【作者】 孙环星；

【导师】 邵荣光；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2008， 博士

【摘要】 癌症的发生与多基因异常有关,包括癌基因的活化和抑癌基因的失活。RNA干扰被认为是一种非常有潜力的肿瘤治疗手段,利用RNA干扰技术抑制癌基因的表达可以逆转肿瘤的恶性表型。但仅就一个基因为靶点往往只能部分逆转肿瘤的恶性表型,治疗效果有限。为寻求更加理想的抗肿瘤效果,可以在两方面进行研究:第一是将RNA干扰技术联合化疗药物,第二是利用RNA干扰技术靶向多个癌基因。我们就这两方面进行了研究。第一部分靶向N-Ras的shRNA联合长春新碱的抗肿瘤活性研究肝癌是常见的恶性肿瘤之一,虽然有手术、放化疗和肝移植等治疗手段,但是肝癌的死亡率仍然很高,5年存活率小于11%,因此有必要寻找新的、更有效的治疗方法。已有研究显示,RNA干扰下调肝癌形成相关基因的表达能有效抑制肝癌细胞的生长。为了取得更好的效果,我们将靶向N-Ras的RNA干扰表达载体(pCSH1-shNR)与化疗药物长春新碱(VCR)联合进行肝癌的治疗研究。一、pCSH1-shNR联合VCR的体外抗肝癌活性SRB法检测pCSH1-shNR联合VCR对HepG2细胞的生长抑制。结果显示,pCSH1-shNR处理的细胞存活率为55.0%,1.5nM VCR的细胞存活率为77.24%,pCSH1-shNR与1.5nM VCR联合处理的细胞存活率为34.60%,两药相互作用系数(CDI)为0.76;VCR浓度为2.5nM时的细胞生存率为46.18%,pCSH1-shNR与2.5nM VCR联合处理的细胞存活率为17.32%,CDI为0.64。结果说明转染pCSH1-shNR联合长春新碱对HepG2细胞的生长有显著协同抑制作用。pCSH1-shNR联合VCR对HepG2细胞克隆形成能力的抑制。单独转染pCSH1-shNR时,克隆形成率为50%,单独VCR(2.5nM)处理时,克隆形成率为13.75%,pCSH1-shNR与VCR联合时,克隆形成率为6.67%,与单独处理比较克隆形成抑制率有明显提高。二、不同处理对增殖相关蛋白水平变化的影响不同处理对蛋白激酶ERK1/2及Akt活性的影响。转染pCSH1-shNR使HepG2细胞中的ERK1/2和Akt的磷酸化水平下降;VCR处理后,ERK1/2和Akt的磷酸化水平也下降;pCSH1-shNR联合VCR组的ERK1/2和Akt的磷酸化水平下降幅度更大,说明pCSH1-shNR与VCR联合可以共同增强对Ras/Raf/MEK/ERK1/2和Ras/P13K/Akt通路的阻遏作用。细胞核因子NF-κB是一种重要的核转录因子,其过表达和活化与癌症的发生相关。ERK1/2及Akt都参与对NF-κB的调节。在人多发性骨髓瘤细胞中,NF-κB的下调可以诱导细胞对VCR的化疗敏感性。我们检测了不同处理对NF-κB通路的影响,结果显示pCSH1-shNR与VCR联合处理组的NF-κB信号通路的活性被整体下调,并且被抑制的程度大于其它处理组。表皮生长因子受体(EGFR)参与Ras信号通路的活性调节及细胞对VCR作用的响应,在HepG2中表达组成型的磷酸化EGFR。Westem Blot检测pCSH1-shNR及VCR处理对EGFR表达及活性的影响,结果显示pCSH1-shNR与VCR联合处理协同下调了EGFR的表达,N-Ras的表达抑制减弱了细胞响应VCR处理时对EGFR的活化。三、pCSH1-shNR联合VCR对HepG2细胞周期分布的影响流式细胞仪检测pCSH1-shNR联合VCR对HepG2细胞周期分布的影响。结果显示,pCSH1-shNR处理对HepG2细胞周期分布的影响不是很明显。用VCR(1.5nM和2.5nM)处理细胞后,低剂量VCR可以引起S期阻滞,高剂量VCR可以引起G2/M期阻滞,结果说明VCR引起的细胞周期阻滞是剂量依赖性的。pCSH1-shNR与VCR联合处理,结果显示pCSH1-shNR可以降低VCR引起的S期或G2/M期阻滞。Western blot检测细胞周期蛋白(Cyclin)和依赖细胞周期蛋白的蛋白激酶(Cdk)的变化。结果显示,pCSH1-shNR、VCR、pCSH1-shNR与VCR联合处理都下调了cyclin D1、Cdk2、Cdk4和Cdk6的表达,与单独处理比较联合处理的下调作用显著提高。此外,pCSH1-shNR可以逆转VCR引起的cyclin E和cyclinB1的上调作用,VCR抑制pCSH1-shNR对Cdc2的上调作用。因此pCSH1-shNR与VCR联合处理使Cyclins和Cdks的整体活性水平趋于下降。Western blot检测Rb磷酸化和E2F-1的表达水平。结果显示pCSH1-shNR使Rb磷酸化水平略有下调,E2F-1表达略有下降;VCR使Rb磷酸化水平大幅提高,E2F-1表达略有下降;pCSH1-shNR联合VCR使Rb磷酸化水平显著高于对照组,但也显著低于VCR组,联合组的E2F-1表达下降最多。结果说明pCSH1-shNR抑制了VCR刺激Rb磷酸化的作用,促进了对E2F-1表达的下调作用。四、pCSH1-shNR联合VCR对HepG2细胞凋亡的诱导作用流式细胞仪检测pCSH1-shNR联合VCR对HepG2细胞的凋亡诱导作用。结果显示,pCSH1-shNR的凋亡诱导率为6.07%,VCR的凋亡诱导率为12.29%,而pCSH1-shNR联合VCR的凋亡诱导率为22.82%,说明pCSH1-shNR联合VCR协同诱导HepG2细胞凋亡。Western blot检测结果显示,pCSH1-shNR联合VCR协同诱导HepG2细胞凋亡与Bax无关,但pCSH1-shNR显著下调凋亡抑制蛋白Survivin的表达量,从而抑制VCR对Survivin的上调,降低了Survivin对凋亡的抑制作用,进一步增强了VCR对HepG2细胞的凋亡诱导作用。五、pCSH1-shNR联合VCR诱导细胞衰老的作用采用SA-β-gal染色法检测转染pCSH1-shNR联合VCR诱导HepG2细胞衰老的作用。结果显示,pCSH1-shNR的细胞衰老诱导率为11.32%,VCR(2.5nm)的细胞衰老诱导率为8.23%;pCSH1-shNR联合VCR的细胞衰老诱导率为18.76%。结果说明pCSH1-shNR联合VCR诱导细胞衰老的能力更强。六、pCSH1-shNR对VCR诱导MDR1基因表达的影响肿瘤细胞对VCR的抗性与MDR1基因表达产物P-糖蛋白(P-gp)的过表达有关,而ras癌基因可以调控MDR1基因的表达。Western Blot检测结果显示,在转染pCSH1-shNR后进行VCR处理的HepG2细胞内P-gp的升高幅度明显小于VCR处理组。综上所述,pCSH1-shNR与VCR联合处理对HepG2细胞的生长产生了显著的抑制效果,细胞凋亡率增多,细胞表面的EGFR的表达和活性都降低,细胞内的ERK1/2和Akt的活性被协同抑制,Cyclin和Cdk的整体活性水平趋于下降,转录因子E2F-1和NF-κB的表达和活性被下调,pCSH1-shNR抑制了VCR引起的Survivin和MDR1基因表达上调。本研究结果提示,靶向特异基因的RNA干扰技术与化疗药物联合应用于肝癌治疗具有好的疗效。第二部分MDM2和Pin1的shRNA表达载体构建及其抗肿瘤活性MDM2的过表达可以抑制p53的功能,促进肿瘤形成。Pin1被认为是肿瘤发生的催化分子,在原癌基因信号转换中是一个不可缺少的翻译者和扩大者。本课题以多基因shRNA表达载体为基础,构建了MDM2和Pin1双干扰的shRNA表达载体,并研究其抗肿瘤活性。一、靶向MDM2和Pin1的shRNA表达载体的构建采用本室已证明可以有效抑制MDM2表达的RNA干扰序列,以及文献报道中已证明可以有效抑制Pin1表达的RNA干扰序列,将这两段序列构建到已有的多基因shRNA表达载体pCSH1-1中,新构建的载体命名为pCSH1-shPM。二、pCSH1-shPM对靶蛋白表达的影响实验选择人的成纤维肉瘤HT1080细胞进行研究。Western blot检测显示,转染pCSH1-shMDM2可以下调MDM2的表达,转染pCSH1-shPin1可以降低Pin1的表达,也可以使得MDM2的表达有所下降。转染pCSH1-shPM可以同时有效降低MDM2和Pin1的表达,对MDM2的下调程度略大于转染pCSH1-shMDM2组,说明抑制Pin1的表达可能下调MDM2的表达。进一步实验证明,在低表达Pin1的HT1080-shPin1稳定细胞系中,MDM2的表达量明显低于对照组,说明在HT1080细胞中抑制Pin1的表达确实可以下调MDM2的表达。三、pCSH1-shPM对细胞生长的影响SRB法检测不同处理对细胞生长的影响。结果显示,转染pCSH1-shPin1和pCSH1-shMDM2都对HT1080细胞的生长有抑制作用,转染pCSH1-shPM对细胞生长的抑制作用强于pCSH1-shPin1或pCSH1-shMDM2单独处理,说明同时抑制MDM2和Pin1的表达更有效地抑制了HT1080细胞的生长。四、pCSH1-shPM对细胞运动能力的影响采用划痕实验检测不同处理对细胞运动能力的影响。结果表明,转染pCSH1-shPin1、pCSH1-shMDM2和pCSH1-shPM都能够减弱HT1080细胞的运动能力,各个处理组间无差异,说明共同抑制Pin1和MDM2表达没有加强对细胞运动能力的抑制。初步研究显示,双干扰载体pCSH1-shPM对HT1080细胞的抑制作用更强,其作用机制有待进一步深入研究。更多还原

【Abstract】 Cancer is a genomic functional disease with features of oncogene activation and tumor suppressor inactivation.RNA interference(RNAi) technology is considered as a very promising cancer therapy.Inhibition the expression of oncogenes by RNAi reversed the malignant morphology of tumors.However,the malignant phenotype of tumor is partially reversed by RNAi.Two parts are needed to study in pursuit of better anti-cancer effect.The first one is RNAi combined with chemotherapy agents.And the second one is targeting numerous oncogenes.Part 1 Antitumor efficacy of combination of Ras shRNA and VCRLiver cancer is one of the commonest malignancies in the world.Although there are surgery,chemotherapy,radiation therapy and liver transplantation,liver cancer is still one of the most fatal cancers,with five-year relative survival rates less than 11%. It has been reported the growth of liver cancer can be effectively controlled by specific gene silencing by RNAi.In pursuit of better anticancer effect,combination of RNAi therapy and chemotherapy may be promising.In the present study,the effect and mechanism of Ras shRNA combined with vincristine(VCR) were investigated.1.Anti-liver cancer activity of combination of pCSH1-shNR and VCR in vitroGrowth inhibition of HepG2 cells was determined by SRB assay.The results showed that the cell survival rates were 77.24%for 1.5nM VCR,55.0%for pCSH1-shNR and 34.60%for pCSH1-shNR combined with 1.5nM VCR,and coefficient of drug interaction(CDI) was 0.76.The cell survival rates were 46.18% for 2.5nM VCR,17.32%for pCSH1-shNR combined with 2.5nM VCR,and CDI was 0.64.These results indicated that the combination of pCSH1-shNR and VCR had remarkable synergistic activity against HepG2 cells.The inhibition of colony formation was induced by combination of pCSH1-shNR and VCR.The rates of colony formation were 50%,13.75%and 6.67%after the treatments of pCSH1-shNR,2.5nM VCR and the combination,respectively. 2.Alteration of proliferation-related proteins by various treatmentsThe effects of various treatments on the protein kinases ERK1/2 and Akt.Both pCSH1-shNR and VCR were down-regulated the levels of phosphorylated ERK1/2 and Akt.And the lowest level was occurred in the treatment of pCSH1-shNR and VCR.The data indicated that the combination of pCSH1-shNR and VCR inhibited the activities of Ras/Raf/MEK/ERK1/2 and Ras/PI3K/Akt pathways.NF-κB is a kind of important nuclear transcription factor.Over expression and activation of NF-κB are connected with tumorigenesis.Both ERK1/2 and Akt participate in the regulation of NF-κB.Down-regulation of NF-κB induces chemosensitivity to VCR in human multiple myeloma.Our results showed that the combination of pCSH1-shNR and VCR down-regulated the activity of NF-κB,and its inhibitory ability was stronger than that ofpCSH1-shNR or VCR alone,respectively.Epidermal growth factor receptor(EGFR) participates in the regulation of Ras signaling pathway.VCR stimulates the phosphorylation of EGFR.Constitutively expression of phosphrylated EGFR is in HepG2 cells.Western blot analysis showed that the treatment of pCSH1-shNR and VCR down-regulated the expression and phosphorylaiton of EGFR,indicating that N-Ras suppression decreased VCRactivated EGFR.3.Effect of pCSH1-shNR and VCR on cell cycle distribution in HepG2 cellsFlow cytometry analysis showed that pCSH1-shNR had less effect of cell cycle distribution in HepG2 cells.Furthermore,1.5nM VCR induced S phase arrest and 2.5nM VCR induced G2/M phase arrest,indicating that VCR-induced cell cycle arrest was in dose-dependent manner.However,pCSH1-shNR abrogated VCR-induced S and G2/M arrest in combination-treated HepG2 cells.Alteration of cyclin and cyclin dependent kinase(CDK) was determined by Western Blot.Results showed that combination of pCSH1-shNR and VCR was markedly down-regulated the expression of cyclin D1,Cdk2,Cdk4 and Cdk6 compared with pCSH1-shNR or VCR alone.Moreover,pCSH1-shNR reversed VCR-induced up-regulations of cyclin E and cyclin B1,and VCR inhibited the expression of cyclin B1 regulated by pCSH1-shNR.Therefore,combination of pCSH1-shNR and VCR decreased the general expressions of Cyclin and Cdk levels.Western blot analysis showed that pCSH1-shNR decreased phosphorylation of Rb and expression of E2F-1.VCR remarkably increased phosphorylation of Rb and decreased expression of E2F-1.Phosphorylation of Rb was obviously higher in the combination-treated cells compared with control cells,but was significantly lower in VCR-treated cells.Expression of E2F-1 was mostly decreased in the combinationtreated cells.These results indicated that pCSH1-shNR inhibited VCR-induced phosphorylation of Rb.4.Combination of pCSH1-shNR and VCR induced apoptosis of HepG2 cellsFlow cytometry analysis showed that the combination of pCSH1-shNR and VCR induced apoptosis.Apoptosis rates of pCSH1-shNR,VCR and the combination were 6.07%,12.29%,and 22.82%,respectively,indicating that pCSH1-shNR and VCR synergistically induced apoptosis of HepG2 cells.Results of Western blot indicated that Bax was not necessary for combinationinduced apoptosis.Survivin is a member of the family of inhibitor of apoptosis proteins(IAPs).pCSH1-shNR remarkably down-regulated the expression of survivin and suppressed the increase of survivin expression in response to VCR treatment.This was a reason why the combination treatment induced apoptosis synergistically.5.Combination of pCSH1-shNR and VCR induced senescence of HepG2 cellsSA-β-gal staining results showed that rates of senescence cells induced by transfected pCSH1-shNR,VCR and the combination were 11.32%,8.23%,and 18.76%respectively,indicating that the combination treatment had stronger senescence-induced ability.6.Effect of pCSH1-shNR on VCR-induced MDR1 expressionThe resistance of tumor cell to VCR is connected with over expression of P-glycoprotein(P-gp) produced by MDR1 gene,and MDR1 gene expression can be regulated by oncogene ras at the same time.Our results showed that the expression of P-gp in HepG2 cells was up-regulated after VCR treatment.However,the up-regulation of P-gp was impaired in the combination-treated cells,indicating that pCSH1-shNR prevented VCR-induced up-regulation of P-gp. In conclusion,the combination of pCSH1-shNR and VCR synergistically inhibited growth of HepG2 cells and enhanced apoptosis through down-regulating the expression and/or activation of EGFR,E2F-1,NF-κB,phosphorylated ERK1/2,Akt, cyclin,Cdk,survivin and P-gp.Above data suggested that RNAi technology combined with appropriate chemotherapeutic agents seems a promising approach for the treatment of human hepatocellular carcinoma.Part 2 Construction and the Antitumor Efficacy of shRNA Expression Vector against MDM2 and Pin1Over expression of MDM2 suppresses function of tumor suppressor p53 and promotes tumorigenesis.Pin1 is considered as a critical catalyst that amplifies and translates multiple oncogenic signaling mechanisms during oncogenesis.It has been reported that suppression of MDM2 or Pin1 could inhibit proliferation and malignant phenotype of tumor.In the present study,a constructed vector with the ability to express several shRNAs was used.We constructed a shRNA expression vector against MDM2 and Pinl,and investigated its anti-cancer activity.1.Construction of MDM2 shRNA and Pin1 shRNA co-expressing vectorThe sequence of MDM2 shRNA with effective inhibition of MDM2 expression was designed in our laboratory.The reported sequence of Pin1 shRNA could inhibit expression of Pin1 effectively.The two sequences were constructed in pCSH1-1 to form a new vector named as pCSH1-shPM.2.pCSH1-shPM inhibited the expression of MDM2 and Pin1Human fibrosarcoma HT1080 cells were used in this study,which was over expressions of MDM2 and Pin1.Western blot analysis showed that pCSH1-shPin1 inhibited not only the expression of Pin1 but also the expression of MDM2. pCSH1-shPM inhibited the expression of both MDM2 and Pin1.Meanwhile,the inhibition of MDM2 expression in pCSH1-shPM-transfected cells was stronger than that in the pCSH1-shMDM2-transfected cells,suggesting the inhibition of Pin1 might down-regulate the expression of MDM2.Furthermore,the expression of MDM2 was significantly reduced in Pin1 knockdown HT1080/shPin1 cells,supporting that inhibition of Pin1 down-regulated the expression of MDM2 in HT1080 cells.3.The effect of pCSH1-shPM on cell growthThe effects of pCSH1-shMDM2,pCSH1-shPin1 and pCSH1-shPM on cell growth were determined by SRB assay.The results showed that pCSH1-shMDM2, pCSHl-shPinl and pCSHI-shPM suppressed the proliferation of HT1080 cells respectively,and pCSH1-shPM was more effective.These data indicated that inhibition of MDM2 and Pin1 at the same time was more effective to suppress the proliferation of HT 1080 cells.4.The effect of pCSH1-shPM on cell mobilityWound healing assay showed that the mobility of HT 1080 cells was reduced by transfection of pCSH1-shMDM2,pCSH1-shPin1 and pCSH1-shPM.The efficacy of mobility inhibition was no difference among pCSH1-shMDM2,pCSH1-shPin1 and pCSH1-shPM,indicating that both MDM2 and Pin1 inhibitions did not augment the inhibition of cell mobility.Taken together,our data suggested that the dual-interfering vector pCSH1-shPM exhibited a stronger inhibition of HT1080 cell growth,and its mechanism might be investigated in the further experiments.更多还原