肝细胞癌中microRNA-19a表达缺失的功能作用及其分子机制研究

The investigator have demonstrated that miR-19a was down-regulated in hepatocellular carcinoma by miRNA microarray and Taqman qPCR and found that down-regulation of miR-19a in primary hepatocellular carcinoma (HCC)correlated with poor prognosis for HCC in preliminary studies. Functional experiments in vitro showed that miR-19a could suppress the proliferation and invasion of HCC cells, However, the mechanism of this phenomenon was not clear yet. Target scan by bioinformatics combined with westen blot analysis displayed that the 3′UTR region of SOX4 gene has a potential binding site of miR-19a and SOX4 was reversely regulated by miR-19a. SOX4 was already proved to stablize cytoplasmic β-catenin and consequently activate the WNT oncogenic signal pathway, and then promoted the progression of hepatocellular carcinoma. Here, we speculate that miR-19a might perform its anti-HCC effect by turn the WNT pathway off through suppressing the SOX4 expression and the subsequent degradation of cytoplasmic β-catenin. So as to verify the above hypothesis, this study aim to deeply discuss the influence of the expression of miR-19a, SOX4 and β-catenin on the proliferation and invasion of HCC cell in vitro and in vivo mainly using gene transfenction and animals living imaging technology. To make it clear that the cascading regulation of miR-19a、SOX4 and β-catenin combining the use of luciferase reporter assay system and HCC tissue microarray and provide futher evidences of mechanism of the hepatocellular carcinoma recurrence.

申请人经miRNA microarray芯片筛选及qPCR验证发现miR-19a肝癌中低表达，且与肝癌的预后相关，功能学实验证实miR-19a可抑制肝癌细胞增殖及侵袭，但分子机制尚待明确。生物信息学靶基因预测结合Western blot分析，提示SOX4基因的3′UTR区存在miR-19a的潜在结合位点并受miR-19a反向调控。SOX4可稳定β-catenin进而激活WNT通路，促进肝癌进展。因此推测，miR-19a可能通过抑制SOX4表达，促进细胞中β-catenin降解，从而失活WNT通路发挥肝癌抑制作用。为证实以上假设，本研究拟应用基因转染、动物活体成像等技术，体外、体内探讨miR-19a、SOX4及β-catenin对肝癌细胞增殖、侵袭能力的影响；结合荧光素酶报告基因及肝癌组织芯片明确miR-19a对SOX4及β-catenin表达的级联调控关系，为阐明肝癌复发机制提供理论依据。

MicroRNA的异常表达及其在肿瘤进展和转移/复发过程中的作用，是近年有关肿瘤分子标志物研究的热点，我们前期应用microRNA microarray 高通量筛选技术，发现 miR-19a是肝癌肝移植术后肿瘤复发相关的重要候选分子。为明确miR-19a在肝癌中的生物学作用，我们应用慢病毒载体转染和miR-19a inhibitor，双向调节不同肝癌细胞株中miR-19a的水平，发现miR-19a可明显抑制肝癌细胞的增殖、凋亡、侵袭、转移等恶性生物学功能；同时，动物活体成像发现miR-19a可抑制肝癌细胞皮下成瘤和肺转移的能力；通过生物信息学分析结合荧光素酶报告基因技术，明确了miR-19a可以明显抑制 SOX4表达，结合肝癌组织芯片，在临床样本中验证了miR-19a与SOX4表达的反向相关性；并进一步通过western blot对miR-19a双向调节后，肝癌细胞中SOX4、β-catenin蛋白对表达及WNT通路下游靶基因如CD44、MMP-2、VEGF等表达进行了检测，发现miR-19a可明显抑制SOX4、β-catenin及WNT通路下游靶基因CD44、MMP-2、VEGF等的表达；为进一步明确miR-19a对肿瘤相关信号通路的影响，我们应用基因表达谱芯片分析miR-19a肿瘤相关基因表达情况，根据KEGG与BIOCARTA中所有pathway的基因信息，将差异基因进行富集分析，发现了与miR-19a相关的肿瘤信号通路，其中包括WNT/β-catenin 信号通路。从而明确了肝癌中存在miR-19a-SOX4-WNT/β-catenin信号通路，miR-19a通过调控SOX4基因的表达影响WNT/β-catenin信号通路发挥抑制肝癌恶性生物学行为的作用。本研究为候选肝癌复发标志物miR-19a的鉴定提供了完善的基础研究数据，具有潜在的临床转化应用价值，对于揭示肝癌转移及复发的机制具有一定的理论意义并为肝癌复发的防治提供新的基因靶点。