ZMIZ2通过SIRT1促进非小细胞肺癌恶性表型的机制研究

The expression of ZMIZ2 in lung cancer specimens and its underlying molecular mechanism remains unclear. In this study, we found that ZMIZ2 was highly expressed in lung cancer specimens, which was closely related to the advanced pTNM stage, lymph node metastasis and poor prognosis of patients with non-small cell lung cancer. Functional experiments in vitro confirmed that the overexpression of ZMIZ2 could promote the proliferation, migration and invasion of lung cancer cells; and immunoprecipitation assay was performed to demonstrate that ZMIZ2 could interact with Sirtuin-1 (SIRT1); Overexpression of ZMIZ2 could lead to the decrease in the acetylation level of β-catenin, and the activity of YAP-TEAD luciferase in Hippo pathway was significantly up-regulated. Therefore, we speculate that ZMIZ2 may regulate the acetylation levels of β-catenin and YAP through interaction with SIRT1, thereby affecting the activities of Wnt and Hippo pathways. We intend to bidirectionally regulate the expression of ZMIZ2 and SIRT1, construct and transfect ZMIZ2-SIRT1 binding site mutants, employ immunoprecipitation, GST-pulldown, dual-luciferase assay and qPCR, to elucidate the effect of ZMIZ2-SIRT1 interaction on the acetylation level of β-catenin and YAP, as well as on Wnt and Hippo activities both in vitro and in vivo. These findings will shed light on the molecular interplay between a yet unknown link between Wnt and Hippo pathways, providing us a potential target of clinical intervention for patients suffering lung cancer.

ZMIZ2在肿瘤中的表达及作用机制尚无报道。我们发现ZMIZ2在非小细胞肺癌中高表达，且与高pTNM分期，淋巴结转移及不良预后密切相关；转染ZMIZ2能够促进肺癌细胞的增殖、迁移和侵袭；免疫共沉淀实验发现ZMIZ2与去乙酰化酶SIRT1相互作用，并下调β-catenin的乙酰化水平、上调YAP-TEAD荧光素酶活性。我们推测ZMIZ2可能通过SIRT1影响Wnt和Hippo通路活性发挥促进肺癌细胞恶性表型的作用。我们拟双向调控ZMIZ2和SIRT1的表达，构建和转染ZMIZ2与SIRT1结合位点突变体等策略，采用免疫共沉淀、GST-pulldown、荧光素酶报告基因和实时定量PCR等技术，在体内外阐明ZMIZ2高表达的原因及与SIRT1的相互作用，激活Wnt和抑制Hippo通路活性，促进肺癌细胞恶性表型的分子机制，为寻找新的非小细胞肺癌生物标记物和开发靶向药物提供实验基础。

本项目通过构建并转染ZMIZ2剪切突变体，双向调控ZMIZ2和SIRT1的表达；结合免疫组织化学、Western blot、Real-time PCR、蛋白质免疫共沉淀、GST-pulldown，免疫荧光共聚焦，裸鼠成瘤以及核/浆蛋白分离等方法，揭示了ZMIZ2在肺癌等恶性上皮性肿瘤中的表达和影响肿瘤细胞生物学行为的具体作用机制。按照原研究计划，我们圆满完成了：1) ZMIZ2在非小细胞肺癌中高表达，并与患者不良预后相关。2）ZMIZ2与SIRT1之间相互作用的验证。3）二者的结合促进了SIRT1对β-catenin及YAP的去乙酰化作用，发挥激活Wnt通路，抑制Hippo通路活性的作用。推断出ZMIZ2是引起Wnt与Hippo两个通路交互作用的重要的下游分子。4）通过功能学实验验证了ZMIZ2对肺癌细胞生物学功能的影响。