乙酰半乳糖氨基转移酶7对前列腺癌细胞迁移、侵袭和增殖的作用及机制

Effect of N-acetyl-galactosaminotransferase 7 on proliferation and migration of prostate cancer cells and mechanism

目的探讨N-乙酰半乳糖氨基转移酶7(GALNT7)对前列腺癌细胞迁移、侵袭和增殖的作用及分子机制。方法选取2019年1月至2022年1月在天津市第五中心医院和延边大学附属医院泌尿外科行前列腺手术的30例前列腺癌患者的癌组织为研究对象,采用免疫组织化学染色、蛋白质免疫印迹(Western blot)和实时荧光定量聚合酶链反应(qRT-PCR)检测前列腺癌组织GALNT7的表达水平。通过细胞计数试剂盒(CCK-8)法、5-溴脱氧尿嘧啶核苷(BrdU)掺入实验、划痕实验检测细胞迁移、侵袭和增殖能力;采用VVA凝集素pull-down实验和Western blot检测GALNT7对表皮生长因子受体(EGFR)磷酸化、O-GlcNAc糖基化修饰的影响,及EGFR/磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号通路的表达水平。组间差异采用配对t检验或方差分析(ANOVA)。结果前列腺癌组织中GALNT7蛋白和RNA表达水平(7.86±1.64、9.49±2.34)明显高于癌旁组织(0.98±0.25、0.99±0.33),差异有统计学意义(t=13.080、11.390,P<0.05)。CCK-8法检测结果显示Ad-GALNT7组细胞增殖能力显著高于Ad-GFP组(1.14±0.35比0.76±0.19,t=8.287,P<0.05)。Ad-GALNT7组BrdU阳性比例明显高于Ad-GFP组(42.36±5.19比14.68±3.12,t=6.091,P<0.05)。划痕实验证明Ad-GALNT7组前列腺癌细胞的迁移侵袭能力明显高于Ad-GFP组(84.80±3.96比58.34±2.19,t=11.860,P<0.05)。Western blot结果显示,si-GALNT7组EGFR磷酸化显著低于si-GFP组(1.03±0.26比2.68±0.57,t=3.693,P<0.05)。VVA凝集素pull-down实验,发现si-GALNT7组EGFR O-GlcNAc糖基化修饰水平显著低于si-GFP组(0.55±0.13比1.00±0.27,t=4.570,P<0.05)。结论GALNT7在前列腺癌中表达显著增加,并通过O-GlcNAc糖基化修饰EGFR,激活EGFR/PI3K/Akt信号通路,促进前列腺癌细胞的迁移、侵袭和增殖。

Objective To investigate the effect of N-acetyl-galactosaminotransferase 7(GALNT7)on the proliferation and migration of prostate cancer cells and its molecular mechanism.Methods The cancer tissues of 30 patients with prostate cancer who underwent prostate surgery in the Department of Urology of Tianjin Fifth Central Hospital and the Affiliated Hospital of Yanbian University from January 2019 to January 2022 were selected as the research objects.Immunohistochemical staining,Western blotting and quantitative real-time polymerase chain reaction(qRT-PCR)were used to detect the expression level of GALNT7 in prostate cancer tissues.Cell counting kit-8(CCK-8)assay,BrdU incorporation assay and wound healing assay were used to detect cell migration,invasion and proliferation.VVA lectin pull-down assay and Western blotting were used to detect the effect of GALNT7 on the phosphorylation and O-GlcNAc modification of epidermal growth factor receptor(EGFR),and the expression level of EGFR/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway.The differences between groups were analyzed by paired t-test or analysis of variance(ANOVA).Results The expression levels of GALNT7 protein and RNA in prostate cancer tissues(7.86±1.64,9.49±2.34)were significantly higher than those in adjacent tissues(0.98±0.25,0.99±0.33,t=13.080,11.390,P<0.05).CCK-8 assay showed that the proliferation ability of Ad-GALNT7 group was significantly higher than that of Ad-GFP group(1.14±0.35 vs.0.76±0.19,t=8.287,P<0.05).The BrdU positive rate in Ad-GALNT7 group was significantly higher than that in Ad-GFP group(42.36±5.19 vs.14.68±3.12,t=6.091,P<0.05).Scratch test showed that the migration and invasion ability of prostate cancer cells in the Ad-GALNT7 group was significantly higher than that in the Ad-GFP group(84.80±3.96 vs.58.34±2.19,t=11.860,P<0.05).Western blotting results showed that the phosphorylation of EGFR in the si-GALNT7 group was significantly lower than that in the si-GFP group(1.03±0.26 vs.2.68±0.57,t=3.693,P<0.05).VVA lectin pull-down assay showed that the level of EGFR O-GlcNAc glycosylation in si-GALNT7 group was significantly lower than that in si-GFP group(0.55±0.13 vs.1.00±0.27,t=4.570,P<0.05).Conclusion The expression of GALNT7 is significantly increased in prostate cancer,and it can modify EGFR through O-GlcNAc glycosylation,activate EGFR/PI3K/Akt signaling pathway,and promote the migration,invasion and proliferation of prostate cancer cells.