SETD5介导AKT1磷酸化调控结肠癌细胞的迁移和5-FU敏感性

SETD5 regulates colon cancer cell migration and 5-FU sensitivity by mediating AKT1 phosphorylation

目的:探讨含SET结构域蛋白5(SETD5)对结肠癌细胞增殖、迁移和对5-氟尿嘧啶(5-FU)药物敏感性的影响及机制。方法:常规培养结肠癌细胞,用Lipofectamine 2000将siSETD5-NC、si-SETD5-1~3质粒转染至HT-29细胞中,将其分为对照组(未处理)、si-SETD5-NC组、si-SETD5组和si-SETD5+SC79组,si-SETD5+SC79组HT-29细胞转染质粒的同时用10µmol/L SC79处理。qPCR法检测NCM460、HT-29和LoVo细胞中SETD5 mRNA表达,流式细胞术、细胞划痕法、WB法和CCK-8法分别检测各组HT-29细胞的凋亡情况、迁移能力、相关蛋白的表达,以及对5-FU的敏感性。结果:SETD5 mRNA在HT-29、LoVo细胞中均呈高表达(均P<0.01)。在HT-29细胞中成功地敲减了SETD5 mRNA(P<0.01)。敲减SETD5 mRNA可明显抑制HT-29细胞的增殖活性(P<0.01)、迁移能力(P<0.01)、相关蛋白(SETD5、p-PI3K、p-AKT1、p-mTOR蛋白)的表达(均P<0.01)、促进细胞凋亡(P<0.01),且提高其对5-FU的敏感性(P<0.01),这些作用均可被AKT激活剂SC79部分阻挡(P<0.05或P<0.01)。结论:SETD5在HT-29、LoVo细胞中高表达,SETD5通过PI3K/AKT1通路促进结肠癌HT-29细胞的增殖、迁移,且降低其对5-FU的敏感性,SETD5是结肠癌临床诊断、治疗的潜在靶点。

Objective:To investigate the effect of SET domain-containing 5(SETD5)on the proliferation,migration and 5-fluorouracil(5-FU)drug sensitivity of colon cancer cells and its mechanism.Methods:Colon cancer cells were cultured routinely,and siSETD5-NC and si-SETD5-1-3 plasmids were transfected into HT-29 cells with Lipofectamine 2000.The cells were divided into the control group(untreated),the si-SETD5-NC group,the si-SETD5 group and the si-SETD5+SC79 group.The HT-29 cells in the si-SETD5+SC79 group were transfected with plasmids and treated simultaneously with 10µmol/L SC79.SETD5 mRNA expression in NCM460,HT-29 and LoVo cells was detected by qPCR.Flow cytometry,cell scratch method,WB method and CCK-8 method were used to detect the apoptosis,migration ability,expression of related proteins and sensitivity to 5-FU of HT-29 cells in each group,respectively.Results:SETD5 mRNA was highly expressed in both HT-29 and LoVo cells(both P<0.01).SETD5 mRNA was successfully knocked down in HT-29 cells(P<0.01).Knockdown of SETD5 mRNA could significantly inhibit the proliferation activity(P<0.01),migration ability(P<0.01),expression of related proteins(SETD5,p-PI3K,p-AKT1,p-mTOR protein)of HT-29 cells(all P<0.01),promote apoptosis(P<0.01),and increase the sensitivity to 5-FU(P<0.01).These effects could be partially blocked by AKT activator SC79(P<0.05 or P<0.01).Conclusion:SETD5 is highly expressed in HT-29 and LoVo cells.SETD5 promotes the proliferation and migration of colon cancer HT-29 cells and reduces sensitivity to 5-FU through PI3K/AKT1 pathway.SETD5 is a potential target for clinical diagnosis and treatment of colon cancer.