本文選題：前列腺癌 + miRNAs�。� 參考：《山東大學(xué)》2016年博士論文

【摘要】：背景目前,發(fā)達(dá)國(guó)家男性最常見(jiàn)的癌癥是前列腺癌,前列腺癌是全世界男性惡性腫瘤相關(guān)死亡的主要原因。我國(guó)近些年來(lái),由于人們生活方式、飲食習(xí)慣的改變和人口老齡化等原因,前列腺癌的發(fā)病率逐年上升。由于前列腺癌無(wú)特異性早期癥狀及腫瘤篩查相對(duì)滯后等原因,我國(guó)很多前列腺癌患者在明確診斷時(shí)往往已是腫瘤晚期。目前臨床上主要應(yīng)用前列腺特異性抗原(prostate-specific antigen, PSA)及前列腺穿刺活檢(prostate biopsy)的病理學(xué)對(duì)前列腺癌診斷、監(jiān)測(cè)進(jìn)展及預(yù)后判斷。Gleason評(píng)分是前列腺腺癌組織學(xué)分級(jí)的方法,被廣泛用于診斷、評(píng)估患者預(yù)后及指導(dǎo)治療方案的選擇。但是目前這些用于診斷及預(yù)后判斷方法的準(zhǔn)確率和有效性仍不很高,需要新的和更特異性的生物標(biāo)志物用于臨床。局限性前列腺癌的標(biāo)準(zhǔn)治療包括根治性手術(shù)和根治性放療,雖然早期前列腺癌通常可以治愈,但在發(fā)現(xiàn)時(shí)往往有20%～30%患者的腫瘤已侵犯到前列腺外,對(duì)于該類患者,目前臨床上常用放療及雄激素去勢(shì)治療(Androgen-deprivation therapy, ADT)改善患者總生存期,盡管大多數(shù)患者起初對(duì)內(nèi)分泌治療有效,但經(jīng)過(guò)中位時(shí)間14～30個(gè)月后,幾乎所有患者都將逐漸由雄激素敏感性前列腺癌轉(zhuǎn)變成去勢(shì)抵抗性前列腺癌(Castrate-resistant Prostate Cancer, CRPC)前列腺癌一旦進(jìn)展到CRPC,尤其是轉(zhuǎn)移性CRPC (metastatic CRPC, mCRPC)則預(yù)后極差,CRPC是前列腺癌患者死亡的主要原因。盡管近幾年新開(kāi)發(fā)的一些抗腫瘤藥物,如多西他賽、醋酸阿比特龍、卡巴他賽、恩扎魯胺和sipuleucel-T等,在一些CRPC患者中顯示出一定臨床療效。然而,對(duì)患者總生存率改善不盡人意。因此,迫切需要了解前列腺癌發(fā)生發(fā)展的潛在分子機(jī)制,以幫助發(fā)現(xiàn)可用于早期診斷、預(yù)后判斷的生物標(biāo)記物,以及發(fā)現(xiàn)更有效的治療方法。小分子核糖核酸MicroRNAs(簡(jiǎn)寫(xiě)成miRNAs)是廣泛存在于多細(xì)胞有機(jī)體如植物、動(dòng)物及人類中的一類內(nèi)源性非蛋白質(zhì)編碼的RNA小分子(包含約22個(gè)核苷酸),miRNAs通過(guò)序列互補(bǔ)識(shí)別并結(jié)合靶基因信使RNA(mRNA),促使靶基因mRNA的降解或翻譯抑制,參與轉(zhuǎn)錄后基因表達(dá)的調(diào)控。miRNAs調(diào)節(jié)多種細(xì)胞功能,包括調(diào)節(jié)細(xì)胞生長(zhǎng)、細(xì)胞和組織分化(與癌癥發(fā)生相關(guān)的細(xì)胞過(guò)程)、凋亡以及抗應(yīng)激等。目前,在所有物種中已確認(rèn)超過(guò)10,000個(gè)miRNAs,人類基因中已發(fā)現(xiàn)超過(guò)1300個(gè)miRNAs。人類miRNAs多數(shù)位于基因間區(qū)或已知的轉(zhuǎn)錄內(nèi)含子內(nèi),大約有1%-5%的基因是miRNAs,它們參與調(diào)節(jié)大約60%的蛋白質(zhì)編碼基因。即使一個(gè)單一的miRNAs異常表達(dá),也可能會(huì)影響到大量的細(xì)胞過(guò)程,據(jù)預(yù)測(cè)每個(gè)miRNAs有可能影響到上百個(gè)蛋白質(zhì)的表達(dá),從而破壞體內(nèi)平衡狀態(tài)。有趣的是,一個(gè)基因編碼序列可由幾個(gè)miRNAs調(diào)控,而一個(gè)miRNAs可調(diào)控多個(gè)靶基因mRNA。越來(lái)越多的證據(jù)表明,miRNAs表達(dá)失調(diào)和功能的改變與人類腫瘤不受控制的生長(zhǎng)有關(guān),它們參與調(diào)控腫瘤細(xì)胞的增殖、分化和凋亡等生物過(guò)程。在人類許多惡性腫瘤中,都觀察到存在miRNAs表達(dá)失調(diào)。同樣在前列腺癌細(xì)胞,也發(fā)現(xiàn)一些miRNAs較前列腺良性組織發(fā)生了改變,例如miR-16、miR-23b、miR-143、 miR-145、let-7、miR-99、miR-125、miR-221、miR-29和miR-30等在前列腺癌表達(dá)下調(diào),而miR-21、miR-20a、miR-32、miR-184、miR-198和miR-370等則在前列腺癌表達(dá)上調(diào)。抑癌miRNAs缺少或致癌miRNAs的表達(dá)增加最終促進(jìn)腫瘤細(xì)胞增殖、浸潤(rùn)和轉(zhuǎn)移。研究發(fā)現(xiàn),作為miRNAs之一的miR-556同樣參與了腫瘤的發(fā)生發(fā)展,2006年在關(guān)于人結(jié)直腸癌細(xì)胞miRNAs表達(dá)譜的研究中首次描述了miR-556,其后的第二年,Landgraf等在對(duì)惡性腫瘤miRNAs分析中miR-556作為致癌miRNAs被描述,隨后在其他惡性腫瘤如宮頸癌和黑色素瘤等亦描述了miR-556。目前對(duì)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這些miRNAs及其調(diào)控的基因,對(duì)進(jìn)一步了解前列腺癌發(fā)生發(fā)展機(jī)制,發(fā)現(xiàn)新的早期診斷及預(yù)后判斷的腫瘤標(biāo)記物,以及開(kāi)發(fā)新的靶向治療藥物具有重要意義。目的前列腺癌進(jìn)展到CRCP后預(yù)后很差,目前尚無(wú)有效的治療方法,急需發(fā)現(xiàn)能夠早期診斷及判斷預(yù)后的生物標(biāo)志物,以及新的治療靶點(diǎn)。目前,microRNAs與前列腺癌發(fā)生發(fā)展的相關(guān)性尚不清楚,需要進(jìn)一步研究以更好地了解miRNAs在前列腺癌的基因調(diào)控網(wǎng)絡(luò)。本課題通過(guò)檢測(cè)作為miRNAs之一的miR-556-5p在前列腺癌的表達(dá)及對(duì)腫瘤細(xì)胞生物學(xué)行為的影響,旨在研究miR-556-5p在前列腺癌發(fā)生發(fā)展中的作用,通過(guò)進(jìn)一步探討miR-556-5p的作用機(jī)制,從而有望為前列腺癌早期診斷、預(yù)后判斷提供新的生物標(biāo)記物,并提供潛在的治療靶點(diǎn)和治療策略。方法為研究miR-556-5p在前列腺癌發(fā)生發(fā)展中的作用及機(jī)制,本課題分三部分進(jìn)行。第一部分：為了檢測(cè)miR-556-5p在前列腺癌的表達(dá)情況,我們?cè)谂R床收集前列腺癌組織和對(duì)應(yīng)的癌旁非腫瘤組織,并培養(yǎng)人類前列腺癌細(xì)胞系M12、 Tsu-Prl、PC3、DU145、22RV1和LNCAP以及非腫瘤前列腺上皮細(xì)胞系RWPE-1,應(yīng)用實(shí)時(shí)定量PCR技術(shù)檢測(cè)miR-556-5p表達(dá)水平,分析miR-556-5p在臨床前列腺腫瘤組織及前列腺癌細(xì)胞系中的表達(dá)有無(wú)失調(diào)。第二部分：為了研究miR-556-5p在前列腺癌細(xì)胞中的作用,我們通過(guò)上調(diào)和下調(diào)前列腺癌PC3細(xì)胞miR-556-5p的水平,應(yīng)用MTT測(cè)定、細(xì)胞集落形成實(shí)驗(yàn)和錨定非依賴性生長(zhǎng)測(cè)定等方法,觀察miR-556-5p對(duì)PC3細(xì)胞生物學(xué)行為的影響。第三部分：為進(jìn)一步探討miR-556-5p在前列腺癌中的作用機(jī)制,我們應(yīng)用生物信息學(xué)算法預(yù)測(cè)miR-556-5p可能的靶基因,然后通過(guò)蛋白印跡技術(shù)(western blotting)和熒光素酶檢測(cè)法(luciferase assay)驗(yàn)證,其后進(jìn)一步研究靶基因在前列腺癌中的作用。結(jié)果miR-556-5p在前列腺癌組織和前列腺癌細(xì)胞系中表達(dá)上調(diào)。與相鄰非腫瘤組織和非腫瘤上皮前列腺細(xì)胞系相比,在8例前列腺癌患者標(biāo)本及6個(gè)前列腺癌細(xì)胞系中,miR-556-5p均明顯高表達(dá),差異有統(tǒng)計(jì)學(xué)意義(P0.05)。miR-556-5p促進(jìn)前列腺癌細(xì)胞增殖。miR-556-5p表達(dá)失調(diào)增加了PC3細(xì)胞的生長(zhǎng)率,細(xì)胞集落形成實(shí)驗(yàn)顯示miR-556-5p的上調(diào)促進(jìn)了PC3細(xì)胞的集落形成能力,錨定非依賴性生長(zhǎng)測(cè)定顯示PC3細(xì)胞的獨(dú)立生長(zhǎng)能力和miR-556-5p水平相關(guān)(P0.05),而下調(diào)miR-556-5p則顯示出相反的作用(P0.05)。PPP2R2A是miR-556-5p直接調(diào)控的下游靶基因。我們用TargetScan算法發(fā)現(xiàn)PPP2R2A mRNA 3’-非編碼區(qū)(3’-UTR)與miR-556-5p“種子”序列互補(bǔ)。為證實(shí)miR-556-5p是否通過(guò)直接結(jié)合3’-UTR區(qū)域抑制PPP2R2A的表達(dá),我們應(yīng)用蛋白印跡技術(shù)和熒光素酶檢測(cè)法,野生型PPP2R2A的3’-UTR克隆進(jìn)熒光素酶基因,熒光素酶基因轉(zhuǎn)染PC3細(xì)胞,PC3細(xì)胞同時(shí)分別被miR-556-5p mimics、 miR-556-5p inhibitor及miRNA negative controls (NC)共轉(zhuǎn)染。蛋白印跡結(jié)果顯示上調(diào)miR-556-5p的PC3細(xì)胞的PPP2R2A蛋白水平顯著降低(P0.05),而下調(diào)miR-556-5p則促進(jìn)PPP2R2A蛋白的表達(dá)。上調(diào)PC3細(xì)胞miR-556-5p后,PPP2R2A 3'-UTR熒光素酶的活性顯著降低(P0.05),而下調(diào)miR-556-5p則出現(xiàn)相反的結(jié)果(P0.05)。此外,與miRNA NC共轉(zhuǎn)染細(xì)胞相比,突變型miR-556-5p共轉(zhuǎn)染細(xì)胞的熒光素酶活性無(wú)明顯改變(P0.05)。這些研究結(jié)果表明miR-556-5p通過(guò)直接結(jié)合到PPP2R2A 3'-UTR對(duì)PPP2R2A進(jìn)行負(fù)調(diào)控。本課題通過(guò)進(jìn)一步研究miR-556-5p對(duì)PPP2R2A下游基因p27、細(xì)胞周期蛋白D1(cyclin D1)表達(dá)的影響發(fā)現(xiàn),在上調(diào)miR-556-5p表達(dá)的PC3細(xì)胞中p27 mRNA及蛋白表達(dá)均下調(diào),而cyclin D1則表達(dá)上調(diào)。反之,在下調(diào)miR-556-5p的PC3細(xì)胞中,p27 mRNA及蛋白表達(dá)上調(diào),而cyclin D1表達(dá)下調(diào)。結(jié)果表明PPP2R2A下游基因p27、cyclin D1參與了miR-556-5p對(duì)前列腺癌PC3細(xì)胞增殖的調(diào)控過(guò)程。本課題在下調(diào)miR-556-5p的PC3細(xì)胞,下調(diào)PPP2R2A的表達(dá),觀察PPP2R2A下調(diào)后對(duì)腫瘤細(xì)胞集落形成能力和錨定非依賴性生長(zhǎng)能力的影響,結(jié)果顯示下調(diào)PPP2R2A可以逆轉(zhuǎn)因miR-556-5p下調(diào)所致的PC3細(xì)胞增殖抑制作用,即在miR-556-5p促進(jìn)前列腺癌PC3細(xì)胞增殖的過(guò)程中,PPP2R2A下調(diào)是必須的。結(jié)論我們的研究顯示miR-556-5p在臨床前列腺癌組織和前列腺癌細(xì)胞系中表達(dá)上調(diào),miR-556-5p這種表達(dá)失調(diào)導(dǎo)致PPP2R2A表達(dá)下調(diào),使p27表達(dá)下調(diào)及周期蛋白D1表達(dá)上調(diào),從而最終促進(jìn)前列腺癌細(xì)胞增殖。總之,我們的研究表明,miR-556-5p作為前列腺的一種致癌miRNAs,通過(guò)抑制PPP2R2A表達(dá),在前列腺癌的發(fā)生和發(fā)展中起重要作用。意義本研究表明miR-556-5p在前列腺癌中表達(dá)上調(diào),是一種致癌miRNAs,而且首次發(fā)現(xiàn)PPP2R2A是miR-556-5p的直接調(diào)控靶基因,miR-556-5p通過(guò)負(fù)性調(diào)控PPP2R2A,促進(jìn)前列腺癌細(xì)胞增殖。因此,本研究將有助于更好地了解前列腺癌的發(fā)病機(jī)制,miR-556-5p有可能作為前列腺癌的一個(gè)早期診斷、預(yù)后判斷的生物標(biāo)記物,以及新的治療靶點(diǎn)。miR-556-5p最令人興奮的臨床應(yīng)用前景是可能有助于前列腺癌患者的精準(zhǔn)治療。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.25

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