调宁蛋白CNN3表达沉默促进肺腺癌细胞生长及EMT机制研究

Lung cancer is one of the highest mortality-malignant cancers worldwide. Intensive investigation is therefore urgently needed to explore the mechanisms of lung cancer and to search for specific diagnostic markers and therapeutic targets. Calponin is a family of actin-associated proteins expressed in smooth muscle and non-muscle cells. To date, three isoforms of calponin (acidic, neutral, and basic calponin), which encoded by three homologous genes were found in the vertebrates. Whereas previous studies of calponin mainly focused on its regulatory role in non-muscle cell motility and the mechanism in regulating smooth muscle contractility has been extensively investigated, the function of calponin in non-muscle cells is less well understood..Our previous comparative analysis of subcellular proteomics between human lung adenocarcinoma cell line A549 and human bronchial epithelial cell line (HBE) revealed the epithelial–mesenchymal transition (EMT) phenotype in lung cancer. Compared with HBE, CNN3 was dramatically down-regulated in but still ubiquitously expressed in lung adenocarcinoma cell lines. Moreover, CNN3 was down-regulated in human lung adenocarcinoma specimens (http://www.proteinatlas.org and http://www.proteinatlas.org)..To investigate its biological roles in lung tumorigenesis in vitro, CNN3 was stably knock down in human lung adenocarcinoma (LADC) cells by lentivirus-mediated delivery of shRNA. Further analysis demonstrated that down-regulation of CNN3 can promote cell proliferation and cell migration, exert an effect on morphologic changes. Moreover, knockdown of CNN3 can enhance epithelial-mesenchymal transition (EMT) in A549 cells which was characterized by low E-cadherin and high vimentin expression..To further investigate the role of CNN3 in LADC cell growth and motility, especially its role in regulation of EMT which may help to reveal the mechanism of tumor metastasis, subcellular fractionation coupled with 2-DE/MS-MS, stable-isotope labeling by amino acids in cell culture (SILAC) based quantitative proteomics/phosphoproteomics will be used to explore the differential proteomic expressions between stable CNN3 knockdown cancer cells and control cells. Meanwhile, research on the function of CNN3 in transcriptional regulatory factors and protein-protein interaction network will be performed to reveal the molecular mechanisms, regulation of pathogenesis in LADC.

发现有效的新治疗靶标，是提高肺癌患者治愈率的关键。调宁蛋白（calponin）是参与平滑肌收缩的肌动蛋白结合蛋白，现有研究表明调宁蛋白与疾病特别是肿瘤发生发展有密切关系。酸性调宁蛋白（即CNN3）在三个调宁蛋白异构体中是被研究最少的，确切作用分子机理未知。我们发现，与正常细胞相比，CNN3在发生EMT的肺腺癌细胞中表达下调，但仍广泛表达于肺腺癌细胞；CNN3在肺腺癌临床组织样品中的表达低于癌旁。前期研究首次发现：沉默CNN3的表达促进了肺腺癌细胞的增殖与迁移，改变了细胞的形态并促进了EMT。提示CNN3可能是一个新肿瘤相关因子。本项目是在前期研究新发现的基础上，利用蛋白质组学技术、报告基因系统等实验方法，系统研究CNN3在肺腺癌细胞中的转录调控过程、CNN3下游调控蛋白及其与细胞增殖和EMT的关系，旨在阐明沉默CNN3促进肺腺的机制，为深入了解肺腺癌发生发展，研发新治疗靶标提供新思路。

Calponin蛋白家族是一类参与平滑肌收缩的细纤维相关结合蛋白，现有研究表明该家族中的酸性蛋白Calponin-3与肿瘤特性相关，但是CNN3在肿瘤发生发展中的具体分子机制尚未明了。本研究开展了以下相关研究工作：⑴利用慢病毒介导，构建CNN3稳定过表达和表达沉默的非小细胞肺癌（NSCLC）细胞株，并在细胞水平和动物水平检测干预CNN3基因表达后的细胞增殖、迁移等肿瘤相关特性的变化，明确CNN3在肿瘤细胞中的功能；⑵ 研究CNN3调控细胞增殖和迁移分子机制。对获得的CNN3过表达/沉默的NSCLC细胞，利用亚细胞蛋白质组学结合基于细胞培养条件下稳定同位素标记 (Stable Isotope Labeling By Amino Acids In Cell Culture, SILAC)的定量蛋白质组学技术，鉴定CNN3调控相关的差异表达蛋白质，获得参与CNN3调控肿瘤细胞特性的基因信息。⑶CNN3及其调控相关蛋白质的临床表达分析，旨在探讨CNN3及其调控相关蛋白与NSCLC发生发展的关系，发现其在临床治疗和诊断中意义，并研究这些关键基因参与CNN3调控细胞增殖和迁移的分子机理。⑷鉴定CNN3相互作用蛋白质并关注CNN3的细胞定位。课题的研究结果：⑴过表达CNN3抑制非小细胞肺癌的增殖和迁移，抑制EMT；敲低CNN3则起到促进作用，表明CNN3是一个肿瘤抑制因子。⑵利用蛋白质组学技术，通过肿瘤细胞中CNN3表达变化的擦蛋白质表达谱，获得CNN3基因调控NSCLC细胞增殖迁移的相关蛋白质信息。⑶经过生物信息学分析于实验验证， DSP和JMJD6这两个基因可能是与CNN3执行功能有关的关键基因；在NSCLC患者中DSP与CNN3的表达显著相关，而JMJD6与CNN3的表达相关性不显著；利用RNA干扰和信号通路等实验技术表明DSP基因参与CNN3调控EMT调控NSCLC细胞的作用机制。④ 利用GST Pull-down技术筛选细胞内与CNN3相互作用的潜在靶蛋白11个，细胞免疫荧光实验 表明CNN3在细胞中与F-actin共定位，还有入核现象。