长链非编码RNA NR_001587在胰腺癌发生发展中的作用和机制研究

Long non-coding RNAs are evolutionary conserved non-coding transcripts which were revealed by gnomonic and functional studies. Recently, lncRNAs were found involved in various tumor developments. However, the functions of lncRNAs were not well studied in pancreatic cancer which is one of cancers have poor prognosis. Based on a high through put expression array analysis of pancreatic cancer samples, we found one lncRNA NR_001587 is highly expressed in the pancreatic cancer and the levels of this lncRNA plays an important role in the progress in pancreatic cancer. We cloned the full length form of lncRNA NR_001587. To test whether this lncRNA has any prognostic value, we will take advantage of our clinic pancreatic cancer sample library, and correlate different clinical indexes with the expression levels of this lncRNA. To study the function of this lncRNA during pancreatic cancer growth and metabasis, we will apply different approaches, like RNA interference to knock-down the expression levels and over-expression with different virual vectors. We will check the cell growth rates and metabasis ability in these cell lines. To further study the targets of this lncRNA, we will firstly check the SMAD4 expression and functions using a serial of methods which are routinely used in a pancreatic cancer lab. We will also try to identify more targeting using RNA immuneprecipitation and Mass Spectrometry. Finally, we will test the function of the lncRNA in vivo in the heterotopic mouse model. Our study of this lncRNA in pancreatic cancer will shed light on the roles of lncRNA during pancreatic cancer development, and more importantly may prove lncRNAs could be attractive cancer therapeutic or prognostic markers.

胰腺癌是目前预后最差的消化道肿瘤，其发生发展的分子机制受广泛关注。长链非编码RNA在肿瘤发生发展中的作用和机制是肿瘤生物学研究的前沿热点问题。我们利用高通量芯片比较胰腺癌组织与癌旁组织的lncRNA表达谱，并结合生物信息学分析筛选差异表达的lncRNA，其中NR_001587的表达水平在胰腺癌组织中上调尤为显著，提示其可能在胰腺癌发生发展过程中起重要作用。据此，我们拟克隆出lncRNA全长，在体内外水平上研究其对胰腺癌恶性生物特征的调控功能及分子机制；鉴定lncRNA可能调控的蛋白编码基因；研究lncRNA通过结合蛋白发挥的功能及机制；通过RNAi干扰，过表达目的lncRNA，免疫共沉淀，裸鼠荷瘤模型等多种功能研究方法，并将其表达水平和临床指标进行关联分析，进而研究目标lncRNA在胰腺癌中的分子机制，为胰腺癌的的临床治疗提供新的理论基础。

课题组在基金项目的支持下，完成了两部分内容：.1）我们首先发现使用qRT-PCR，western印迹分析，Oncomine数据挖掘和免疫组织化学分析，在胰腺癌细胞和组织中PSME3在mRNA和蛋白质水平上都显着上调。高PSME3表达与肿瘤大小和pM阶段正相关，并且与通过Kaplan-Meier分析揭示的胰腺癌患者中的不良预后显着相关。基因组富集分析表明，与细胞增殖和转移相关的基因集与升高的PSME3表达呈正相关。沉默PSME3抑制胰腺癌的细胞增殖和侵袭能力。机制上，PSME3抑制c-Myc的降解，从而增强糖酵解，最终导致PSME3对胰腺癌的致癌作用。总之，我们的数据表明PSME3通过抑制c-Myc降解以促进糖酵解在胰腺癌中发挥致癌作用，并且可以作为胰腺癌治疗的新的治疗靶标。.2）完成了5对（10例）胰腺癌样本中全基因组lncRNA和mRNA表达水平的表达谱检测，并对差异表达的mRNA进行了GO和Pathway功能注释，结果发现pathway in cancer，cell cycle等通路的mRNA被显著异常表达；进一步的我们根据差异表达倍数和lncRNA可能参与的功能挑选了15条lncRNA通过荧光定量PCR进行验证，结果发现有9条lncRNA表达显著上调，与芯片结果趋势一致；接下来我们对这9条lncRNA构建沉默表达shRNA并包装慢病毒感染胰腺癌细胞株PANC-1和sw1990，并通过MTT细胞增殖实验检测检测细胞在lncRNA表达沉默后增殖能力变化，结果发现有3条lncRNA：BC072678，RP11-142L1.3和RP11-375B1.3在表达被沉默后细胞增殖能力显著下降（P<0.05），接下来我们进一步检测这三条lncRNA在表达沉默后细胞侵袭能力、克隆形成和细胞凋亡能力的变化，结果发现RP11-142L1.3不仅影响到胰腺癌细胞的增殖，在表达沉默后细胞的侵袭和迁徙能力显著下降；而BC072678和RP11-375B1.3仅抑制细胞增殖，在表达被沉默后，细胞周期被阻滞，细胞凋亡比例显著升高。接下来，我们重点对lncRNA RP11-142L1.3沉默组和对照组影响的基因进行PCR Array检测，结果发现wnt信号通路、Jak-stat3通路异常调控，进一步通过WB对Wnt和stat3进行检测验证了以上结果。