PDGF-D调控miR-106a介导了吉西他滨耐药肝癌细胞上皮间质样转化的机制研究

Drug resistance is the main reason for failure to chemotherapy in patients with hepatocellular carcinoma. In our previous studies, confirmatory evidences for the epithelial-mesenchymal transition (EMT) in a gemcitabine-resistant hepatocellular carcinoma cells have been documented. The aberrant activation of platelet-derived growth factor D (PDGF-D) pathway has been involved in the development of EMT. Based on the cell models built by use of gene overexpression and gene silencing technology, miR-106a was primarily determined to act on the PDGF-D pathway by using bioinformatics analysis on miRNA chips. The down-regulation of miR-106a could significantly up-regulated the expression of several target genes related to EMT. Based on these interesting facts, a hypothesis was proposed that the down-regulation of miR-106a by PDGF-D contributes to the EMT in gemcitabine-resistant hepatocellular carcinoma cells. To confirm the hypothesis, hepatocellular carcinoma cell lines and their corresponding gemcitabine-resistant cells were used as the modes and a series of molecular biological methods such as gene transfection, qRT-PCR, Western Blot, luciferase reporter gene assay, small interfering RNA, and cell biological assay were employed in the present study. The regulating effects of PDGF-D on the expression of miR-106a and subsequently, miR-106a on the expression of its target genes and the EMT phenotype in gemcitabine-resistant cells were investigated. Moreover, the association between miR-106 target genes and the EMT phenotype in drug resistant cells was also revealed. This study plays significant roles for exploring the possible mechanisms of chemoresistance and the useful ways to overcome it in hepatocellular carcinoma.

耐药是肝细胞癌化疗失败的主要原因。我们前期工作表明吉西他滨（GEM）耐药肝癌细胞发生上皮间质样转化（EMT），这个过程涉及血小板源性生长因子D（PDGF-D）信号通路异常激活。利用PDGF-D过表达和基因沉默细胞模型，结合微小RNA（miRNA）芯片和生物信息学分析，我们确定miR-106a可能参与其中，后者低表达能显著上调与EMT相关的靶基因表达。基于此，本课题提出“PDGF-D下调miR-106a介导了GEM耐药肝癌细胞发生EMT”的学术假说，拟通过基因转染、qRT-PCR、Western Blot、报告基因、小干扰RNA和细胞生物学分析等方法，在肝癌亲本和GEM耐药细胞上，考察PDGF-D对miR-106a表达的调节，分析miR-106a对靶基因和GEM耐药细胞EMT表型的影响，揭示靶基因与GEM耐药细胞EMT表型之间的关系。本研究探索了肝癌耐药机制和克服耐药的方法，具有重要意义。

全身化疗是晚期肝细胞肝癌（HCC）重要的治疗方法。然而，HCC的耐药性严重制约着细胞毒药物的疗效，已成为HCC化疗的瓶颈。因此，深入研究新型细胞毒药物的耐药机制具有重要的科学价值和临床意义。为了研究新型细胞毒药物在HCC中的耐药机制，课题组前期构建了肝癌细胞系HepG2、SMMC-7721和Huh-7的吉西他滨（GEM）耐药细胞模型。前期研究表明GEM耐药肝癌细胞发生上皮间质样转化（EMT），这个过程涉及血小板源性生长因子D（PDGF-D）信号通路异常激活。我们利用PDGF-D过表达和基因沉默细胞模型，结合微小RNA（miRNA）芯片和生物信息学分析，确定miR-106a参与其中，miR-106a低表达能显著上调与EMT相关的靶基因表达。本研究在前期工作基础上，提出“PDGF-D下调miR-106a介导了GEM耐药肝癌细胞发生EMT”的学术假说。本研究通过生物信息学分析出miR-106a下游靶基因，应用荧光素酶报告实验验证了Twist1为miR-106a下游靶基因。通过转染miR-106a模拟物和阻遏物，可调控Twist1和EMT相关分子表达以及影响GEM耐药肝癌细胞生物学行为。其次，在GEM耐药肝癌细胞中过表达和沉默PDGF-D，可调控Twist1和EMT相关分子表达以及影响GEM耐药肝癌细胞生物学行为，而沉默Twist1表达同样影响其细胞生物学行为和EMT相关分子表达。在肝癌组织样本中，发现PDGF-D和Twist1表达呈正相关，而miR-106a和Twist1表达呈负相关。除此之外，课题组做了一些探索性研究，发现miR-130a-3p通过调控靶基因SMAD4表达也参与了GEM耐药肝癌细胞EMT的发生过程。本研究部分揭示了肝癌细胞对GEM耐药的机制，提出外源性干预手段逆转EMT的方法，为克服肝癌对GEM耐药提供了理论依据和策略，具有重要的临床意义。