摘要
β-1,3-半乳糖-O-糖基-糖蛋白β-1,6-N-乙酰氨基葡萄糖转移酶(β-1,3-galactosyl-O-glycosyl-glycoproteinβ-1,6-N-acetylglucosaminyltransferase,GCNT3)是黏液蛋白质生物合成中不可缺少的一类N-乙酰葡萄糖转移酶。越来越多的证据表明,GCNT3的异常表达与肿瘤的侵袭以及病人的生存率有关,然而相关的研究仍较少报道。本研究旨在揭示GCNT3在调控肝细胞癌进程中的潜在机制。本研究首先利用高通量基因表达数据库(Gene Expression Omnibus,GEO)和癌症基因组图谱(Cancer Genome Atlas databases,TCGA)数据库分析肝癌和正常组织中GCNT3的mRNA表达水平,结果表明,肝癌组织中GCNT3的mRNA表达水平高于正常组织(P≤0.001)。同时利用免疫组化、Western印迹进一步分析了肝癌组织、癌旁组织、正常组织中GCNT3蛋白的表达,发现有30%肝癌组织GCNT3表达水平高于癌旁组织和正常组织。随后,在肝癌细胞系HCCLM3和Huh7细胞中,采用CCK-8、平板克隆、划痕实验、Transwell实验分析GCNT3对肝癌细胞增殖、迁移和侵袭能力的影响,结果显示,敲低GCNT3可抑制肝癌细胞的增殖、迁移和侵袭,而过表达GCNT3发挥相反作用。细胞周期和Western印迹结果显示,GCNT3调控G_(0/)G_1期关键蛋白质的表达来促进肝癌细胞周期G_1/S的转换。进一步探究发现,GCNT3可能通过激活PI3K/AKT/mTOR信号通路促进细胞增殖;以及GCNT3上调RhoA/ROCK/Cofilin通路关键蛋白质的表达来促进F-肌动蛋白(F-actin)的形成,从而参与细胞的迁移和侵袭。总之,本研究通过对GCNT3在肝癌细胞中的功能分析,初步证明,GCNT3与肝癌细胞增殖、迁移和侵袭中的功能有关,证实了GCNT3在肝癌临床治疗中的潜在价值,为肝癌治疗和诊断提供了新思路。
β-1,3-galactosyl-O-glycosyl-glycoproteinβ-1,6-N-acetylglucosaminyltransferase(GCNT3)is a type of N-acetylglucosamine transferase that is essential in mucus biosynthesis.Increasing evidence has shown that the aberrant expression of GCNT3 is related to the aggressiveness of certain malignant tumors and a reduction in survival rate.The present work intended to reveal the role of GCNT3 in hepatocellular carcinoma.First,data showed increased GCNT3 expression levels in hepatocellular carcinoma samples as compared with normal tissues through the Gene Expression Omnibus(GEO)and Cancer Genome Atlas databases(TCGA).GCNT3 expression in 10 pairs of hepatocellular carcinoma tissue samples was detected via immunohistochemistry,and Western blot(WB)assays,which indicated that 30%of hepatocellular carcinoma tissues expressed significantly higher levels of GCNT3 than matched paracancerous and normal tissues.Whereafter,Cell Counting Kit-8,colony formation,wound healing and transwell assays were applied to detect the effects of GCNT3 on cell proliferation,migration and invasion.The results revealed that knockdown of GCNT3 inhibited the proliferation,migration and invasion of HCCLM3 cells,and overexpressing GCNT3 induced the proliferative and aggressive ability of Huh7 cells.Studies also revealed that GCNT3 accelerated G 1/S phase progression within HCC cells by regulating critical cell cycle-related protein via cell cycle and WB assays.Furthermore,the upregulation of GCNT3 in HCC cells activate the PI3K/Akt/mTOR pathway via WB assays,leading to the promotion of cell proliferation.Meanwhile,GCNT3 may activate the RhoA/ROCK/Cofilin pathway to promote the formation of F-actin in HCC cells and participate in tumor metastasis,as shown by immunofluorescence and WB tests.In conclusion,in vitro data proved that GCNT3 induced the proliferation,migration and invasion of HCC cells through both the PI3K/Akt/mTOR and RhoA/ROCK/Cofilin pathways,which may be a potential candidate target for the treatment of HCC.
作者
刘丽
王哲近
潘邦伦
刘林
王刚林
李伟
金晶
LIU Li;WANG Zhe-Jin;PAN Bang-Lun;LIU Lin;WANG Gang-Lin;LI Wei;JIN Jing(Key Laboratory of Laboratory Medicine,Ministry of Education of China,School of Laboratory Medicine and Life Sciences,Wenzhou Medical University,Wenzhou 325035,Zhejiang,China;Department of Hepatobiliary Surgery,Wenzhou Central Hospital,The Second Affiliated Hospital of Shanghai University,Wenzhou 325000,Zhejiang,China;Zhejiang Provincial Key Laboratory of Medical Genetics,Wenzhou Medical University,Wenzhou 325035,Zhejiang,China)
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2023年第4期562-572,共11页
Chinese Journal of Biochemistry and Molecular Biology
基金
Supported by National Natural Science Foundation of China (No.31300676)。
