[摘 要] 目的：探讨RNA结合蛋白15（RBM15）通过三磷酸腺苷酶家族蛋白3A（ATAD3A）调控Wnt/β-catenin通路对宫颈癌细 胞恶性生物学行为的影响。方法：利用TCGA数据库分析宫颈癌组织中RBM15 mRNA表达水平及其与患者预后的关系。收集 2024年1月至10月期间在赣州市人民医院手术切除的 32 例宫颈癌组织及癌旁组织标本 ，以及宫颈癌细胞HeLa、MS-751、 C-33A 和 SiHa，通过免疫组化、WB 法检测宫颈癌组织和细胞中 RBM15 的表达水平。利用 SRAMP 在线数据库筛选 ATAD3A mRNA的m6 A修饰位点。通过RNA免疫沉淀实验、RNA衰变实验及挽救实验鉴定RBM15与ATAD3A mRNA的相互作用。采用 RNA干扰技术和病毒感染技术，在宫颈癌HeLa和SiHa细胞敲低或过表达RBM15和ATAD3A，qPCR和WB法检测mRNA和蛋 白表达，CCK-8法、划痕实验和Transwell实验检测各组细胞的增殖、迁移和侵袭能力。结果：宫颈癌组织中RBM15 mRNA和蛋 白阳性率均显著高于癌旁组织（均P < 0.001）。宫颈癌HeLa、MS-751、C-33A和SiHa细胞RBM15蛋白表达水平显著高于正常宫 颈细胞Ect1/E6E7（均P < 0.01）。RBM15 mRNA高表达组患者5年无进展生存率低于低表达组患者（P < 0.01）。宫颈癌组织中 ATAD3A的表达水平显著高于癌旁组织（P < 0.001），RBM15 mRNA与ATAD3A mRNA呈正相关（r = 0.601，P < 0.05）。ATAD3A mRNA的501、5 312、12 137位点存在高可信度的m6 A修饰位点。HeLa、SiHa细胞中过表达RBM15后，ATAD3A mRNA和蛋白表 达升高，而敲低RBM15后，ATAD3A mRNA和蛋白表达降低（均P < 0.001）。RNA免疫沉淀实验显示，与IgG组相比，RBM15抗 体的免疫沉淀中ATAD3A mRNA显著富集（均P < 0.001）。MeRIP-qPCR实验显示，ATAD3A mRNA 501、5 312、12 137位点均存 在明显的m6 A甲基化富集（均P < 0.001）。RNA衰变实验显示，敲低RBM15能降低HeLa、SiHa细胞ATAD3A mRNA的半衰期和 稳定性（均P < 0.001）。敲低HeLa、SiHa细胞RBM15的表达，显著抑制癌细胞的增殖、迁移及侵袭，Wnt/β-catenin通路相关蛋白 Wnt3、β-catenin、vimentin表达均显著降低（均P < 0.001）；而过表达ATAD3A可完全逆转上述抑制作用（均P < 0.001）。结论： RBM15通过m6 A修饰ATAD3A mRNA调控Wnt/β-catenin信号通路，从而促进宫颈癌细胞增殖、迁移及侵袭。

[Abstract] Objective: To investigate the effects of RNA binding protein 15 (RBM15) on the malignant biological behaviors of cervical cancer cells by regulating the Wnt/β-catenin pathway through ATPase family AAA-domain containing 3A (ATAD3A). Methods: TCGA database was used to analyze the expression level of RBM15 mRNA in cervical cancer tissues and its relationship with patient prognosis. 32 samples of cervical cancer tissues and adjacent para-cancerous tissues surgically removed in Ganzhou People's Hospital between January and October 2024, as well as cervical cancer cells HeLa, MS-751, C-33A, and SiHa, were collected. The expression levels of RBM15 in cervical cancer tissues and cells were detected by immunohistochemistry and WB assay. m6 A modification sites in ATAD3A mRNA were screened by the SRAMP online database. The interaction between RBM15 and ATAD3A mRNA were identified by RNA immunoprecipitation assay, RNA decay assay, and salvage assay. Knockdown or overexpression of RBM15 and ATAD3A in cervical cancer HeLa and SiHa cells were conducted by RNA interference and viral infection. The expressions of mRNA and protein were detected by qPCR and WB methods, while the proliferation, migration, and invasion abilities of cells in each group were assessed by CCK-8 method, scratch assay, and Transwell assay. Results: The positivity rates of RBM15 mRNA and protein in cervical cancer tissues were significantly higher than those in adjacent para-cancerous tissues (both P < 0.001). The protein expression levels of RBM15 in cervical cancer cell lines HeLa, MS-751, C-33A, and SiHa were significantly higher than those in normal cervical cell lines Ect1/E6E7 (all P < 0.001). The 5-year progression free survival rate of the RBM15 mRNA high expression group was lower than that of the low expression group (P < 0.001). Compared with that in adjacent para-cancerous tissues, the expression level of ATAD3A was significantly upregulated in cervical cancer tissues (P < 0.001). RBM15 mRNA was positively correlated with ATAD3A mRNA (r = 0.601, P < 0.05). There were highly reliable m6 A modification sites at position 501, 5 312, 12 137 in ATAD3A mRNA. Overexpression of RBM15 in HeLa and SiHa cells led to an increase in ATAD3A mRNA and protein expressions, while knockdown of RBM15 resulted in a decrease in ATAD3A mRNA and protein expression (all P < 0.001). RNA immunoprecipitation experiment showed that compared with the IgG group, ATAD3A mRNA was significantly enriched in the immunoprecipitation of RBM15 antibody (both P < 0.001). MeRIP-qPCR experiment showed that there was significant m6 A methylation enrichment at positions ATAD3A mRNA 501, 5 312 and 12 137 (all P < 0.001). RNA decay experiments showed that knocking down RBM15 in HeLa and SiHa cells could reduce the half-life and stability of ATAD3A mRNA (all P < 0.001). Knocking down the expression of RBM15 in HeLa and SiHa cells could significantly inhibit the proliferation, migration, and invasion of cancer cells and significantly reduce the expressions of Wnt/β-catenin pathway related proteins Wnt3, β-catenin, and vimentin, while overexpression of ATAD3A could completely reverse the above inhibiting effects (all P < 0.001). Conclusion: RBM15 can modify ATAD3A mRNA and regulate the Wnt/β-catenin pathway through m6 A, and thus promote the proliferation, migration and invasion of cervical cancer cells.

[基金项目] 江西省卫生健康委科研项目（No. 202410848）