临床治疗技术仍不完善,因此,开发研究进展期结直肠癌的治疗方法,干预结直肠癌进一步发展,提高晚期结直肠癌患者的生存率,延长其生存期势在必行,是结直肠癌治疗领域的重点。迄今为止,研究报道提示SHC家族共有四个基因成员构成,分别是ShcA, ShcB, ShcC, ShcD。其中,在哺乳动物的ShcA蛋白家族编码三个主要成员,分别是p46Shc, p52Shc, p66Shc,三个亚单位接受不同的细胞信号的调控,执行不同的生理功能,三者均有高度保守的C端区域,都有磷酸酪氨酸结合区(PTB),富含脯氨酸的同源结构域(CH1) ,Src同源结构域(SH2),除却上述三个相同的结构特点外,p66Shc还有不同于其它两者的独有结构,其所特有的结构是N端脯氨酸富集区CH2,该区域包含细胞色素C的结合域,在36位含有磷酸丝氨酸位点,该位点是一个潜在的磷酸化位点,已有研究表明,3种蛋白(p46Shc, p52Shc, p66Shc)均具有调节酪氨酸激酶受体信号传导通路的作用,该功能极有可能在促进细胞有丝分裂及恶性肿瘤的发生、发展中承担重要作用,而p66Shc因其独特的CH2区域,因此具有更为独特的功能。已有的研究证实p66Shc主要存在于胞质中,小部分定位于线粒体,p66Shc在多种恶性肿瘤细胞中均呈高表达状态,如前列腺癌、乳腺癌、卵巢癌等。在肿瘤与信号通路的研究提示,p66Shc适配蛋白参与多条信号通路,诸如激活Ras有丝分裂信号通路,参与磷脂酰肌醇3-激酶/苏氨酸激酶(phosphoinositide 3-kinase/protein kinase B,PI3K/Akt)信号通路等。在过去的20年里,对Shc蛋白家族的亚型、结构、信号通路、致癌分子机制等生理功能的研究取得了重大进展,但仍有许多问题尚待解决。众所周知,PI3K/Akt通路是一条被广泛认可的重要的信号通路,在此信号通路中的PI3K,其属于磷脂酰肌醇3-激酶(PI3Ks)家族,是该家族中研究最广泛的一个成员。在哺乳动物细胞中,PI3K参与多种细胞内生物过程,如细胞周期进程、细胞生长、运动、粘附及存活,其与下游的调控分子Akt蛋白共同构成PI3K/Akt信号通路,该条通路与人类恶性肿瘤密切相关,在该信号通路中任何参与调控信号传导的蛋白质发生异常都可能会导致细胞恶性转化,同时研究发现,上述过程的失控与肿瘤细胞的远处转移、微环境黏附、肿瘤血管生成以及细胞外基质的降解等密切相关。与所有的信号传导通路相同的是,PI3K /Akt信号通路接受到信号后,PI3K被激活,活化后的PI3K首先在细胞膜上产生第二信使PIP3,然后PIP3与细胞内的下游信号蛋白Akt和磷酸化依赖型蛋白激酶1(PDK1)结合,之后Akt蛋白的S308发生磷酸化最终引起Akt的活化,活化的Akt可以通过磷酸化作用激活或抑制其下游靶蛋白Bad、Caspase-9等效应蛋白,进而发挥调节细胞的生理过程的作用。Akt也能通过直接磷酸化促凋亡蛋白Bad来促进细胞的生存。Bad是Bcl-2家族中的一个促凋亡蛋白,它通过结合和拮抗Bcl-2来促进细胞凋亡(Apoptosis)。Akt能直接使Bad与胞质中的蛋白螯合,从而终止Bad在线粒体膜上对Bcl-2的拮抗作用,使得被释放后的Bcl-2,恢复抗细胞凋亡的功能。最近的研究提示,Akt通路通过Mdm-2-p53功能通路参与细胞周期的调节,众所周知,p53是经典的抑癌基因,然而当Mdm-2以单体形式进入细胞核后可与p53结合,促进其降解,因此Mdm-2可以抑制p53的功能,从而进一步影响细胞周期的进行,然而Mdm-2是Akt的下游作用底物,因此推测PI3K/Akt/Mdm-2/p53通路是一条调节细胞周期的重要通路。众多的研究表明,PI3K/Akt通路在多种恶性肿瘤组织中被激活及过表达,诸如胃癌、乳腺癌、胰腺癌等,PI3K/Akt通路能够抑制许多肿瘤抑制蛋白的表达,诸如Bad, FOXO转录因子等,Akt可以通过磷酸化作用活化Bad,活化后的Bad与Bcl-2解离,从而使Bcl-2发挥抗凋亡作用,因此,阻断PI3K/Akt通路可能对于抑制肿瘤细胞增殖、促进其凋亡有重要意义。在关于乳腺癌、前列腺癌的研究中表明,p66Shc参与了细胞的线粒体介导的细胞凋亡过程,多数报道认为p66Shc通过氧化应激过程参与了肿瘤细胞抗凋亡过程,在我们的前期实验中表明,p66Shc有促进结直肠癌细胞增殖及抗凋亡作用,而且预实验结果提示p66Shc的表达水平与Bcl-2蛋白家族、蛋白水解酶存在相关关系,因此提出假说考虑p66Shc的通过PI3K/ Akt/Mdm-2/p53通路参与调控结直肠癌的增殖和凋亡过程。在我们的实验研究中,首先检测验证了p66Shc在结直肠癌组织及结直肠细胞株中的表达水平,其次通过干扰其表达进一步研究p66Shc沉默后对结直肠癌细胞的增殖、凋亡的影响,最后检测PI3K/Akt/Mdm-2/p53通路中的凋亡相关蛋白的表达变化,旨在探讨p66Shc对结直肠癌细胞增殖、凋亡的影响及信号调控通路的调控机制,为临床干预进展期结直肠癌的增殖、凋亡提供理论实验基础。研究方法1. p66Shc在结直肠癌组织及细胞株中的表达采用实时定量PCR技术(RT-PCR)与免疫印迹(Western blot)技术检测30例结直肠癌组织和癌旁组织中p66Shc的表达。检测细胞株(NCM460. HCT8、HCT116、SW620、CaC02)中p66Shc的表达水平。(NCM460是正常结肠上皮细胞,其余四株是结直肠癌细胞)。2.干扰结肠癌细胞株HCT8中的p66Shc的表达(1)利用生物信息在线网站Ambion、Genesil、Genecard设计RNAi序列。(2)生物技术公司合成si-p66Shc,运用RNA干扰技术沉默HCT8中的p66Shc的表达。(3) RT-PCR、Western blot技术检测干扰效果。3.干扰p66Shc的表达对结直肠细胞增殖、凋亡的影响(1)运用CCK-8细胞增殖实验,检测干扰p66Shc对HCT8细胞增殖的影响。(2)运用流式细胞技术AnnexinV-FITC方法,检测干扰p66Shc对HCT8细胞凋亡的影响。4. p66Shc调节结直肠癌细胞信号通路的初步研究(1)干扰p66Shc对结直肠癌细胞中凋亡相关蛋白的影响运用Western blot法检测HCT-8干扰后的细胞中细胞凋亡蛋白caspase-3、 caspase-9、Bax、Bcl-2的表达变化。(2)干扰p66Shc对PI3K-AKT信号通路相关蛋白的影响运用Western blot法检测干扰后p-PI3K、PI3K、p-AKT、AKT、p-Mdm-2、 p53表达变化。5.流式细胞仪检测干扰p66Shc后对HCT-8细胞周期的影响6.统计学方法研究中所有数据均采用SPSS 13.0统计学软件进行统计学分析。定量数值采用均数±标准差(standard deviation,SD)表示。其中组织的RT-PCR结果ACt的比较采用配对样本t检验(Paired-Sample t text)。实验过程中RT-PCR实验结果采用单因素方差分析(One-Way ANOVA)法进行统计分析。CCK-8体外增殖实验采用方差分析和单因素方差分析法进行分析。所有统计学结果P<0.05为差异有统计学意义。研究结果1. p66Shc在结直肠癌组织和细胞中呈高表达状态与肿瘤的增殖、生长有关(1) 实时定量PCR技术(RT-PCR)检测结果显示30例结直肠癌组织中p66Shc的表达高于癌旁组织(P<0.05)。(2) 实时定量PCR技术(RT-PCR)、免疫印迹(Western blot)技术检测细胞株(NCM460、HCT8、HCT116、SW620、CaCO2)中的p66Shc的表达,在结直肠癌细胞株中的p66Shc的表达高于正常结肠上皮细胞(NCM460)(P<0.05),其中HCT8细胞株中p66Shc的表达最为显著(P<0.01)。2.干扰p66Shc的表达对结直肠癌细胞增殖、凋亡的影响(1) 成功干扰HCT-8细胞中p66Shc的表达后,CCK-8体外增殖实验表明,Control组、si-scramble组、si-p66Shc组各组的细胞增殖能力有显著差异(P<0.01),si-p66Shc组在干扰后的第3-6天,细胞的增殖被显著抑制(P<0.05)。(2) 干扰HCT-8细胞中p66Shc的表达后,与si-scramble相比si-p66Shc组的活细胞数有所减少(si-scramble组97.0%,si-p66Shc组84.8%,P<0.05),两组间凋亡细胞差异显著(si-scramble组0.112%,si-p66Shc组0.159%,P<0.05),数据表明干扰p66Shc的表达促进结直肠癌细胞凋亡。3.p66Shc调节结直肠癌细胞信号通路的初步研究(1) 流式细胞仪分析干扰p66Shc对HCT8细胞周期的影响在本部分实验中运用流式细胞仪技术检测了干扰p66Shc后,对HCT8的细胞周期的影响,研究结果表明,干扰后的细胞较之对照组G0/G1期细胞比例明显增加(P<0.01),而G2/M期细胞比例显著减少(P<0.05),由此印证了干扰p66Shc可以使结直肠癌细胞停滞在G0/G1期。(2) Western blot法检测p66Shc干扰后的HCT8中凋亡相关蛋白表达Western blot法检测Control、si-scramble、si-p66Shc三组结直肠癌细胞中蛋白水解酶(Caspase-3, Caspase-9)、促凋亡蛋白Bax、抗凋亡蛋白Bcl-2的表达,在实验结果中我们可以看到,当结直肠癌细胞株中的p66Shc被干扰后,Caspase-3, Caspase-9,促凋亡蛋白Bax表达水平显著增加(P<0.01),同时,观察到抗凋亡蛋白Bcl-2的表达水平显著下降(P<0.01)。(3) p66Shc干扰对PI3K-AKT信号通路相关蛋白的影响在本部分的研究中发现,在p66Shc被干扰后,HCT8中PI3K、AKT的磷酸化作用被抑制,同时发现AKT的下游因子Mdm-2明显受到抑制,然而,相反的p53的表达明显增加。结果证实干扰p66Shc后通过PI3K/AKT/Mdm-2 /p53信号通路发挥抑制HCT8细胞的增殖的作用。结论1. p66Shc在结直肠癌组织中呈高表达,同样在结直肠癌细胞株用亦呈高表达,初步推断p66Shc在结直肠癌的发病过程中发挥促癌作用。2.干扰p66Shc表达可以抑制结直肠癌细胞的增殖,同时促进结直肠癌细胞的凋亡。提示通过干扰p66Shc对于控制肿瘤细胞的增殖具有重要意义。3.干扰p66Shc表达可以使结直肠癌细胞周期停滞在G1期,印证了p66Shc对于结直肠癌细胞增殖过程的作用。4.干扰p66Shc表达后HCT8中Caspase-3, Caspase-9,促凋亡蛋白Bax表达水平显著增加,PI3K、AKT的磷酸化作用被抑制,AKT的下游因子Mdm-2表达下调,然而,p53的表达明显增加。因此分析认为,干扰p66Shc表达后在HCT8中通过PI3K/AKT/Mdm-2/p53信号通路实现了细胞调控。更多还原

【Abstract】 Background and ObjectiveColorectal cancer is a kind of high-incidence carcinoma, it is the third most common cancer in worldwide. Mortality rates of colorectal cancer is the second in the all of carcinoma. Colorectal cancer is the third most common cancer in men and the second in women in worldwide. In 2014, International Agency for Research on Cancer reported on the latest investigate datas about the colorectal cancer in 184 countries. In 2012, There were 96,830 new cancer cases,39,590 cancer deaths in USA. Obviously, the therapeutic effect of colorectal cancer in the developed region still is not satisfying. In our country, colorectal cancer has become the fifth most common cancer and its incidence has shown an significant increasing trend over the past decade. At present, early stage colorectal cancer patients can be treated successfully with surgical resection, but owing to the non-invision examination are low sensitivity on the hollow organ and most of peoples can’t accept the colonoscopy before symptom arise.So most of patients were diagnosed in the late stage. Now patients with terminal stage are reftactory. Therefore the effective therapeutic approaches for advanced colorectal cancer patients are still needed.SHC family was consist of four gene members,including Shc A,ShcB, Shc C, Shc D. In mammalian, ShcA adaptor protein family encode three isoforms, p66Shc, p46Shc, p52Shc.Three isforms were controlled by different cell signal transduction pathway, and play different physiological functions. All three isoforms of the Shc family consist of the N-terminal phosphotyrosine binding damain (PTB),the middle collagen homology domain(CH1),and the C-terminal Src-homology domain (SH2). p66Shc contains unique amino-terminal structure (CH2) which distinguish this isoform from the others. CH2 domain is rich in glycine and proline which contain the cytochrome C binding domain.CH2 domain contains a very crucial S36 residue which is a potential phosphorylation site. Some studies have shown that p66Shc, p46Shc, p52Shc plays the roles of mitogenic and carcinoma arise by regulate the tyrosine kinase receptor. Because of the distinguish p66Shc from the p46Shc and p52Shc, p66Shc has some particular functions. Some research indicated that p66Shc was mainly find in cytoplasm, small part located in mitochondria. Several lines of evidences show that p66Shc is high-level expression in a variety of carcinomas, such as prostate cancer, breast cancer,ovarian cancer. In the fields about cancer and signal transduction pathway, p66Shc adaptor protein involved in a various of signal transduction pathway. Such as it can activate the RAS mitogenic signal pathway and take part in the phosphoinositide 3-kinase/protein kinase B(PI3K/Akt) signal pathway.In the past decades, many of researches which including isoform,stuctures, signal pathway, molecular mechanisms of She family have been gained significant progress.But the mechanism are incompletely understood. It is well known that phosphatidylinositol 3-kinase (PI3K)-AKT pathway is a important transduction pathway which involve in cell proliferation, tumor differentiation, metastasis, apoptosis and so on. PI3K (IA type) and Akt are correlated with tumor arise in human. This pathway was associated with regulation of tumor proliferation and survival. If the pathway out of control which not only lead to cell malignant transformation, also influence the process of cell migration, adhesion, tumor angiogenesis, degradation of extracellular matrix. Therefore,PI3K/Akt signal pathway has been regarded as a potential target for cancer therapy. Activated PI3K can bring out second messenger (PIP3) on the cytomembrane, PIP3 can combined the Akt with phosphoinositi-dedependent kinase-1,PDK1 and phosphorylated the Ser 308,finally induced the process of Akt Activation. Activated Akt can invoke or inhibit the protein factor such as Bad,Caspase-9,GSK-3,FKHR, p21Cip1,p27Kipl which can regulate the tumor cell proliferation, differentiation, apoptosis, metastasis. In addition,Akt can phosphorylate and inhibit BAD. BAD is a pro-apoptosis protein,it belong to Bcl-2 family. Generally, BAD can combined with the Bcl-2,and antagonized anti-apoptosis of Bcl-2.Akt can make the BAD depolymerized with Bcl-2, and induced Bcl-2 recover its anti-apoptosis function. Recent studies suggest that Akt signal pathway involve in regulating cell cycle by Mdm-2-p53,p53 is a classic tumor suppressor gene. When the dissociative Mdm-2 enters the nucleus and combines with p53 to form a Mdm-2-p53 compound, thus inhibited the transcriptional activity of p53 and induce p53 degradation. So many studies demonstrated that Mdm-2 can inhibit the functions of p53 and influenced the cell cycle. However,Mdm-2 is the downstream molecule of Akt,so we speculated that PI3K-Akt-Mdm-2-p53 is a important pathway for cell cycle. A mass of prevous studies demonstrated that PI3K-Akt signaling was activated and excessive expressed in multi cancer tissue, such as gastroenteric tumor,breast cancer and pancreatic cancer. This pathway serves to inhibit many tumor suppressor proteins such as the BAD,FOXO transcription factors. Akt can activated the BAD by phosphorylation, then BAD depolymerized with Bcl-2, and induced Bcl-2 recover its anti-apoptosis function.Thus blocking this pathway could therefore simultaneously inhibit the proliferation of tumor cells and sensitize them toward apoptosis. Some researches on breast cancer and prostate cancer have been demonstrate that p66Shc involve in the oxygen stress in mitochondrial which can induce cell apoptosis. In this experiment, p66Shc can promotes cell proliferation and has anti-apoptosis roles. In pilot trial, we found that p66Shc is associated with Bcl-2 and caspase. So we hypothesized that p66Shc involve in the colorectal cancer cell proliferation and apoptosis by PI3K-Akt-Mdm-2-p53 pathway.In this experiment, firstly, we detected the expression level of p66Shc in colorectal cancer tissue and cell lines.Secondly, we also detected the effects of silenced p66Shc in HCT8 cells on the proliferation, apoptosis. Lastly, we determined the pro-apoptotic and anti-apoptotic expression and the cell cycle distribution. This research aim at illustrating the mechanism of p66Shc on proliferation and apoptosis of colorectal cancer cells, and providing the theory basis for the research on colorectal cancer.Methods1. The expression of p66Shc in colorectal cancer tissue and cell lines was detected using quantitative RT-PCR and Western blotQuantitative reverse transcription-PCR(qRT-PCR) was employed to detecte the expression of p66Shc in colorectal cancer tissue and adjacent tissue. Meanwhile detect the expression of p66Shc in colorectal cancer cell lines(NCM460、HCT8 HCT116、SW620、CaCO2). (NCM460 normal colonic epithelial cells, others are colorectal cancer cell lines)2. To interfere the expression of P66Shc in colorectal cancer cell lines(1) Disigned the RNAi sequence by bioinformatics database(Ambion, Genesil, Genecard).(2) RNAi was synthesized by biotechnology company, the interfere the expression of P66Shc in colorectal cancer cell lines by RNA interference.(3)To detect the outcome of RNA interference by RT-PCR and western blot.3. Effect of silence the P66Shc in HCT-8 on cell proliferation and apoptosis(1) To detect the effect of silence the P66Shc in HCT-8 on cell proliferation by CCK8 assay.(2) To detect the effect of silence the p66Shc in HCT-8 on cell apoptosis by AnnexinV-FITC.4.Research on p66Shc cell signal pathway on colorectal cancer cell(1) To detect the effect of silence the p66Shc on the expression of caspase-3,caspase-9,Bax, Bcl-2 in HCT8 by western blot.(2) To detect the effect of silence the p66Shc on the expression of PI3K、AKT、 Mdm-2、p53 in HCT8 by western blot.5. To detect the effect on the cell cycle distribution by infere p66Shc in HCT8 cells 6.Statistical analysisSPSS 13.0 software was used for statistical analysis. Quantitative values of all experiments are expressed as the mean±standard deviation(SD).Relative quantific-ation value of qRT-PCR in cells were analysed by One-way ANOVA.It also analysed through two-tailed independent-samples t-test.The data of CCK8 assay was analysed by Factorial design analysis of variance.Differences were considered significant if P<0.05.Results1. p66Shc overexpression in CRC tissue and cell lines correlates with proliferation and growth.(1) We explored the expression of p66Shc in colon cancer tisuue by RT-PCR. The rusults demonstrated that p66Shc was highly expressed in colon cancer tissue than adjacent tissue(P<0.05).(2) There was also a statistic difference in the expreesion of p66Shc between colon cancer cell line(HCT8、HCT116、SW620、CaCO2) and NCM460 cells(P<0.05).2. Effect of silence the p66Shc in HCT-8 on cell proliferation and apoptosis(1) After knockdown p66Shc in colon cancer cells, There was also a statistic difference on proliferative abilities in the control group, si-scramble group、 si-p66Shc group(P<0.01).HCT8 cells was significantly prohibited from day3 to day6 when compared to the control and scramble siRNA groups.(2) After silence p66Shc in colon cancer cells, the datas shown that amount of live cells were decreased compared with control group and si-scramble group (si-scramble group is 97.0%, si-p66Shc group is 84.8%,P<0.05). There was also a statistic difference on the apoptosis cells between the si-p66Shc group with si-scramble group(si-scramble group is 0.112%, si-p66Shc group is 0.159%,P<0.05). The apoptosis of HCT8 cells was dramatically increased in p66Shc siRNA treated group.3. Research on p66Shc cell signal pathway on colorectal cancer cell(1) Definite the effect on the cell cycle distribution by infere p66Shc in HCT8 cells with flow cytometry.In this part, the results refer to the control and scramble siRNA treated group,the proportion of cells in G0/G1 phase(P<0.01) was increased and the proportion of cells in G2/M phase (P<0.05)was declined in p66Shc siRNA treated group, which suggests the silence of p66Shc induces cell cycle arrest in G0/G1 phase.(2) The effect of silence the p66Shc on the expression of apoptosis proteins in HCT8 by western blot.The expression of caspase-3,caspase-9,Bax was significantly enhanced in si-p66Shc groups than the control and scramble siRNA treated group (P<0.01).On the contrary, the expression of Bcl-2 was declined in si-p66Shc groups by western blot(P<0.01).(3)The effect of silence the p66Shc on the expression of PI3K-AKT signal pathway proteins in HCT8 by western blot.In this part,after silence the p66Shc in the HCT8,we found that the phosphorylation of PI3K and AKT was significantly suppressed in si-p66Shc group when compared to the control and si-scramble group. Also the express of Mdm-2, a downstream of AKT,was obviously prohibited when compared to the other groups. In contrast, the expression of p53 was enhanced in p66Shc siRNA treated group than the control and scramble siRNA group. The gray scale value analysis of PI3K, AKT, Mdm-2, p53 expression further confirmed this result. The results revealed that the silence of p66Shc played an important role in the viability inhibition in HCT8 cells via PI3K/AKT/Mdm-2/p53 signaling pathway.Conclusion1. p66Shc protein highly expressed in colon cancer tissue and colon cancer cell line cells. So we infer that p66Shc is able to promote colon cancer cell proliferation.2. Silence the p66Shc can inhibited the colon cancer cell proliferation and induced cell apoptosis. These datas demonstrated that infere the p66Shc is important to regulate colon cancer cell proliferation.3. Silence the p66Shc can induces cell cycle arrest in G0/G1 phase.4. Silence the p66Shc can promoted the expression of caspase-3,caspase-9,Bax, reduced the phosphorylation of PI3K and AKT.Based on the above, we can draw a conclusion that the silence of p66Shc played an important role in the viability inhibition in HCT8 cells via PI3K/AKT /Mdm-2/p53 signaling pathway.更多还原