长链非编码RNA UCA1靶向调控PTEN在胰腺癌细胞恶性转化中的作用和机制研究

We have recently identified a novel long non-coding RNA (lncRNA) urothelial carcinoma antigen 1 (UCA1) by lncRNA microarray assay, and suggested that lncRNA-UCA1 may play a pivotal role in pancreatic cancer progression. The previous results showed that lncRNA-UCA1 expression was significantly up-regulated in the pancreatic cancer cell lines and pancreatic cancer tissues，compared with corresponding non-cancerous pancreatic tissues. In addition, the knock-down of lncRNA-UCA1 obviously suppressed the proliferative and invasive behaviors of pancreatic cancer cell lines and influenced PTEN expression. To further detect whether lncRNA-UCA1 can facilitate the progression of pancreatic cancer and the underlying molecular mechanisms, in present study, we first need to analyze the relationship of lncRNA-UCA1 expression with clinicopathologic parameters in pancreatic cancers. Moreover, down-regulation of lncRNA-UCA1 expression by shRNA and ectopic expression of lncRNA-UCA1 will be carried out to explore whether lncRNA-UCA1 alteration can influence the malignant behavior of pancreatic cancer in vitro. Furthermore, nude mouse xenograft model will be established to investigate the regulatory role of UCA1 in tumorigenicity of pancreatic cancer cells in vivo. Last，we need to confirm whether the expression of UCA1 RNA leads to the change of PTEN mRNA and protein expression. Since promoter methylation and histone deacetylase activities are epigenetic mechanisms regulating PTEN expression, 5-aza-dC and TSA treatments will be used to investigate whether lncRNA-UCA1 affects PTEN expression through such mechanisms. In a word, detection of the lncRNA-UCA1 function in pancreatic cancer progression will be critical for understanding the oncogenesis and development of pancreatic cancer.

UCA1是与胰腺癌相关的长链非编码RNA（lncRNA），课题组的前期研究表明lncRNA-UCA1在胰腺癌组织和胰腺癌细胞株中的表达显著上调，下调UCA1的表达能抑制胰腺癌细胞的增殖和侵袭能力，并且改变PTEN的活性。为进一步明确lncRNA-UCA1在胰腺癌发生发展中的作用及其分子机制，本课题在研究lncRNA-UCA1在胰腺癌组织的表达并分析其与临床病理特征的关系的基础上，构建UCA1真核表达载体和shRNA干扰载体，阐述调控UCA1表达对胰腺癌细胞恶性生物学特征的影响，并通过裸鼠胰腺癌模型观察改变UCA1表达前后对胰腺癌细胞体内成瘤性的影响。最后，阐明重新表达或沉默UCA1后PTEN mRNA和蛋白表达的变化，并通过5-杂氮-2-脱氧胞苷及曲古抑菌素A处理探讨lncRNA-UCA1对PTEN启动子区甲基化的影响。该研究将对进一步揭示胰腺癌发病的分子机制提供充分的科学依据。

胰腺癌是一种恶性程度很高的消化系统肿瘤，总体5 年生存率低于5%。UCA1是与胰腺癌相关的长链非编码RNA（lncRNA），我们通过lncRNA芯片发现与正常胰腺组织相比，UCA1在胰腺癌组织中的表达明显升高，在胰腺癌细胞株中同样印证了这一点，并且UCA1在胰腺癌细胞株PANC-1中的表达最高。我们构建UCA1的shRNA干扰载体，筛选出稳定低表达UCA1的细胞株。常规培养对照组细胞和shRNA干扰的PANC-1细胞，realtime PCR检测两组细胞中UCA1的表达，结果提示干扰组中UCA1的表达明显降低。采用MTT法检测细胞的增殖情况，结果发现培养的第4天和第5天UCA1低表达细胞的增殖明显低于正常对照组；流式细胞仪检测发现在24、48和72小时，UCA1低表达细胞细胞较正常值相比，subG1期细胞比例明显升高；划痕实验结果发现UCA1低表达细胞的迁移能力低于正常对照组；采用transwell小室检测两组细胞之间侵袭能力的变化，结果发现降低UCA1的表达能抑制胰腺癌细胞的侵袭能力。将对照组细胞和shRNA干扰的PANC-1细胞进行基因芯片检测，发现许多和细胞增殖、凋亡和迁移相关的信号通路分子的改变，其中我们发现DDIT3的表达明显升高，western blot方法验证了DDIT3的变化。将对照组细胞和shRNA干扰的PANC-1细胞分别接种于裸鼠右侧背部皮下，结果发现4周后低表达UCA1的裸鼠组移植瘤的体积均小于阴性对照组裸鼠移植瘤的体积，差异有统计学意义。因此，该研究结果有助于进一步探索胰腺癌的发生发展机制。