miR-556-5p负调控PPP2R2A促进前列腺癌细胞增殖的实验研究

发布时间：2018-06-28 05:25

本文选题：前列腺癌 + miRNAs　；参考：《山东大学》2016年博士论文

【摘要】：背景目前,发达国家男性最常见的癌症是前列腺癌,前列腺癌是全世界男性恶性肿瘤相关死亡的主要原因。我国近些年来,由于人们生活方式、饮食习惯的改变和人口老龄化等原因,前列腺癌的发病率逐年上升。由于前列腺癌无特异性早期症状及肿瘤筛查相对滞后等原因,我国很多前列腺癌患者在明确诊断时往往已是肿瘤晚期。目前临床上主要应用前列腺特异性抗原(prostate-specific antigen, PSA)及前列腺穿刺活检(prostate biopsy)的病理学对前列腺癌诊断、监测进展及预后判断。Gleason评分是前列腺腺癌组织学分级的方法,被广泛用于诊断、评估患者预后及指导治疗方案的选择。但是目前这些用于诊断及预后判断方法的准确率和有效性仍不很高,需要新的和更特异性的生物标志物用于临床。局限性前列腺癌的标准治疗包括根治性手术和根治性放疗,虽然早期前列腺癌通常可以治愈,但在发现时往往有20%～30%患者的肿瘤已侵犯到前列腺外,对于该类患者,目前临床上常用放疗及雄激素去势治疗(Androgen-deprivation therapy, ADT)改善患者总生存期,尽管大多数患者起初对内分泌治疗有效,但经过中位时间14～30个月后,几乎所有患者都将逐渐由雄激素敏感性前列腺癌转变成去势抵抗性前列腺癌(Castrate-resistant Prostate Cancer, CRPC)前列腺癌一旦进展到CRPC,尤其是转移性CRPC (metastatic CRPC, mCRPC)则预后极差,CRPC是前列腺癌患者死亡的主要原因。尽管近几年新开发的一些抗肿瘤药物,如多西他赛、醋酸阿比特龙、卡巴他赛、恩扎鲁胺和sipuleucel-T等,在一些CRPC患者中显示出一定临床疗效。然而,对患者总生存率改善不尽人意。因此,迫切需要了解前列腺癌发生发展的潜在分子机制,以帮助发现可用于早期诊断、预后判断的生物标记物,以及发现更有效的治疗方法。小分子核糖核酸MicroRNAs(简写成miRNAs)是广泛存在于多细胞有机体如植物、动物及人类中的一类内源性非蛋白质编码的RNA小分子(包含约22个核苷酸),miRNAs通过序列互补识别并结合靶基因信使RNA(mRNA),促使靶基因mRNA的降解或翻译抑制,参与转录后基因表达的调控。miRNAs调节多种细胞功能,包括调节细胞生长、细胞和组织分化(与癌症发生相关的细胞过程)、凋亡以及抗应激等。目前,在所有物种中已确认超过10,000个miRNAs,人类基因中已发现超过1300个miRNAs。人类miRNAs多数位于基因间区或已知的转录内含子内,大约有1%-5%的基因是miRNAs,它们参与调节大约60%的蛋白质编码基因。即使一个单一的miRNAs异常表达,也可能会影响到大量的细胞过程,据预测每个miRNAs有可能影响到上百个蛋白质的表达,从而破坏体内平衡状态。有趣的是,一个基因编码序列可由几个miRNAs调控,而一个miRNAs可调控多个靶基因mRNA。越来越多的证据表明,miRNAs表达失调和功能的改变与人类肿瘤不受控制的生长有关,它们参与调控肿瘤细胞的增殖、分化和凋亡等生物过程。在人类许多恶性肿瘤中,都观察到存在miRNAs表达失调。同样在前列腺癌细胞,也发现一些miRNAs较前列腺良性组织发生了改变,例如miR-16、miR-23b、miR-143、 miR-145、let-7、miR-99、miR-125、miR-221、miR-29和miR-30等在前列腺癌表达下调,而miR-21、miR-20a、miR-32、miR-184、miR-198和miR-370等则在前列腺癌表达上调。抑癌miRNAs缺少或致癌miRNAs的表达增加最终促进肿瘤细胞增殖、浸润和转移。研究发现,作为miRNAs之一的miR-556同样参与了肿瘤的发生发展,2006年在关于人结直肠癌细胞miRNAs表达谱的研究中首次描述了miR-556,其后的第二年,Landgraf等在对恶性肿瘤miRNAs分析中miR-556作为致癌miRNAs被描述,随后在其他恶性肿瘤如宫颈癌和黑色素瘤等亦描述了miR-556。目前对于很多miRNAs,尤其是miR-556在前列腺癌中的作用尚不清楚,更加全面深入的研究这些miRNAs及其调控的基因,对进一步了解前列腺癌发生发展机制,发现新的早期诊断及预后判断的肿瘤标记物,以及开发新的靶向治疗药物具有重要意义。目的前列腺癌进展到CRCP后预后很差,目前尚无有效的治疗方法,急需发现能够早期诊断及判断预后的生物标志物,以及新的治疗靶点。目前,microRNAs与前列腺癌发生发展的相关性尚不清楚,需要进一步研究以更好地了解miRNAs在前列腺癌的基因调控网络。本课题通过检测作为miRNAs之一的miR-556-5p在前列腺癌的表达及对肿瘤细胞生物学行为的影响,旨在研究miR-556-5p在前列腺癌发生发展中的作用,通过进一步探讨miR-556-5p的作用机制,从而有望为前列腺癌早期诊断、预后判断提供新的生物标记物,并提供潜在的治疗靶点和治疗策略。方法为研究miR-556-5p在前列腺癌发生发展中的作用及机制,本课题分三部分进行。第一部分：为了检测miR-556-5p在前列腺癌的表达情况,我们在临床收集前列腺癌组织和对应的癌旁非肿瘤组织,并培养人类前列腺癌细胞系M12、 Tsu-Prl、PC3、DU145、22RV1和LNCAP以及非肿瘤前列腺上皮细胞系RWPE-1,应用实时定量PCR技术检测miR-556-5p表达水平,分析miR-556-5p在临床前列腺肿瘤组织及前列腺癌细胞系中的表达有无失调。第二部分：为了研究miR-556-5p在前列腺癌细胞中的作用,我们通过上调和下调前列腺癌PC3细胞miR-556-5p的水平,应用MTT测定、细胞集落形成实验和锚定非依赖性生长测定等方法,观察miR-556-5p对PC3细胞生物学行为的影响。第三部分：为进一步探讨miR-556-5p在前列腺癌中的作用机制,我们应用生物信息学算法预测miR-556-5p可能的靶基因,然后通过蛋白印迹技术(western blotting)和荧光素酶检测法(luciferase assay)验证,其后进一步研究靶基因在前列腺癌中的作用。结果miR-556-5p在前列腺癌组织和前列腺癌细胞系中表达上调。与相邻非肿瘤组织和非肿瘤上皮前列腺细胞系相比,在8例前列腺癌患者标本及6个前列腺癌细胞系中,miR-556-5p均明显高表达,差异有统计学意义(P0.05)。miR-556-5p促进前列腺癌细胞增殖。miR-556-5p表达失调增加了PC3细胞的生长率,细胞集落形成实验显示miR-556-5p的上调促进了PC3细胞的集落形成能力,锚定非依赖性生长测定显示PC3细胞的独立生长能力和miR-556-5p水平相关(P0.05),而下调miR-556-5p则显示出相反的作用(P0.05)。PPP2R2A是miR-556-5p直接调控的下游靶基因。我们用TargetScan算法发现PPP2R2A mRNA 3’-非编码区(3’-UTR)与miR-556-5p“种子”序列互补。为证实miR-556-5p是否通过直接结合3’-UTR区域抑制PPP2R2A的表达,我们应用蛋白印迹技术和荧光素酶检测法,野生型PPP2R2A的3’-UTR克隆进荧光素酶基因,荧光素酶基因转染PC3细胞,PC3细胞同时分别被miR-556-5p mimics、 miR-556-5p inhibitor及miRNA negative controls (NC)共转染。蛋白印迹结果显示上调miR-556-5p的PC3细胞的PPP2R2A蛋白水平显著降低(P0.05),而下调miR-556-5p则促进PPP2R2A蛋白的表达。上调PC3细胞miR-556-5p后,PPP2R2A 3'-UTR荧光素酶的活性显著降低(P0.05),而下调miR-556-5p则出现相反的结果(P0.05)。此外,与miRNA NC共转染细胞相比,突变型miR-556-5p共转染细胞的荧光素酶活性无明显改变(P0.05)。这些研究结果表明miR-556-5p通过直接结合到PPP2R2A 3'-UTR对PPP2R2A进行负调控。本课题通过进一步研究miR-556-5p对PPP2R2A下游基因p27、细胞周期蛋白D1(cyclin D1)表达的影响发现,在上调miR-556-5p表达的PC3细胞中p27 mRNA及蛋白表达均下调,而cyclin D1则表达上调。反之,在下调miR-556-5p的PC3细胞中,p27 mRNA及蛋白表达上调,而cyclin D1表达下调。结果表明PPP2R2A下游基因p27、cyclin D1参与了miR-556-5p对前列腺癌PC3细胞增殖的调控过程。本课题在下调miR-556-5p的PC3细胞,下调PPP2R2A的表达,观察PPP2R2A下调后对肿瘤细胞集落形成能力和锚定非依赖性生长能力的影响,结果显示下调PPP2R2A可以逆转因miR-556-5p下调所致的PC3细胞增殖抑制作用,即在miR-556-5p促进前列腺癌PC3细胞增殖的过程中,PPP2R2A下调是必须的。结论我们的研究显示miR-556-5p在临床前列腺癌组织和前列腺癌细胞系中表达上调,miR-556-5p这种表达失调导致PPP2R2A表达下调,使p27表达下调及周期蛋白D1表达上调,从而最终促进前列腺癌细胞增殖。总之,我们的研究表明,miR-556-5p作为前列腺的一种致癌miRNAs,通过抑制PPP2R2A表达,在前列腺癌的发生和发展中起重要作用。意义本研究表明miR-556-5p在前列腺癌中表达上调,是一种致癌miRNAs,而且首次发现PPP2R2A是miR-556-5p的直接调控靶基因,miR-556-5p通过负性调控PPP2R2A,促进前列腺癌细胞增殖。因此,本研究将有助于更好地了解前列腺癌的发病机制,miR-556-5p有可能作为前列腺癌的一个早期诊断、预后判断的生物标记物,以及新的治疗靶点。miR-556-5p最令人兴奋的临床应用前景是可能有助于前列腺癌患者的精准治疗。
[Abstract]:Against background, the most common cancer in men in developed countries is prostate cancer. Prostate cancer is the main cause of the death of male malignant tumors all over the world. In recent years, the incidence of prostate cancer has risen year by year because of people's lifestyle, changes in eating habits and aging of the population. Many of the prostate cancer patients in our country are often advanced in diagnosis. The main clinical application of prostate specific antigen (prostate-specific antigen, PSA) and prostate biopsy (prostate biopsy) in the diagnosis of prostate cancer, monitoring progress and prognosis Judging the histological grading of the prostate adenocarcinoma, the.Gleason score is widely used to diagnose, evaluate the patient's prognosis and guide the treatment options. However, the accuracy and effectiveness of these methods for diagnosis and prognosis are still not very high, and new and more specific biomarkers are needed to be used in clinical. Localized prostate The standard treatment of cancer includes radical surgery and radical radiation therapy. Although early prostate cancer is usually cured, 20% to 30% patients often have tumors that have been infringed on the prostate. For this type of patients, the current clinical use of radiotherapy and androgenic Androgen-deprivation therapy (ADT) improves the total life of the patients. Although most patients were initially effective in endocrinology, after 14~30 months of median time, almost all patients gradually changed from androgen sensitive prostate cancer to Castrate-resistant Prostate Cancer (CRPC) before adenocarcinoma progressed to CRPC, especially metastatic CRPC (metas). Tatic CRPC, mCRPC) has a poor prognosis, and CRPC is the main cause of death in patients with prostate cancer. Although some newly developed antitumor drugs in recent years, such as docetaxel, amiseton acetate, kappasai, inzi, and sipuleucel-T, have been shown to have a certain clinical effect in some CRPC patients. However, the overall survival rate of patients is not improved. It is desirable. Therefore, there is an urgent need to understand the potential molecular mechanisms of the development of prostate cancer to help discover biomarkers that can be used for early diagnosis and prognosis, and to find a more effective treatment. Small molecular ribonucleic acid MicroRNAs (miRNAs) is widely stored in multicellular organisms such as plants, animals and humans. A class of endogenous non protein encoded RNA small molecules (including about 22 nucleotides), miRNAs through sequence complementation and binding target gene messenger RNA (mRNA) to promote the degradation of target gene mRNA or translation inhibition. The regulatory.MiRNAs involved in post transcriptional gene expression regulates a variety of cell functions, including cell growth, cell and tissue differentiation. At present, more than 10000 miRNAs have been identified in all species, and more than 1300 human miRNAs in human genes have been found to be in the intergenic or known transcriptional introns of more than 1300 human miRNAs, and about 1%-5% genes are miRNAs, and they are involved in regulating about 60% of the protein. Even a single miRNAs abnormal expression may affect a large number of cellular processes. It is predicted that each miRNAs may affect the expression of hundreds of proteins and thus disrupt the state of balance in the body. Interestingly, a gene coding sequence can be regulated by several miRNAs, and one miRNAs can regulate multiple target genes, M RNA. increasing evidence suggests that miRNAs expression disorders and changes in function are associated with uncontrolled growth of human tumors. They are involved in the regulation of biological processes such as proliferation, differentiation and apoptosis of tumor cells. In many human malignant tumors, there is a loss of miRNAs expression in many human malignant tumors. Also, some miRNAs are also found in prostate cancer cells. Compared with benign prostatic tissues, such as miR-16, miR-23b, miR-143, miR-145, let-7, miR-99, miR-125, miR-221, miR-29 and miR-30, the expression of prostate cancer was down. Tumor cell proliferation, infiltration and metastasis. The study found that miR-556, one of the miRNAs, was also involved in the development of the tumor. In 2006, miR-556 was first described in the study of miRNAs expression profiles in human colorectal cancer cells. After second years, Landgraf was described as a carcinogenic miRNAs in the miRNAs analysis of malignant swollen tumors. Subsequently, other malignant tumors, such as cervical cancer and melanoma, have also described miR-556.'s current role in many miRNAs, especially miR-556 in prostate cancer, and a more comprehensive study of these miRNAs and its regulated genes, to further understand the mechanism of the development of prostate cancer, and to discover new early diagnosis and prediagnosis. After the progression of the prostate cancer to CRCP, the prognosis of the prostate cancer is very poor. There is no effective treatment. It is urgent to find biomarkers for early diagnosis and prognosis, as well as new therapeutic targets. At present, the development of microRNAs and prostate cancer is developing. The correlation is still unclear. Further research is needed to better understand the gene regulatory network of miRNAs in prostate cancer. The purpose of this study is to investigate the role of miR-556-5p in the development of adenocarcinoma of the prostatic carcinoma by detecting the expression of miR-556-5p as one of the miRNAs in prostate cancer and the biological behavior of the tumor cells. To explore the mechanism of miR-556-5p, which is expected to provide new biomarkers for the early diagnosis and prognosis of prostate cancer, and provide potential therapeutic targets and therapeutic strategies. The method is to study the role and mechanism of miR-556-5p in the development of prostate cancer. This subject is divided into three parts. The first part: to detect miR-556-5 P in the expression of prostate cancer, we collect the prostate cancer tissue and the corresponding non cancer tissue, and cultivate the human prostate cancer cell line M12, Tsu-Prl, PC3, DU145,22RV1 and LNCAP, and the non tumor prostate epithelial cell line RWPE-1. We use real-time quantitative PCR to detect the miR-556-5p expression level and analyze miR-556-5p in the prostate cancer cell line. The second part: in order to study the role of miR-556-5p in prostate cancer cells, we use MTT assay, cell colony formation test and anchoring non dependent growth assay by up and down the level of miR-556-5p in prostate cancer cell PC3 cells. The effect of miR-556-5p on the biological behavior of PC3 cells was observed. Third: to further explore the mechanism of miR-556-5p in prostate cancer, we use bioinformatics algorithm to predict the possible target genes of miR-556-5p, and then verify by the Western blot (Western blotting) and luciferase assay (luciferase assay). The role of target gene in prostate cancer was studied. Results miR-556-5p was up-regulated in prostate cancer tissue and prostate cancer cell lines. Compared with adjacent non tumor and non tumor epithelial cell lines, the miR-556-5p expression was significantly higher in 8 cases of prostate cancer and 6 prostate cancer cells. Statistically significant (P0.05).MiR-556-5p promoted the proliferation of.MiR-556-5p in the proliferation of prostate cancer cells and increased the growth rate of PC3 cells. Cell colony formation experiments showed that the up regulation of miR-556-5p promoted the colony formation ability of PC3 cells. Anchoring non dependent growth assay showed the independent growth and miR-556-5p level of PC3 cells. P0.05, and the downregulation of miR-556-5p showed the opposite effect (P0.05).PPP2R2A was the downstream target gene directly regulated by miR-556-5p. We found that the PPP2R2A mRNA 3 '- non coding region (3' -UTR) complemented with the miR-556-5p "seed" sequence by TargetScan algorithm. We used Western blot and luciferase assay. The 3 '-UTR of wild type PPP2R2A was cloned into luciferase gene, the luciferase gene was transfected into PC3 cells, and PC3 cells were co transfected by miR-556-5p mimics, miR-556-5p inhibitor and miRNA negative controls (NC). The level of PPP2R2A protein in C3 cells decreased significantly (P0.05), while down regulation of miR-556-5p promoted the expression of PPP2R2A protein. The activity of PPP2R2A 3'-UTR luciferase decreased significantly (P0.05) after up regulation of miR-556-5p in PC3 cells. There was no significant change in the luciferase activity of the transfected cells (P0.05). These results showed that miR-556-5p was negatively regulated by the direct combination of PPP2R2A 3'-UTR to PPP2R2A. This subject further studied the effect of miR-556-5p on the expression of the D1 (cyclin D1) of the downstream PPP2R2A gene p27 and the cyclin protein D1 (cyclin D1). The expression of p27 mRNA and protein in C3 cells were all down regulated, while cyclin D1 was up regulated. On the other hand, the expression of p27 mRNA and protein was up regulated in the PC3 cells of miR-556-5p reduction, while cyclin D1 was down regulated. The results showed that the downstream PPP2R2A genes were involved in the regulation of the proliferation of prostate cancer cells. The PC3 cells of -556-5p down regulated the expression of PPP2R2A and observed the effect of down regulation of PPP2R2A on the colony forming ability and anchoring non dependent growth ability of the tumor cells. The results showed that the down regulation of PPP2R2A could reverse the proliferation inhibition of PC3 cells caused by the downregulation of miR-556-5p, that is, PPP in the process of promoting the proliferation of PC3 cells in prostate cancer, PPP. The down-regulation of 2R2A is necessary. Conclusion our study showed that the expression of miR-556-5p was up-regulated in the clinical prostate cancer tissues and the prostate cancer cell lines. The imbalance of miR-556-5p expression led to the downregulation of the expression of PPP2R2A, the down regulation of p27 expression and the up regulation of the cyclin D1 expression, thus promoting the proliferation of prostate cancer cells. MiR-556-5p, a carcinogenic miRNAs of the prostate, plays an important role in the development and development of prostate cancer by inhibiting the expression of PPP2R2A. The significance of this study indicates that the up regulation of miR-556-5p in prostate cancer is a carcinogenic miRNAs, and it is the first time that PPP2R2A is a direct target gene for the regulation of miR-556-5p and miR-556-5p is negative. Regulation of PPP2R2A to promote the proliferation of prostate cancer cells. Therefore, this study will help to better understand the pathogenesis of prostate cancer. MiR-556-5p may be an early diagnosis of prostate cancer, biomarkers for prognostic judgment, and the most exciting clinical applications of new therapeutic targets,.MiR-556-5p, may be helpful to the future. Accurate treatment of patients with adenocarcinoma.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.25

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