【作者】 郝骏；

【导师】 胡先贵；

【作者基本信息】 第二军医大学 ， 普通外科学， 2011， 博士

【摘要】 胰腺癌是恶性程度最高的肿瘤之一,近20年来治疗效果无明显提高,5年生存率仍然低于5%。探索胰腺癌的侵袭转移机制、寻找新的诊断标记物和治疗靶点是提高治疗效果的关键。胰腺癌的发生发展也是“多步骤、多基因”突变的结果,其分子机制是癌基因的激活和抑癌基因的功能丧失。胰腺癌中常见的癌基因K-ras,抑癌基因INK4A, TP53及DPC4/SMAD4的突变均与胰腺癌密切相关。miRNA是一类长度约为21～13个核苷酸大小的非编码单链小RNA分子,在细胞内由约70nt的pre-miRNA经Dicer酶剪切而来,和靶基因结合介导其mRNA的降解或抑制其翻译,实现对靶基因的负调控。越来越多的研究证明miRNAs在细胞增殖、分化凋亡、压力抵抗等多种生理病理过程中有重要作用。已有多项研究证实miRNAs在肿瘤中发挥着类似于癌基因与抑癌基因的作用,肿瘤中的miRNAs表达具有组织特异性。miRNAs通过影响重要的肿瘤相关基因的表达,从而对肿瘤发生,发展,转移及恶性程度等性状进行调控。已经有多项研究证实胰腺癌组织和正常胰腺组织的miRNAs表达谱存在显著差异,如miR-21、miR-155、miR-29b、miR-34家族and let-7家族。最近还发现了调控KRAS的miR-96在胰腺癌中起抑癌基因作用。但还不清楚是否其他miRNA能够调节胰腺癌中的其他的重要基因。本实验将在筛选胰腺癌组织miRNA的基础上,通过多种研究方法,探讨miRNA对胰腺癌特异相关抑癌基因DPC4/Smad4基因的转录后调控和功能机制,为胰腺癌的诊疗寻找新的靶点。一、在胰腺癌/癌旁组织中差异表达的miRNAs的筛选和鉴定本实验选用高通量的miRCURY LNA?基因芯片(CAT#208021,丹麦Exipon)检测3对重复胰腺癌/癌旁组织样本,发现胰腺癌组织中miR-21、135b、199a-3p,221、222,29a,31等88个miRNAs比癌旁组织显著高表达,miR-126、103、132、141,148a等101个miRNAs显著低表达。为验证芯片筛选结果,实验中还用茎环法实时定量PCR实验对芯片筛选到的胰腺癌/癌旁组织miRNAs表达谱差异进行验证。结果表明验证了miR-34a、145、621等在胰腺癌中筛选结果提示高表达的miRNAs。表明实时定量PCR的结果虽然在数值上和芯片筛选结果有一定差异,但总体趋势是一致的。表明芯片筛选结果是准确可靠的,证实了胰腺癌组织和正常胰腺组织有表达差异,提示差异表达的miRNAs在胰腺癌的发生发展过程中可能起到重要作用。二、miR-483-3p的在胰腺癌中的表达及功能研究miRNAs通过调控靶基因参与生命活动。DPC4/SMAD4和胰腺癌的发生发展特异相关,是和胰腺癌密切相关的抑癌基因,其突变、缺失及表达下调等改变均会导致肿瘤的发生。我们利用常见的靶标基因预测软件PicTar、TargetScan、miRanda、RNAhybrid、RNA22、DIANA-microT预测了上述差异表达的microRNA作用的下游靶基因。经各个数据库之间的查询比较,在候选的miRNA预测到miR-483-3p可能对DPC4/Smad4 mRNA 3’UTR可能有直接调控作用。采用了茎环法实时定量PCR方法检测miR-483-3p在胰腺癌组织中的相对表达量,其中miR-483-3p在胰腺癌中的表达上调显著上调,进一步证实了芯片筛选结果,证实了胰腺癌组织和正常胰腺组织有表达差异。我们通过在胰腺癌PANC 1和SW 1990细胞株中转染miR-483-3p mimics和inhibitor的方法研究miR-483-3p的作用。MTT方法是通过快速简便的颜色反应来检测细胞存活数量及活性,实验显示转染miR-483-3p mimics组的胰腺癌细胞株生长加快,转染inhibitor的细胞生长变缓。克隆形成率反映细胞群体依赖性和增殖能力两个重要性状,实验中,转染miR-483-3p mimics组的胰腺癌细胞株克隆形成数高于转染无功能对照组,而转染,转染inhibitor的实验组的克隆形成数明显少于对照组。Western blot显示,转染miR-483-3p能够降低SMAD4的蛋白表达水平。由于SMAD4是TGF-β和BMP通路共用的下游关键蛋白,miR-483-3p能够抑制SMAD4蛋白的表达,必将对其通路下游产生影响。因此我们进一步探索miR-483-3p对SMAD4下游基因及TGF-beta/BMP通路的影响。结果表明,转染miR-483-3p后,P21、Msx1以及PAI的mRNA水平均显著下调,表明miR-483-3p能影响SMAD4下游基因的转录。DPC4/Smad4参与了TGF-β引起的EMT途径,为了探索miR-483-3p是否影响EMT,我们检测了TGF-β处理后转染miR-483-3p mimics相关蛋白的变化。Western blot检测表明转染miR-483-3p mimics能增加E-cadherin水平,同时降低fibronectin的表达,表明miR-483-3p影响EMT。在人胰腺癌细胞系SW 1990和PANC 1中转染miR-483-3p minics后培养48小时,Western Blot结果显示,miR-483-3p minics可抑制胰腺癌细胞株SW1990和PANC 1中DPC4/SMAD4蛋白水平的表达。miR-483-3p能够有效抑制SMAD4表达水平,表明DPC4/SMAD4的mRNA 3’UTR是miR-483-3p的直接作用靶点,miR-483-3p对其有直接负调控作用,进一步验证了信息生物学预测的结果。双荧光素酶报告基因法结果显示,miR-483-3p minics可以显著下调含有DPC4/SMAD4靶基因3’UTR的荧光素酶报告基因pGL3-3’UTR的活性,(p=0.005) ,而对含突变位点的pGL3-3’UT R-mut的荧光素酶活性无明显改变。表明miR-483-3p对DPC4/SMAD4 3’UTR有直接负转录调控作用,上述结果可以表明,miR-483-3p在胰腺癌中起促癌作用,DPC4/SMAD4 mRNA 3’UTR是其直接作用的下游靶基因。miR-483-3p通过抑制SMAD4蛋白,进而加速细胞生长,并影响TGF-β和BMP下游基因mRNA的表达,可成为胰腺癌治疗的新的靶点。三、miR-421的在胰腺癌中的表达及功能研究每个miRNA可以有多个靶基因,而几个miRNAs也可以调节同一个基因。这种复杂的调节网络既可以通过一个miRNA来调控多个基因的表达,也可以通过几个miRNAs的组合来精细调控某个基因的表达。随着miRNA调控基因表达的研究的逐步深入,将帮助我们理解高等真核生物的基因组的复杂性和复杂的基因表达调控网络。在前面的胰腺癌miRNAs表达差异筛选和miR-483-3p功能验证的基础上,经生物信息学分析,在芯片筛选结果中预测到miR-421可能对DPC4/Smad4可能同样有直接调控作用。茎环法实时定量PCR方法验证miR-421在胰腺癌组织中的相对表达量。在胰腺癌PANC 1和SW 1990细胞株中转染miR-421 mimics和inhibitor,结果显示:MTT方法结果显示转染miR-421 mimics组的胰腺癌细胞株生长加快,转染inhibitor的细胞生长变缓;转染miR-421 mimics组的胰腺癌细胞株克隆形成数高于转染无功能对照组,而转染,转染inhibitor的实验组的克隆形成数明显少于对照组。Western blot显示,转染miR-421 mimics能够降低SMAD4的蛋白表达水平。结果表明,转染miR-421mimics后,P21、Msx1以及PAI的mRNA水平均显著下调,表明miR-421能影响SMAD4下游基因的转录。双荧光素酶报告基因法结果显示DPC4/SMAD4 3’UTR是miR-421的直接作用靶点。miR-421在胰腺癌中起促癌作用,DPC4/SMAD4 mRNA 3’UTR是其直接作用的下游靶基因。miR-421通过抑制SMAD4蛋白,进而加速细胞生长上述结果可以表明,miR-421在胰腺癌中起促癌作用,DPC4/SMAD4 mRNA 3’UTR是其直接作用的下游靶基因。miR-421在胰腺癌中通过和miR483-3p相似的机理起作用,可成为胰腺癌治疗的新的靶点。更多还原

【Abstract】 Pancreatic cancer is an aggressive malignancy with one of the worst mortality. Despite the extensive research efforts, the prognosis for pancreatic cancer is the worst among all cancers due to minimal improvement in its prevention and treatment and the 5-year relative survival rate is only 5% for all stages of this disease over the last decades. Therefore, the quest for new associated factors and novel therapeutic targets for pancreatic cancer remains an imperative clinical issue.In recent years, there have been important advances in understanding of the molecular biology of pancreatic cancer and genetic analysis showed that the genetic basis of this dismal disease is extremely complex and heterogeneous. It has been suggested that pancreatic cancer is associated with the successive accumulation of gene mutations such as oncogene KRAS2 and tumor-suppressor genes INK4A, TP53 and DPC4/Smad4.Recently, microRNAs (miRNA) have emerged as a critical class of negative regulators of gene expression. miRNAs are endogenous small 21-23 nucleotide noncoding RNAs that possess remarkable evolutionary conservation. These small molecules elicit their regulatory effects by base-pairing to partially complementary mRNAs and function by at least two mechanisms: degradation of target mRNA transcripts and inhibition of mRNA translation. Emerging evidence indicates that miRNAs play critical roles in the regulation of various biological and pathologic processes, involving cell proliferation, differentiation, apoptosis, and stress resistance. More importantly, it has been suggested that the development and progression of cancer are associated with aberrant upregulation or downregulation of specific miRNAs and their targets in various types of cancer. And certain cancer histotypes can be classified based on miRNA expression profiles. In pancreatic cancer a multitude of misexpressed miRNAs have been reported, such as‘onco(genic)-miRs’, miR-21 and miR-155, and‘tumor suppressor miRs’, miR-29b and the miR-34 and let-7 families. Moreover, it was recently found that miR-96 is a tumor-suppressor gene in pancreatic cancer by repressing KRAS expression. However, it is less clear whether there is any other miRNA targeting these key regulatory genes in pancreatic cancer.In this study, using a miRNA array-based differential analysis, we explore the function and mechanism of miRNAs relate in the post transcriptional gene regulation of pancreatic cancerr-specific tumor suppressor gene DPC4/Smad4 to find a new target point for the treatment of pancreatic cancer by a variety of research methods.一、Screening and identification of differential expression of miRNAs in pancreatic cancerTo search for miRNAs which may be involved in the development of pancreatic cancer especially among Chinese patients, we collected three different human clinical specimens and then used miRCURY LNA? miRNA-array analysis to screen differentially expressed miRNAs between pancreatic cancer tissues and respective side normal tissues. 88 miRNAs are overexpressed in pancreatic cancer, such as miR-21、135b、199a-3p,221、222,29a,31, whereas 101 miRNAs are down-regulated,such as miR-126、103、132、141,148a.We next use q-PCR methods to validate that The most significantly overexpressed miRNA in pancreatic were miR-762, miR-17-92 cluster, miR-106-363 cluster and miR-23a/b; whereas miR-200c, miR-486, miR-199a, miR-143, miR-145, miR-125b, miR-31, miR-26,miR-100 and let-7 were among the most down-modulated miRNA.二、The expression and function of microRNA 483-3p in pancreatic cancermiRNAs elicit their regulatory effects by base-pairing to partially complementary mRNAs and function by at least two mechanisms: degradation of target mRNA transcripts and inhibition of mRNA translation. It has been suggested that pancreatic cancer is associated with the successive accumulation of gene mutations. DPC4/Smad4, a critical effector in TGF-βsignaling pathway, was a regulatory factor in pancreatic cancer progression and approximately 55% cases of pancreatic adenocarcinomas involve inactivation or lost expression of the DPC4/Smad4 gene .To explore the mechanism(s) by which miR-483-3p executes its function in pancreatic cancer, we firstly applied common used bioinformatic algorithms (TargetScan, PicTar and miRanda) to identify its potential target genes. Among these many candidates, DPC4/Smad4 was selected for further analysis. DPC4/Smad4, a critical effector in TGF-β signaling pathway, was a regulatory factor in pancreatic cancer progression and approximately 55% cases of pancreatic adenocarcinomas involve inactivation or lost expression of the DPC4/Smad4 gene.To further confirm the miRNA array results, Real-time PCR analysis showed that miR-483-3p was increased significantly in cancer tissues compared with matched side tissues, suggesting that miR-483-3p may be a novel factor associated with the development of pancreatic cancer.we next tried to examine whether miR-483-3p has oncogenic functions in pancreatic cancer cells. The MTT proliferation assay showed that miR-483-3p mimics significantly promoted the proliferation of both SW1990 and PANC1 cells. Consistent with that, the colony formation analysis also showed that miR-483-3p stimulated SW1990 and PANC1 cells to grow more and larger colonies. Conversely, anti-miR-483-3p transfection in SW1990 and PANC1 cells significantly reduced cell proliferation and resulted in fewer SW1990 or PANC1 colonies. These results suggest that miR-483-3p promotes the proliferation and anchorage-independent growth of pancreatic cancer cells in vitro.We overexpressed miR-483-3p mimics in SW1990 cells and Western blot assays, and they indicated that the level of DPC4/Smad4 protein was reduced significantly by miR-483-3p overexpression compared with control treatment. The same results were observed using another human pancreatic cancer cell line PANC1. As Smad4 is the common-Smad protein for the transduction of TGF-βand BMP signals, the repression of DPC4/Smad4 by miR-483-3p should impair these two signaling pathways. Next, we examined the effect of miR-483-3p on the expression of these pathways’transcriptional targets in PANC1 cells. Real-time PCR analysis showed that the expression levels of TGF-βsignaling target genes PAI and p21, as well as BMP signaling targets Msx2, were suppressed by miR-483-3p overexpression, suggesting that miR-483-3p can regulate DPC4/Smad4-regulated signaling pathways. It is known that DPC4/Smad4 is involved in TGF-β-induced epithelial-mesenchymal transition (EMT) , and that pancreatic cancers with wild type DPC4/Smad4 show frequent EMT. To test the possibility that miR-483-3p could regulate the DPC4/Smad4-mediated EMT process, we transfected PANC1 cells with miR-483-3p mimics or scramble oligos and simultaneously treated them with TGF-β. Western blot analysis showed that compared with TGF-βtreatment alone, transfection of miR-483-3p mimics increased E-cadherin levels while decreasing fibronectin expression, suggesting that miR-483-3p could antagonize DPC4/Smad4-mediated TGF-βinduction of EMT. Taken together, these data suggest that miR-483-3p may exert its biological function through regulating the expression of its direct target DPC4/Smad4.To test the hypothesis that DPC4/Smad4 might be a target of miR-483-3p, we constructed three reporters by putting the wild-type fragments covering position 1-2050, 2201-4105 or 4200-6000 from the 3’-UTR region of DPC4/Smad4 to the downstream of the luciferase coding region. Oligonucleotides of miR-483-3p mimics or scramble were cotransfected with these reporter plasmids into human pancreatic cancer cells SW1990 and PANC1. Results showed higher luciferase activities than the respective wild-type ones, suggesting that they are indeed regulated by endogenous miR-483-3p. Moreover, transfection of miR-483-3p mimics had no interference with activities of the two reporters. These results suggest that DPC4/Smad4 may be a target of miR-483-3p for its regulatory function in pancreatic cancer.Taken together, our results suggest that the upregulated miR-483-3p targets tumor-suppressor gene DPC4/Smad4 in pancreatic cancer and that miR-483-3p may be a potential target for pancreatic cancer therapy.三、The expression and function of microRNA 421 in pancreatic cancerEach miRNA can have multiple target genes, while a number of miRNAs can also regulate the same gene. This complex regulatory network either through a miRNA to regulate the expression of multiple genes, miRNAs can also be a combination of several fine regulation of the expression of a gene. MiRNA regulation of gene expression with the gradual deepening of the research will help us understand the genome of higher eukaryotes, the complexity and complex regulation of gene expression networks.we have found that miR-483-3p is involved in pancreatic cancer by repressing DPC4/Smad4. However, it is still unknown that whether there is other miRNAs regulating DPC4/Smad4 expression in pancreatic cancer.In our previous miRNA array, we compared differentially expressed miRNAs between pancreatic cancer tissues and respective side normal tissues. We determined that miR-421, which had not been suggested to be involved in pancreatic cancer in previous studies, harbored four predicted binding sites in the 3’-UTR of DPC4/Smad4 mRNA and was aberrantly upregulated in malignant tissues. To further confirm this array result, Real-time PCR analysis showed that expression levels of miR-421 were increased significantly in cancer tissues compared with matched side tissues, while DPC4/Smad4 expression was pronouncedly inhibited in pancreatic cancer tissues.This finding led us to examine whether miR-421 overexpression could reduce endogenous DPC4/Smad4 protein levels in human pancreatic cancer cell lines. We ectopically expressed miR-421 mimics in SW1990 cells.Using Western blot assays, we found that the level of DPC4/Smad4 protein was reduced significantly by miR-421 overexpression compared with control treatment. The same results were observed in another human pancreatic cancer cell line PANC1. As DPC4/Smad4 is the common Smad protein for the transduction of TGF-βand BMP signals, the repression of DPC4/Smad4 by miR-421 should impair activation of downstream target genes of the two signaling pathways. We then examined the effect of miR-421 on the expression of these pathway’s transcriptional targets in PANC1 cells. Real-time PCR analysis showed that the expression levels of the TGF-βtarget genes p21and p15, as well as the BMP target gene Id3, were suppressed by miR-421 overexpression, suggesting that miR-421 can regulate DPC4/Smad4-mediated signaling pathways. Collectively, these data suggest that miR-421 indeed directly targets DPC4/Smad4 to exert its biological function in pancreatic cancer.We then tried to examine whether or not miR-421 has oncogenic functions in pancreatic cancer cells. The MTT proliferation assay showed that miR-421 mimics significantly promoted the proliferation of both SW1990 and PANC1 cells. Colony formation analysis consistently showed that miR-421 increased the quantity and size of SW1990 and PANC1 cell colonies. Conversely, the miR-421 inhibitor significantly reduced cell proliferation and resulted in fewer SW1990 and PANC1 colonies. These results suggest that miR-421 promotes the proliferation and anchorage-independent growth of pancreatic cancer cells in vitro.To test the above hypothesis, we sought to confirm the possible regulation of DPC4/Smad4 by miRNA-421. Based on the bioinformatics prediction that there are four potential positions, 438-444, 722-728, 2799-2785 and 5252-5258, in the 3’-UTR of DPC4/Smad4 mRNA targeted by miRNA-421, Mimics or scramble oligonucleotides of miR-421 were cotransfected with these reporter plasmids into the human pancreatic cancer cell lines SW1990 and PANC1, both of which contain the wild-type DPC4/Smad4 gene. Remarkably, luciferase analyses showed that miR-421 mimics repressed the activities of pGL3-UTR1-WT, pGL3-UTR2-WT and pGL3-UTR3-WT, all of which harbor miR-421 binding sites. In contrast, pGL3-UTR1-MUT, pGL3-UTR2-MUT and pGL3-UTR3-MUT, in which the putative binding sites of miR-421 are mutated, showed higher luciferase activities than their respective wild-type constructs, suggesting that they are indeed regulated by endogenous miR-421. Moreover, transfection of miR-421 mimics had no suppression or activation of the three reporters. These results suggest that DPC4/Smad4 may targeted by miR-421 for their regulatory functions in pancreatic cancer.In continuation of our previous study, we found that DPC4/Smad4 is regulated by miR-421 in pancreatic tumors and their expression levels are inversely correlated. There are four predicted binding sites of miR-421 in the 3’-UTR of DPC4/Smad4, and ectopic expression of miR-421 represses DPC4/Smad4 protein level. Besides, its overexpression inhibits downstream targets of DPC4/Smad4, such as p21, p15 and Id3 . Altogether, miR-421 may be used as a novel biomarker and therapeutic target for pancreatic and other human adult cancers.更多还原