【作者】 刘平平；

【导师】 陈杰；

【作者基本信息】 北京协和医学院 ， 病理学与病理生理学， 2014， 博士

【摘要】 胰腺导管腺癌(PDAC)在世界范围内是死亡率最高的恶性肿瘤之一,在过去的三十年,PDAC患者的预后没有明显改善,五年生存率只有6%。近年来长链非编码RNA(lncRNAs)是研究的热点,lncRNA的突变及异常表达与包括肿瘤在内的多种人类疾病有密切联系,但lncRNAs在胰腺癌中的作用尚不清楚。本课题通过分析胰腺导管腺癌表达谱数据,筛选在胰腺癌中差异表达且可能与患者预后相关的lncRNAs。我们从Pubmed基因表达文库(GEO)数据库筛选出3个高质量的胰腺癌表达芯片数据,分析差异表达基因。选取在三个表达谱数据中表达变化一致的lncRNAs,根据胰腺癌患者各个差异lncRNA表达水平和患者生存资料,分析其是否影响预后。在筛选出的lncRNAs中,研究已经发现lncRNA MALAT1(metastasis-associated lung adenocarcinoma transcript1)在非小细胞肺癌、胰腺癌、前列腺癌等肿瘤中表达上调,且与总生存时间负相关,而其在胰腺癌中发挥的作用未知,因此本课题将重点研究MALAT1在胰腺癌中的表达及功能。研究表明lncRNAs可以受到microRNA(miRNA)的调控,机制与调控蛋白编码基因类似,因此,我们将研究胰腺癌中的muRNA是否可以调控MALAT1。分析表达谱数据发现,与癌旁组织相比MALAT1在PDAC组织中高表达,并且在转移发生后,表达水平进一步升高；与永生化胰腺导管上皮细胞HPDE相比MALAT1在20种PDAC细胞系的表达水平均有不同程度的上调。将GSE21501胰腺癌患者根据MALAT1表达水平分为MALAT1高表达组(高15%)和低表达组(低85%),Kaplan-Meier生存分析结果显示,与MALAT1高表达组相比,低表达组术后生存时间更长,两组术后中位生存时间分别是19个月95%CI[15.6,22.4]和13个月95%CI[7.9,18.0],Log-rank检测差异有统计学意义(p=0.0014)。通过分析MALAT1高表达组及低表达组的差异表达基因,做基因功能分析,发现上调表达基因主要与正向调控细胞生长/增殖、负向调控细胞死亡/凋亡、细胞定位及代谢等生物学过程相关。MALAT1基因位于11q13.1,其临时转录本长8708bp,共有4种不同的剪接形式。在转录后水平,MALAT1RNA3’端经RNaseP和RNase Z剪接修饰产生一条长61nt的tRNA样分子,被分泌至胞浆,称为nascRNA。在胰腺癌细胞系及组织中用qPCR和ISH实验验证MALAT1的表达水平,发现与HPDE细胞相比,MALAT1在ANC1、AsPC1及BxPC3细胞中均表达上调,这与PDAC细胞系表达谱数据结果一致。与癌旁正常组织相比,MALAT1在PDAC组织肿瘤细胞核中表达上调。Northern blot实验用一段MALAT1的反义RNA为探针检测到两个MALAT1条带,长转录本位于相当于MALAT1全长转录本的位置,另一条短转录本长度在28S RNA(4700nt)与18S RNA(1900nt)之间。胰腺癌细胞系中长转录本的丰度要高于短转录本。我们用RNAi技术敲低胰腺癌PANC1和AsPCl细胞中MALAT1表达水平,分别用CCK-8、EdU实验、集落形成实验、细胞划痕实验、Transwell小室侵袭实验和流式细胞术证实敲低MALATl可以抑制细胞增殖、抑制细胞的迁移和侵袭能力、诱导细胞发生G0/G1到S期阻滞和促进细胞凋亡。MALAT1敲低PANC1细胞的小鼠移植瘤生长缓慢。转染miR-217mimic可以下调MALAT1RNA表达水平。采用qPCR技术同时检测miR-217及MALAT1在7种胰腺癌细胞系：PANC1、AsPC1、BxPC3、MIA PaCa2、P3、P4、P7及永生化胰腺导管上皮细胞系HPDE中的表达,经Kendall’s tau-b相关分析发现,miR-217和MALAT1的表达之间存在负相关关系。构建包含miR-217结合位点的MALAT1片段的pmirGLO双荧光素酶报告基因载体,报告基因实验显示过表达miR-217使pmirGLO-MALATl-WT报告基因的荧光素酶活性降低,将miR-217结合位点突变后,降低荧光素酶活性作用消失；说明niR-217是通过MALAT1的miR-217结合位点发挥直接调控作用。实验室前期研究发现miR-217可以直接靶向调控KRAS mRNA,提示KRAS与MALAT1可能为竞争性内源性RNA。Western blot实验发现敲低MALAT1可以下调KRAS蛋白表达并且下调pRL-TK-KRAS-3’UTR-WT报告基因载体荧光素酶活性。证实MALAT1是通过竞争结合niR-217来调控KRAS蛋白质表达。综上所述,MALAT1在胰腺癌细胞系及组织中高表达,敲低MALAT1抑制胰腺癌细胞增殖,促进细胞凋亡,使细胞周期发生G0/G1到S期阻滞,抑制细胞迁移侵袭。MALAT1是miR-217的一个新的靶基因,并且通过miR-217影响KRAS蛋白水平。提示MALAT1可能在胰腺癌的发生发展过程中发挥重要作用,本研究可能为胰腺癌的诊断和靶向治疗提供新的线索。更多还原

【Abstract】 Pancreatic ductal adenocarcinoma(PDAC) is one of the most lethal malignancies worldwide. There has been little change in survival rates over the past30years, with an overall5-year survival rate of6%. Long noncoding RNAs (lncRNAs) have recently been discovered to play important regμlatory roles in various kinds of tμmors and a variety of diseases. However, the functional roles of these transcripts in PDAC are not thorouμghly understood.In this study, we selected lncRNAs that were differentially expressed and correlated with overall survival in PDAC throμgh re-analysis publically available databases. We analyzed three sets of high-quality pancreatic expression microarray data from the Pub-Med Gene Expression Omnibus datasets and found out a group of lncRNAs that were consistent differential in all the3expression sets of data. Data was compared with sur-vival data using the Kaplan-Meier method and compared between groups by the log-rank test. As a resμlt, MALAT1showed correlated with overall survival. Researchers con-firmed MALAT1was up regμlated in non-small cell lung cancer, pancreatic cancer, prostate cancer and other tμmors, and negatively correlated with overall survival. How-ever, the role of MALAT1in pancreatic tμmorgenesis was uncovered. Additionlly, It has been founded that microRNAs(miRN As) co μld regμlate lncRNAs in a way similar to the miRN A-mediated silencing of target protein-coding genes. So, we woμld find if there were any miRNAs regμlating MALAT1.Data-mining studies of publically available databases showed that MALAT1was overexpressed in pancreatic tμmors compared with the normal pancreas and was more highly expressed in advanced tμmors. MALAT1exhibited elevated expression levels in the20kinds of pancreatic cancer cells compared to immortalized pancreatic duct cell HPDE. In GSE21501gene profiling resμlts, Kaplan-Meier survival analysis showed that patients with low MALAT1expression (bottom85%) had significantly increased overall survival compared with patients with high MALAT1expression (top15%), The median time of overall survival of low MALAT1expression and high was19months95%CI [15.6,22.4] and13months95%CI [7.9,18.0] separately.Gene Ontology analysis using the same study showing that gene set differences in MALAT1high vs. tow patients indicated that MALAT1regμlates gene sets mainly asso- ciated with positive regμlation of cell growth and proliferation, negative regμlation of apoptotic process and cell death, cellμlar localization and metabolic process.MALAT1DNA localizes on11ql3.1, with a provisional RNA RefSeq as long as8708nt and4variant transcripts. At the posttranscriptional level, a processing mechanism executed by two endogenous RNases-RNase P and RNase Z can modify the3’-end of MALATl. The processing step generates a61nt long tRNA-like noncoding RNA termed mascRNA that localizes to the cytoplasm. Then we validated the expression level of se-lected lncRNAs in pancreatic cancer cell lines and paired pancreatic cancerous and nor-mal tissues using real-time qPCR and ISH. Compared to HPDE cell, MALATl was highly expressed in PANC1, AsPCl and BxPC3cells. These resμlts overlapped with data mining from microarray data from20pancreatic cancer cell lines which showed that MALATl was overexpressed in all PDAC cell lines. Compared to adjacent normal tis-sues, MALAT1was highly expressed in pancreatic cancerous tissues. In Northern blot analysis using a antisense sequence of MALAT1as probe, two RNA bands were detected with the long band at a size in accordance to the fμll length size of the MALAT1tran-script and the short band between28S RNA (4700nt) and18S RNA (1900nt).The long transcript was more abundance than the short transcript in PDAC cell lines.Knockdown of MALAT1in PANC1and AsPCl cells by RNAi showed that MALAT1knockdown was associated with inhibited cell proliferation, cell migration and invasion, G0/G1to S phase arrest and induction of apoptosis using CCK-8assay, EdU incorporation assays, anchorage-independent colony formation assay, wound healing test, transwell chamber assay and flow cytometry. MALAT1knockdown in PANC1cells also inhibited tμmor growth in a mouse xenograft model in nude mice. miR-217overexpression resμlted in a significant downregμlation of MALAT1. We detected the level of MALAT1and miR-217from HPNE cell and PDAC cell lines, PANC1,AsPC1, BxPC3,MIA PaCa2,P3,P4and P7. Kendall’s tau-b rank correlation analysis showed a negative correlation between them We cloned the fragment of MALAT1encompassing the miR-217binding sites into pmirGlo dual-luciferase reporter plasmids and performed luciferase assays in PANC1cells. miR-217over-expression resμted in a significant decrease in luciferase activity, however, directed mutagenesis of the predicted miR-217binding sites abolished this effect. The resμlts indicated MALAT1expression is regμlated throμgh direct miR-217binding. We had found KRAS is a direct target of miR-217. This s u ggested MALATl and KRAS were competing endogenous RNAs. Knockdown MALATl respμed in a significant downregμation of KRAS protein. RLuc-KRAS-3’UTR-WT. constructs were subsequently transfected in PANC1cell after knockdown MALATl, Luciferase assays indicate that knockdown MALATl resμlted in a decrease in luciferase activity, moreover, directed mutagenesis of the miR-217binding sites abolished this effect. This indicated MALATl regulate KRAS protein throμgh miRNA recognition elements.To sμm up, this study confirms that MALATl is up regμlated in PDAC tissue and cells. MALAT1knockdown was associated with inhibited cell proliferation, cell migra-tion and invasion, G0/G1to S phase arrest and induction of apoptosis. We identify MALATl as a bona fide target of miR-217and regμlate KRAS protein thro μgh miRNA recognition elements. MALATl was a potential oncogene in PDAC development. Therefore, MALAT1may serve as a usefμl therapeutic agent for PDAC therapy.更多还原