MiR-221在前列腺癌雄激素非依赖转变过程中作用的研究
摘要
背景
前列腺癌(Prostate cancer, PCa)在许多西方国家是男性最常见的恶性肿瘤之占男性癌症死因的第二位。在我国,近年来随着生活水平的提高及人均寿命的延长,前列腺癌发病率也呈现明显上升趋势。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。这种方法早期一般是有效的,此时称激素敏感型前列腺癌(androgen-dependent ostate cancer, ADPC),其有效率一般可达70%左右,然而,经过12～16个月病情缓解期后,几乎无一例外的会进展为激素非敏感的前列腺癌(androgen-independent prostate cancer, AIPC),此期患者病情不可遏制的急剧恶化,肿瘤迅速转移侵袭其它器官,最终因此导致患者死亡。治疗失败的原因是缺乏对于激素非依赖型前列腺癌的有效治疗方法,此外,也因为缺乏简便准确的早期前列腺肿瘤分期判定手段,医生只有在多次穿刺,结合ADT治疗行进过程中PSA观察,定夺治疗方案,这样不仅增加了患者痛苦,也容易导致患者错过最佳治疗时期。因此,探索敏感、特异、准确的前列腺癌分子标志物,实现肿瘤的早期诊断,以及寻找遏制前列腺癌进入激素非依赖期的药物,成为泌尿外科研究迫切需要解决的问题。
以往利用基因芯片和蛋白组学等方法,科研工作者对AIPC机制已经开展大量研究,极大推动了对AIPC机制的了解。例如发现对前列腺癌具有代表性的分子除了PSA,还包括OC, PSMA, PAP, PSMA,这些分子都是治疗前列腺癌治疗潜在的靶标,但是深入研究表明,虽然这些分子联合PSA检测,提高了PCa的分期诊断,特异性和敏感性却始终不能突破PSA对Pca的意义,对于AIPC的诊治始终缺乏有力的突破。
近年来microRNA(miRNA)在肿瘤发生发展及耐药性中发挥重要调控作用相应得以证实,部分miRNA在相应肿瘤中作用甚至申请国际专利保护,其对肿瘤的重要性迅速引起研究者兴趣。通过计算机分析,人类约30%的编码蛋白基因被miRNA所调控,已经发现的人类miRNA大部分(52%)都位于已知的肿瘤相关基因组区域内或已知的基因脆性位点,改变特异性miRNA的表达导致象肿瘤样疾病的触发和进展。逐渐明确的miRNA表达谱研究使其迅速成为新的临床辅助手段：1、miRNA表达谱为诊断工具。不明原发部位的转移性癌是癌症诊断中常见的一种情况。2、miRNA表达谱做为预后工具。值得关注是,miRNA表达的变化,尤其对低分化的肿瘤具有重要指导意义,而前列腺癌正属于低分化肿瘤的范畴,提示miRNA检测可能为前列腺癌AIPC研究提供了新的突破口。这引起了众多前列腺癌科研工作者的关注,miRNA针对PCa表达谱以及前列腺良性增生的表达谱近年累计有更新报道,虽然如此,针对PIN发展成癌变以及针对ASPC进展为AIPC其中具有重要调控作用的miRNA,实验性研究才刚刚起步。
本研究获得AIPC 5例标本,和ADPC 5例标本一同送往通过国际认证的miRNA芯片公司,在原位组织筛选获得珍贵的miRNA差异信息。并后期Nothern验证,确证miRNA-221表达差异显著。而我们的结果提示,miR-221可能在晚期前列腺癌的雄激素治疗非依赖化进程中发挥重要调控作用,其具体作用机制和表达量可能跟前列腺癌细胞命运甚至疾病进程密切相关,为了进一步明确miR-221对AIPC发生发展的生物学意义,进行细胞学功能学检测。以求寻找理想的诊治晚期前列腺癌的有效靶标。
研究目的
针对激素治疗敏感和失效的病例标本,进行miRNA芯片筛选,建立前列腺癌雄激素阻断治疗后,两个重要阶段的miRNA表达谱数据库。通过细胞模型,明确miR-221主要参与调控细胞哪些生物学功能。寻找miR-221作用的靶蛋白,探索miR-221作用机制。探测分析miR-221及其调控的靶蛋白与雄激素非依赖前列腺癌产生及进展的关系。
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-AIi(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。
研究方法
1、针对ADPC和AIPC的病人组织标本各5例,筛选差异miRNA,鉴定miRNA表达差异通过Northen和RT-PCR方法,鉴定组织样本中miRNA芯片筛选的miRNA表达存在差异并探测差异大小,明确后续工作有研究价值。
2、建立雄激素非依赖细胞模型,根据组织学筛选的miRNA表达谱,鉴定并比较差异miRNA在LNCaP/AI-LNCaP细胞模型和ASPC/AIPC组织表达是否平行,明确细胞模型可以用于生物学功能研究,选取表达差异大的miRNA(miR-221)进行生物学功能探索。
3、建立前列腺癌细胞侵袭模型,使用细胞转染探测分析雄激素对miR-221细胞定位及细胞生物学功能的影响,深入探讨miR-221在激素非依赖化转变中的作用机制。
4、通过生物信息学和miRNA数据库,筛选miR-221作用的靶蛋白基因,确定靶基因确切结合区域或位点,经过western blot和RT-PCR实验,进一步明确miR-221及其调控的靶基因与疾病的关系。
结果
1、利用Northern及RT-PCR方法检测,ADPC及AIPC患者标本中差异表达的microRNA,其中表达下调的有miR-15a和miR-101,上调有：miR-221, miR-222 miR-21,miR-205和miR-125b,其中miR-221上调明显。
2、利用RT-PCR检测LNCaP-AI, LNCaP细胞系中miR-221的表达明显升高,达10倍左右。证明miR-221在前列腺癌的雄激素非依赖转化过程中发挥作用。
3、CCK-8方法检测转染了miR-221的LNCaP及LNCaP-AI细胞能够在无激素的环境下迅速增殖,并且转染了anti-miR-221的LNCaP及LNCaP-AI细胞生长则明显受到抑制。通过流式细胞学检测发现转染了miR-221的LNCaP及LNCaP-AI细胞的S期细胞明显升高,证明miR-221有可能促进前列腺癌细胞的分裂生长。
4、通过转染miR-221至LNCaP细胞发现转染3天后LNCaP细胞出现神经内分泌型表现,RT-PCR检测转染了miR-221的LNCaP细胞中NSE的水平较对照组明显升高,Western在蛋白水平检测NSE蛋白也明显高表达。提示miR-221促进前列腺癌细胞的神经内分泌转化。
5、划痕实验检测转染了miR-221的LNCaP-AI细胞迁移至空白区的细胞数量较对照组明显增加,证实其迁移能力明显增强。
6、Transwell侵袭实验发现转染了anti-miR-221的LNCaP-AI细胞穿过基质胶的细胞数量较对照组明显升高,提示miR-221提高了LNCaP-AI细胞的侵袭能力。
7、通过检索microrna. sanger. ac. uk针对miR-221寻找相关调控的靶基因DVL2,提示miR-221通过调控DVL2从而影响前列腺癌细胞的侵袭能力。qRT-PCR方法对比LNCaP-AI与LNCaP细胞中DVL2明显上调。通过Western检测发现转染了miR-221的LNCaP-AI细胞中DVL2相对于对照组明显升高。提示miR-221通过调节DVL2从而改变LNCaP-AI的侵袭能力。
8、通过收集ADPC及AIPC期患者的全血,利用RT-PCR检测miR-221的水平,发现AIPC患者中miR-221的水平明显低于ADPC患者。可以用来进行作为前列腺癌患者进展的指标。
结论
1、本研究通过对于miR-221在不同前列腺癌细胞系中的功能的研究,发现miR-221通过增加S期细胞的数量,从而促进LNCaP及LNCaP-AI细胞的增殖。因此miR-221会在前列腺癌的生长及进展期发挥作用。同时我们也证实miR-221通过增加NSE的表达促进LNCaP细胞的神经内分泌化的转变。而NE的转变则被认为是前列腺癌雄激素非依赖性转变的重要原因。
2、通过对于LNCaP及LNCaP-AI细胞转染不同miR-221模拟物进行检测,发现miR-221可以明显提升LNCaP-AI细胞的迁移和侵袭能力。这也在一定程度上证明miR-221可以促进晚期前列腺癌的扩散。并且通过对于其靶基因的寻找发现DVL2可能是改变LNCaP-AI细胞侵袭能力的重要通路。然而miR-221往往作用于多个通路因此,作用机制仍然需要进一步的研究。
3、通过对于AIPC和ADPC患者血液中miR-221的检测发现AIPC患者血中miR-221的含量明显低于ADPC患者,并且其差异明显,因此其结果有希望可以做为将来研究前列腺癌进展阶段的重要靶标。并为寻找控制晚期前列腺癌的进展提供治疗的新途径。
Background
Prostate cancer is the most common cancer in man and the second leading cause of death in European counties. Prostate cancer (CaP) is becoming the increasing malignant tumor in China when the quality of Chinese people lives and the number of the people increase greatly. Since androgen ablation is administrated to treat advanced prostate cancer (Pca) and gains significant therapeutic efficacy by Huggins in 1941, hormone therapy has been an important therapy for PCa. In this stage called androgen-dependent prostate cancer (ADPC) the treatment is effective, and the valid rate is up to 70%. After androgen ablation therapy for 12-16 months, androgen- dependent cancer cells inevitably progress to an androgen-independent status (AIPC).And the cancer cells spread into other organs rapidly, and lead to death finally. But no effective treatment has been developed. The treatments of CaP are decided after punatura examation of PSA, which greatly raise the patients'pains. Thus specific, sensitive marker focused on diagnosis and treatments of CaP are expected. So it is become a main task to find an effective medicine to prevent CaP from becoming AIPC.
Many studies have been done on the roles of AIPC by microarray and proteomics.Some molecules such as OC, PSAM, PAP, PSMA are the targets in order to treating prostate cancer. But the lower specificity and sensitivity compared with PSA confine them to a small scope, and it is difficult to improve their function.
The reason for the occurrence and progression of CaP remain largely unknown. Recent studies suggest that microRNAs (miRNAs) are involved in human tumori genesis. Some aberrantly- expressed miRNAs have been discovered in CaP cell lines and clinical tissues and these CaP-related miRNAs may play critical roles in the pathogenesis
Currently, the studies of microRNA focus on the tumour development, progression and drug resistance. Some microRNAs studies have applied for patients, and the importance of microRNA attract many researchers attentions. Up to 30% of all gene coding proteins are negatively regulated by miRNAs in human cells. It is now evident that 52% of miRNAs located in the fragile site of genes associated with cancers.The regulations of miRNAs expressions can lead to the cancers development and progression.MicroRNAs are coming to be the new clinical assistant methods.1、MicroRNAs are diagnosis tools. The diagnosis of metastatic carcinoma is the main purpose.2、MicroRNAs are prognosis tools. It attracted more attention that poorly differentiated tumours are suitable for the application of microRNAs.And prostate cancers belong to poorly differentiated cancers. And more reports on miRNA differential expression in the CaP and BPH. Thus the study aimed on the transition of PIN into CaP is in the early stage.And the miRNA that works in the progression of ADPC into AIPC need to be deeply studied.
In this study we collected 5 ADPC specimens and 5 AIPC specimens,and in gene chip company the differential expression of microRNAs were got by ISH. The results were confirmed through Northern blotting, and the expression of miR-221was the most different one. In our study we conclud that miR-221 plays an important role in the progression of androgen-independence.The function and expression of miR-221 is closely related with the fate of prostate cancer cells.In order to identifying the function of miR-221,we detect the contribution of miR-221 in prostate cancer cell lines. We can find some significant objectives in the treatment of the advanced prostate cancer.
Objective
Gene screening was operated through miRNA chip on the basis of 5 ADPC specimen and 5 AIPC specimen.And miRNA expression database was constructed. The regulation mechanism of miR-221 was identified through prostate cancer cell lines. Seeking the target proteins that were regulated by miR-221, and elucidating the relationship with the AIPC progression.
Methods
1.MiRNAs screening were operated on the basis of 5 ADPC specimen and 5 AIPC specimen,and the different expression of miRNAs were confirmed through Northern and
RT-PCR.
2.Constructing AIPC cell lines and verifying the differential expression of miRNA in LNCaP cell lines and LNCaP-AI cell lines. Confirming the prostate cancer cell lines used in study of the biological function. And selecting miR-221 to be studied.
3. Constructing the prostate cancer cells invasive model and confirming the effect of androgen on miR-221 and the biological function of miR-221.Studying the roles of miR-221 in the AIPC progression of prostate cancer.
4. Indentifying accurate binding site of miR-221 target protein in the gene through bioinformatics and miRNA datebase.Verifying the relationship of the disease with the target gene by Western blot and RT-PCR.
Results
1. It was found in our study that most AIPC LNCaP-AI lines increased miRNA expression compared with ADPC line, and that five miRNAs (miR-221, miR-222 miR-21, miR-205 and miR-125b) were up-regulated, while two miRNAs (miR-15a and miR-101) were down-regulated in AI LNCaP-AI cell lines compared with their parental AD LNCaP line, by Northern and RT-PCR.And miR-221 was upregulated significantly.
2. qRT-PCR was performed to evaluate miR-221 expression in LNCaP, LNCaP-AI and PC3 cell lines. It was found that miR-221 expression was up-regulated in LNCaP-AI independently derived AIPC cell lines and down-regulated in LNCaP dependently derived ADPC cell lines, suggesting that these differentially expressed miRNAs might contribute to the progression of CaP cells.
3. Cell growth was assessed by using the CCK-8 cell proliferation assay. Treatment with miR-221 significantly stimulated the growth of LNCaP cells and LNCaP-AI cells, while the miRNA negative control (miR-NC) without treatment failed to affect cell growth. The influence of miR-221m on the S-phase fraction was also upregulated by flow cytometry.
4. NE differentiation was studied in LNCaP cells stimulated with increased miR-221 levels.NSE mRNA levels increased rapidly on day.We examined the level of NSE protein in miR-221-and miR-NC-treated LNCaP cells by Western blot. Transfection with miR-221 led to remarkable up-regulation of NSE in LNCaP cell lines on day 3,
5. LNCaP cells transiently transfected with miR-221 or miR-NC were grown until confluence in a wound healing assay. Migration of LNCaP-AI cells transfected with anti-miR-221 migrated into the open area was significantly increased.and the ability of migration was upregulated.
6. A Matrigel invasion assay was also performed to obtain a measure of the migration of these transfected cells. The results clearly revealed a significant increase in migration and invasiveness in anti-miR-221-treated LNCaP-AI cells. It was concluded that miR-221 was an important determinant of motility and invasiveness of CaP.
7. Analyses of the migration relative genes were performed using the Sanger miRNA database target search program.RT-PCR analysis of RNA extracted from anti-miR-221-treated LNCaP-AI cells showed that there was a significant increase in the expression of DVL2. Treatment of LNCaP-AI cells with anti-miR-221 caused a great increase in DVL2 protein in LNCaP-AI cells by Western blot. And DVL2 may play a role in the invasive ability of LNCaP-AI cells.
8. We next analyzed the level of miR-221 in the plasma samples from ADPC and AIPC patients by TaqMan qRT-PCR.MiR-221 expression was upregulated in ADPC patients compared with AIPC patients.MiR-221 could be decided as a new tumor-derived marker.
Conclusions
1. In our study miR-221 promotes the growth of LNCaP and LNCaP-AI cells. Consistently, ectopic introduction of miR-221 in low expressers of LNCaP cells strongly increased their growth potential by inducing a G1-S shift in the cell cycle. In this study, we showed that miR-221 could promote neuroendocrine differentiation of LNCaP cells in an androgen deprived environment. Thus miR-221 plays an important role in the progression of androgen independence.
2. Down-regulation of miR-221 in LNCaP-AI cells greatly increased the number of cells migrating through the transwell lambers. We therefore conclude that miR-221 might not affect the ability of migration of LNCaP cells significantly; Rather, a poor invasive CaP cell line and miR-221 level might inversely correlate with the invasiveness in androgen independence CaP. We have demonstrated the importance of miR-221 in the migration on LNCaP-AI cells that are strongly invasive and androgen-independent in nature. In the present study, we chose one target mRNA DVL2 that was correlated with the migration. As expected, the expression of DVL2 was up-regulated in LNCaP-AI cells transfected with anti-miR-221.And the reason for it need to be investigated deeply.
3. We analyzed the level of miR-221 in the plasma samples from ADPC and AIPC patients by TaqMan qRT-PCR.MiR-221 expression was upregulated in ADPC patients compared with AIPC patients. These results suggest that tumor-derived miR-221 in plasma would be a blood-based miRNA biomarker candidates for CaP.
前列腺癌(Prostate cancer, PCa)在许多西方国家是男性最常见的恶性肿瘤之占男性癌症死因的第二位。在我国,近年来随着生活水平的提高及人均寿命的延长,前列腺癌发病率也呈现明显上升趋势。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。这种方法早期一般是有效的,此时称激素敏感型前列腺癌(androgen-dependent ostate cancer, ADPC),其有效率一般可达70%左右,然而,经过12～16个月病情缓解期后,几乎无一例外的会进展为激素非敏感的前列腺癌(androgen-independent prostate cancer, AIPC),此期患者病情不可遏制的急剧恶化,肿瘤迅速转移侵袭其它器官,最终因此导致患者死亡。治疗失败的原因是缺乏对于激素非依赖型前列腺癌的有效治疗方法,此外,也因为缺乏简便准确的早期前列腺肿瘤分期判定手段,医生只有在多次穿刺,结合ADT治疗行进过程中PSA观察,定夺治疗方案,这样不仅增加了患者痛苦,也容易导致患者错过最佳治疗时期。因此,探索敏感、特异、准确的前列腺癌分子标志物,实现肿瘤的早期诊断,以及寻找遏制前列腺癌进入激素非依赖期的药物,成为泌尿外科研究迫切需要解决的问题。
以往利用基因芯片和蛋白组学等方法,科研工作者对AIPC机制已经开展大量研究,极大推动了对AIPC机制的了解。例如发现对前列腺癌具有代表性的分子除了PSA,还包括OC, PSMA, PAP, PSMA,这些分子都是治疗前列腺癌治疗潜在的靶标,但是深入研究表明,虽然这些分子联合PSA检测,提高了PCa的分期诊断,特异性和敏感性却始终不能突破PSA对Pca的意义,对于AIPC的诊治始终缺乏有力的突破。
近年来microRNA(miRNA)在肿瘤发生发展及耐药性中发挥重要调控作用相应得以证实,部分miRNA在相应肿瘤中作用甚至申请国际专利保护,其对肿瘤的重要性迅速引起研究者兴趣。通过计算机分析,人类约30%的编码蛋白基因被miRNA所调控,已经发现的人类miRNA大部分(52%)都位于已知的肿瘤相关基因组区域内或已知的基因脆性位点,改变特异性miRNA的表达导致象肿瘤样疾病的触发和进展。逐渐明确的miRNA表达谱研究使其迅速成为新的临床辅助手段：1、miRNA表达谱为诊断工具。不明原发部位的转移性癌是癌症诊断中常见的一种情况。2、miRNA表达谱做为预后工具。值得关注是,miRNA表达的变化,尤其对低分化的肿瘤具有重要指导意义,而前列腺癌正属于低分化肿瘤的范畴,提示miRNA检测可能为前列腺癌AIPC研究提供了新的突破口。这引起了众多前列腺癌科研工作者的关注,miRNA针对PCa表达谱以及前列腺良性增生的表达谱近年累计有更新报道,虽然如此,针对PIN发展成癌变以及针对ASPC进展为AIPC其中具有重要调控作用的miRNA,实验性研究才刚刚起步。
本研究获得AIPC 5例标本,和ADPC 5例标本一同送往通过国际认证的miRNA芯片公司,在原位组织筛选获得珍贵的miRNA差异信息。并后期Nothern验证,确证miRNA-221表达差异显著。而我们的结果提示,miR-221可能在晚期前列腺癌的雄激素治疗非依赖化进程中发挥重要调控作用,其具体作用机制和表达量可能跟前列腺癌细胞命运甚至疾病进程密切相关,为了进一步明确miR-221对AIPC发生发展的生物学意义,进行细胞学功能学检测。以求寻找理想的诊治晚期前列腺癌的有效靶标。
研究目的
针对激素治疗敏感和失效的病例标本,进行miRNA芯片筛选,建立前列腺癌雄激素阻断治疗后,两个重要阶段的miRNA表达谱数据库。通过细胞模型,明确miR-221主要参与调控细胞哪些生物学功能。寻找miR-221作用的靶蛋白,探索miR-221作用机制。探测分析miR-221及其调控的靶蛋白与雄激素非依赖前列腺癌产生及进展的关系。
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-AIi(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。
研究方法
1、针对ADPC和AIPC的病人组织标本各5例,筛选差异miRNA,鉴定miRNA表达差异通过Northen和RT-PCR方法,鉴定组织样本中miRNA芯片筛选的miRNA表达存在差异并探测差异大小,明确后续工作有研究价值。
2、建立雄激素非依赖细胞模型,根据组织学筛选的miRNA表达谱,鉴定并比较差异miRNA在LNCaP/AI-LNCaP细胞模型和ASPC/AIPC组织表达是否平行,明确细胞模型可以用于生物学功能研究,选取表达差异大的miRNA(miR-221)进行生物学功能探索。
3、建立前列腺癌细胞侵袭模型,使用细胞转染探测分析雄激素对miR-221细胞定位及细胞生物学功能的影响,深入探讨miR-221在激素非依赖化转变中的作用机制。
4、通过生物信息学和miRNA数据库,筛选miR-221作用的靶蛋白基因,确定靶基因确切结合区域或位点,经过western blot和RT-PCR实验,进一步明确miR-221及其调控的靶基因与疾病的关系。
结果
1、利用Northern及RT-PCR方法检测,ADPC及AIPC患者标本中差异表达的microRNA,其中表达下调的有miR-15a和miR-101,上调有：miR-221, miR-222 miR-21,miR-205和miR-125b,其中miR-221上调明显。
2、利用RT-PCR检测LNCaP-AI, LNCaP细胞系中miR-221的表达明显升高,达10倍左右。证明miR-221在前列腺癌的雄激素非依赖转化过程中发挥作用。
3、CCK-8方法检测转染了miR-221的LNCaP及LNCaP-AI细胞能够在无激素的环境下迅速增殖,并且转染了anti-miR-221的LNCaP及LNCaP-AI细胞生长则明显受到抑制。通过流式细胞学检测发现转染了miR-221的LNCaP及LNCaP-AI细胞的S期细胞明显升高,证明miR-221有可能促进前列腺癌细胞的分裂生长。
4、通过转染miR-221至LNCaP细胞发现转染3天后LNCaP细胞出现神经内分泌型表现,RT-PCR检测转染了miR-221的LNCaP细胞中NSE的水平较对照组明显升高,Western在蛋白水平检测NSE蛋白也明显高表达。提示miR-221促进前列腺癌细胞的神经内分泌转化。
5、划痕实验检测转染了miR-221的LNCaP-AI细胞迁移至空白区的细胞数量较对照组明显增加,证实其迁移能力明显增强。
6、Transwell侵袭实验发现转染了anti-miR-221的LNCaP-AI细胞穿过基质胶的细胞数量较对照组明显升高,提示miR-221提高了LNCaP-AI细胞的侵袭能力。
7、通过检索microrna. sanger. ac. uk针对miR-221寻找相关调控的靶基因DVL2,提示miR-221通过调控DVL2从而影响前列腺癌细胞的侵袭能力。qRT-PCR方法对比LNCaP-AI与LNCaP细胞中DVL2明显上调。通过Western检测发现转染了miR-221的LNCaP-AI细胞中DVL2相对于对照组明显升高。提示miR-221通过调节DVL2从而改变LNCaP-AI的侵袭能力。
8、通过收集ADPC及AIPC期患者的全血,利用RT-PCR检测miR-221的水平,发现AIPC患者中miR-221的水平明显低于ADPC患者。可以用来进行作为前列腺癌患者进展的指标。
结论
1、本研究通过对于miR-221在不同前列腺癌细胞系中的功能的研究,发现miR-221通过增加S期细胞的数量,从而促进LNCaP及LNCaP-AI细胞的增殖。因此miR-221会在前列腺癌的生长及进展期发挥作用。同时我们也证实miR-221通过增加NSE的表达促进LNCaP细胞的神经内分泌化的转变。而NE的转变则被认为是前列腺癌雄激素非依赖性转变的重要原因。
2、通过对于LNCaP及LNCaP-AI细胞转染不同miR-221模拟物进行检测,发现miR-221可以明显提升LNCaP-AI细胞的迁移和侵袭能力。这也在一定程度上证明miR-221可以促进晚期前列腺癌的扩散。并且通过对于其靶基因的寻找发现DVL2可能是改变LNCaP-AI细胞侵袭能力的重要通路。然而miR-221往往作用于多个通路因此,作用机制仍然需要进一步的研究。
3、通过对于AIPC和ADPC患者血液中miR-221的检测发现AIPC患者血中miR-221的含量明显低于ADPC患者,并且其差异明显,因此其结果有希望可以做为将来研究前列腺癌进展阶段的重要靶标。并为寻找控制晚期前列腺癌的进展提供治疗的新途径。
Background
Prostate cancer is the most common cancer in man and the second leading cause of death in European counties. Prostate cancer (CaP) is becoming the increasing malignant tumor in China when the quality of Chinese people lives and the number of the people increase greatly. Since androgen ablation is administrated to treat advanced prostate cancer (Pca) and gains significant therapeutic efficacy by Huggins in 1941, hormone therapy has been an important therapy for PCa. In this stage called androgen-dependent prostate cancer (ADPC) the treatment is effective, and the valid rate is up to 70%. After androgen ablation therapy for 12-16 months, androgen- dependent cancer cells inevitably progress to an androgen-independent status (AIPC).And the cancer cells spread into other organs rapidly, and lead to death finally. But no effective treatment has been developed. The treatments of CaP are decided after punatura examation of PSA, which greatly raise the patients'pains. Thus specific, sensitive marker focused on diagnosis and treatments of CaP are expected. So it is become a main task to find an effective medicine to prevent CaP from becoming AIPC.
Many studies have been done on the roles of AIPC by microarray and proteomics.Some molecules such as OC, PSAM, PAP, PSMA are the targets in order to treating prostate cancer. But the lower specificity and sensitivity compared with PSA confine them to a small scope, and it is difficult to improve their function.
The reason for the occurrence and progression of CaP remain largely unknown. Recent studies suggest that microRNAs (miRNAs) are involved in human tumori genesis. Some aberrantly- expressed miRNAs have been discovered in CaP cell lines and clinical tissues and these CaP-related miRNAs may play critical roles in the pathogenesis
Currently, the studies of microRNA focus on the tumour development, progression and drug resistance. Some microRNAs studies have applied for patients, and the importance of microRNA attract many researchers attentions. Up to 30% of all gene coding proteins are negatively regulated by miRNAs in human cells. It is now evident that 52% of miRNAs located in the fragile site of genes associated with cancers.The regulations of miRNAs expressions can lead to the cancers development and progression.MicroRNAs are coming to be the new clinical assistant methods.1、MicroRNAs are diagnosis tools. The diagnosis of metastatic carcinoma is the main purpose.2、MicroRNAs are prognosis tools. It attracted more attention that poorly differentiated tumours are suitable for the application of microRNAs.And prostate cancers belong to poorly differentiated cancers. And more reports on miRNA differential expression in the CaP and BPH. Thus the study aimed on the transition of PIN into CaP is in the early stage.And the miRNA that works in the progression of ADPC into AIPC need to be deeply studied.
In this study we collected 5 ADPC specimens and 5 AIPC specimens,and in gene chip company the differential expression of microRNAs were got by ISH. The results were confirmed through Northern blotting, and the expression of miR-221was the most different one. In our study we conclud that miR-221 plays an important role in the progression of androgen-independence.The function and expression of miR-221 is closely related with the fate of prostate cancer cells.In order to identifying the function of miR-221,we detect the contribution of miR-221 in prostate cancer cell lines. We can find some significant objectives in the treatment of the advanced prostate cancer.
Objective
Gene screening was operated through miRNA chip on the basis of 5 ADPC specimen and 5 AIPC specimen.And miRNA expression database was constructed. The regulation mechanism of miR-221 was identified through prostate cancer cell lines. Seeking the target proteins that were regulated by miR-221, and elucidating the relationship with the AIPC progression.
Methods
1.MiRNAs screening were operated on the basis of 5 ADPC specimen and 5 AIPC specimen,and the different expression of miRNAs were confirmed through Northern and
RT-PCR.
2.Constructing AIPC cell lines and verifying the differential expression of miRNA in LNCaP cell lines and LNCaP-AI cell lines. Confirming the prostate cancer cell lines used in study of the biological function. And selecting miR-221 to be studied.
3. Constructing the prostate cancer cells invasive model and confirming the effect of androgen on miR-221 and the biological function of miR-221.Studying the roles of miR-221 in the AIPC progression of prostate cancer.
4. Indentifying accurate binding site of miR-221 target protein in the gene through bioinformatics and miRNA datebase.Verifying the relationship of the disease with the target gene by Western blot and RT-PCR.
Results
1. It was found in our study that most AIPC LNCaP-AI lines increased miRNA expression compared with ADPC line, and that five miRNAs (miR-221, miR-222 miR-21, miR-205 and miR-125b) were up-regulated, while two miRNAs (miR-15a and miR-101) were down-regulated in AI LNCaP-AI cell lines compared with their parental AD LNCaP line, by Northern and RT-PCR.And miR-221 was upregulated significantly.
2. qRT-PCR was performed to evaluate miR-221 expression in LNCaP, LNCaP-AI and PC3 cell lines. It was found that miR-221 expression was up-regulated in LNCaP-AI independently derived AIPC cell lines and down-regulated in LNCaP dependently derived ADPC cell lines, suggesting that these differentially expressed miRNAs might contribute to the progression of CaP cells.
3. Cell growth was assessed by using the CCK-8 cell proliferation assay. Treatment with miR-221 significantly stimulated the growth of LNCaP cells and LNCaP-AI cells, while the miRNA negative control (miR-NC) without treatment failed to affect cell growth. The influence of miR-221m on the S-phase fraction was also upregulated by flow cytometry.
4. NE differentiation was studied in LNCaP cells stimulated with increased miR-221 levels.NSE mRNA levels increased rapidly on day.We examined the level of NSE protein in miR-221-and miR-NC-treated LNCaP cells by Western blot. Transfection with miR-221 led to remarkable up-regulation of NSE in LNCaP cell lines on day 3,
5. LNCaP cells transiently transfected with miR-221 or miR-NC were grown until confluence in a wound healing assay. Migration of LNCaP-AI cells transfected with anti-miR-221 migrated into the open area was significantly increased.and the ability of migration was upregulated.
6. A Matrigel invasion assay was also performed to obtain a measure of the migration of these transfected cells. The results clearly revealed a significant increase in migration and invasiveness in anti-miR-221-treated LNCaP-AI cells. It was concluded that miR-221 was an important determinant of motility and invasiveness of CaP.
7. Analyses of the migration relative genes were performed using the Sanger miRNA database target search program.RT-PCR analysis of RNA extracted from anti-miR-221-treated LNCaP-AI cells showed that there was a significant increase in the expression of DVL2. Treatment of LNCaP-AI cells with anti-miR-221 caused a great increase in DVL2 protein in LNCaP-AI cells by Western blot. And DVL2 may play a role in the invasive ability of LNCaP-AI cells.
8. We next analyzed the level of miR-221 in the plasma samples from ADPC and AIPC patients by TaqMan qRT-PCR.MiR-221 expression was upregulated in ADPC patients compared with AIPC patients.MiR-221 could be decided as a new tumor-derived marker.
Conclusions
1. In our study miR-221 promotes the growth of LNCaP and LNCaP-AI cells. Consistently, ectopic introduction of miR-221 in low expressers of LNCaP cells strongly increased their growth potential by inducing a G1-S shift in the cell cycle. In this study, we showed that miR-221 could promote neuroendocrine differentiation of LNCaP cells in an androgen deprived environment. Thus miR-221 plays an important role in the progression of androgen independence.
2. Down-regulation of miR-221 in LNCaP-AI cells greatly increased the number of cells migrating through the transwell lambers. We therefore conclude that miR-221 might not affect the ability of migration of LNCaP cells significantly; Rather, a poor invasive CaP cell line and miR-221 level might inversely correlate with the invasiveness in androgen independence CaP. We have demonstrated the importance of miR-221 in the migration on LNCaP-AI cells that are strongly invasive and androgen-independent in nature. In the present study, we chose one target mRNA DVL2 that was correlated with the migration. As expected, the expression of DVL2 was up-regulated in LNCaP-AI cells transfected with anti-miR-221.And the reason for it need to be investigated deeply.
3. We analyzed the level of miR-221 in the plasma samples from ADPC and AIPC patients by TaqMan qRT-PCR.MiR-221 expression was upregulated in ADPC patients compared with AIPC patients. These results suggest that tumor-derived miR-221 in plasma would be a blood-based miRNA biomarker candidates for CaP.
引文
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