前列腺癌细胞LNCaP激素非依赖性转变过程中microRNA的变化研究
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摘要
一、研究背景
自1941年Huggins等采用雄激素剥夺疗法治疗晚期前列腺癌取得显著疗效后,激素治疗逐渐成为治疗前列腺癌的一项重要的方法。但随着激素治疗在前列腺癌治疗中的广泛应用,人们发现雄激素剥夺疗法的效果平均只有12～16个月,此后前列腺癌继续生长、恶化,一年生存率仅为50%,临床上将这种情况称为激素非依赖性前列腺癌。因此,激素非依赖性前列腺癌的研究成为泌尿外科学中一个重要课题。
目前对激素非依赖前列腺癌的研究热点主要集中在雄激素受体、细胞凋亡及细胞周期的调控、多肽生长因子、肿瘤血管形成和一些与激素非依赖密切相关的基因等多个方面。
虽然这些研究从不同的角度为激素非依赖前列腺癌的发生机制,提供了许多有价值的信息。但是,人们对前列腺癌从激素依赖向激素非依赖转变过程的分子生物学改变仍未能透彻了解。主要的研究障碍就是缺乏理想的细胞模型,来模拟体内前列腺癌患者由激素依赖向激素非依赖转变的过程。一个理想的前列腺癌细胞模型应该能够表现出激素依赖性的特性,表达野生型雄激素受体,能够在激素剥夺的环境下转变为激素非依赖性。已有的前列腺癌细胞系,如PC-3、DU145均不表达雄激素受体,CWR22R表达的是突变的雄激素受体,而且这些细胞系均能够在激素剥夺的环境下生长。目前仅有LNCaP细胞系保留了人前列腺癌的激素依赖性的特征,能够分泌PSA、PSMA及雄激素受体,能够在具有雄激素样活性的甾体激素的刺激下生长。最近的研究表明LNCaP细胞系能够在激素剥夺的环境下逐渐转变为激素非依赖的LNCaP前列腺癌细胞亚系。这种前列腺癌细胞模型可以模拟临床内分泌治疗过程中,前列腺癌由激素依赖发展为雄激素非依赖的过程。同时,由于是从LNCaP前列腺癌细胞系经过干预建立的细胞亚系,两者具有可比性,可以用来观察前列腺癌由激素依赖向非依赖转变过程的分子生物学变化,是一个理想的研究激素非依赖前列腺癌机制的模型。
近几年随着对microRNA(miRNA)重视,越来越多的与细胞分化成熟障碍、增殖失控、细胞永生化为本质的肿瘤疾病密切相关的miRNA被发现,在组织和细胞不同的分化时相中,miRNA有着本身独特的表达方式,即不同时相有不同的表达。目前认为,miRNA在肿瘤发生巾的作用主要在于3个方面:①有些编码miRNA的基因可能起着癌基因的作用;②有些miRNA基因则可能起到抑癌基因的作用;③一些致瘤病毒编码的miRNA也可能参与相关肿瘤的发生。通过临床研究发现,某些特异性miRNA的变化能够对肿瘤诊断及提示预后有帮助,甚至于与临床分期分级有关。并且在急性白血病中应用mir-16和mir-15基因治疗取得有意义的进展,开拓了肿瘤治疗的新纪元。提出重新使miRNA的表达平衡(把升高的miRNA降低、降低的予以升高)可能是肿瘤治疗的一个方向。由于对前列腺癌研究microRNA的方法不同,可能有不同的结果,甚至相反。也由于对microRNA功能及机理还没有完全了解,microRNA对靶基因的调控不是一对一的关系,而是一个microRNA可以调控多个靶基因,在不同的细胞条件,其功能可能变化等复杂情况,还不能解释其中的差异。对前列腺癌相关的microRNA还有大量没有明确。系统、全面的研究其功能及表达,从而利用microRNA来对肿瘤定性分级及寻找出治疗靶点,是将来的一个新方向。尽管有初步研究提示一些microRNA与前列腺癌非雄依赖转化有关,但还有许多没有系统的研究,及早明确哪些microRNA参入了非雄依赖转化及其功能,是研究前列腺癌非雄依赖转化的一个新起点。可以相信,microRNA将是肿瘤(包括前列腺癌)研究的新热点领域。
二、研究目的
本研究利用激素依赖的LNCaP前列腺癌细胞系在体外诱导下建立激素非依赖LNCaP前列腺癌细胞亚系,模拟前列腺癌在激素剥夺治疗后由激素依赖性向非依赖性转变的过程,对其发生机制进行初步探讨,并为进一步深入研究激素非依赖前列腺癌的发生机理,提供一个可靠的平台。通过对比LNCaP-AI(雄激素非依赖细胞)与LNCaP细胞中microRNA的变化,进一步阐述非雄转化的机制。
三、主要研究内容
1、体外长期在去激素的血清环境下培养LNCaP细胞,培养出能够适应无激素环境的LNCaP细胞亚系(LNCaP-AI细胞系)。
2、通过CCK-8方法验证LNCaP-AI细胞系在无激素环境下的增殖能力;荧光免疫的方法检测LNCaP-AI细胞系分泌PSA的情况。
3、RT-PCR的方法检测LNCaP-AI细胞系AR基因表达情况,利用Agilent microRNA寡核苷酸基因芯片对LNCaP及LNCaP-AI细胞检测microRNA的变化,并用RT-PCR方法对6个microRNA进行验证,验证过程中,对比利用PC-3细胞及PC-3细胞去雄一周7个microRNA的变化。
4、通过检索已有的非雄依赖转化过程中上调表达的基因,对比我们已测定的microRNA,联系microrna.sanger.ac.uk针对靶基因寻找相关调控的microRNA,来阐述microRNA的变化与已上调基因的关系,从而说明microRNA的变化参入前列腺癌激素非依赖转化。
5、半定量PCR法测定LNCaP及LNCaP-AI细胞促红细胞生成素(EPO)及促红细胞生成素受体(EPOR)的变化,来推断EPO及EPOR是否参入非雄依赖转化的过程。
6、利用P,E标记c-kit抗体及流式细胞仪测定LNCaP及LNCaP-AI细胞膜原癌受体c-kit的表达情况。
四、结果
1、利用活性炭处理的血清培养模拟去雄细胞环境,逐步递减培养基中激素10天,后完全在非雄培养,共连续培养3个月,一周左右开始处于自分泌状态,3个月后逐渐适应了无激素的环境,能够正常增殖,成为激素非依赖的LNCaP-AI细胞亚系。
2、CCK-8方法检测LNCaP-AI细胞能够在无激素的环境下迅速增殖,LNCaP细胞生长则明显受到抑制。荧光免疫法检测在无激素的环境下LNCaP-AI细胞培养液中PSA的分泌水平能够随着时间的推移而逐渐增加,而LNCaP细胞的PSA的分泌则明显受到抑制,始终处于极低的水平。RT-PCR法检测LNCaP-AI细胞表达AR的水平要明显高于LNCaP细胞,表达水平是后者7倍。
3、通过LNCaP-AI细胞和LNCaP细胞Agilent microRNA芯片方法检测发现,LNCaP-AI细胞有37个microRNA降低,有11个microRNA升高。选出6个microRNA验证结果与Agilent microRNA芯片检测结果一致。通过PC-3细胞去雄一周对比前后6个microRNA的变化,提示5个变化与去雄处理相关。
4、通过检索microrna.sanger.ac.uk针对microRNA调控的靶基因,参考已发现与非雄依赖相关的基因,发现LNCaP转化为激素非依赖的LNCaP-AI细胞中,明显变化的microRNA主要调控AR、EGFR、MMP9、c-kit、bcl-2和雄激素相关代谢酶。认为非雄依赖转化过程中microRNA的变化,通过调高AR、AR旁路信号通路、金属酶、抗凋亡基因及雄激素相关代谢酶基因的表达,参入了前列腺癌非雄依赖转化。
5、半定量PCR法没有发现LNCaP及LNCaP-AI EPO及EPOR有明显的变化,说明EPO及EPOR可能没有参入LNCaP非雄依赖转化。
6、利用P,E标记c-kit抗体及流式细胞仪测定LNCaP及LNCaP-AI细胞膜原癌受体c-kit的表达情况。发现LNCaP-AI细胞膜原癌受体c-kit的表达高于LNCaP,认为通过microRNA的变化,能够调高c-kit的表达,参入非雄依赖的转化。
五、结论
1、本研究利用LNCaP前列腺癌细胞系,在去激素的培养液中长期培养,培养出激素非依赖性的LNCaP-AI前列腺癌细胞亚系。这种前列腺癌细胞模型可以模拟临床上在内分泌治疗过程中,前列腺癌由激素依赖发展为雄激素非依赖的过程。同时,由于是从LNCaP前列腺癌细胞系经过干预建立的细胞亚系,两者有可比性,可以用来观察前列腺痛由激素依赖向非依赖转变过程的分子生物学变化,是一个理想的激素非依赖前列腺癌的细胞模型。
2、通过LNCaP-AI细胞和LNCaP细胞Agilent microRNA芯片方法检测发现,LNCaP-AI细胞有37个microRNA降低,有11个microRNA升高。通过检索microrna.sanger.ac.uk针对microRNA调控的靶基因,参考已发现与非雄依赖相关的基因,发现LNCaP转化为激素非依赖的LNCaP-AI细胞中,明显变化的microRNA主要调控AR、EGFR、MMP9、c-kit、bcl-2和雄激素相关代谢酶。其中,AR、EGFR、bcl-2、MMP9和雄激素相关代谢酶在非雄依赖转化中都是高表达的。发现这些上调的基因相关的microRNA大部分是降低的,根据microRNA调控基因的原理,可以推测这些microRNA可能通过转录后抑制的降低,调高相关基因的表达。认为非雄依赖转化过程中microRNA的变化,通过调高AR、AR旁路信号通路、金属酶、抗凋亡基因及雄激素相关代谢酶基因的表达,参入了前列腺癌非雄依赖转化。利用P,E标记c-kit抗体及流式细胞仪测定LNCaP及LNCaP-AI细胞膜原癌受体c-kit的表达情况。发现LNCaP-AI细胞膜原癌受体c-kit的表达高于LNCaP,认为通过microRNA的变化,能够调高c-kit的表达,参入非雄依赖的转化。
MicroRNAs alterations associated with the prostate cancer LNCaP cell progression to the androgen-independenceBackground
Since androgen ablation was administrated to treat late-stage prostate cancer(Pca) and gained significant therapeutic efficacy by Huggins in 1941, hormone therapy had been an important therapy for PCa. However, with the spreading administration of hormone therapy, people found the effect of androgen ablation remained only 12-16 months, and then PCa cell continued to grow and deteriorate. After that, only about half of the patients could survive beyond 1 year. This situation was called clinically as androgen-independent prostate cancer (AIPC). The study of AIPC had been a significant topic of urology.
Currently, the study of AIPC focus on certain ascpects such as the androgen receptor, tumor cell apoptosis and the regulation of cell cycle, polypeptide growth factor (PGF), angiopoiesis of tumor and genes strongly related with AIPC etc.
Although those studies have provided us lots of valuable information from different aspects of the pathogenesis of AIPC, the molecular biological change from ADPC to AIPC remains unclear. The major obstacle is lacking of ideal cell model that can imitates the transition from ADPC to AIPC in vitro. An ideal Pca cell line shall fulfill following criteria such as androgen-dependent,express a wild-tye AR and would progress to androgen independence after chronic androgen withdrawal. Among the known PCa cell lines,neither PC-3 nor DU-145 expresses AR,while CWR22R expresses a mutant AR.Only LNCaP cell remains androgen dependent characteristics and express AR. LNCaP cell also secretes PSA and PSMA and grow in the presence of steroid hormone. Recently some stuidies demonstrated that LNCaP could be transformed from androgen dependent status to androgen independent status under the circumstance of androgen ablation. This kind of PCa cell model could imitate the process from ADPC to AIPC during endocrine therapy of PCa. Because LNCaP-AI was developed from LNCaP cell inferred, it could be compared with LNCaP cell. The cell model could be used to observe the molecular biological change from ADPC to AIPC and is an ideal model to study the pathogenesis of AIPC.
MicroRNAs (miRNAs) are endogenous short noncoding RNA molecules (20 - 23 nucleotides) that regulate cell differentiation, cell proliferation, andapoptosis through post-transcriptional suppression of gene expression by binding to the complementary sequence in the 3' untranslated region (3'UTR) of target messenger RNAs (mRNAs). Recently, It has been revealed that the change of miRNA expressions contributes to the initiation and progression of cancer. Some recent studies indicate that miRNAs can function as tumor suppressors and oncogenes,and the miRNAs expression profiling of human malignancies has identified signatures involving in cancer development, progression, diagnosis and prognosis. Using miR-15a and miR-16 to control the B-cell chronic lymphocytic leukemia (CLL) recently lead to a new era of novel cancer therapies.Up to date the miRNAs relating prostate cancer showing differential expression were either not the same ones or showed the opposite results of up-regulation or down-regulation. This discrepancy may be due to the fundamental methodologic differences used in the studies. It is well known and widely predicted that the relationship between microRNAs and target mRNAs is not a "one to one" connection, as the same mRNA can be regulated by more than one miRNA, and that the choice of how many and which miRNAs target one 3'UTR is strongly determined by the specific cellular environment. An miRNA that regulates targets playing opposite roles in the control of cell proliferation may act as a tumor suppressor in some cancers and as an oncogene in others, depending on which targets are driving tumorigenesis in that specific cellular milieu. And it still is known that the functional of miRNA dysregulation in cancer has not been well understood.Those aspects contribute to the discrepancy of the microRNAs relating the prostate cancer by now.As there is no systematically study on the alteraions between the LNCaP and LNCaP-AI cell lines by today and also there are still much microRNAs have not been elucidated in the progression of Pca,it is important to identify how many microRNAs functions in development of the Pca and the transition of androgen independence. The change of microRNAs associated with prostate cancer progression may reveal some aspect of the androgen independence biological behavior,providing a new strategy for controlling and treating prostate cancer.
Objective
To construct an androgen independent LNCaP cell line ,we imitated the process from ADPC to AIPC by androgen ablation and investigated the pathogenesis of this process. The cell model provided us a reliable platform for further investigating the pathogenesis of AIPC. By comparing the microRNAs between the LNCaP and LNCaP-AI cell lines the mechanism of androgen independence can be further elucidated in some aspects.
Methods
1. Culturing LNCaP cells in the medium with the steroids deprived step by step for 10 days and then culturing the cells in the medium with 10% FBS depleted of steroids by charcoal stripped for 3 months and gain LNCaP-AI cell line that can accommodate the circumstance without hormone.
2. Verify the reproductive activity of LNCaP and LNCaP-AI cell lines in the absence of hormone by CCK-8 method. Detect PSA level secreted by using immun fluorescence method. Detect AR expression by using RT-PCR.
3.The microRNA alterations between the LNCaP and LNCaP-AI cell lines were detected by Agilent's microassay.The microRNAs alteration were verificated by RT-PCR.The EPO,EPOR expression were detected in the LNCaP and LNCaP-AI cell lines using RT-PCR and the c-kit expression by PE anti-human antibody immunofluorescent staining with flow cytometric analysis.
4.The functions of microRNAs alterations during the transition of androgen independence were eludicated by a search with miRBase software (http://microrna.sanger.ac.uk)
Results
1.Initially,the proliferation of LNCaP cells were suppressed rapidly in the absence of hormone and laid in autocrine status after about 1 week. Three months later, the cells gradually adapted for the no hormone circumstance and began to proliferate. At this time the cells become androgen independent LNCaP-AI cell line.
2. LNCaP-AI cells proliferated rapidly in the no hormone circumstance; In contrast, LNCaP cells were suppressed significantly in the no hormone circumstance. PSA level in LNCaP-AI cell culture supernatant increased with time. However, the PSA expression of LNCaP-AI cell was suppressed as of that in LNCaP cell. The expression level of AR gene level in LNCaP-AI cell inecreased significantly comparing with LNCaP.
3.The Aglint's microRNA microassay showed there were 11 microRNAs upreglulations and 37 microRNAs downregulations during the LNCaP progression into the androgen independence.The verification of the microRNAs changes between the two cells by RT-PCR indicated the Agilen's microassay chips was reliable.
4. There was no significant difference of the EPO and EPOR between LNCaP and LNCaP-AI cell. Expression of c-kit protein elevated in LNCaP-AI cell comparing to LNCaP cell by the PE anti-human antibody immunofluorescent staining with flow cytometric analysis.
5.By looking through the regulating targets of microRNAs using the miRBase software (http://microrna.sanger.ac.uk).It shows those downregulation of the microRNAs prompt the upregulation of the genes including AR,MMP9,bcl-2,c-kit,EGFR and the multiple genes mediating androgen metabolism which involved in the androgen independent transition.
Conclusions
1.Androgen independent LNCaP-AI cell line can be induced from parental cell line LNCaP in the absence of hormone.The resulted cell line imitates the process from ADPC to AIPC during endocrine therapy of PCa. The direct comparasion between LNCaP and its parental strain LNCaP can provide us invaluable insights regarding to the molecular biological change from ADPC to AIPC and the pathogenesis of AIPC.
2. Those alterations of the microRNAs play roles in the progression of LNCaP into androgen independence. The increase in the expression of c-kit may be a new reasons that androgen-stimulated PCa gained the ability of resistance to androgen ablation.
自1941年Huggins等采用雄激素剥夺疗法治疗晚期前列腺癌取得显著疗效后,激素治疗逐渐成为治疗前列腺癌的一项重要的方法。但随着激素治疗在前列腺癌治疗中的广泛应用,人们发现雄激素剥夺疗法的效果平均只有12～16个月,此后前列腺癌继续生长、恶化,一年生存率仅为50%,临床上将这种情况称为激素非依赖性前列腺癌。因此,激素非依赖性前列腺癌的研究成为泌尿外科学中一个重要课题。
目前对激素非依赖前列腺癌的研究热点主要集中在雄激素受体、细胞凋亡及细胞周期的调控、多肽生长因子、肿瘤血管形成和一些与激素非依赖密切相关的基因等多个方面。
虽然这些研究从不同的角度为激素非依赖前列腺癌的发生机制,提供了许多有价值的信息。但是,人们对前列腺癌从激素依赖向激素非依赖转变过程的分子生物学改变仍未能透彻了解。主要的研究障碍就是缺乏理想的细胞模型,来模拟体内前列腺癌患者由激素依赖向激素非依赖转变的过程。一个理想的前列腺癌细胞模型应该能够表现出激素依赖性的特性,表达野生型雄激素受体,能够在激素剥夺的环境下转变为激素非依赖性。已有的前列腺癌细胞系,如PC-3、DU145均不表达雄激素受体,CWR22R表达的是突变的雄激素受体,而且这些细胞系均能够在激素剥夺的环境下生长。目前仅有LNCaP细胞系保留了人前列腺癌的激素依赖性的特征,能够分泌PSA、PSMA及雄激素受体,能够在具有雄激素样活性的甾体激素的刺激下生长。最近的研究表明LNCaP细胞系能够在激素剥夺的环境下逐渐转变为激素非依赖的LNCaP前列腺癌细胞亚系。这种前列腺癌细胞模型可以模拟临床内分泌治疗过程中,前列腺癌由激素依赖发展为雄激素非依赖的过程。同时,由于是从LNCaP前列腺癌细胞系经过干预建立的细胞亚系,两者具有可比性,可以用来观察前列腺癌由激素依赖向非依赖转变过程的分子生物学变化,是一个理想的研究激素非依赖前列腺癌机制的模型。
近几年随着对microRNA(miRNA)重视,越来越多的与细胞分化成熟障碍、增殖失控、细胞永生化为本质的肿瘤疾病密切相关的miRNA被发现,在组织和细胞不同的分化时相中,miRNA有着本身独特的表达方式,即不同时相有不同的表达。目前认为,miRNA在肿瘤发生巾的作用主要在于3个方面:①有些编码miRNA的基因可能起着癌基因的作用;②有些miRNA基因则可能起到抑癌基因的作用;③一些致瘤病毒编码的miRNA也可能参与相关肿瘤的发生。通过临床研究发现,某些特异性miRNA的变化能够对肿瘤诊断及提示预后有帮助,甚至于与临床分期分级有关。并且在急性白血病中应用mir-16和mir-15基因治疗取得有意义的进展,开拓了肿瘤治疗的新纪元。提出重新使miRNA的表达平衡(把升高的miRNA降低、降低的予以升高)可能是肿瘤治疗的一个方向。由于对前列腺癌研究microRNA的方法不同,可能有不同的结果,甚至相反。也由于对microRNA功能及机理还没有完全了解,microRNA对靶基因的调控不是一对一的关系,而是一个microRNA可以调控多个靶基因,在不同的细胞条件,其功能可能变化等复杂情况,还不能解释其中的差异。对前列腺癌相关的microRNA还有大量没有明确。系统、全面的研究其功能及表达,从而利用microRNA来对肿瘤定性分级及寻找出治疗靶点,是将来的一个新方向。尽管有初步研究提示一些microRNA与前列腺癌非雄依赖转化有关,但还有许多没有系统的研究,及早明确哪些microRNA参入了非雄依赖转化及其功能,是研究前列腺癌非雄依赖转化的一个新起点。可以相信,microRNA将是肿瘤(包括前列腺癌)研究的新热点领域。
二、研究目的
本研究利用激素依赖的LNCaP前列腺癌细胞系在体外诱导下建立激素非依赖LNCaP前列腺癌细胞亚系,模拟前列腺癌在激素剥夺治疗后由激素依赖性向非依赖性转变的过程,对其发生机制进行初步探讨,并为进一步深入研究激素非依赖前列腺癌的发生机理,提供一个可靠的平台。通过对比LNCaP-AI(雄激素非依赖细胞)与LNCaP细胞中microRNA的变化,进一步阐述非雄转化的机制。
三、主要研究内容
1、体外长期在去激素的血清环境下培养LNCaP细胞,培养出能够适应无激素环境的LNCaP细胞亚系(LNCaP-AI细胞系)。
2、通过CCK-8方法验证LNCaP-AI细胞系在无激素环境下的增殖能力;荧光免疫的方法检测LNCaP-AI细胞系分泌PSA的情况。
3、RT-PCR的方法检测LNCaP-AI细胞系AR基因表达情况,利用Agilent microRNA寡核苷酸基因芯片对LNCaP及LNCaP-AI细胞检测microRNA的变化,并用RT-PCR方法对6个microRNA进行验证,验证过程中,对比利用PC-3细胞及PC-3细胞去雄一周7个microRNA的变化。
4、通过检索已有的非雄依赖转化过程中上调表达的基因,对比我们已测定的microRNA,联系microrna.sanger.ac.uk针对靶基因寻找相关调控的microRNA,来阐述microRNA的变化与已上调基因的关系,从而说明microRNA的变化参入前列腺癌激素非依赖转化。
5、半定量PCR法测定LNCaP及LNCaP-AI细胞促红细胞生成素(EPO)及促红细胞生成素受体(EPOR)的变化,来推断EPO及EPOR是否参入非雄依赖转化的过程。
6、利用P,E标记c-kit抗体及流式细胞仪测定LNCaP及LNCaP-AI细胞膜原癌受体c-kit的表达情况。
四、结果
1、利用活性炭处理的血清培养模拟去雄细胞环境,逐步递减培养基中激素10天,后完全在非雄培养,共连续培养3个月,一周左右开始处于自分泌状态,3个月后逐渐适应了无激素的环境,能够正常增殖,成为激素非依赖的LNCaP-AI细胞亚系。
2、CCK-8方法检测LNCaP-AI细胞能够在无激素的环境下迅速增殖,LNCaP细胞生长则明显受到抑制。荧光免疫法检测在无激素的环境下LNCaP-AI细胞培养液中PSA的分泌水平能够随着时间的推移而逐渐增加,而LNCaP细胞的PSA的分泌则明显受到抑制,始终处于极低的水平。RT-PCR法检测LNCaP-AI细胞表达AR的水平要明显高于LNCaP细胞,表达水平是后者7倍。
3、通过LNCaP-AI细胞和LNCaP细胞Agilent microRNA芯片方法检测发现,LNCaP-AI细胞有37个microRNA降低,有11个microRNA升高。选出6个microRNA验证结果与Agilent microRNA芯片检测结果一致。通过PC-3细胞去雄一周对比前后6个microRNA的变化,提示5个变化与去雄处理相关。
4、通过检索microrna.sanger.ac.uk针对microRNA调控的靶基因,参考已发现与非雄依赖相关的基因,发现LNCaP转化为激素非依赖的LNCaP-AI细胞中,明显变化的microRNA主要调控AR、EGFR、MMP9、c-kit、bcl-2和雄激素相关代谢酶。认为非雄依赖转化过程中microRNA的变化,通过调高AR、AR旁路信号通路、金属酶、抗凋亡基因及雄激素相关代谢酶基因的表达,参入了前列腺癌非雄依赖转化。
5、半定量PCR法没有发现LNCaP及LNCaP-AI EPO及EPOR有明显的变化,说明EPO及EPOR可能没有参入LNCaP非雄依赖转化。
6、利用P,E标记c-kit抗体及流式细胞仪测定LNCaP及LNCaP-AI细胞膜原癌受体c-kit的表达情况。发现LNCaP-AI细胞膜原癌受体c-kit的表达高于LNCaP,认为通过microRNA的变化,能够调高c-kit的表达,参入非雄依赖的转化。
五、结论
1、本研究利用LNCaP前列腺癌细胞系,在去激素的培养液中长期培养,培养出激素非依赖性的LNCaP-AI前列腺癌细胞亚系。这种前列腺癌细胞模型可以模拟临床上在内分泌治疗过程中,前列腺癌由激素依赖发展为雄激素非依赖的过程。同时,由于是从LNCaP前列腺癌细胞系经过干预建立的细胞亚系,两者有可比性,可以用来观察前列腺痛由激素依赖向非依赖转变过程的分子生物学变化,是一个理想的激素非依赖前列腺癌的细胞模型。
2、通过LNCaP-AI细胞和LNCaP细胞Agilent microRNA芯片方法检测发现,LNCaP-AI细胞有37个microRNA降低,有11个microRNA升高。通过检索microrna.sanger.ac.uk针对microRNA调控的靶基因,参考已发现与非雄依赖相关的基因,发现LNCaP转化为激素非依赖的LNCaP-AI细胞中,明显变化的microRNA主要调控AR、EGFR、MMP9、c-kit、bcl-2和雄激素相关代谢酶。其中,AR、EGFR、bcl-2、MMP9和雄激素相关代谢酶在非雄依赖转化中都是高表达的。发现这些上调的基因相关的microRNA大部分是降低的,根据microRNA调控基因的原理,可以推测这些microRNA可能通过转录后抑制的降低,调高相关基因的表达。认为非雄依赖转化过程中microRNA的变化,通过调高AR、AR旁路信号通路、金属酶、抗凋亡基因及雄激素相关代谢酶基因的表达,参入了前列腺癌非雄依赖转化。利用P,E标记c-kit抗体及流式细胞仪测定LNCaP及LNCaP-AI细胞膜原癌受体c-kit的表达情况。发现LNCaP-AI细胞膜原癌受体c-kit的表达高于LNCaP,认为通过microRNA的变化,能够调高c-kit的表达,参入非雄依赖的转化。
MicroRNAs alterations associated with the prostate cancer LNCaP cell progression to the androgen-independenceBackground
Since androgen ablation was administrated to treat late-stage prostate cancer(Pca) and gained significant therapeutic efficacy by Huggins in 1941, hormone therapy had been an important therapy for PCa. However, with the spreading administration of hormone therapy, people found the effect of androgen ablation remained only 12-16 months, and then PCa cell continued to grow and deteriorate. After that, only about half of the patients could survive beyond 1 year. This situation was called clinically as androgen-independent prostate cancer (AIPC). The study of AIPC had been a significant topic of urology.
Currently, the study of AIPC focus on certain ascpects such as the androgen receptor, tumor cell apoptosis and the regulation of cell cycle, polypeptide growth factor (PGF), angiopoiesis of tumor and genes strongly related with AIPC etc.
Although those studies have provided us lots of valuable information from different aspects of the pathogenesis of AIPC, the molecular biological change from ADPC to AIPC remains unclear. The major obstacle is lacking of ideal cell model that can imitates the transition from ADPC to AIPC in vitro. An ideal Pca cell line shall fulfill following criteria such as androgen-dependent,express a wild-tye AR and would progress to androgen independence after chronic androgen withdrawal. Among the known PCa cell lines,neither PC-3 nor DU-145 expresses AR,while CWR22R expresses a mutant AR.Only LNCaP cell remains androgen dependent characteristics and express AR. LNCaP cell also secretes PSA and PSMA and grow in the presence of steroid hormone. Recently some stuidies demonstrated that LNCaP could be transformed from androgen dependent status to androgen independent status under the circumstance of androgen ablation. This kind of PCa cell model could imitate the process from ADPC to AIPC during endocrine therapy of PCa. Because LNCaP-AI was developed from LNCaP cell inferred, it could be compared with LNCaP cell. The cell model could be used to observe the molecular biological change from ADPC to AIPC and is an ideal model to study the pathogenesis of AIPC.
MicroRNAs (miRNAs) are endogenous short noncoding RNA molecules (20 - 23 nucleotides) that regulate cell differentiation, cell proliferation, andapoptosis through post-transcriptional suppression of gene expression by binding to the complementary sequence in the 3' untranslated region (3'UTR) of target messenger RNAs (mRNAs). Recently, It has been revealed that the change of miRNA expressions contributes to the initiation and progression of cancer. Some recent studies indicate that miRNAs can function as tumor suppressors and oncogenes,and the miRNAs expression profiling of human malignancies has identified signatures involving in cancer development, progression, diagnosis and prognosis. Using miR-15a and miR-16 to control the B-cell chronic lymphocytic leukemia (CLL) recently lead to a new era of novel cancer therapies.Up to date the miRNAs relating prostate cancer showing differential expression were either not the same ones or showed the opposite results of up-regulation or down-regulation. This discrepancy may be due to the fundamental methodologic differences used in the studies. It is well known and widely predicted that the relationship between microRNAs and target mRNAs is not a "one to one" connection, as the same mRNA can be regulated by more than one miRNA, and that the choice of how many and which miRNAs target one 3'UTR is strongly determined by the specific cellular environment. An miRNA that regulates targets playing opposite roles in the control of cell proliferation may act as a tumor suppressor in some cancers and as an oncogene in others, depending on which targets are driving tumorigenesis in that specific cellular milieu. And it still is known that the functional of miRNA dysregulation in cancer has not been well understood.Those aspects contribute to the discrepancy of the microRNAs relating the prostate cancer by now.As there is no systematically study on the alteraions between the LNCaP and LNCaP-AI cell lines by today and also there are still much microRNAs have not been elucidated in the progression of Pca,it is important to identify how many microRNAs functions in development of the Pca and the transition of androgen independence. The change of microRNAs associated with prostate cancer progression may reveal some aspect of the androgen independence biological behavior,providing a new strategy for controlling and treating prostate cancer.
Objective
To construct an androgen independent LNCaP cell line ,we imitated the process from ADPC to AIPC by androgen ablation and investigated the pathogenesis of this process. The cell model provided us a reliable platform for further investigating the pathogenesis of AIPC. By comparing the microRNAs between the LNCaP and LNCaP-AI cell lines the mechanism of androgen independence can be further elucidated in some aspects.
Methods
1. Culturing LNCaP cells in the medium with the steroids deprived step by step for 10 days and then culturing the cells in the medium with 10% FBS depleted of steroids by charcoal stripped for 3 months and gain LNCaP-AI cell line that can accommodate the circumstance without hormone.
2. Verify the reproductive activity of LNCaP and LNCaP-AI cell lines in the absence of hormone by CCK-8 method. Detect PSA level secreted by using immun fluorescence method. Detect AR expression by using RT-PCR.
3.The microRNA alterations between the LNCaP and LNCaP-AI cell lines were detected by Agilent's microassay.The microRNAs alteration were verificated by RT-PCR.The EPO,EPOR expression were detected in the LNCaP and LNCaP-AI cell lines using RT-PCR and the c-kit expression by PE anti-human antibody immunofluorescent staining with flow cytometric analysis.
4.The functions of microRNAs alterations during the transition of androgen independence were eludicated by a search with miRBase software (http://microrna.sanger.ac.uk)
Results
1.Initially,the proliferation of LNCaP cells were suppressed rapidly in the absence of hormone and laid in autocrine status after about 1 week. Three months later, the cells gradually adapted for the no hormone circumstance and began to proliferate. At this time the cells become androgen independent LNCaP-AI cell line.
2. LNCaP-AI cells proliferated rapidly in the no hormone circumstance; In contrast, LNCaP cells were suppressed significantly in the no hormone circumstance. PSA level in LNCaP-AI cell culture supernatant increased with time. However, the PSA expression of LNCaP-AI cell was suppressed as of that in LNCaP cell. The expression level of AR gene level in LNCaP-AI cell inecreased significantly comparing with LNCaP.
3.The Aglint's microRNA microassay showed there were 11 microRNAs upreglulations and 37 microRNAs downregulations during the LNCaP progression into the androgen independence.The verification of the microRNAs changes between the two cells by RT-PCR indicated the Agilen's microassay chips was reliable.
4. There was no significant difference of the EPO and EPOR between LNCaP and LNCaP-AI cell. Expression of c-kit protein elevated in LNCaP-AI cell comparing to LNCaP cell by the PE anti-human antibody immunofluorescent staining with flow cytometric analysis.
5.By looking through the regulating targets of microRNAs using the miRBase software (http://microrna.sanger.ac.uk).It shows those downregulation of the microRNAs prompt the upregulation of the genes including AR,MMP9,bcl-2,c-kit,EGFR and the multiple genes mediating androgen metabolism which involved in the androgen independent transition.
Conclusions
1.Androgen independent LNCaP-AI cell line can be induced from parental cell line LNCaP in the absence of hormone.The resulted cell line imitates the process from ADPC to AIPC during endocrine therapy of PCa. The direct comparasion between LNCaP and its parental strain LNCaP can provide us invaluable insights regarding to the molecular biological change from ADPC to AIPC and the pathogenesis of AIPC.
2. Those alterations of the microRNAs play roles in the progression of LNCaP into androgen independence. The increase in the expression of c-kit may be a new reasons that androgen-stimulated PCa gained the ability of resistance to androgen ablation.
引文
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1.Tsuchiyal S,Okuno Y,Tsujimoto G.MicroRNA:Biogenetic and Functional Mechanisms and Involvements in Cell Differentiation and Cancer.Journal of Pharmacological Sciences,2006;101:267-270.
2.Lu J,Getz G,Miska EA,et al.MicroRNA expression profiles classify human cancers.Nature 2005;435:834-838.
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5.Calin GA,Cimmino A,Fabbri M,et al.MiR-15a and miR-16-1 cluster functions in human leukemia.PNAS,2008;105(13):5166-5171
6.Porkka KP,Pfeiffer M J,Waltering KK,et al.MicroRNA expression profiling in prostate cancer.Cancer Res.2007;Jul 1;67(13):6130-6135.
7.Ozen M,Creighton CJ,Ozdemir M,Ittmann M.Widespread deregulation of microRNA expression in human prostate cancer.Oncogene.2008;Mar 13;27(12):1788-93.
8.Shi XB,Xue L,Yang J,et al.An androgen-regulated miRNA suppresses Bakl expression and induces androgen-independent growth of prostate cancer cells.PNAS December 11,2007;104(50):19983-19988
9.Holzbeierlein J,Lal P,LaTulippe E,Smith A,et al.Gene expression analysis of human prostate carcinoma during hormonal therapy identifies androgen-responsive genes and mechanisms of therapy resistance.American Journal of Pathology.2004;164(1):217-227.
10.Chen Q,Watson JT,Marengo SR,Decker KS,et al.Gene expression in the LNCaP human prostate cancer progression model:progression associated expression in vitro corresponds to expression changes associated with prostate cancer progression in vivo. Cancer Letters.2006;244 (2):274-288
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