m6Am修饰酶PCIF1调控靶基因ACOT8参与胃癌进展的机制研究

The mechanism of m6Am-modifying enzyme PCIF1 regulating target gene ACOT8 in gastric cancer progression

背景与目的:最新证据显示N6, 2’-O-二甲基腺苷(N6, 2’-O-dimethyladenosine,m6Am)修饰酶磷酸化C末端结构域相互作用因子1(phosphorylated c-terminal domain-interacting factor 1,PCIF1)可能是胃癌的重要生物标志物及治疗靶点。然而,这种新的PCIF1分子机制与胃癌进展的关系仍有待进一步探索。本研究分析PCIF1对胃癌细胞增殖、迁移和侵袭的调控作用及其调控靶基因酰基辅酶A硫代酯酶8(acyl-CoA thioesterase 8,ACOT8)在胃癌进展中的机制。方法:使用基因表达谱交互分析(gene expression profiling interactive analysis,GEPIA)分析胃癌患者的胃癌组织和非胃癌组织中的PCIF1表达,并分析了PCIF1表达与胃癌患者总生存率的相关性。收集2019年——2021年长沙市第一医院消化内科就诊患者的89对胃癌组织和匹配的癌旁组织。通过实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)和蛋白质印迹法(Western blot)分析PCIF1表达。在体外实验中,将SNU5细胞分为PCIF1敲低(shPCIF1)组和相应对照(sh-NC)组,将AGS细胞分为载体对照组(normal control,NC)组和PCIF1过表达(PCIF1)组。使用细胞计数试剂盒-8(cell counting kit-8,CCK-8)、5-乙炔基-2’-脱氧尿苷(5-ethynyl-2’-deoxyuridine,EdU)和transwell法分析了PCIF1对胃癌细胞增殖、侵袭和迁移的影响。此外,在PCIF1过表达的AGS细胞中敲低ACOT8并进行了拯救实验。采用皮下异种移植瘤模型来测定PCIF1在胃癌中的生物学效应。结果:PCIF1在胃癌组织和细胞系中呈高表达,并且PCIF1高表达胃癌患者的预后较差。与sh-NC组相比,sh-PCIF1组的细胞活力、EdU阳性细胞、迁移和侵袭细胞数均显著降低(P<0.05)。与NC组相比,PCIF1组的细胞活力、EdU阳性细胞、迁移和侵袭细胞数均显著增加(P<0.05)。在PCIF1过表达的AGS细胞中,敲低ACOT8的表达可降低细胞活力、EdU阳性细胞、迁移和侵袭细胞数。在体内实验中,与NC组相比,PCIF1过表达组裸鼠的移植瘤体积和重量均显著增加(P<0.05)。结论:PCIF1在胃癌细胞系和组织中上调。此外,PCIF1/ACOT8轴参与介导胃癌细胞的恶性行为产生。

Background and purpose:Recent evidence suggests that N6,2'-O-dimethyladenosine(m6Am)modifying enzyme phosphorylated C-terminal domain-interacting factor 1(PCIF1)may be an important biomarker/therapeutic target in gastric cancer.However,the relationship between this novel PCIF1 molecular mechanism and gastric cancer progression remains to be further explored.In this study,we analyzed the regulatory role of PCIF1 on gastric cancer proliferation,migration and invasion and the mechanism of its regulatory target gene ACOT8 in gastric cancer progression.Methods:PCIF1 expression in gastric cancer and non-gastric cancer tissues of gastric cancer patients was analyzed using Gene Expression Profile Interaction Analysis(GEPIA),and the correlation of PCIF1 expression with overall survival in gastric cancer patients was analyzed.We collected all gastric cancer tissues and matched para-cancerous tissues from 89 primary gastric cancer patients treated in Department of Gastroenterology of Changsha First Hospital from 2019 to 2021.PCIF1 expression was analyzed by real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR)and Western blot.In in vitro experiments,SNU5 cells were divided into PCIF1 knockdown(sh-PCIF1)group and corresponding control(sh-NC)group,and AGS cells were divided into vehicle as normal control(NC)group and PCIF1 overexpression(PCIF1)group.The effect of PCIF1 on gastric cancer cell proliferation,invasion and migration was investigated using cell counting kit-8(CCK-8),EdU and transwell assays.Furthermore,ACOT8 expression was knocked down in PCIF1-overexpressing AGS cells,and rescue experiments were performed.A subcutaneous xenograft model was used to determine the biological effects of PCIF1 in gastric cancer in vivo.Results:PCIF1 expression was abnormally high in gastric cancer tissues and cell lines,with significant correlation between PCIF1 expression and the prognosis of gastric cancer patients.Compared with the sh-NC group,the cell viability,EdU positive cells,migration and invasion cell numbers in the sh-PCIF1 group were significantly decreased(P<0.05).Compared with the NC group,the cell viability,EdU positive cells,migration and invasion cell numbers in the PCIF1 group were significantly increased(P<0.05).In PCIF1-overexpressing AGS cells,knockdown of ACOT8 expression decreased cell viability,EdU-positive cells and migrating and invasive cell numbers.In vivo experiments showed,compared with the NC group,the tumor volume and weight of the nude mice in the PCIF1-overexpressing group were significantly increased(P<0.05).Conclusions:PCIF1 is upregulated in gastric cancer cell lines and tissues.In addition,the PCIF1/ACOT8 axis is involved in mediating the malignant behavior of gastric cancer cells.