【作者】 谢棒祥；

【导师】 周建光；

【作者基本信息】 中国人民解放军军事医学科学院 ， 遗传学， 2008， 博士

【摘要】 目的:近年来,我国男性前列腺癌发病率呈明显上升趋势,在欧美国家,前列腺癌已连续15年居男性恶性肿瘤发病率首位,其发病率超过肺癌,死亡率在男性恶性肿瘤中居第二位。前列腺癌是一个起源于多病灶和随机进展模式的异质性疾病。到目前为止对晚期和转移性尤其是激素非依赖型、放疗和化疗抵抗型前列腺癌依然没有治愈的疗法。研究前列腺癌进展相关的基因可能揭示其机制或找出分新的治疗靶标。本研究拟应用基因芯片、RT-PCR、Realtime-PCR、免疫组化等技术筛选出前列腺癌分子标志物候选基因、雄激素反应性基因及电离辐射诱导基因从而为探索前列腺癌的进展、雄激素非依赖的产生及辐射抗性相关的分子机制提供线索和依据,为临床诊断与治疗提供新的途径和可能。结果:1、应用Affymetrix公司的人类全基因组U133A芯片筛选出LNCaP与C4-2细胞系间差异表达的基因/转录本1053个;仅在LNCaP或C4-2中独立表达的基因/转录本分别是739和835个。根据差异倍数、染色体定位及研究进展确定104个候选基因/转录本进行RT-PCR验证获得76个基因/转录本与芯片结果一致的候选基因/转录本。2、为了筛选出前列腺癌分子标志物候选基因,用RT-PCR检测上述76个候选差异基因/转录本在BPH1、LNCaP、C4-2、C4-2B、PC—3、DUl45、MCF-7、HEPG2、Hela、PC12、293T 11种细胞系中的表达谱。其中与前列腺癌进展程度呈正相关特异性较好的基因/转录本26个,与前列腺癌进展程度呈负相关特异性较好的基因/转录本17个。3、为了筛选出雄激素调控基因,分别用1nM R1881和20μM Casodex处理LNCaP和C4-2,用RT-PCR检测76个候选基因/转录本在LNCaP、C4-2中处理前后的表达。其中在LNCaP细胞中R1881应答基因49个,Casodex应答基因42个。在C4-2细胞中R1881应答基因16个,Casodex应答基因15个。4、为了探索适合电离辐射诱导基因筛选的遗传背景相同的辐射敏感性和辐射抗性细胞模型,分别用MTT、平板克隆形成、软琼脂克隆形成、周期变化检测了LNCaP和C4-2细胞系对5 Gy辐射的反应,结果证明了LNCaP的辐射敏感性和C4-2的辐射抗性。用Real-time PCR检测LNCaP和C4-2间p53、p21、Chek1、Chek2、Bcl-2、ATR、MRE11间表达的差异,结果:C4-2与LNCaP相比,p53、Chekl、Bcl-2、ATR、MRE11皆下调表达,同时检测了5、10Gy辐射后24小时LNCaP和C4-2上述7基因的表达差异,结果:LNCaP细胞5Gy辐射后24小时,p21上调表达,Chek1、Chek2、Bcl-2、MRE11下调表达,10Gy辐射后24小时,p21上调表达、Chek1、Chek2下调表达。C4-2细胞5Gy辐射后24小时,p53、p21、Chek1、Bcl-2、ATR、MRE11上调表达,10Gy辐射后24小时p21、Chek1、Bcl-2、ATR、MRE11上调表达。以上基因的差异表达是LNCaP辐射敏感性和C4-2辐射抗性的分子基础。5、为了筛选出电离辐射诱导基因分别用0、2.5、5、10Gy ⁶⁰Coγ射线辐射处理LNCaP和C4-2细胞,用RT-PCR检测76个候选基因/转录本在上述处理后6、12、24、48、72小时的表达,其中在LNCaP细胞中辐射应答31个基因/转录本。6、根据上述前列腺癌分子标志物、雄激素反应性基因及电离辐射诱导基因3方面的筛选结果综合考虑再次筛选出25个候选基因/转录本,用Real-timeRT-PCR再次验证它们在LNCaP与C4-2细胞间的差异表达。25个基因Real-time RT-PCR验证结果与芯片结果、RT-PCR验证结果一致。7、为了验证上述筛出的前列腺癌分子标志物的可靠和实用性,用组织切片和组织芯片做免疫组化对AGR2和RPS4Y1临床表达进行了检测,其中AGR2胞浆阳性,其表达与恶性程度呈正相关,这与表达谱结果一致,RPS4Y1胞浆阳性,表达与恶性程度呈负相关,这与表达谱结果相符。该结果说明上述筛出的前列腺癌分子标志物候选基因有可能成为真正的前列腺癌分子标志物应用到临床。8、为了探索上述筛出的雄激素调控基因及电离辐射诱导基因在前列腺癌雄激素非依赖和辐射抗性产生中扮演的角色,首先建立了ELF5-2b和ACADL的前列腺癌细胞系高表达稳定克隆并且得到了ELF5-2b和ACADL高表达的Real-time RT-PCR结果。接着,为了研究ELF5-2b和ACADL高表达对前列腺癌细胞生长、克隆形成能力、恶性进展、雄激素非依赖性、辐射抗性的影响,用MTT、平板克隆形成、软琼脂克隆形成、Casodex和辐射处理后MTT对此开展研究,结果:ELF5-2b的高表达促进了LNCaP和C4-2细胞的生长、克隆形成能力及恶性程度进展,提高了LNCaP细胞雄激素的非依赖性/Casodex耐受能力及抗辐射能力。ACADL的高表达促进了C4-2细胞的生长、克隆形成能力及恶性程度进展,提高了C4-2细胞雄激素的非依赖性/Casodex耐受能力及抗辐射能力。为了探讨ELF5-2b和ACADL高表达诱导或促进前列腺癌细胞雄激素非依赖性/Casodex耐受能力及辐射抗性进展的分子基础,用Real-timeRT-PCR分别检测了ELF5-2b和ACADL高表达对AR、PSA、p53、p21、Chek1、hek2、Bcl-2、ATR、MRE11表达的影响,结果发现ELF5-2b在LNCaP中高表达分别促进了PSA、p53、、ATR、MRE11的表达却抑制了Chek2的表达。ELF5-2b在C4-2中高表达分另0促进了AR、PSA、p21、Chek2、Bcl-2、ATR、MRE11的表达。ACADL在C4-2中高表达分别促进了p21、Bcl-2、ATR、MRE11的表达却抑制了PSA、Chek1的表达。ELF5-2b和ACADL高表达对这些分子表达的影响与ELF5-2b和ACADL高表达促进了前列腺癌细胞雄激素非依赖性/Casodex耐受能力及辐射抗性的进展是相吻合的。从而证实了上述筛出的雄激素调控基因及电离辐射诱导基因的可靠性,提示前列腺癌雄激素非依赖和辐射抗性产生可能与雄激素调控基因及电离辐射诱导基因密切相关。结论:1、LNCaP与C4-2间存在差异表达基因/转录本1023个:其中在C4-2中高表达的690个,低表达的333个,经RT-PCR验证的76个,经过Real time RT-PCR验证的25个。2、筛选出分子标志物候选基因43个,受雄激素调控候选基因62个,受辐射诱导的候选基因31。3、前列腺癌细胞系C4-2具有辐射抗性,LNCaP和C4-2具有相同的遗传背景,不但能够用作研究前列腺癌雄激素依赖和非依赖进展的细胞模型,还可以用作研究前列腺癌辐射敏感和抗性进展的细胞模型。4、AGR2和RPS4Y1的表达与前列腺癌的进展程度分别呈正相关和负相关。5、ELF5-2b的高表达能够促进前列腺癌细胞的生长、存活及恶性进展,诱导前列腺癌细胞雄激素非依赖性和辐射抗性的发生和进展而且具有前期阶段性。6、ACADL的高表达能够促进前列腺癌细胞的生长、存活及恶性进展,诱导前列腺癌细胞雄激素非依赖性和辐射抗性的进展而且具有后期阶段性。更多还原

【Abstract】 Objective:The incidence of prostate cancer has been increasing in recent years in Chinese males,In the western countries,the incidence of prostate cancer has been remained in the first position over lung cancer for continuous 15 yeas in male malignant carcinomas,and prostate cancer has become the second leading cause of death in male malignant carcinomas.Prostate cancer is a kind of alloplastic disease derived from multiple nidus and developing in random mode.Effective therapeutic methods remain to be found for the advanced prostate cancer with metastasis, especially the androgen-independent and chemical and radio therapy resistant prostate cancer.Our investigation was designed to find novel molecular biomarkers, androgen-responsive genes and radiation induced genes using Cdna microarray, RT-PCR,Real time PCR and immunohistochemistry methods to provide clues and foundation for exploring the molocular mechanism mediating prostate cancer development to androgen-independence and radiation resistence and provide novel insight for clinical diagnosis and therapy.Methods and Results:1.1053 genes/transcripts were screened out to be differently expressed in LNCaP and C4-2 using U133A microarray chip from Affymetrix company.739 and 835 genes/transcripts were screened out to be expressed solely in LNCaP and C4-2 respectively.104 genes/transcripts were selected and 76 genes/transcripts differential expression were confirmed to be consistent with microarray results using RT-PCR.2.To screen out the molecular biomarker of PCa,RT-PCR was performed to examine the expression pattern of the 76 genes described above in BPH1、LNCaP、C4-2、C4-2B、PC-3、DU145、MCF-7、HEPG2、Hela、PC12、293T cell lines.26 genes expression were screened out to be positively associated with prostate cancer development and prostate tissue specific.17 genes expression were found out to be negatively associated with prostate cancer progression and possess prostate tissue specific characteristic.3.To screen out androgen responsive genes,1nM R1881 and 20μM Casodex were used to treat LNCaP and C4-2.RT-PCR were performed to examine the androgen responsive characteristics in the 76 genes described above.As the result,49 R1881 responsive genes were screened out and 42 genes were found to be responsive to Casodex in LNCaP;In C4-2 cell,16 genes were found to be responsive to R1881 and 15 responsive to Casodex.4.To investigate whether LNCaP and C4-2 are suitable to be radiation sensitive/resistant model cell lines for screening the radiation induced genes,MTT, soft agar colony formation and cell cycle assays were performed to detect the 5 Gy radiation response in LNCaP and C4-2 respectively.As the result,LNCaP and C4-2 were found to be radiation sensitive and resistant cell lines respectively. Real-time PCR were performed to examine the differential expression of p53、p21、Chek1、Chek2、Bcl-2、ATR、MRE11 in LNCaP and C4-2.As the result, expression of p53、Chek1、Bcl-2、ATR、MRE11 genes were found to be up-regulated in LNCaP cells compared with C4-2 cells.Then the radiation responses were examined in the 7 genes described above using 5 or 10 Gy radiation in LNCaP and C4-2.As the result,in LNCaP cell line,p21 expression was up-regulated in 24hs post-5Gy-radiation and the expression of Chek1,Chek2, Bcl-2,MRE11 were found to be down-regulated in the same condition.In 24hs post-10Gy-radiation p21 expression was up-regulated and the expression of Chek1,Chek2 were found to be down-regulated in the same condition.In C4-2 cell line,p53、p21、Chek1、Bcl-2、ATR、MRE11 expression were enhanced in 24hs post-5Gy radiation.The expression of p21、Chek1、Bcl-2、ATR、MRE11 were found to be increased in 24hs post-10Gy radiation.The genes expression differentiation provide the molecular foundation for the radiation sensitive LNCaP and radiation resistant C4-2.5.To screen out the radiation induced genes,LNCaP and C4-2 cells were treated with 0,2.5,5,10Gy Co60 radiation and RT-PCR were performed to examine the expression of the 76 candidate genes described above 6,12,24,72 hours post radiation.As the result,31genes/transcript were found to be responsive to the radiation.6.Base on the results of the screening ofbiomarker,androgen response and radiation response described above,25 genes/transcript were selected and real-time PCR were performed to confirm the differential expression pattern of these genes in LNCaP and C4-2.The results were demonstrated to be consistent with the microarray and RT-PCR assays.7.To confirm the reliability and practicality of the results from PCa molecular marker screening,immunohistochemistry were performed using the antibodies against AGR2 and RPS4Y1 respectively.The results revealed that AGR2 expression was positive in cytoplasma and in positive relation to the malignant level,which is consistent with the expression pattern analysis.Moreover,RPS4Y1 expression was also found to in cytoplasma and negatively associated with the malignant level,consistent with the expression pattern analysis.These results revealed that the molecular screened may be applied to the clinic as molecular marker for PCa. 8.To evaluate the role of the androgen and radiation responsive genes in the androgen independent and radiation resistant development of prostate cancer,the prostate cancer cell lines stably overexpressing ELF5-2 and ACADL respectively were constructed.The MTT,plate colony formation assay,soft agar colony formation assay and Casodex or radiation treatment were performed to investigate the effect of ELF5-2 and ACADL overexpression on the growth ability, anchorage-independent growth ability and androgen-dependence,radiation resistence and malignant progress in prostate cancer.The results demonstrated that ELF5-2 overexpression promoted the growth ability,colony formation and malignant progress in LNCaP and C4-2 and augmented the androgen-independence,Casodex and radiation resistence in LNCaP.Moreover, ACADL expression increased the growth ability,colony formation and malignant progress in C4-2,and promoted the androgen-independence,Casodex and radiation resistence in C4-2.In order to investigate the molecular mechanism mediating ELF5-2 and ACADL function,real-time PCR were performed to examine the expression changes of AR,PSA,p53,p21,Chek1,Chek2,Bcl-2, ATR,MRE11 mediated by ELF5-2 and ACADL overexpression.The results revealed that ELF5-2 expression up-regulated the expression of PSA、p53、、ATR、MRE11 and inhibited the expression of Chek1 in LNCaP.ELF5-2 overexpression in C4-2 increased the expression of AR、PSA、p21、Chek2、Bcl-2、ATR、MRE11. Moreover,ACADL expression in C4-2 promoted the expression of p21、Bcl-2、ATR、MRE11 and inhibited the expression of PSA、Chekl.These molecular results are consistent with the function of ELF5-2 and ACADL in promoting androgen-independence,Casodex and radiation resistence.These results also show the reliability of the screening assay,indicating that the androgen-independent and radiation resistant progress of PCa may be significantly associated with androgen and radiation responsive genes.Conclusions:1.1023 genes/transcripts were screened out to be differentially expressed between LNCaP and C4-2.690 genes/transcripts expression are up-regulated and 333 are down-regulated in C4-2.76 genes/transcripts expression differentiation were confirmed using RT-PCR,and 25 were confirmed using Real-time PCR.2.43 candidate molecular markers,62 androgen induced and 31 radiation induced genes/transcripts were screened out.3.C4-2 possesses radiation resistant property compared with parent LNCaP cell line. LNCaP and C4-2 prostate cancer progress model can be used not only for investigation of androgen-independent progress,but also for research of radiation resistence in prostate cancer. 4.AGR2 and RPS4Y1 were demonstrated to be positively and negatively associated with prostate cancer progress respectively.5.ELF5-2b overexpression promotes cell growth,survival and malignant progress, moreover induces androgen independence and radiation resistence in early stage of prostate cancer development.6.ACADL overexpression enhances cell growth,survival and malignant progress, moreover induces androgen independence and radiation resistence in late stage of prostate cancer development.更多还原