miR-125b调控TAZ在肺癌侵袭转移中的作用机制研究

Invasion and metastasis is the characteristic of lung cancer, with high rate of recurrence rate and and poor prognosis. TAZ is one of the main downstream transcriptional co-activators. We firstly demonstrated that TAZ expression was closely correlated with poor prognosis in resected non-small cell lung cancer patients. Our previous sudy showed that RNA interference of TAZ resulted in increased expression of E-Cadherin and decreased expressions of Vimentin and Snail, suggesting that TAZ may promote tumor invasion and metastasis via epithelial-mesenchymal transition (EMT) in lung cancer. To date,microRNA involved in the regulation of TAZ remains unclear. We found that the TAZ was direct target of TAZ by bioinformatics analysis software. miR-125b was overexpressed in advanced lung cancer. Transfecting pre-miR-125b in lung cancer cells significantly enhanced TAZ level in lung cancer cells. The aim of this study is to investigate the mechanisms of miR-125b targeting TAZ during the process of invasion and metastasis in lung cancer. Our study herein plans to construct luciferase reporter plasmid, Anoikis assay, in situ hybridization, Matrigel invasion assay and PCR-based site-directed mutagenesis to investigate the interaction and relationship between miR-125b, TAZ, EMT and lung cancer metastasis. Furthermore, the inhibiton of TAZ expression and ability of invasion and metastasis will be studied by targeting miR-125b overexpression in xenograft lung cancer nude mice models.Taken together, this study will further elucidate the mechanisms of metastasis and identify miR-125b as target for a novel therapy in lung cancer.

肺癌侵袭转移力强，术后复发转移率高，预后差。TAZ是Hippo信号通路下游主要的转录共激活因子。我们已证实肺癌TAZ过表达与根治切除术不良预后密切相关；前期研究提示TAZ可能通过介导上皮-间质转型（EMT）促进肺癌体外侵袭力；生物学信息软件预测TAZ是miR-125b的靶基因；miR-125b在进展期肺癌高表达；过表达miR-125b使肺癌TAZ 表达明显增加。目前直接调控肺癌TAZ的microRNA及其机制尚不明确。miR-125b是否通过靶向调控TAZ参与肺癌EMT及侵袭转移呢？我们拟进一步构建荧光素酶报告载体、Anoikis实验、原位杂交、Matrigel 侵袭实验、PCR定点突变等方法研究miR-125b与TAZ、EMT及肺癌侵袭转移之间的关系。在裸鼠肺癌模型中研究靶向抑制miR-125b对逆转肺癌EMT及侵袭转移的作用。本研究将为阐明肺癌转移机制、寻找新的肺癌靶向治疗提供依据。

肺癌侵袭转移力强，术后复发转移率高，预后差。TAZ是Hippo信号通路下游主要的转录共激活因子。我们已证实肺癌TAZ过表达与根治切除术不良预后密切相关；前期研究提示TAZ可能通过介导上皮-间质转型（EMT）促进肺癌体外侵袭力；生物学信息软件预测TAZ是miR-125b的靶基因；miR-125b在进展期肺癌高表达；过表达miR-125b使肺癌TAZ 表达明显增加。目前直接调控肺癌TAZ的microRNA及其机制尚不明确。. 我们进一步通过免疫组化方法检测肺癌细胞株和组织芯片，证实TAZ在肺癌细胞株和肺癌组织中高表达。TAZ siRNA转染肺癌细胞株使TAZ表达下调。Matrigel 侵袭实验发现 TAZ RNA干扰显著抑制A549和HCC827细胞体外侵袭力 (P < 0.05)。软琼脂集落形成实验发现TAZ RNA干扰同样明显减弱A549和HCC827细胞株锚定-不依赖性生长能力，表现为明显减少和缩小的细胞克隆。TAZ siRNA转染肺癌细胞株A549和HCC827后E-Cadherin 表达升高，Vimentin 和Snail 表达减弱。miR-125b表达水平在分化程度低的肺癌组织中表达水平较高分化者更高。通过使用miRNA qPCR试剂盒检测正常支气管上皮细胞株以及各肺癌细胞株miR-125b表达水平，结果发现miR-125b在正常支气管上皮细胞表达水平明显低于肺癌细胞株，在肺癌细胞株NCI-H292、NCI-H1299和NCI-358中miR-125b表达较低。. 使用荧光素酶报告基因检测发现抑制miR-125b的表达水平使肺癌细胞TAZ.3'-UTR荧光酶活性减弱，miR-125b可直接作用于TAZ 3'-UTR。我们进一步研究裸鼠皮下肺癌移植瘤模型过表达 miR-125b 对成瘤大小、肿瘤转移的影响。结果发现过表达miR-125b的裸鼠皮下移植瘤生长加速，肺转移瘤增多。. RNA-Seq（转录组测序）发现miR-125b使E-cadherin表达下降、Vimentin表达增高。热点图（Heat map) 显示miR-125b诱导MET与MET相关标志物Twist, Snail, ZEB1表达水平相关。