Objectives: MICAL-L2, a member of the molecules interacting with the CasL (MICAL) family, was reported to be highly expressed in several types of cancers, however, the roles of MICAL-L2 in NSCLC pathogenesis remain to be explored. This study is designed to clarify the mechanisms by which MICAL-L2 participates in NSCLC cell proliferation.

Materials and Methods: The expression levels of MICAL-L2 in human lung cancer samples were assessed by immunohistochemical staining. Cells were transfected with siRNA or plasmids to regulate MICAL-L2 expression. Cell proliferation was measured by EdU staining and CCK-8 assays. MICAL-L2 and phosphorylated/total c-Myc expression were examined by Western blotting analysis. Interaction between MICAL-L2 and c-Myc was assessed by immunofluorescence staining, Western blotting and co-immunoprecipitation assays. Western blotting, polyubiquitylation detection and protein stability assays were used to assess whether MICAL-L2 exerts its oncogenic effect via c-Myc.

Results: We found that MICAL-L2 was highly expressed in human NSCLC. While overexpressing MICAL-L2 increased NSCLC cell proliferation, MICAL-L2 depletion decreased the proliferation of NSCLC cells, an effect that was linked to cell cycle arrest. MICAL-L2 physically interacted with the c-Myc protein and functioned to maintain nuclear c-Myc levels and prolonged its half-life. Knockdown of MICAL-L2 expression led to decreased c-Myc protein stability through accelerating polyubiquitylation of c-Myc and gave rise to c-Myc degradation. We further found that MICAL-L2 deubiquitinated c-Myc and blocked its degradation, presumably by inhibiting c-Myc phosphorylation at threonine residue 58.

Conclusions: These results indicate that MICAL-L2 is a key regulator of c-Myc deubiquitination and stability in the nucleus, and this activity may be involved in promoting NSCLC cell proliferation.

目标：据报道，与CasL（MICAL）家族相互作用的分子成员MICAL-L2在几种类型的癌症中高表达，然而，MICAL-L2在NSCLC发病机理中的作用仍有待探索。这项研究旨在阐明MICAL-L2参与NSCLC细胞增殖的机制。

材料和方法：通过免疫组织化学染色评估人肺癌样品中MICAL-L2的表达水平。用siRNA或质粒转染细胞以调节MICAL-L2表达。通过EdU染色和CCK-8测定法测量细胞增殖。通过蛋白质印迹分析检查了MICAL-L2和磷酸化/总c-Myc表达。MICAL-L2和c-Myc之间的相互作用通过免疫荧光染色，Western印迹和免疫共沉淀法进行评估。使用蛋白质印迹，多泛素化检测和蛋白质稳定性分析来评估MICAL-L2是否通过c-Myc发挥其致癌作用。

结果：我们发现，MICAL-L2在人类NSCLC中高度表达。虽然过表达MICAL-L2会增加NSCLC细胞的增殖，但MICAL-L2耗竭会降低NSCLC细胞的增殖，这与细胞周期停滞有关。MICAL-L2与c-Myc蛋白发生物理相互作用，并起维持核c-Myc水平的作用，并延长了其半衰期。抑制MICAL-L2表达通过加速c-Myc的多泛素化作用导致c-Myc蛋白稳定性降低，并导致c-Myc降解。我们进一步发现，MICAL-L2可以通过抑制苏氨酸残基58处的c-Myc磷酸化来使c-Myc去泛素化并阻止其降解。

结论： 这些结果表明，MICAL-L2是c-Myc去泛素化和细胞核稳定性的关键调节剂，该活性可能与促进NSCLC细胞增殖有关。