钙调蛋白样蛋白3通过调控外泌体途径影响肝癌发生发展的机制研究

Exosomes released by secretory vesicles impact carcinogenesis and progression of hepatocellular carcinoma (HCC). Previously, we found that the expression of calcium sensor CALML3 is reduced in clinical HCC tissues and chemical-induced HCC model. Loss of CALML3 expression in HCC tissues is associated with poor overall survival and high recurrence. Moreover, CALML3 expression in HCC cells could significantly reduce the number of secretory vesicles and the release of exosomes. We further conducted co-immunoprecipitation combined with mass spectrum assay, and found that CALML3 could interact with Rab11. We further conducted liver-specific deletion of CALML3 mice. Indeed, the phosphorylation level of Rab11 is remarkably increased after hepatocyte-specific deletion of CALML3. These results proposed a previously unrecognized role for CALML3 in carcinogenesis and development, which CALML3 may regulate exosome pathway via Rab11 signal. In the present study, we aim to further delineate the role of CALML3 in carcinogenesis and development of HCC through exosome pathway by primary hepatocyte, CALML3 gene conditional knockout mice and clinical materials, in cells, in animals and at clinical level. Our study will provide new target and new clues for targeted therapy for HCC.

分泌性囊泡释放的外泌体是影响肝癌发生、发展的重要因素。前期研究发现，钙离子感受蛋白CALML3在人肝细胞癌组织和化学诱导肝癌动物模型中，表达均显著降低，且癌组织中CALML3的降低与肝癌的预后不良相关；肝癌细胞中CALML3的表达能够显著减少分泌性囊泡的数量和外泌体释放；利用免疫沉淀联合质谱筛选，找到可与CALML3相互作用的候选蛋白——调控胞吐途径的重要Ras亚家族成员Rab11，进一步构建了肝脏条件性敲除CALML3基因小鼠，发现Rab11在肝脏条件性敲除CALML3基因小鼠中磷酸化表达水平显著升高。提示肝脏表达的CALML3可能通过调控Rab11水平，进而调控外泌体分泌。本课题拟利用原代肝细胞、条件性敲除基因小鼠和临床肝癌资料，在细胞水平、动物水平和人体水平，探讨肝癌发生、发展的机制，课题的完成将为肝癌靶向治疗提供新靶点和新策略。

分泌性囊泡释放的外泌体是影响肝癌发生、发展的重要因素。课题组前期发现，钙离子感受蛋白CALML3在人肝细胞癌组织和化学诱导肝癌动物模型中，表达均显著降低，且癌组织中CALML3的降低与肝癌的预后不良相关。然而CALML3如何影响肝癌细胞的发生发展，其机制尚不清楚。在本研究中，我们利用Tet-on技术行体外实验证实CALML3的表达能够显著抑制囊泡的分泌和外泌体释放，从而抑制肝癌细胞的增殖及迁移等肿瘤生物学行为，并抑制EMT相关蛋白；机制研究表明，利用免疫沉淀联合质谱筛选到与CALML3相互作用的候选蛋白——调控胞吐途径的重要Ras亚家族成员Rab11，免疫共沉淀实验证实了二者之间的相互作用；进一步体内DEN诱癌模型发现，与野生型小鼠相比，肝脏条件性敲除CALML3基因小鼠的肝癌更为严重，Rab11磷酸化表达水平显著升高，并与CALML3表达负性相关；人肝癌临床样本中CALML3亦与Rab11磷酸化表达负相关。本研究将CALML3和肝癌的病理生理发生过程联系在一起，为肝癌靶向治疗提供新靶点和新策略。