目的·探讨E3泛素连接酶膜相关锌指蛋白9（membrane-associated RING-CH 9，MARCH9）调控受体型蛋白质酪氨酸磷酸酶α（receptor protein tyrosine phosphatase alpha，RPTPα）泛素化修饰的分子机制及其生物学功能。方法·采用Western blotting鉴定RPTPα的泛素化修饰类型及MARCH9对其泛素化水平的调控作用；比较MARCH9野生型、MARCH9酶活突变体（MARCH9 S198A或MARCH9-HC/CC），以及shRNA介导的内源MARCH9敲低对RPTPα蛋白稳定性的影响；利用蛋白酶体抑制剂MG132、自噬抑制剂3-MA和溶酶体抑制剂氯喹（chloroquine，CQ）鉴定MARCH9介导的RPTPα泛素化降解类型；探究43 ℃热激促进RPTPα蛋白降解的可能机制；利用慢病毒载体构建MARCH9单敲低（H1299-shMARCH9）及MARCH9/RPTPα双敲低（H1299-shMARCH9-shRPTPα）的肺癌稳转细胞株，通过CCK-8细胞增殖、平板克隆形成和软琼脂克隆形成实验，评估MARCH9或RPTPα对肺癌细胞增殖与集落形成能力的影响；通过血管拟态形成实验和划痕实验检测MARCH9或RPTPα对肺癌细胞侵袭、迁移能力的影响；建立裸鼠皮下移植瘤模型评价MARCH9或RPTPα对肺癌细胞体内成瘤能力的影响；通过生物信息学方法比较MARCH9和RPTPα在肺癌患者中的表达差异与预后相关性。结果·RPTPα主要发生K63连接型多聚泛素化修饰，E3泛素连接酶MARCH9过表达显著增强其泛素化水平；过表达野生型MARCH9而非酶活突变体能显著降低RPTPα蛋白稳定性，而shRNA敲低内源MARCH9后RPTPα蛋白水平又会明显上升；CQ而非MG132或3-MA处理能增加RPTPα蛋白稳定性，提示MARCH9主要介导RPTPα的泛素化-溶酶体降解途径；43 ℃热激可以特异增强MARCH9-RPTPα相互作用并促进RPTPα蛋白降解。功能实验显示：与对照H1299细胞相比，H1299-shMARCH9细胞的RPTPα蛋白水平升高，细胞增殖速率加快，克隆形成和细胞侵袭能力均明显增强，裸鼠中成瘤能力也明显增强，而双敲低MARCH9/RPTPα的稳转细胞株又能逆转上述表型（均P<0.05）。生物信息学分析肺癌患者相关数据，显示RPTPα高表达与肺癌患者不良预后及肿瘤转移呈正相关，而MARCH9则为负相关。结论·MARCH9介导了磷酸酶RPTPα的K63型泛素化依赖的溶酶体降解过程。这一发现为开发靶向RPTPα的肿瘤治疗策略提供了新的依据。

Objective ·To investigate the molecular mechanisms and biological functions of the E3 ubiquitin ligase membrane-associated RING-CH 9 (MARCH9) in regulating the ubiquitination of receptor protein tyrosine phosphatase alpha (RPTPα). Methods ·Western blotting was employed to identify the ubiquitination type of RPTPα and to evaluate the regulatory effect of MARCH9 on its ubiquitination level; Comparative analysis of RPTPα protein stability was performed among wild-type MARCH9, catalytically inactive MARCH9 mutants (MARCH9 S198A or MARCH9-HC/CC), and endogenous MARCH9 knockdown via shRNA. Proteasome inhibitor MG132, autophagy inhibitor 3-MA, and lysosomal inhibitor chloroquine (CQ) were used to determine the degradation pathway of MARCH9-mediated RPTPα ubiquitination. The mechanism underlying 43 ℃ heat shock-induced RPTPα degradation was explored. Stable lung cancer cell lines with MARCH9 single-knockdown (H1299-shMARCH9) and MARCH9/RPTPα double-knockdown (H1299-shMARCH9-shRPTPα) were established using lentiviral vectors. CCK-8 proliferation assay, colony formation assay, and soft agar assay were conducted to evaluate the effects of MARCH9 or RPTPα on lung cancer cell proliferation and clonogenicity. Vasculogenic mimicry formation assay and scratch wound healing assay were performed to assess the impacts on tumor cell invasion and migration. Subcutaneous xenograft models in nude mice were established to examine in vivo tumorigenicity. Bioinformatics analysis was used to compare the expression differences and prognostic correlations of MARCH9 and RPTPα in lung cancer patients. Results ·RPTPα predominantly underwent K63-linked poly-ubiquitination, which was significantly enhanced by MARCH9 overexpression. Wild-type MARCH9, but not its catalytic mutants, markedly reduced RPTPα protein stability, while endogenous MARCH9 knockdown increased RPTPα levels. CQ, not MG132 or 3-MA, restored RPTPα stability, indicating that MARCH9 mediated lysosomal degradation of RPTPα through ubiquitination. Heat shock at 43 ℃ specifically enhanced MARCH9-RPTPα interaction, promoting RPTPα degradation. Functional assays revealed that, compared to control H1299 cells, MARCH9-knockdown cells exhibited elevated RPTPα levels, accelerated proliferation, enhanced clonogenicity and invasive capacity, and increased tumorigenicity in nude mice. These phenotypes could be reversed by double knockdown of MARCH9/RPTPα. Bioinformatics analysis demonstrated that high RPTPα expression correlated with poor prognosis and tumor metastasis in lung cancer patients, while MARCH9 showed inverse correlations. Conclusion ·MARCH9 mediates K63-linked ubiquitination-dependent lysosomal degradation of phosphatase RPTPα, providing new insights into developing RPTPα-targeted cancer therapeutic strategies.