FHL2与LDHA相互作用促进胶质瘤细胞的增殖

FHL2 interacts with LDHA to promote glioma cell proliferation

目的:探讨四个半LIM结构域2(FHL2)蛋白对胶质瘤细胞增殖的影响及其分子机制。方法:利用TCGA和CGGA数据库分析胶质瘤组织中FHL2 mRNA表达水平与患者预后的关系。通过WB法检测人胶质瘤组织标本及人胶质瘤细胞U87、T98G、U251、SNB19、GSC23、A172、LN229、G267和星形胶质细胞NHA中的FHL2蛋白表达水平。利用慢病毒载体构建稳定敲低FHL2的U87细胞和过表达FHL2的SNB19细胞,即U87-shGFP、U87-shFHL2-1#、U87-shFHL2-4#和SNB19-3flag、SNB19-3flag-FHL2组。通过CCK-8法、克隆形成实验检测敲低和过表达FHL2对细胞增殖的影响,免疫共沉淀(Co-IP)和液相色谱-串联质谱(LC/MS)法筛选FHL2在胶质瘤细胞中的相互作用蛋白,并用Co-IP和免疫荧光法验证它们的结合作用和共定位情况。使用酶标仪检测敲低和过表达FHL2细胞内乳酸产量和乳酸脱氢酶(LDH)活性的变化,WB法分析FHL2、LDHA及p-LDHA在正常脑组织和胶质瘤组织中的蛋白表达差异及其相互关系。在过表达FHL2的SNB19细胞中使用LDHA的小分子抑制剂AT-101,通过CCK-8实验和酶标仪比色法验证FHL2在胶质瘤乳酸代谢中的作用,验证AT-101在胶质瘤中潜在的治疗效果。结果:Co-IP和LC/MS检测发现,FHL2与LDHA在胶质瘤细胞中存在相互作用。FHL2过表达可提高LDHA活性和乳酸生成(均P<0.001),进而促进胶质瘤细胞增殖(P<0.001)。相反,敲低FHL2会降低LDHA活性和乳酸产量(P<0.001或P<0.05)并抑制细胞增殖(P<0.001)。AT101能抑制LDHA活性,并显著抑制FHL2促进胶质瘤细胞的增殖,同时恢复磷酸化LDHA(Y10)水平(P<0.01或P<0.001)。结论:FHL2与LDHA蛋白相互作用,FHL2通过激活p-LDHA(Y10)的表达促进LDHA活性和乳酸产生,进而促进胶质瘤细胞的增殖,靶向这种相互作用可能成为治疗胶质瘤的潜在策略。

Objective:To investigate the effects of four-and-a-half LIM domains 2(FHL2)on glioma cell proliferation and its molecular mechanisms.Methods:The relationship between FHL2 mRNA expression levels in gliomas and patient prognosis was analyzed using TCGA and CGGA databases.The protein expression levels of FHL2 in collected human glioma tissue samples and human glioma cell lines(U87,T98G,U251,SNB19,GSC23,A172,LN229,G267),and astrocyte NHA were analyzed using Western blot(WB).Lentiviral vecors were used to construct U87 cells with stable FHL2 knockdown and SNB19 cells overexpressing FHL2,namely U87-shGFP,U87-shFHL2-1#,U87-shFHL2-4#,and SNB19-3flag,SNB19-3flag-FHL2 groups.The effects of FHL2 knockdown and overexpression on cell proliferation were assessed using CCK-8 assays and colony formation assay.Co-immunoprecipitation(Co-IP)and liquid chromatography-tandem mass spectrometry(LC/MS)were employed to identify proteins interacting with FHL2 in glioma cells,and Co-IP and immunofluorescence were used to verify their binding and co-localization.Changes in intracellular lactate production and lactate dehydrogenase(LDH)activity following FHL2 knockdown and overexpression were measured using a microplate reader.WB was used to analyze the protein expression levels of FHL2,LDHA,and p-LDHA in normal brain tissues and glioma tissues,as well as their relationships.The small molecule inhibitor of LDHA,AT-101,was used to treat SNB19 cells overexpressing FHL2.The role of FHL2 in glioma lactate metabolism and the potential therapeutic effect of AT-101 in glioma were validated using CCK-8 assays and colorimetric assays with a microplate reader.Results:Co-IP and LC/MS analyses revealed an interaction between FHL2 and LDHA in glioma cells.Overexpression of FHL2 increased LDHA activity and lactate production(all P<0.001),thereby promoting glioma cell proliferation(P<0.001 or P<0.001).Conversely,knockdown of FHL2 reduced LDHA activity and lactate production(P<0.001,P<0.05)and inhibited cell growth(P<0.01).AT101 inhibited LDHA activity and significantly suppressed FHL2-induced glioma cell proliferation while restoring phosphorylated LDHA(Y10)levels(P<0.01,P<0.001).Conclusion:FHL2 interacts with LDHA protein,and FHL2 promotes LDHA activity and lactic acid production by activating the expression of p-LDHA(Y10),thus facilitating the proliferation of glioma cells.Targeting this interaction may become a potential strategy for treating gliomas.