目的：探究甲基转移酶样蛋白3（METTL3）修饰的RNASEH1-AS1 通过BUD13/膜联蛋白A2/ Wnt/β-连环蛋白（BUD13/ANXA2/Wnt/β-catenin）轴调控结直肠癌细胞 SW480 恶性生物学行为的分子机制。方法：选取2022 年6 月至2022 年11月间在复旦大学附属中山医院厦门医院手术治疗的24例CRC患者，收集患者的CRC组织和对应的癌旁组织，用qPCR法检测其中METTL3、RNASEH1-AS1、BUD13、ANXA2、β-catenin、GSK-3β mRNA的表达，用Pearson法分析CRC 组织中RNASEH1-AS1表达分别与METTL3和BUD13 表达的相关性。常规培养结直肠癌细胞SW480，分为sh-NC 组、sh-RNASEH1-AS1组、NC 组、 sh-METTL 组 、si-NC 组、si-BUD13 组、sh-RNASEH1-AS1+pc-NC 组、sh-RNASEH1-AS1+pc-ANXA2 组、sh-METTL+pc-NC 组、 sh-METTL+pc-ASEH1-AS1组，用Lipofectamine? 2000 转染试剂将sh-NC、sh-RNASEH1-AS1、sh-NC、sh-METTL、si-NC、si-BUD13 、sh-RNASEH1-AS1+pc-NC 、sh-RNASEH1-AS1+pc-ANXA2 、sh-METTL+pc-NC 、sh-METTL+pc-ASEH1-AS1 分别转染 SW480细胞，用qPCR 法检测后SW480 细胞中METTL3、RNASEH1-AS1、BUD13、ANXA2 的表达，细胞克隆形成实验检测转染后各组SW480 细胞的增殖能力，FCM术检测转染后各组SW480 细胞的凋亡情况，细胞划痕实验检测转染后各组SW480 细胞的迁移能力，WB法检测转染后各组SW480 细胞中ANXA2、β-catenin、p-β-catenin、GSK-3β、p-GSK-3β、c-Myc、cyclinD1 蛋白表达，RNA免疫沉淀（RIP）法检测RNASEH1-AS1与BUD1、BUD1与ANXA2的靶向关系。结果：数据库CRC数据分析和中国人CRC组织和癌旁组织的qPCR 法检测结果均显示，与癌旁组织相比CRC 组织中METTL3、RNASEH1-AS1、BUD13、ANXA2、β-catenin、GSK-3β 均呈明显高表达（均P<0.01），且RNASEH1-AS1 表达与METTL3（r=0.698，P<0.01）、BUD13（r=0.784，P<0.01）的表达呈正相关。在结直肠癌各细胞中METTL3、RNASEH1-AS1、BUD13、ANXA2 mRNA均呈高表达（均P<0.05）；敲减RNASEH1-AS1或METTL3后SW480 细胞中RNASEH1-AS1、BUD13、ANXA2表达显著降低（均P<0.05），而过表达RNASEH1-AS1后上述分子表达明显上调（均P<0.05）；敲减RNASEH1-AS1 或METTL3 可抑制SW480 的增殖、迁移和p-β-catenin、p-GSK-3β、c-Myc、 cyclinD1蛋白的表达，促进其凋亡（均P<0.05），而过表达RNASEH1-AS1 则可促进SW480 增殖、迁移和p-β-catenin、p-GSK-3β、c-Myc、cyclinD1蛋白的表达和抑制其凋亡（均P<0.05）；RNASEH1-AS1通过招募BUD13靶向促进ANXA2的表达（均P<0.05）；过表达ANXA2 可部分逆转敲减RNASEH1-AS1 对SW480 细胞的影响（均P<0.05）。结论：METTL3 修饰的RNASEH1-AS1 通过BUD13/ANXA2/Wnt/β-catenin轴调控SW480细胞的恶性生物学行为。

Objective: To explore the molecular mechanism by which methyltransferase-like protein 3 (METTL3) -modified RNASEH1-AS1 regulates the malignant biological behaviors of colorectal cancer cells SW480 through the BUD13/membrane associated protein A2/Wnt/β-catenin (BUD13/ANXA2/Wnt/β-catenin) axis. Methods: Twenty-four CRC patients who were surgically treated in our hospital from June 2022 to November 2022 were selected as the study subjects, and the patients' CRC tissues and corresponding paracancerous tissues were collected, and the qPCR method was used to detect the METTL3, RNASEH1-AS1, BUD13,ANXA2, β -catenin, GSK-3β mRNA in CRC tissues and paracancerous tissues expression, and the correlation of RNASEH1-AS1 expression with METTL3 expression and BUD13 expression in CRC tissues was analyzed by Pearson's method. Colorectal cancer cells SW480 were routinely cultured, and the experiments were divided into sh-NC group, sh-RNASEH1-AS1 group, NC group, sh-METTL group, si-NC group, si-BUD13 group, sh-RNASEH1-AS1+pc-NC group, sh-RNASEH1-AS1+pc-ANXA2 group, sh-METTL+pc-NC group, sh-METTL+pc-ASEH1-AS1 group, and sh-NC, sh-RNASEH1-AS1, sh-NC, sh-METTL, si-NC, si-BUD13, sh-RNASEH1-AS1 and pc-NC, sh-RNASEH1-AS1 and pc- ANXA2, sh-METTL and pc-NC, sh-METTL and pc-ASEH1-AS1 were transfected in SW480 cells. The expression of METTL3, RNASEH1-AS1, BUD13, and ANXA2 in SW480 cells was detected by qPCR. The cell clone formation assay detected the proliferative ability of SW480 cells in the transfected groups; FCM assay detected the apoptosis of SW480 cells in the transfected groups; cell scratch assay detected the migratory ability of SW480 cells in the transfected groups; and WB assay detected the migration of SW480 cells in the transfected groups. ANXA2, β-catenin, p-β-catenin, GSK-3β, p-GSK-3β, c-Myc, cyclinD1 protein expression in each group of SW480 cells; RNA immunoprecipitation (RIP) assay to detect the targeting relationship between RNASEH1-AS1 and BUD1, and BUD1 and ANXA2. Results: Both database analysis and qPCR assay results of CRC tissues and cancerous tissues of nationals showed that METTL3, RNASEH1-AS1, BUD13, ANXA2, β -catenin, and GSK-3β were significantly highly expressed in CRC tissues compared with paracancerous tissues (all P<0.01), and RNASEH1-AS1 expression was positively correlated with the METTL3 (r=0.698, P<0.01) and BUD13 (r=0.784, P<0.01) expression were positively correlated. METTL3, RNASEH1-AS1, BUD13, and ANXA2 mRNA were highly expressed in colon cancer cells (all P<0.05), and the expression of RNASEH1-AS1, BUD13, and ANXA2 was significantly decreased in SW480 cells after knockdown of RNASEH1-AS1 or METTL3 (all P<0.05), while overexpression of RNASEH1-AS1 significantly upregulated the expression of the above molecules (all P<0.05).Knockdown of RNASEH1-AS1 or METTL3 inhibited the proliferation, migration and expression of p-β-catenin, p-GSK-3β, c-Myc,cyclinD1 proteins, and promoted apoptosis of SW480 (all P<0.05), whereas over-expression of RNASEH1-AS1 promoted the proliferation, migration and expression of p-β-catenin p-β-catenin, p-GSK-3β, c-Myc, cyclinD1 protein expression and inhibited its apoptosis (all P<0.05). RNASEH1-AS1 promoted the expression of ANXA2 by recruiting BUD13 targeting (all P<0.05); over-expression of ANXA2 partially reversed the knockdown of the effect of RNASEH1-AS1 on SW480 cells (all P<0.05). Conclusion: METTL3-modified RNASEH1-AS1 regulates the malignant biological behavior of SW480 cells through the BUD13/ANXA2/Wnt/β-catenin axis.

福建省科技厅引导性科研项目（No. 2022R0027）