胰腺癌特异性microRNAs的筛选及机制研究

【作者】 刘雯静；

【导师】 赵玉沛；

【作者基本信息】 北京协和医学院 ， 外科学， 2011， 博士

【摘要】 背景：胰腺癌恶性程度极高,是消化系统第二大恶性肿瘤,在肿瘤死亡原性诱因中列第四位,是5年生存率最低的恶性肿瘤,而缺乏早期诊断手段和化疗耐药是导致胰腺癌高死亡率的主要原因。胰腺癌早期诊断率低,导致95%以上的胰腺癌患者在明确诊断时肿瘤已进入进展期,手术虽是根治胰腺癌的唯一方法,但根治性手术切除率仅15-20%；化疗是胰腺癌主要的辅助治疗手段,然而,肿瘤细胞对化疗药物产生的耐药性严重地影响了疗效。因此,提高早期胰腺癌的诊断率、扭转胰腺癌耐药,是提高胰腺癌的治疗效果和改善预后的关键。近年来,人们在研究中发现,microRNAs (miRNAs)是稳定存在于外周血中一种候选的肿瘤标志物,同时与肿瘤耐药密切相关。miRNAs是近年来发现的一系列内源性单链非编码的小RNAs分子,长度约22个核苷酸。其广泛分布于真核生物中。miRNAs常以基对相互原则与靶信使RNA (mRNA)的3’端的非翻译区完全或不完全配对,降解靶信使RNA或阻遏其转录后翻译。对组织分化、细胞分化、凋亡等正常的生理过程起到重要的调控作用。最近多项研究发现,若干miRNAs在各种肿瘤组织中的表达水平有不同程度上调或下调,这一现象初步揭示了肿瘤发生、进展、耐药与miRNAs表达量及种类之间存在相关性,为逆转现状,从根本上改善肿瘤预后提供新的途径。目的：在胰腺癌中,寻找具有潜在诊断价值的外周血特异性miRNAs,探索胰腺癌耐药相关的miRNAs,并通过调控miR-155在胰腺癌早期抑制]hMLH1表达改善胰腺癌预后。探索miRNAs对于胰腺癌早期诊断、治疗和指导预后的价值。方法：1、人胰腺癌组织和外周血特异性miRNAs表达谱的建立：①建立利用实时定量PCR(Real-time PCR)对临床血浆标本中循环miRNAs进行定量测定的技术平台；②采用基于qPCR(quantitative PCR)的TaqMan低密度阵列(TaqMan low density array, TLDA)技术,对健康个体、胰腺癌和胰腺炎患者血浆标本中的循环miRNAs进行初步分析,建立人胰腺癌、胰腺炎外周血特异性miRNAs表达谱；③在筛选结果中,选择5种在胰腺癌中呈差异表达的miRNAs进行验证(let-7b、miR-miR-363、miR-942、miR-645、miR-190b)。2、耐药相关miRNAs筛选：①cck-8法验证胰腺癌耐药(耐健泽、耐5-fu、耐阿霉素)细胞株的耐药性；②利用miRNAs芯片技术比较人胰腺癌耐药(耐健泽、耐5-fu、耐阿霉素)细胞株与非耐药细胞株的miRNA表达差异；③根据表达谱结果,应用real-time PCR对部分差异表达的miRNA进行验证。3、探索与胰腺癌早期事件相关的miR-155的作用机制及其指导治疗、改善预后的意义：①应用双荧光素酶报告基因及Western Blot的方法探索miR-155下游潜在靶基因hMLH1;②应用免疫组化方法研究其与胰腺癌临床预后的关系。结果：1、①建立了循环miRNAs定量检测方法；②在胰腺癌、胰腺炎、正常人的血浆中共检测到188种miRNAs。胰腺癌与胰腺炎相比,有6条显著上调、1条显著下调(P<0.01)；胰腺癌和胰腺炎相比,有10条显著上调,9条显著下调(P<0.01)；③验证结果显示,miR-942在胰腺癌与健康人外周血中表达呈显著差异(P=0.023)。2、①SW1990的阿霉素、氟尿嘧啶和吉西他滨的耐药株的耐药性分别是亲本株的12.57、133.68和438.96倍；②与亲本株相比,在SW1990/Gem细胞株中差异表达显著的miRNAs有58条,在SW1990/Adm细胞株中有59条,在SW1990/5-Fu细胞株中有47条。在SW1990/Gem、5-Fu两个耐药株中同时呈现显著差异的miRNAs14条,在SW1990/Gem、Adm两个耐药株中同时呈现显著差异的miRNAs22条,在SW1990/5-Fu、Adm两个耐药株中同时呈现显著差异的miRNAs 12条；其中3个耐药株中同时呈现显著差异的miRNAs7条;即miR-33a、33b、1285、486-5p、99b*、125a-3p、200c*在吉西他滨、氟尿嘧啶、阿霉素3株耐药株中的表达与亲本株相比差异具有显著性(P<0.05)；③文献检索胰腺癌耐药相关基因,及在其它肿瘤中调控上述基因的miRNAs,并与本研究的筛选结果进行比对。其中miR-34a、424、449a、497、451、21和miR-101在胰腺癌耐药株中呈差异表达；④应用实时荧光定量(Real Time-PCR)的方法对miR-486-5p、miR-125a-3p、miR-99b*、miR-1285进行验证,结果与芯片的结果一致,且均存在显著差异(P<0.05)3、①hMLH1是miR-155的靶基因；②miR-155通过与hMLH1的3’-UTR区不完全配对,抑制mRNA翻译；③hMLH1与胰腺癌的恶性进展及预后相关。结论：1、建立成熟的循环miRNAs定量检测方法；初步建立胰腺癌miRNAs表达谱；has-mir-942可能成为胰腺癌血清特异性肿瘤标记物,具有潜在的早期诊断价值。2、初步建立胰腺癌耐药相关miRNAs表达谱,其中miR-486-5p、miR-99b*、miR-125a-3p、miR-1285是潜在的与胰腺癌多药耐药相关的上游调控基因,验证其差异表达可能成为对耐药人群早期诊断的依据,进而通过指导个体化治疗改善预后；明确与其他肿瘤耐药相关的miRNAs (miR-34a、424、449a、497、451、21和miR-101)亦在胰腺癌miRNAs耐药谱中呈现差异表达,为进一步验证其在胰腺癌耐药机制中的作用提供线索。3、MiR-155通过下调靶基因hMLH1调控胰腺癌的恶性进展。更多还原

【Abstract】 Background Pancreatic cancer is one of the most aggressive tumor and continues to be a major unsolved health problem. It is the second most frequent gastrointestinal malignancy with poor prognosis, and the fourth leading cause of cancer deaths in America and has the lowest 5-year survival rate among all malignancies. The high mortality rate for pancreatic cancer is primarily due to diagnosis mostly at advanced stage no sensitive and specific tools of early detection. Furthermore, the conventional treatment approaches, such as surgery, chemotherapy, or combinations of both, have had little impact on the natural course of this aggressive neoplasm. Although surgical resection remains the only curative treatment, around 15-20% of patients have resectable disease, only around 20% of them survive to 5 years. For locally advanced, unresectable, and metastatic disease, chemotherapy seems to hold the greatest promise, but the dismal prognosis is partly due to its intrinsic and extrinsic drug resistance characteristics. Pancreatic cancer remain a challenging problem during the 21st century. However, improvements in early detection and increasing the drug sensitive could be novel strategies to prolong substantially rvival of patients with this disease.The microRNAs (miRNAs) are a group of small RNAs, which are single-stranded and consist of 18-25 nucleotides (～22 nt). They do not code for any protein or peptide; however, they regulate gene expression by multiple mechanisms. It is commonly accepted that mature miRNAs regulate gene expression by binding to the 3’-untranslated region (3’-UTR) of target mRNA, causing degradation of mRNA or inhibition of their translation to functional proteins. miRNAs play important roles in many normal biological processes; however, the aberrant miRNA expression and its correlation with the development and progression of cancers is an emerging field.Recently, miRNAs have received increasing attention in the field of cancer research. miRNA alterations are involved in the initiation and progression of human cancer. MiRNA-expression profiling of human tumours has identified signatures associated with diagnosis, staging, progression, prognosis and response to treatment. In addition, profiling has been exploited to identify miRNA genes that might represent downstream targets of activated oncogenic pathways, or that target proteincoding genes involved in cancer. miRNAs could be used as biomarkers for diagnosis of cancer, prediction of prognosis and to constitute a novel target for cancer treatment. Importantly, some miRNAs could regulate the formation of cancer stem cells and the acquisition of epithelial-mesenchymal transition, which are critically associated with drug resistance. Moreover, some miRNAs could target genes related to drug sensitivity, resulting in the altered sensitivity of cancer cells to anti-cancer drugs. Therefore, the study of miRNAs holds much promise for improving diagnosis and treatment of pancreatic cancer.Objective 1、To indentify and validate the expression patterns of miRNAs associated pancreatic cancer’s diagnosis and drug resistance.2、To validate targeting of miR-155 by hMLHl induced cancer advance.Methods 1.Detection of Human pancreatic cancer based on measurement of a pancreatic cancer-expressed miRNAs in Plasma①To investigate the feasibility of identifing specific circulating miRNAs as Potential markers for diagnosis of pancreas-related disease and to establish the approaches of blood-based miRNAs cancer detection by using Real-time PCR;②Using qPCR-based expression profiling (TaqMan low density arrays, TLDA) followed by Real-Time quantitative Polymerase Cycle Reaction (RT-qPCR) validation, we compared the levels of circulating miRNAs in plasma samples from 20 pancreatic cancer patients,6 Pancreatitis patients and 5 matched healthy controls.③To further explore the potential roles of these circulating miRNAs, let-7b、miR-miR-363、miR-942、miR-645、miR-190b, using qPCR between pancreatic cancer and healthy control.2. To investigate the involvement of miRNAs in drug-resistant of pancreatic cancer cell lines①The drug resistance of SW1990/ ADM, SW1990/ 5Fu and SW1990/ GEM cells was evaluated using CCK-8 assay.②③High-throughput analysis of the miRNAs profile in a panel of adriamycin- (SW1990/ADM), 5-fluorouracil- (SW1990/5Fu) and gemcitabine resistant (SW1990/GEM) cells was assessed using a microarray platform and subsequent validation with qPCR.3.To confirm the possible mechanisms of inducing mortality in pancreatic cance①To explore the miR-155 target gene hMLH1 using dual luciferase reporter assay validation, PCR and Western Blot.②A total of 43 patients with pancreatic cancer underwent surgical resection were included. Expression of hMLH1 in tumor and para-tumor tissues was determined by immunohistochemistry. The relationship between hMLH1 expression and clinicopathological and prognostic variables were evaluated.Results 1.①We established a novel approach to test the miRNAs in plasma.②We established a list of likely blood-based miRNAs biomarker candidates for pancreatic cancer and Pancreatitis patients. The data indicated that a total of 188 miRNAs could be detected. Compared the levels of circulating miRNAs from pancreatic cancer and Pancreatitis group or from pancreatic and healthy group, there are 7 and 19 miRNAs dysregulating significantly respective (P<0.01)③We chose to analyze 5 candidates in a case-control cohort of plasma samples collected from 20 individuals with pancreatic cancer and 22healthy age-matched control individuals. Of all of the candidates, miR-942 showed the greatest differential expression in the pancreatic cancer compared with the healthy control (P=0.023)2.①The drug resistance index of SW1990/ADM, SW1990/5Fu, SW1990/ GEM ells relative to the SW1990 cells was 12.57、133.68 and 438.96, respectively.②The microarray indicated that 59,47,58 miRNAs were differentially expressed in ADM,5Fu, GEM resistant cells, respectively, with a>2-fold change. Seven miRNAs (hsa-miR-33a、33b、1285、486-5p、99b*、125a-3p、200c*) were always diversely expressed in all the resistant cell lines. Another seven miRNAs (miR-34a、424、449a、497、451、21、101), inducing drug resistance in other tumors, were diversely expressed in resistant cell lines with a>1-fold change.③To further validate our results, miR-486-5p、miR-99b*、miR-125a-3p, four of the most differentially expressed miRNAs, performed frequent deregulation as same as the microarray.3.①Dual luciferase reporter assay was carried out and showed that Hmlh1 is regulated by miR-155directly.②hMLH1 induced the development and associated with the prognosis in pancreatic cancer.Conclusion 1. We established a novel approach to test the miRNAs in plasma and established a list of likely blood-based miRNAs biomarker candidates for pancreatic cancer and pancreatitis patients. has-mir-942 identified as a candidate biomarker of pancreatic neoplasia. In summary, we show the feasibility of developing plasma miRNAs profiling as a sensitive and specific blood-based biomarker assay for pancreatic cancer that has the potential of translation to the clinic with additional improvements in the future.2. Drug resistance cell lines showed a different miRNAs expression profile from its parental SW1990 cells, suggesting the involvement of miRNAs in tumor cells drug resistance. Four miRNAs, miR-486-5p, miR-99b*, miR-125a-3p and miR-1285, identified as a potential biomarker of drug resistance in pancreatic cancer. This finding provides a experimental basis for further study of mechanism underlying the drug resistance of pancreatic cancer.3. hMLH1 was confirmed as the target of miR-155 in pancreatic cancer cell line. The result of modulation of hMLH1 by miR-155 induced mortality of pancreatic cancer. miR-155 is an potential treatment target to prolong the life of patients with pancreatic cancer.更多还原