shRNA表达载体介导的谷胱甘肽S-转移酶P1基因沉默对前列腺癌DU145细胞影响的实验研究
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摘要
shRNA表达载体介导的谷胱甘肽S-转移酶P1基因沉默对前列腺癌DU145细胞影响的实验研究摘要
研究背景：目前对于激素难治型前列腺癌缺乏有效治疗方法,研究开发对该类患者的治疗新方法已成为当前国内外众多学者研究的热点。经过持续内分泌治疗后病变复发、进展的前列腺癌,包括雄激素非依赖性前列腺癌(androgen-independent prostate cancer, AIPC)和激素难治性前列腺癌(hormone-refractory prostate cancer, HRPC)。在激素非依赖发生的早期有些患者对二线内分泌治疗仍有效,称为雄激素非依赖性前列腺癌(AIPC),而对二线内分泌治疗无效或二线内分泌治疗过程中病变继续发展的则称为激素难治性前列腺癌(HRPC)。在美国每年因HRPC死亡的人数达28,900人。随着生活水平提高和人均寿命延长,我国前列腺癌发病率不断增加,在男性泌尿、生殖系统恶性肿瘤中发病率跃居第三位。2/3的前列腺癌患者一旦确诊已失去根治时机,只能采用雄激素全阻断为主的内分泌治疗。12-18个月后,70%-80%转化为雄激素非依赖性,出现复发或远处转移,平均生存期小于1年。由于HRPC缺乏有效的治疗手段,因此成为近年来肿瘤研究的热点。
谷胱甘肽巯基转移酶(Glutathione S-transferases, GS-Ts)是一种二聚体同工酶,具有强有力的催化作用,能催化底物分子,亲电子中心与还原型谷胱甘肽的轭合,促进其代谢、失活,是体内生物转化最重要的Ⅱ相代谢酶之一。GSTs超家族按其染色体定位和序列同源性,主要分为α、μ、π、θ四个亚家族。GSTP1基因编码的蛋白GST-π由于与恶性肿瘤发生发展密切相关,近年来成为研究热点。胃肠道肿瘤研究中有报道,抑制GSTP1的活性可以降低肿瘤的耐药性,其主要方法包括使用信号通路药物抑制其表达和RNA干扰降低其表达水平。有关GSTP1与前列腺癌的关系近年来报道较多,在前列腺癌形成和发生早期,GSTP1基因启动子序列发生甲基化,导致GSTP1表达下降或缺失从而参与促进前列腺癌的发生和发展。可将GSTP1基因启动子甲基化检测作为前列腺癌的早期诊断方法。抗雄激素治疗对前列腺癌中GSTP1基因表达存在影响,在激素难治性前列腺癌患者其原发病灶和转移病灶中表达发现有GSPT1表达。前列腺癌在进展成为激素非依赖性的过程中,由于组织对于损伤应激反应恢复GSTP1的表达。而GSTP1的恢复表达可能导致了激素难治性前列腺癌的耐药性产生。
RNA干扰(RNAi)是1998年提出,近年快速发展的一种基因沉默技术,具有高度特异性、作用效率高和干扰作用可扩散的特点,具有很强的抑制目的基因的作用。RNA干扰是一种有效基因沉默过程,其主要机制是转录后抑制,基本可分为启动和效应两个阶段。目前RNA干扰治疗激素难治性前列腺癌还处于实验阶段,转染表达载体包括脂质体转染和病毒介导。
本研究以GSTP1作为基因治疗靶点,运用短发夹状RNA(short hairpin RNA)介导RNA干扰沉默GSTP1基因观察其对于雄激素非依赖性前列腺癌细胞株DU145的增殖活性,细胞周期和凋亡率的影响,并观察转染前后化疗药物敏感性的变化。
第一部分GSTP1在前列腺癌组织及血清中表达水平研究
目的：分析和比较GSTP1在激素难治型前列腺癌与早期局限性前列腺癌组织和血清中表达情况及其意义。
方法：用免疫组化的方法检测GSTP1在10例早期局限性前列腺癌(PCa)组织,10例激素难治型前列腺癌(HRPC)组织中的表达情况,取前列腺增生(BPH)组织10例做对照。采用酶联免疫吸附剂测定(Enzyme-Linked Immunosorbent Assay, ELISA)方法检测GSTP1在10例早期局限性前列腺癌患者,10例激素难治型前列腺癌患者血清中的表达情况,取前列腺增生患者10例做对照。
结果：10例早期局限性前列腺癌组织中GSTP1蛋白的表达情况：阴性8例,弱阳性1例,中等阳性1例,阳性率20%。10例激素难治型前列腺癌组织中GSTP1蛋白的表达情况：阴性2例,弱阳性1例,中等阳性4例,强阳性3例,阳性率80%。10例前列腺增生组织中GSTP1蛋白的表达情况：阴性0例,弱阳性1例,中等阳性2例,强阳性7例,阳性率100%。Wilcoxon秩和检验免疫组化结果示HRPC组织中GSTP1蛋白阳性率高于PCa组织,P<0.01；而HRPC组织中GSTP1蛋白阳性率低于BPH组织,P<0.01。10例早期局限性前列腺癌患者血清中GSTP1含量2033.39±232.75pg/mL,10例激素难治型前列腺癌患者血清中GSTP1含量3879.89±210.32pg/mL,两组之间比较,激素难治型组GSTP1含量高于早期局限性前列腺癌组,差异有统计学意义,P<0.01。10例前列腺增生患者血清中GSTP1含量4019.07±170.33pg/mL,高于早期局限性前列腺癌组。,差异有统计学意义,P<0.01。激素难治型组与前列腺增生组之间差异不显著,P=0.862。
结论：1.激素难治性前列腺癌组织中GSTP1蛋白表达高于早期局限性前列腺癌组织。
2.激素难治性前列腺癌患者血清中GSTP1蛋白浓度高于早期局限性前列腺癌患者的血清浓度。
第二部分DU145, PC3, LNCaP三种前列腺癌细胞株GSTP1基因mRNA和蛋白表达水平检测
目的：DU145、PC3和LNCaP三种前列腺癌细胞株进行对比,检测其GSTP1基因mRNA含量和蛋白表达水平。
方法：采用RPMI1640培养基培养人前列腺癌细胞株DU145、PC3和LNCaP。采用逆转录聚合酶链式反应(Reverse transcription-Ploymerase chain reaction, RT-PCR)检测三种前列腺癌细胞株中GSTP1基因mRNA含量,免疫印迹Western Blot检测三种前列腺癌细胞株中GSTP1蛋白含量。
结果：半定量RT-PCR检测三种细胞株中GSTP1基因mRNA含量,结果以吸光度表示。雄激素非依赖性前列腺癌细胞株DU145中GSTP1基因mRNA含量346.7±7.3,PC3中GSTP1基因mRNA含量229.0±1.4,雄激素依赖性前列腺癌细胞株LNCaP中GSTP1基因mRNA含量73.5±2.2。DU145与LNCaP比较,DU145中GSTP1基因mRNA含量高于LNCaP,差异有统计学意义,P<0.01；PC3与LNCaP比较,PC3中GSTP1基因mRNA含量高于LNCaP,差异有统计学意义,P<0.01;DU145与PC3比较,DU145中GSTP1基因mRNA含量高于PC3,差异有统计学意义,P<0.01。Western Blot检测三种细胞株中GSTP1蛋白含量,结果以吸光度表示。雄激素非依赖性前列腺癌细胞株DU145中GSTP1蛋白含量103.1±1.1,PC3中GSTP1蛋白含量78.5±0.5,雄激素依赖性前列腺癌细胞株LNCaP中GSTP1蛋白含量24.4±1.2。DU145与LNCaP比较,DU145中GSTP1蛋白含量高于LNCaP,差异有统计学意义,P<0.01；PC3与LNCaP比较,PC3中GSTP1蛋白含量高于LNCaP,差异有统计学意义,P<0.01；DU145与PC3比较,DU145中GSTP1蛋白含量高于PC3,差异有统计学意义,P<0.01。
结论：1、雄激素非依赖性前列腺癌细胞株DU145、PC3中GSTP1基因mRNA含量和蛋白表达水平均高于雄激素依赖性前列腺癌细胞株LNCaP。
2、DU145中GSTP1基因mRNA含量和蛋白表达水平高于PC3。GSTP1基因表达升高可能与激素非依赖性前列腺癌的发生发展有关。
第三部分shRNA沉默GSTP1基因对雄激素非依赖性前列腺癌细胞株DU145的影响
目的：设计特异性shRNA干扰序列转染雄激素非依赖性前列腺癌细胞株DU145,诱导GSTP1基因沉默,观察其对DU145增殖活性和对化疗药物敏感性的影响。
方法：通过软件设计与参考相关文献,选取靶序列形成shRNA的DNA模板并克隆到shRNA表达载体pGPU6/GFP/Neo载体中,获得靶向抑制GSTP1基因表达的干涉质粒。经酶切、测序鉴定。筛选出转染率最高,基因沉默效果最好的表达载体介导shRNA用作RNA干扰。用脂质体介导该质粒转染DU145细胞,通过逆转录PCR、Western blotting方法检测GSTP1基因的mRNA、蛋白水平的表达情况,评价RNA干扰的效果。观察转染后细胞增殖活性的变化,应用MTT法、流式细胞术初步评价转染后对DU145细胞生物学效应的影响。比较RNA干扰前后DU145细胞对化疗药物5-FU、PA敏感性的变化,应用MTT法、流式细胞术初步评价转染前后对不同浓度5-FU、PA对DU145细胞增殖活性的影响。
结果：设计的三条表达载体和一条阴性对照载体：pGPU6/GFP/Neo-shRNA255, pGPU6/GFP/Neo-shRNA554 pGPU6/GFP/Neo-shRNA593,和Negative-shRNA。通过预实验,shRNA255、shRNA554和shRNA593转染率分别为63.3±1.04%,76.2±0.68%,72.7±0.33%,比较得出shRNA554转染率最高,P<0.01。通过预实验RT-PCR和Western Blot检测,shRNA255、shRNA554和shRNA593转染Du145细胞后GSTP1基因mRNA含量分别为128.31±2.5,43.24±4.3和85.62±6.3,GSTP1蛋白含量分别为163.92±12.4,65.38±9.3和114.25±16.7,比较得出shRNA554转染后GSTP1基因mRNA和蛋白含量均最低,P<0.01。转染shRNA554后,DU145细胞第2、4、6天GSTP1蛋白表达水平检测结果分别为125.44±10.85,106.52±11.2和56.43±8.76,而空白质粒组为174.35±7.2,168.09±6.54和171.72±8.25。转染后GSTP1蛋白表达水平降低(P<0.01)且随转染时间延长递减,具有时间依赖性(P<0.01)。转染shRNA554后第2、4、6天MTT检测细胞存活率低于对照组(P<0.01),具有时间依赖性(P<0.01)。流式细胞仪检测目的基因转染组处于亚G1期细胞率高于空白质粒组(P<0.01),且增殖指数降低(P<0.01)。MTT检测转染前不同浓度5-FU(μg/ml)作用后细胞存活率为95.6±2.11%(30),90.2±0.86%(60),83.1±3.12%(120)和74.6±1.32%(240)；转染后不同浓度5-FU(μg/ml)作用后细胞存活率为91.3±1.43%(30),84.6±2.13%(60),73.2±1.52%(120)和65.5±0.94%(240)。流式细胞术检测转染前不同浓度5-FU(μg/ml)作用后亚G1期比率(凋亡期)为5.37±0.43%(30),6.49±2.06%(60),9.02±0.34%(120)和12.33±2.46%(240)。增殖指数为47.54±2.33%(30),46.69±2.12%(60),46.01±1.87%(120)和44.29±1.78%(240)。而转染后不同浓度5-FU(μg/ml)作用后亚G1期比率(凋亡期)为7.69±1.04%(30),11.44±0.98%(60),13.57±3.44%(120)和22.42±0.84%(240)。增殖指数为40.97±0.92%(30),38.51±2.03%(60),36.39±2.67%(120)和34.04±3.21%(240)。统计学分析显示转染后相同5-FU浓度下细胞存活率降低,凋亡期比率上升,增殖指数下降,差异均具有统计学意义(P<0.01)。MTT检测转染前不同浓度PA(μg/ml)作用后细胞存活率为98.5±2.34%(0.2),95.2±1.32%(2),89.4±0.68%(10)和82.7±1.73%(20)；转染后不同浓度PA(μg/ml)作用后细胞存活率为94.2±0.78%(0.2),86.5±2.13%(2),78.7±1.34%(10)和70.1±0.76%(20)；。流式细胞术检测转染前不同浓度PA(μg/ml)作用后亚G1期比率(凋亡期)为3.51±0.35%(0.2),5.41±1.03%(2),9.48±1.09%(10)和10.91±1.03%(20)。增殖指数为46.25±1.27%(0.2),46.05±1.98%(2),41.75±2.44%(10)和41.04±1.71%(20)。而转染后不同浓度PA(μg/ml)作用后亚G1期比率(凋亡期)为5.66±1.32%(0.2),11.02±0.87%(2),19.53±1.23%(10)和26.31±2.01%(20)。增殖指数为44.20±2.52%(0.2),41.37±2.51%(2),35.04±2.41%(10)和32.97±2.89%(20)。统计学分析显示转染后相同PA浓度下细胞存活率降低,凋亡期比率上升,增殖指数下降,差异均具有统计学意义(P<0.01)。
结论：1、表达载体介导GSTP1-shRNA554转染后可降低雄激素非依赖型前列腺癌DU145细胞GSTP1基因mRNA、GSTP1蛋白水平,抑制体外培养DU145增殖活性,并且呈现作用时间依赖性。
2、表达载体介导GSTP1-shRNA554转染后可将雄激素非依赖型前列腺癌DU145细胞诱导凋亡,增殖指数下降。
3、表达载体介导GSTP1-shRNA554转染后提高雄激素非依赖型前列腺癌DU145细胞对化疗药物的敏感性,相同药物浓度下转染后DU145细胞存活率降低,凋亡期比率上升,增殖指数下降。
第四部分ERK抑制剂PD98059对DU145细胞中GSTP1表达及细胞凋亡的影响研究
目的：检测细胞外信号激酶ERK抑制剂PD98059对DU145细胞GSTP1蛋白水平的影响,及其对细胞存活率和凋亡水平的影响。
方法：将细胞外信号激酶ERK抑制剂PD98059作用DU145细胞,通过Western Blot检测不同浓度PD98059对于P-ERK、GSTP1蛋白水平的影响,采用MTT法检测细胞存活率,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记技术(terminal de-oxynucleotidyl transferase-mediated dUTP nick end labeling, TUNEL)检测DU145细胞凋亡水平。
结果：不同浓度ERK抑制剂PD98059作用后P-ERK和GSTP1蛋白表达水平以吸光度表示,当PD98059作用浓度分别为0μmol/L, 5μmol/L,10μmol/L,20μmol/L,40μmol/L时, Western Blot检测P-ERK含量为485.93±12.1,397.02±7.8,351.11±7.2,230.54±6.5,,142.97±13.2。GSTP1蛋白表达水平为380.74±14.4,285.57±9.87,231.45±6.43,190.87±11.2,102.08±14.4。PD98059作用后P-ERK和GSTP1蛋白表达水平随浓度增高而下降,各浓度组间差别具有统计学意义,P<0.01。ERK抑制剂PD98059作用后TUNEL检测DU145细胞凋亡率,当PD98059作用浓度分别为0μmol/L,5μmol/L,10μmol/L PD98059, 20μmol/L,40μmol/L时,凋亡率为5.54±0.39%,10.46±0.88%,19.8±1.64%,36.2±2.39%,49.2±2.59%。随PD98059浓度增加细胞凋亡率升高,不同浓度组间差异具有统计学意义,P<0.01。
结论：1、ERK抑制剂PD98059作用于DU145细胞后,对P-ERK、GSTP1蛋白表达呈抑制效应,并且呈明显的剂量效应关系。
2、ERK抑制剂PD98059作用于DU145细胞后诱导细胞凋亡,凋亡的诱导作用呈显著剂量依赖性。
3、ERK信号通路可能参与调控GSTP1蛋白的表达。
Background Hormone-refractory prostate cancer (HRPC) has no effective treatment at present, and the new therapy for HRPC is a focus of research around the world. After endocrine treatment of androgen deprivation the prostate carcinoma will usually get the progression of androgen-independent prostate cancer (AIPC) or even hormone-refractory prostate cancer. The AIPC is responsive to some secondary endocrine treatment, but the HRPC is irresponsive to any endocrine treatment and the disease gets development during the endocrine treatment. About 28,900 people died of HRPC annually in America. The incidence of prostate cancer increases is increasing rapidly in China as the improvement of living quality and prolongation in life span. In our country the prostate cancer took the third place in common malignant tumor in the genitourinary system. Two thirds of the prostate cancer patients lose the chance of radical cure, for whom the maximum androgen blockade (MAB) is the only choice. But after a median period of 12-18 months,70%-80% converted to androgen independent prostate cancer and recurrence or metastasis occurs. The mean survival time is about 1 year. As lack of efficient treatment for HRPC, it is the focus in carcinoma research.
Glutathione S-transferases (GSTs) is a kind of dimeride isoenzyme, which has powerful catalytic action. The GSTs could catalyse electron binding centre of the substrate molecule to conjugate with reduced glutathione to promote the metabolism and inactivation of the substrate. According to the location in chromosome and the sequence homology, the superfamily of GSTs consists ofα、μ、π、θfour subfamily. The GST-πprotein produced by GSTP1 gene has close relation with the malignant tumors, which became the focus of research. The reports of research in gastrointestinal tumor show that inhibition of GSTP1 could reduce the drug resistance of tumors. The main inhibitive methods include the signal path block drug and RNA interference to lower the expression level. During the early stage of prostate cancer, the promoter sequence of GSTP1 got hypermethylation, which caused the deletion of GSTP1 expression. As a result the prostate cancer took place and progressed. The detection of GSTP1 promoter methylation condition could be an early diagnostic method for prostate cancer. The effect of androgen deprivation on the expression of GSTP1 has been reported that after androgen deprivation therapy, the tissue got stress reaction and regained the expression of GSTP1. Research reports shew that GSTP1 expressed in HRPC primary lesion and the metastatic lesion. The recovery expression of GSTP1 may result in the resistance of chemotherapy of HRPC.
RNA interference (RNAi) is the technique that invented in 1998 and the development of RNAi is rapid. RNAi is a kind of gene silencing technique, which has high degree of specificity, efficiency and the interfere effect is diffusive. As a result the inhibitive effect on target gene is powerful. RNA interference is an effective gene silencing process, and the mechanism is post transcription inhibition. The process could divide into initiation and effector stage. In initiation stage the RNAaseⅢfabricated the lipofectamine or virus imported dsRNA in to siRNA of 21-23nt. In the effector phase the double strand of the siRNA unrolled and the anti-sense siRNA combined the related protein to form the active RNA induced silencing complex (RISC). RISC then recognize the mRNA with the completely complementary sequences and make the mRNA degrade. The process has cascade amplification. At present the RNAi via lipofectamine or virus to treat the prostate cancer is in the experiment stage.
In this experiment GSTP1 is taken as the target of gene therapy, and the application of short hairpin RNA is taken to silence the GSTP1 gene. The GSTP1 shRNA is constructed and transfected to androgen independent prostate cancer cell line DU145. Then RT-PCR, Western Blotting are used to detect the mRNA and protein level of GSTP1 to evaluate the effect of RNA interference. After transfection the MTT, flow cytometry are used to assess the RNAi effect on the biological behavior of DU145. The sensitivity to chemotherapeutics of DU145 before and after the transfecion are compared. TUNEL was used to detect the effect of ERK inhibitor PD98059 on the apoptosis of prostate cancer cell line.
Part I
Study on the expression of GSTP1 in the prostate cancer tissue and plasma
Objective Analyze and compare the expression level of GSTP1 in the prostate cancer tissue and plasma of the hormone-refractory prostate cancer and prophase localized prostate cancer.
Methods Immunohistochemistry analysis is used to detect the expression of GSTP1 in 10 cases of prophase localized prostate cancer(PCa) tissue, and 10 cases of hormone-refractory prostate cancer(HRPC) tissue. In control 10 cases of benign prostate hyperplasia(BPH) tissue were analyzed.
Enzyme-linked immunosorbent assay (ELISA) is used to detect the expression of GSTP1 in plasma of 10 prophase localized prostate cancer patients, and 10 cases of HRPC patients. In control 10 cases of BPH patients'plasma were analyzed.
Results The expression result of GSTP1 in 10 cases of prophase localized prostate cancer tissue:Negative 8 cases, weakly positive 1 case, middling positive 1 case, the positive ratio was 20%. The expression result of GSTP1 in 10 cases of HRPC tissue:Negative 2 cases, weakly positive 1 case, middling positive 4 cases, obviously positive 3 cases, the positive ratio was 80%. In control 10 cases of BPH tissue:no negative case,1 case weakly positive,2 cases middling positive and 7 cases obviously positive, the positive ratio was 100%. Wilcoxon rank test shew that the GSTP1 positive ratio of HRPC is higher than that of PCa, P <0.01, but lower than that of BPH, P<0.01. The concentration of GSTP1 in plasma was 2033.39±232.75pg/mL in 10 cases of PCa patients; 3879.89±210.32pg/mL in 10 cases of HRPC patients; 4019.07±170.33pg/mL in 10 cases of BPH patients. The HRPC group had higher GSTP1 concentration than the PCa group, P<0.01; the HRPC group had lower GSTP1 concentration than the BPH group, P<0.01.
Conclusion
1. The prostate cancer tissue of hormone-refractory prostate cancer has higher GSTP1 expression than that of prophase localized prostate cancer.
2. The concentration of GSTP1 in plasma of patients with hormone-refractory prostate cancer is higher than that of patients with prophase localized prostate cancer.
PartⅡ
Detection of the mRNA and protein expression level of gene GSTP1 in the prostate cancer cells line DU145, PC3 and LNCaP
Objective Compare the mRNA and protein expression level of gene GSTP1 among the prostate cell line DU145, PC3 and LNCaP.
Methods RPMI 1640 nutrient medium is used to culture the prostate cancer cell line DU145, PC3 and LNCaP. RT-PCR is used to detect the mRNA level of gene GSTP1 of the three cell lines, and western blot is used to detect the GSTP1 protein level.
Results Semi-quantitive RT-PCR was used to detect the level of the GSTP1 mRNA and the absorbance of light indicate the expression ratio. Of the androgen independent prostate cancer cell line DU145 the GSTP1 mRNA was 346.7±7.3; in PC3 229.0±1.4; of the androgen dependent prostate cancer cell line LNCaP the GSTP1 mRNA was 73.5±2.2. Compared with the androgen dependent prostate cancer cell line LNCaP, the androgen independent prostate cancer cell line DU145 and PC3 had higher level of GSTP1 mRNA, P<0.01. DU145 had higher level of GSTP1 mRNA than that of PC3, P<0.01. Western blot was used to detect the level of GSTP1 protein level in the three cell lines. The absorbance of light indicated the GSTP1 protein level. Of the androgen independent prostate cancer cell line DU145 the GSTP1 protein was 103.1±1.1; PC3 78.5±0.5; of the androgen dependent prostate cancer cell line LNCaP the GSTP1 protein was 24.4±1.2. Compared with the androgen dependent prostate cancer cell line LNCaP, the androgen independent prostate cancer cell line DU145 and PC3 had higher level of GSTP1 protein, P<0.01. DU145 had higher level of GSTP1 protein than that of PC3,P<0.01.
Conclusion
1. The androgen independent prostate cancer cell lines DU145 and PC3 have higher level of GSTP1 mRNA and GSTP1 protein, compared with that of androgen dependent prostate cancer cell line LNCaP.
2. DU145 has higher level of GSTP1 mRNA and GSTP1 protein than that of PC3.
PartⅢ
The effect of gene GSTP1 silencing via shRNA transfection on the androgen independent prostate cancer cell line DU145
Objective To design special shRNA interference sequence for transfection to the androgen independent prostate cancer cell line DU145 to silence the gene GSTP1, and then to investigate the effect on proliferate activity and sensitivity to chemotherapeutics.
Methods Through software design and review of related references, the target sequence was picked up to form the shRNA, and the DNA template was cloned to the shRNA expression vector pGPU6/GFP/Neo to construct the plasmid with the targeting inhibitive shRNA of gene GSTP1. The shRNA was identified by gene sequencing. The screening experiment was taken to pick up the shRNA expression vector with highest transfection ratio and best gene silencing result. Lipofectamine was used to mediate transfection of plasmid to DU145, and RT-PCR, western blot was used to detect the level of mRNA and protein of GSTP1 which indicated the effect of transfection. MTT analysis and flow cytometry were used to evaluate the effectiveness of shRNA transfection. TO compare the sensitivity to chemotherapeutics before and after transfecion, MTT analysis and flow cytometry were used to detect the proliferate activity of DU145 after the 5-FU or PA of different concentration added.
Results The constructed vectors including three shRNA expression vector and the negative vector. They were pGPU6/GFP/Neo-shRNA255, pGPU6/GFP/Neo-shRNA554, pGPU6/GFP/Neo-shRNA593 and Negative-shRNA. In preliminary experiment, the transfection efficiency of shRNA255、shRNA554 and shRNA593 was 63.3±1.04%,76.2±0.68% and 72.7±0.33% respectively. In which the shRNA554 had highest transfection efficiency, P< 0.01. In preliminary experiment, after transfection of shRNA255, shRNA554 and shRNA593 the gene GSTP1 mRNA level detected by RT-PCR of DU145 was 128.31±2.5,43.24±4.3 and 85.62±6.3 respectively, and the GSTP1 protein level detected by western blot of DU145 was 163.92±12.4,65.38±9.3 and 114.25±16.7 respectively. Statistical analysis indicated that shRNA554 of highest transfection ratio and best gene silencing result, P< 0.01. After transfection of shRNA554 to DU145, on the 2 day,4 day and 6 day the level of GSTP1 protein was 125.44±10.85,106.52±11.2 and 56.43±8.76 respectively. While the level after the transfection of blank plasmid was 174.35±7.2,168.09±6.54 and 171.72±8.25 respectively. The data indicated that after transfection of shRNA554 to DU145 the GSTP1 protein level decreased(P<0.01), and decreased progressively as the extension of transfection time, which indicated the time dependence(P< 0.01). Similarly MTT analysis shew that after 2 days,4 days and 6 days after transfection the survival ratio of DU145 decreased progressively as the extension of transfection time, which indicated the time dependence(P < 0.01). Flow cytometry indicated that the ratio of subGl phase transfected with shRNA554 was higher than that of blank plasmid(P< 0.01), and the proliferate ratio decreased(P< 0.01). MTT analysis indicated that before transfection, the survival ratio of DU145 added different concentration of 5-FU (μg/ml) were:95.6±2.11%(30), 90.2±0.86%(60),83.1±3.12%(120) and 74.6±1.32%(240); while after transfection the survival ratio of DU145 added different concentration of 5-FU (μg/ml) were:91.3±1.43%(30),84.6±2.13%(60),73.2±1.52%(120) and 65.5±0.94%(240). Flow cytometry indicated that before transfection, the subGl phase(apoptosis cell) ratio of DU145 with different concentration of 5-FU (μg/ml) added were:5.37±0.43%(30), 6.49±2.06%(60),9.02±0.34%(120) and 12.33±2.46%(240); the proliferate index were:47.54±2.33%(30),46.69±2.12%(60), 46.01±1.87%(120) and 44.29±1.78%(240); while after transfection the subGl phase(apoptosis cell) ratio of DU145 with different concentration of 5-FU (μg/ml) added were:7.69±1.04%(30),11.44±0.98%(60), 13.57±3.44%(120) and 22.42±0.84%(240); the proliferate index were: 40.97±0.92%(30),38.51±2.03%(60),36.39±2.67%(120) and 34.04±3.21%(240). Compared with the data before transfection, statistical analysis indicated that after transfection, under the same concentration of 5-FU, the survival ratio decreased, apoptosis increased and the proliferate index decreased of statistical significance, P<0.01. MTT analysis indicated that before transfection, the survival ratio of DU145 added different concentration of PA (μg/ml) were:98.5±2.34%(0.2), 95.2±1.32%(2),89.4±0.68%(10) and 82.7±1.73%(20); while after transfection the survival ratio of DU145 added different concentration of PA (μg/ml) were:94.2±0.78%(0.2),86.5±2.13%(2),78.7±1.34%(10) and 70.1±0.76%(20). Flow cytometry indicated that before transfection, the subG1 phase(apoptosis cell) ratio of DU145 with different concentration of PA (μg/ml) added were:3.51±0.35%(0.2),5.41±1.03%(2), 9.48±1.09%(10) and 10.91±1.03%(20); the proliferate index were: 46.25±1.27%(0.2),46.05±1.98%(2),41.75±2.44%(10) and 41.04±1.71%(20); while after transfection the subGl phase(apoptosis cell) ratio of DU145 with different concentration of PA(μg/ml) added were: 5.66±1.32%(0.2),11.02±0.87%(2),19.53±1.23%(10) and 26.31±2.01%(20); the proliferate index were:44.20±2.52%(0.2), 41.37±2.51%(2),35.04±2.41%(10) and 32.97±2.89%(20). Compared with the data before transfection, statistical analysis indicated that after transfection, under the same concentration of PA, the survival ratio decreased, apoptosis increased and the proliferate index decreased of statistical significance, P<0.01.
Conclusion
1. The shRNA expression vector-transfected to androgen independent prostate cancer cell line DU145 could reduce the level of GSTP1 mRNA and GSTP1 protein, in vitro inhibition of the proliferate activity of DU145 in a manor of time dependent.
2. The shRNA expression vector-transfected to androgen independent prostate cancer cell line DU145 could induce apoptosis and decrease the proliferate index.
3. The shRNA expression vector-transfected to androgen independent prostate cancer cell line DU145 could raise the sensitivity to chemotherapeutics, under the same concentration of chemotherapeutics, the survival ratio of DU145 decreased, apoptosis increased and the proliferate index decreased.
Part IV
The effect of ERK inhibitor PD98059 on the expression of GSTP1 protein and apoptosis of DU145
Objective To detect the effect of the extracellular regulated kinase (ERK) inhibitor PD98059 on the expression of GSTP1 of DU145, and the effect on the survival ratio and apoptosis level of DU145.
Methods ERK inhibitor PD98059 was added to DU145, then western blot was used to detect the effect of PD98059 with different concentration on the level of P-ERK and GSTP1. MTT analysis was used to detect the cell survival ratio and TUNEL was used to detect the apoptosis level of DU145 when treated with PD98059.
Results After treated with different concentration of PD98059, the protein level of P-ERK and GSTP1 protein were detected by western blot and the absorbance of light indicated the protein level. The DU145 was treated with different concentration of PD98059 0μmol/L,5μmol/L, 10μmol/L,20μmol/L,40μmol/L; the P-ERK level was 485.93±12.1, 397.02±7.8,351.11±7.2,230.54±6.5,142.97±13.2 respectively; the GSTP1 protein level was 380.74±14.4,285.57±9.87,231.45±6.43, 190.87±11.2,102.08±14.4 respectively. The level of P-ERK and GSTP1 protein decreased progressively as the concentration of PD98059 incresed, and the difference between every two different concentration group is statistically significant, P<0.01. The DU145 was treated with different concentration of PD98059 0μmol/L,5μmol/L,10μmol/L,20μmol/L, 40μmol/L; the apoptosis ratio detected by TUNEL was 5.54±0.39%, 10.46±0.88%,19.8±1.64%,36.2±2.39%,49.2±2.59% respectively. The apoptosis ratio increased progressively as the concentration of PD98059 incresed, and the difference between every two different concentration group is statistically significant, P<0.01.
Conclusion
1. The ERK inhibitor PD98059 has inhibitive effect on the expression of P-ERK, GSTP1 of DU145 in the manner of obvious dose-effect relationship.
2. ERK inhibitor PD98059 induced the DU145 apoptosis in the manner of significant dose-dependent.
3. The ERK pathway may participate in regulation of GSTP1 protein expression.
研究背景：目前对于激素难治型前列腺癌缺乏有效治疗方法,研究开发对该类患者的治疗新方法已成为当前国内外众多学者研究的热点。经过持续内分泌治疗后病变复发、进展的前列腺癌,包括雄激素非依赖性前列腺癌(androgen-independent prostate cancer, AIPC)和激素难治性前列腺癌(hormone-refractory prostate cancer, HRPC)。在激素非依赖发生的早期有些患者对二线内分泌治疗仍有效,称为雄激素非依赖性前列腺癌(AIPC),而对二线内分泌治疗无效或二线内分泌治疗过程中病变继续发展的则称为激素难治性前列腺癌(HRPC)。在美国每年因HRPC死亡的人数达28,900人。随着生活水平提高和人均寿命延长,我国前列腺癌发病率不断增加,在男性泌尿、生殖系统恶性肿瘤中发病率跃居第三位。2/3的前列腺癌患者一旦确诊已失去根治时机,只能采用雄激素全阻断为主的内分泌治疗。12-18个月后,70%-80%转化为雄激素非依赖性,出现复发或远处转移,平均生存期小于1年。由于HRPC缺乏有效的治疗手段,因此成为近年来肿瘤研究的热点。
谷胱甘肽巯基转移酶(Glutathione S-transferases, GS-Ts)是一种二聚体同工酶,具有强有力的催化作用,能催化底物分子,亲电子中心与还原型谷胱甘肽的轭合,促进其代谢、失活,是体内生物转化最重要的Ⅱ相代谢酶之一。GSTs超家族按其染色体定位和序列同源性,主要分为α、μ、π、θ四个亚家族。GSTP1基因编码的蛋白GST-π由于与恶性肿瘤发生发展密切相关,近年来成为研究热点。胃肠道肿瘤研究中有报道,抑制GSTP1的活性可以降低肿瘤的耐药性,其主要方法包括使用信号通路药物抑制其表达和RNA干扰降低其表达水平。有关GSTP1与前列腺癌的关系近年来报道较多,在前列腺癌形成和发生早期,GSTP1基因启动子序列发生甲基化,导致GSTP1表达下降或缺失从而参与促进前列腺癌的发生和发展。可将GSTP1基因启动子甲基化检测作为前列腺癌的早期诊断方法。抗雄激素治疗对前列腺癌中GSTP1基因表达存在影响,在激素难治性前列腺癌患者其原发病灶和转移病灶中表达发现有GSPT1表达。前列腺癌在进展成为激素非依赖性的过程中,由于组织对于损伤应激反应恢复GSTP1的表达。而GSTP1的恢复表达可能导致了激素难治性前列腺癌的耐药性产生。
RNA干扰(RNAi)是1998年提出,近年快速发展的一种基因沉默技术,具有高度特异性、作用效率高和干扰作用可扩散的特点,具有很强的抑制目的基因的作用。RNA干扰是一种有效基因沉默过程,其主要机制是转录后抑制,基本可分为启动和效应两个阶段。目前RNA干扰治疗激素难治性前列腺癌还处于实验阶段,转染表达载体包括脂质体转染和病毒介导。
本研究以GSTP1作为基因治疗靶点,运用短发夹状RNA(short hairpin RNA)介导RNA干扰沉默GSTP1基因观察其对于雄激素非依赖性前列腺癌细胞株DU145的增殖活性,细胞周期和凋亡率的影响,并观察转染前后化疗药物敏感性的变化。
第一部分GSTP1在前列腺癌组织及血清中表达水平研究
目的：分析和比较GSTP1在激素难治型前列腺癌与早期局限性前列腺癌组织和血清中表达情况及其意义。
方法：用免疫组化的方法检测GSTP1在10例早期局限性前列腺癌(PCa)组织,10例激素难治型前列腺癌(HRPC)组织中的表达情况,取前列腺增生(BPH)组织10例做对照。采用酶联免疫吸附剂测定(Enzyme-Linked Immunosorbent Assay, ELISA)方法检测GSTP1在10例早期局限性前列腺癌患者,10例激素难治型前列腺癌患者血清中的表达情况,取前列腺增生患者10例做对照。
结果：10例早期局限性前列腺癌组织中GSTP1蛋白的表达情况：阴性8例,弱阳性1例,中等阳性1例,阳性率20%。10例激素难治型前列腺癌组织中GSTP1蛋白的表达情况：阴性2例,弱阳性1例,中等阳性4例,强阳性3例,阳性率80%。10例前列腺增生组织中GSTP1蛋白的表达情况：阴性0例,弱阳性1例,中等阳性2例,强阳性7例,阳性率100%。Wilcoxon秩和检验免疫组化结果示HRPC组织中GSTP1蛋白阳性率高于PCa组织,P<0.01；而HRPC组织中GSTP1蛋白阳性率低于BPH组织,P<0.01。10例早期局限性前列腺癌患者血清中GSTP1含量2033.39±232.75pg/mL,10例激素难治型前列腺癌患者血清中GSTP1含量3879.89±210.32pg/mL,两组之间比较,激素难治型组GSTP1含量高于早期局限性前列腺癌组,差异有统计学意义,P<0.01。10例前列腺增生患者血清中GSTP1含量4019.07±170.33pg/mL,高于早期局限性前列腺癌组。,差异有统计学意义,P<0.01。激素难治型组与前列腺增生组之间差异不显著,P=0.862。
结论：1.激素难治性前列腺癌组织中GSTP1蛋白表达高于早期局限性前列腺癌组织。
2.激素难治性前列腺癌患者血清中GSTP1蛋白浓度高于早期局限性前列腺癌患者的血清浓度。
第二部分DU145, PC3, LNCaP三种前列腺癌细胞株GSTP1基因mRNA和蛋白表达水平检测
目的：DU145、PC3和LNCaP三种前列腺癌细胞株进行对比,检测其GSTP1基因mRNA含量和蛋白表达水平。
方法：采用RPMI1640培养基培养人前列腺癌细胞株DU145、PC3和LNCaP。采用逆转录聚合酶链式反应(Reverse transcription-Ploymerase chain reaction, RT-PCR)检测三种前列腺癌细胞株中GSTP1基因mRNA含量,免疫印迹Western Blot检测三种前列腺癌细胞株中GSTP1蛋白含量。
结果：半定量RT-PCR检测三种细胞株中GSTP1基因mRNA含量,结果以吸光度表示。雄激素非依赖性前列腺癌细胞株DU145中GSTP1基因mRNA含量346.7±7.3,PC3中GSTP1基因mRNA含量229.0±1.4,雄激素依赖性前列腺癌细胞株LNCaP中GSTP1基因mRNA含量73.5±2.2。DU145与LNCaP比较,DU145中GSTP1基因mRNA含量高于LNCaP,差异有统计学意义,P<0.01；PC3与LNCaP比较,PC3中GSTP1基因mRNA含量高于LNCaP,差异有统计学意义,P<0.01;DU145与PC3比较,DU145中GSTP1基因mRNA含量高于PC3,差异有统计学意义,P<0.01。Western Blot检测三种细胞株中GSTP1蛋白含量,结果以吸光度表示。雄激素非依赖性前列腺癌细胞株DU145中GSTP1蛋白含量103.1±1.1,PC3中GSTP1蛋白含量78.5±0.5,雄激素依赖性前列腺癌细胞株LNCaP中GSTP1蛋白含量24.4±1.2。DU145与LNCaP比较,DU145中GSTP1蛋白含量高于LNCaP,差异有统计学意义,P<0.01；PC3与LNCaP比较,PC3中GSTP1蛋白含量高于LNCaP,差异有统计学意义,P<0.01；DU145与PC3比较,DU145中GSTP1蛋白含量高于PC3,差异有统计学意义,P<0.01。
结论：1、雄激素非依赖性前列腺癌细胞株DU145、PC3中GSTP1基因mRNA含量和蛋白表达水平均高于雄激素依赖性前列腺癌细胞株LNCaP。
2、DU145中GSTP1基因mRNA含量和蛋白表达水平高于PC3。GSTP1基因表达升高可能与激素非依赖性前列腺癌的发生发展有关。
第三部分shRNA沉默GSTP1基因对雄激素非依赖性前列腺癌细胞株DU145的影响
目的：设计特异性shRNA干扰序列转染雄激素非依赖性前列腺癌细胞株DU145,诱导GSTP1基因沉默,观察其对DU145增殖活性和对化疗药物敏感性的影响。
方法：通过软件设计与参考相关文献,选取靶序列形成shRNA的DNA模板并克隆到shRNA表达载体pGPU6/GFP/Neo载体中,获得靶向抑制GSTP1基因表达的干涉质粒。经酶切、测序鉴定。筛选出转染率最高,基因沉默效果最好的表达载体介导shRNA用作RNA干扰。用脂质体介导该质粒转染DU145细胞,通过逆转录PCR、Western blotting方法检测GSTP1基因的mRNA、蛋白水平的表达情况,评价RNA干扰的效果。观察转染后细胞增殖活性的变化,应用MTT法、流式细胞术初步评价转染后对DU145细胞生物学效应的影响。比较RNA干扰前后DU145细胞对化疗药物5-FU、PA敏感性的变化,应用MTT法、流式细胞术初步评价转染前后对不同浓度5-FU、PA对DU145细胞增殖活性的影响。
结果：设计的三条表达载体和一条阴性对照载体：pGPU6/GFP/Neo-shRNA255, pGPU6/GFP/Neo-shRNA554 pGPU6/GFP/Neo-shRNA593,和Negative-shRNA。通过预实验,shRNA255、shRNA554和shRNA593转染率分别为63.3±1.04%,76.2±0.68%,72.7±0.33%,比较得出shRNA554转染率最高,P<0.01。通过预实验RT-PCR和Western Blot检测,shRNA255、shRNA554和shRNA593转染Du145细胞后GSTP1基因mRNA含量分别为128.31±2.5,43.24±4.3和85.62±6.3,GSTP1蛋白含量分别为163.92±12.4,65.38±9.3和114.25±16.7,比较得出shRNA554转染后GSTP1基因mRNA和蛋白含量均最低,P<0.01。转染shRNA554后,DU145细胞第2、4、6天GSTP1蛋白表达水平检测结果分别为125.44±10.85,106.52±11.2和56.43±8.76,而空白质粒组为174.35±7.2,168.09±6.54和171.72±8.25。转染后GSTP1蛋白表达水平降低(P<0.01)且随转染时间延长递减,具有时间依赖性(P<0.01)。转染shRNA554后第2、4、6天MTT检测细胞存活率低于对照组(P<0.01),具有时间依赖性(P<0.01)。流式细胞仪检测目的基因转染组处于亚G1期细胞率高于空白质粒组(P<0.01),且增殖指数降低(P<0.01)。MTT检测转染前不同浓度5-FU(μg/ml)作用后细胞存活率为95.6±2.11%(30),90.2±0.86%(60),83.1±3.12%(120)和74.6±1.32%(240)；转染后不同浓度5-FU(μg/ml)作用后细胞存活率为91.3±1.43%(30),84.6±2.13%(60),73.2±1.52%(120)和65.5±0.94%(240)。流式细胞术检测转染前不同浓度5-FU(μg/ml)作用后亚G1期比率(凋亡期)为5.37±0.43%(30),6.49±2.06%(60),9.02±0.34%(120)和12.33±2.46%(240)。增殖指数为47.54±2.33%(30),46.69±2.12%(60),46.01±1.87%(120)和44.29±1.78%(240)。而转染后不同浓度5-FU(μg/ml)作用后亚G1期比率(凋亡期)为7.69±1.04%(30),11.44±0.98%(60),13.57±3.44%(120)和22.42±0.84%(240)。增殖指数为40.97±0.92%(30),38.51±2.03%(60),36.39±2.67%(120)和34.04±3.21%(240)。统计学分析显示转染后相同5-FU浓度下细胞存活率降低,凋亡期比率上升,增殖指数下降,差异均具有统计学意义(P<0.01)。MTT检测转染前不同浓度PA(μg/ml)作用后细胞存活率为98.5±2.34%(0.2),95.2±1.32%(2),89.4±0.68%(10)和82.7±1.73%(20)；转染后不同浓度PA(μg/ml)作用后细胞存活率为94.2±0.78%(0.2),86.5±2.13%(2),78.7±1.34%(10)和70.1±0.76%(20)；。流式细胞术检测转染前不同浓度PA(μg/ml)作用后亚G1期比率(凋亡期)为3.51±0.35%(0.2),5.41±1.03%(2),9.48±1.09%(10)和10.91±1.03%(20)。增殖指数为46.25±1.27%(0.2),46.05±1.98%(2),41.75±2.44%(10)和41.04±1.71%(20)。而转染后不同浓度PA(μg/ml)作用后亚G1期比率(凋亡期)为5.66±1.32%(0.2),11.02±0.87%(2),19.53±1.23%(10)和26.31±2.01%(20)。增殖指数为44.20±2.52%(0.2),41.37±2.51%(2),35.04±2.41%(10)和32.97±2.89%(20)。统计学分析显示转染后相同PA浓度下细胞存活率降低,凋亡期比率上升,增殖指数下降,差异均具有统计学意义(P<0.01)。
结论：1、表达载体介导GSTP1-shRNA554转染后可降低雄激素非依赖型前列腺癌DU145细胞GSTP1基因mRNA、GSTP1蛋白水平,抑制体外培养DU145增殖活性,并且呈现作用时间依赖性。
2、表达载体介导GSTP1-shRNA554转染后可将雄激素非依赖型前列腺癌DU145细胞诱导凋亡,增殖指数下降。
3、表达载体介导GSTP1-shRNA554转染后提高雄激素非依赖型前列腺癌DU145细胞对化疗药物的敏感性,相同药物浓度下转染后DU145细胞存活率降低,凋亡期比率上升,增殖指数下降。
第四部分ERK抑制剂PD98059对DU145细胞中GSTP1表达及细胞凋亡的影响研究
目的：检测细胞外信号激酶ERK抑制剂PD98059对DU145细胞GSTP1蛋白水平的影响,及其对细胞存活率和凋亡水平的影响。
方法：将细胞外信号激酶ERK抑制剂PD98059作用DU145细胞,通过Western Blot检测不同浓度PD98059对于P-ERK、GSTP1蛋白水平的影响,采用MTT法检测细胞存活率,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记技术(terminal de-oxynucleotidyl transferase-mediated dUTP nick end labeling, TUNEL)检测DU145细胞凋亡水平。
结果：不同浓度ERK抑制剂PD98059作用后P-ERK和GSTP1蛋白表达水平以吸光度表示,当PD98059作用浓度分别为0μmol/L, 5μmol/L,10μmol/L,20μmol/L,40μmol/L时, Western Blot检测P-ERK含量为485.93±12.1,397.02±7.8,351.11±7.2,230.54±6.5,,142.97±13.2。GSTP1蛋白表达水平为380.74±14.4,285.57±9.87,231.45±6.43,190.87±11.2,102.08±14.4。PD98059作用后P-ERK和GSTP1蛋白表达水平随浓度增高而下降,各浓度组间差别具有统计学意义,P<0.01。ERK抑制剂PD98059作用后TUNEL检测DU145细胞凋亡率,当PD98059作用浓度分别为0μmol/L,5μmol/L,10μmol/L PD98059, 20μmol/L,40μmol/L时,凋亡率为5.54±0.39%,10.46±0.88%,19.8±1.64%,36.2±2.39%,49.2±2.59%。随PD98059浓度增加细胞凋亡率升高,不同浓度组间差异具有统计学意义,P<0.01。
结论：1、ERK抑制剂PD98059作用于DU145细胞后,对P-ERK、GSTP1蛋白表达呈抑制效应,并且呈明显的剂量效应关系。
2、ERK抑制剂PD98059作用于DU145细胞后诱导细胞凋亡,凋亡的诱导作用呈显著剂量依赖性。
3、ERK信号通路可能参与调控GSTP1蛋白的表达。
Background Hormone-refractory prostate cancer (HRPC) has no effective treatment at present, and the new therapy for HRPC is a focus of research around the world. After endocrine treatment of androgen deprivation the prostate carcinoma will usually get the progression of androgen-independent prostate cancer (AIPC) or even hormone-refractory prostate cancer. The AIPC is responsive to some secondary endocrine treatment, but the HRPC is irresponsive to any endocrine treatment and the disease gets development during the endocrine treatment. About 28,900 people died of HRPC annually in America. The incidence of prostate cancer increases is increasing rapidly in China as the improvement of living quality and prolongation in life span. In our country the prostate cancer took the third place in common malignant tumor in the genitourinary system. Two thirds of the prostate cancer patients lose the chance of radical cure, for whom the maximum androgen blockade (MAB) is the only choice. But after a median period of 12-18 months,70%-80% converted to androgen independent prostate cancer and recurrence or metastasis occurs. The mean survival time is about 1 year. As lack of efficient treatment for HRPC, it is the focus in carcinoma research.
Glutathione S-transferases (GSTs) is a kind of dimeride isoenzyme, which has powerful catalytic action. The GSTs could catalyse electron binding centre of the substrate molecule to conjugate with reduced glutathione to promote the metabolism and inactivation of the substrate. According to the location in chromosome and the sequence homology, the superfamily of GSTs consists ofα、μ、π、θfour subfamily. The GST-πprotein produced by GSTP1 gene has close relation with the malignant tumors, which became the focus of research. The reports of research in gastrointestinal tumor show that inhibition of GSTP1 could reduce the drug resistance of tumors. The main inhibitive methods include the signal path block drug and RNA interference to lower the expression level. During the early stage of prostate cancer, the promoter sequence of GSTP1 got hypermethylation, which caused the deletion of GSTP1 expression. As a result the prostate cancer took place and progressed. The detection of GSTP1 promoter methylation condition could be an early diagnostic method for prostate cancer. The effect of androgen deprivation on the expression of GSTP1 has been reported that after androgen deprivation therapy, the tissue got stress reaction and regained the expression of GSTP1. Research reports shew that GSTP1 expressed in HRPC primary lesion and the metastatic lesion. The recovery expression of GSTP1 may result in the resistance of chemotherapy of HRPC.
RNA interference (RNAi) is the technique that invented in 1998 and the development of RNAi is rapid. RNAi is a kind of gene silencing technique, which has high degree of specificity, efficiency and the interfere effect is diffusive. As a result the inhibitive effect on target gene is powerful. RNA interference is an effective gene silencing process, and the mechanism is post transcription inhibition. The process could divide into initiation and effector stage. In initiation stage the RNAaseⅢfabricated the lipofectamine or virus imported dsRNA in to siRNA of 21-23nt. In the effector phase the double strand of the siRNA unrolled and the anti-sense siRNA combined the related protein to form the active RNA induced silencing complex (RISC). RISC then recognize the mRNA with the completely complementary sequences and make the mRNA degrade. The process has cascade amplification. At present the RNAi via lipofectamine or virus to treat the prostate cancer is in the experiment stage.
In this experiment GSTP1 is taken as the target of gene therapy, and the application of short hairpin RNA is taken to silence the GSTP1 gene. The GSTP1 shRNA is constructed and transfected to androgen independent prostate cancer cell line DU145. Then RT-PCR, Western Blotting are used to detect the mRNA and protein level of GSTP1 to evaluate the effect of RNA interference. After transfection the MTT, flow cytometry are used to assess the RNAi effect on the biological behavior of DU145. The sensitivity to chemotherapeutics of DU145 before and after the transfecion are compared. TUNEL was used to detect the effect of ERK inhibitor PD98059 on the apoptosis of prostate cancer cell line.
Part I
Study on the expression of GSTP1 in the prostate cancer tissue and plasma
Objective Analyze and compare the expression level of GSTP1 in the prostate cancer tissue and plasma of the hormone-refractory prostate cancer and prophase localized prostate cancer.
Methods Immunohistochemistry analysis is used to detect the expression of GSTP1 in 10 cases of prophase localized prostate cancer(PCa) tissue, and 10 cases of hormone-refractory prostate cancer(HRPC) tissue. In control 10 cases of benign prostate hyperplasia(BPH) tissue were analyzed.
Enzyme-linked immunosorbent assay (ELISA) is used to detect the expression of GSTP1 in plasma of 10 prophase localized prostate cancer patients, and 10 cases of HRPC patients. In control 10 cases of BPH patients'plasma were analyzed.
Results The expression result of GSTP1 in 10 cases of prophase localized prostate cancer tissue:Negative 8 cases, weakly positive 1 case, middling positive 1 case, the positive ratio was 20%. The expression result of GSTP1 in 10 cases of HRPC tissue:Negative 2 cases, weakly positive 1 case, middling positive 4 cases, obviously positive 3 cases, the positive ratio was 80%. In control 10 cases of BPH tissue:no negative case,1 case weakly positive,2 cases middling positive and 7 cases obviously positive, the positive ratio was 100%. Wilcoxon rank test shew that the GSTP1 positive ratio of HRPC is higher than that of PCa, P <0.01, but lower than that of BPH, P<0.01. The concentration of GSTP1 in plasma was 2033.39±232.75pg/mL in 10 cases of PCa patients; 3879.89±210.32pg/mL in 10 cases of HRPC patients; 4019.07±170.33pg/mL in 10 cases of BPH patients. The HRPC group had higher GSTP1 concentration than the PCa group, P<0.01; the HRPC group had lower GSTP1 concentration than the BPH group, P<0.01.
Conclusion
1. The prostate cancer tissue of hormone-refractory prostate cancer has higher GSTP1 expression than that of prophase localized prostate cancer.
2. The concentration of GSTP1 in plasma of patients with hormone-refractory prostate cancer is higher than that of patients with prophase localized prostate cancer.
PartⅡ
Detection of the mRNA and protein expression level of gene GSTP1 in the prostate cancer cells line DU145, PC3 and LNCaP
Objective Compare the mRNA and protein expression level of gene GSTP1 among the prostate cell line DU145, PC3 and LNCaP.
Methods RPMI 1640 nutrient medium is used to culture the prostate cancer cell line DU145, PC3 and LNCaP. RT-PCR is used to detect the mRNA level of gene GSTP1 of the three cell lines, and western blot is used to detect the GSTP1 protein level.
Results Semi-quantitive RT-PCR was used to detect the level of the GSTP1 mRNA and the absorbance of light indicate the expression ratio. Of the androgen independent prostate cancer cell line DU145 the GSTP1 mRNA was 346.7±7.3; in PC3 229.0±1.4; of the androgen dependent prostate cancer cell line LNCaP the GSTP1 mRNA was 73.5±2.2. Compared with the androgen dependent prostate cancer cell line LNCaP, the androgen independent prostate cancer cell line DU145 and PC3 had higher level of GSTP1 mRNA, P<0.01. DU145 had higher level of GSTP1 mRNA than that of PC3, P<0.01. Western blot was used to detect the level of GSTP1 protein level in the three cell lines. The absorbance of light indicated the GSTP1 protein level. Of the androgen independent prostate cancer cell line DU145 the GSTP1 protein was 103.1±1.1; PC3 78.5±0.5; of the androgen dependent prostate cancer cell line LNCaP the GSTP1 protein was 24.4±1.2. Compared with the androgen dependent prostate cancer cell line LNCaP, the androgen independent prostate cancer cell line DU145 and PC3 had higher level of GSTP1 protein, P<0.01. DU145 had higher level of GSTP1 protein than that of PC3,P<0.01.
Conclusion
1. The androgen independent prostate cancer cell lines DU145 and PC3 have higher level of GSTP1 mRNA and GSTP1 protein, compared with that of androgen dependent prostate cancer cell line LNCaP.
2. DU145 has higher level of GSTP1 mRNA and GSTP1 protein than that of PC3.
PartⅢ
The effect of gene GSTP1 silencing via shRNA transfection on the androgen independent prostate cancer cell line DU145
Objective To design special shRNA interference sequence for transfection to the androgen independent prostate cancer cell line DU145 to silence the gene GSTP1, and then to investigate the effect on proliferate activity and sensitivity to chemotherapeutics.
Methods Through software design and review of related references, the target sequence was picked up to form the shRNA, and the DNA template was cloned to the shRNA expression vector pGPU6/GFP/Neo to construct the plasmid with the targeting inhibitive shRNA of gene GSTP1. The shRNA was identified by gene sequencing. The screening experiment was taken to pick up the shRNA expression vector with highest transfection ratio and best gene silencing result. Lipofectamine was used to mediate transfection of plasmid to DU145, and RT-PCR, western blot was used to detect the level of mRNA and protein of GSTP1 which indicated the effect of transfection. MTT analysis and flow cytometry were used to evaluate the effectiveness of shRNA transfection. TO compare the sensitivity to chemotherapeutics before and after transfecion, MTT analysis and flow cytometry were used to detect the proliferate activity of DU145 after the 5-FU or PA of different concentration added.
Results The constructed vectors including three shRNA expression vector and the negative vector. They were pGPU6/GFP/Neo-shRNA255, pGPU6/GFP/Neo-shRNA554, pGPU6/GFP/Neo-shRNA593 and Negative-shRNA. In preliminary experiment, the transfection efficiency of shRNA255、shRNA554 and shRNA593 was 63.3±1.04%,76.2±0.68% and 72.7±0.33% respectively. In which the shRNA554 had highest transfection efficiency, P< 0.01. In preliminary experiment, after transfection of shRNA255, shRNA554 and shRNA593 the gene GSTP1 mRNA level detected by RT-PCR of DU145 was 128.31±2.5,43.24±4.3 and 85.62±6.3 respectively, and the GSTP1 protein level detected by western blot of DU145 was 163.92±12.4,65.38±9.3 and 114.25±16.7 respectively. Statistical analysis indicated that shRNA554 of highest transfection ratio and best gene silencing result, P< 0.01. After transfection of shRNA554 to DU145, on the 2 day,4 day and 6 day the level of GSTP1 protein was 125.44±10.85,106.52±11.2 and 56.43±8.76 respectively. While the level after the transfection of blank plasmid was 174.35±7.2,168.09±6.54 and 171.72±8.25 respectively. The data indicated that after transfection of shRNA554 to DU145 the GSTP1 protein level decreased(P<0.01), and decreased progressively as the extension of transfection time, which indicated the time dependence(P< 0.01). Similarly MTT analysis shew that after 2 days,4 days and 6 days after transfection the survival ratio of DU145 decreased progressively as the extension of transfection time, which indicated the time dependence(P < 0.01). Flow cytometry indicated that the ratio of subGl phase transfected with shRNA554 was higher than that of blank plasmid(P< 0.01), and the proliferate ratio decreased(P< 0.01). MTT analysis indicated that before transfection, the survival ratio of DU145 added different concentration of 5-FU (μg/ml) were:95.6±2.11%(30), 90.2±0.86%(60),83.1±3.12%(120) and 74.6±1.32%(240); while after transfection the survival ratio of DU145 added different concentration of 5-FU (μg/ml) were:91.3±1.43%(30),84.6±2.13%(60),73.2±1.52%(120) and 65.5±0.94%(240). Flow cytometry indicated that before transfection, the subGl phase(apoptosis cell) ratio of DU145 with different concentration of 5-FU (μg/ml) added were:5.37±0.43%(30), 6.49±2.06%(60),9.02±0.34%(120) and 12.33±2.46%(240); the proliferate index were:47.54±2.33%(30),46.69±2.12%(60), 46.01±1.87%(120) and 44.29±1.78%(240); while after transfection the subGl phase(apoptosis cell) ratio of DU145 with different concentration of 5-FU (μg/ml) added were:7.69±1.04%(30),11.44±0.98%(60), 13.57±3.44%(120) and 22.42±0.84%(240); the proliferate index were: 40.97±0.92%(30),38.51±2.03%(60),36.39±2.67%(120) and 34.04±3.21%(240). Compared with the data before transfection, statistical analysis indicated that after transfection, under the same concentration of 5-FU, the survival ratio decreased, apoptosis increased and the proliferate index decreased of statistical significance, P<0.01. MTT analysis indicated that before transfection, the survival ratio of DU145 added different concentration of PA (μg/ml) were:98.5±2.34%(0.2), 95.2±1.32%(2),89.4±0.68%(10) and 82.7±1.73%(20); while after transfection the survival ratio of DU145 added different concentration of PA (μg/ml) were:94.2±0.78%(0.2),86.5±2.13%(2),78.7±1.34%(10) and 70.1±0.76%(20). Flow cytometry indicated that before transfection, the subG1 phase(apoptosis cell) ratio of DU145 with different concentration of PA (μg/ml) added were:3.51±0.35%(0.2),5.41±1.03%(2), 9.48±1.09%(10) and 10.91±1.03%(20); the proliferate index were: 46.25±1.27%(0.2),46.05±1.98%(2),41.75±2.44%(10) and 41.04±1.71%(20); while after transfection the subGl phase(apoptosis cell) ratio of DU145 with different concentration of PA(μg/ml) added were: 5.66±1.32%(0.2),11.02±0.87%(2),19.53±1.23%(10) and 26.31±2.01%(20); the proliferate index were:44.20±2.52%(0.2), 41.37±2.51%(2),35.04±2.41%(10) and 32.97±2.89%(20). Compared with the data before transfection, statistical analysis indicated that after transfection, under the same concentration of PA, the survival ratio decreased, apoptosis increased and the proliferate index decreased of statistical significance, P<0.01.
Conclusion
1. The shRNA expression vector-transfected to androgen independent prostate cancer cell line DU145 could reduce the level of GSTP1 mRNA and GSTP1 protein, in vitro inhibition of the proliferate activity of DU145 in a manor of time dependent.
2. The shRNA expression vector-transfected to androgen independent prostate cancer cell line DU145 could induce apoptosis and decrease the proliferate index.
3. The shRNA expression vector-transfected to androgen independent prostate cancer cell line DU145 could raise the sensitivity to chemotherapeutics, under the same concentration of chemotherapeutics, the survival ratio of DU145 decreased, apoptosis increased and the proliferate index decreased.
Part IV
The effect of ERK inhibitor PD98059 on the expression of GSTP1 protein and apoptosis of DU145
Objective To detect the effect of the extracellular regulated kinase (ERK) inhibitor PD98059 on the expression of GSTP1 of DU145, and the effect on the survival ratio and apoptosis level of DU145.
Methods ERK inhibitor PD98059 was added to DU145, then western blot was used to detect the effect of PD98059 with different concentration on the level of P-ERK and GSTP1. MTT analysis was used to detect the cell survival ratio and TUNEL was used to detect the apoptosis level of DU145 when treated with PD98059.
Results After treated with different concentration of PD98059, the protein level of P-ERK and GSTP1 protein were detected by western blot and the absorbance of light indicated the protein level. The DU145 was treated with different concentration of PD98059 0μmol/L,5μmol/L, 10μmol/L,20μmol/L,40μmol/L; the P-ERK level was 485.93±12.1, 397.02±7.8,351.11±7.2,230.54±6.5,142.97±13.2 respectively; the GSTP1 protein level was 380.74±14.4,285.57±9.87,231.45±6.43, 190.87±11.2,102.08±14.4 respectively. The level of P-ERK and GSTP1 protein decreased progressively as the concentration of PD98059 incresed, and the difference between every two different concentration group is statistically significant, P<0.01. The DU145 was treated with different concentration of PD98059 0μmol/L,5μmol/L,10μmol/L,20μmol/L, 40μmol/L; the apoptosis ratio detected by TUNEL was 5.54±0.39%, 10.46±0.88%,19.8±1.64%,36.2±2.39%,49.2±2.59% respectively. The apoptosis ratio increased progressively as the concentration of PD98059 incresed, and the difference between every two different concentration group is statistically significant, P<0.01.
Conclusion
1. The ERK inhibitor PD98059 has inhibitive effect on the expression of P-ERK, GSTP1 of DU145 in the manner of obvious dose-effect relationship.
2. ERK inhibitor PD98059 induced the DU145 apoptosis in the manner of significant dose-dependent.
3. The ERK pathway may participate in regulation of GSTP1 protein expression.
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