Downregulation of N-Acetylglucosaminyltransferase GCNT3 by miR-302b-3p Decreases Non-Small Cell Lung Cancer (NSCLC) Cell Proliferation, Migration and Invasion
Downregulation of N-Acetylglucosaminyltransferase GCNT3 by miR-302b-3p Decreases Non-Small Cell Lung Cancer (NSCLC) Cell Proliferation, Migration and Invasion
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miR-302b-3p 下调 N-乙酰氨基葡萄糖转移酶 GCNT3 可减少非小细胞肺癌 (NSCLC) 细胞增殖、迁移和侵袭
DOI：
10.1159/000494482
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发表时间：
2018-01-01
影响因子：
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通讯作者：
Xiao, Chunjie
中科院分区：
文献类型：
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作者：
Li, Qian;Ran, Pengzhan;Xiao, Chunjie
背景/目的：GCNT 3是参与粘蛋白生物合成的N-乙酰葡糖胺转移酶家族的成员。已知GCNT 3异常表达促进几种人类癌症的进展。然而，其在肿瘤发生和非小细胞肺癌（NSCLC）进展中的作用尚未得到充分表征。我们的研究探讨了GCNT 3在NSCLC中由microRNAs（miRNAs）调控的功能机制。研究方法：采用mRNA芯片技术筛选云南宣威地区NSCLC组织及相应癌旁肺组织中mRNA的差异表达。在92例配对的肿瘤组织和癌旁正常组织中检测GCNT 3的表达及其与NSCLC进展的相关性。使用siRNA和GCNT 3表达载体测量GCNT 3在NSCLC细胞中的功能及其潜在机制。miRNA免疫沉淀（miRIP）方法用于鉴定靶向GCNT 3的miRNA。蛋白质印迹法检测蛋白质表达，实时荧光定量PCR（qRT-PCR）检测mRNA表达。采用细胞计数试剂盒-8（CCK-8）和集落形成试验检测细胞增殖;采用24孔Transwell小室和8 μm微孔滤膜进行细胞迁移和侵袭试验，并通过流式细胞术分析细胞周期和凋亡。进行双荧光素酶报告基因测定以确认GCNT 3基因是否为miR-302 b-3 p的直接靶点。结果如下：发现GCNT 3在NSCLC组织和细胞系中均高表达，并且高表达与晚期肿瘤淋巴结转移（TNM）阶段、阳性淋巴结转移和较差的总生存率显著相关。GCNT 3的敲低抑制了NSCLC细胞的增殖、迁移和侵袭能力，而过表达促进了这些活动。使用miRIP和双荧光素酶报告基因测定的进一步机制实验显示，GCNT 3是miR-302 b-3 p的直接靶标。miR-302 b-3 p在NSCLC细胞中的低表达与GCNT 3水平呈负相关，而miR-302 b-3 p过表达则抑制NSCLC细胞的增殖、迁移和侵袭。与miR-302 b-3 p和GCNT 3表达载体共转染消除了mir-302 b-3 p的作用，证实miR-302 b-3 p通过靶向GCNT 3抑制NSCLC进展。Western blotting结果显示E-cadherin、N-cadherin、vimentin、p-Erk和cyclin D1是miR-302 b-3 p/GCNT 3通路的下游分子。结论：miR-302 b-3 p/GCNT 3轴通过激活Erk信号通路和上皮-间质转化（EMT）来调节细胞增殖、迁移和侵袭，其被确定为NSCLC的潜在治疗靶点。
Background/Aims: GCNT3 is a member of N-acetylglucosaminyltransferase family involved with mucin biosynthesis. GCNT3 aberrant expression is known to promote the progression of several human cancers. However, its role in tumorigenesis and the progression of non-small cell lung cancer (NSCLC) has not been well-characterized. Our study investigated the functional mechanisms of GCNT3 regulated by microRNAs (miRNAs) in NSCLC. Methods: The differential expression of mRNAs in NSCLC tissues and matched adjacent non-cancerous lung tissues from patients in Xuanwei, Yunnan province, China, was screened via mRNA microarray. The expression of GCNT3 and its correlation with NSCLC progression was measured in 92 paired tumor tissues and adjacent normal tissues. The functions of GCNT3 in NSCLC cells and its underlying mechanisms were measured using siRNA and GCNT3-expression vectors. The miRNA immunoprecipitation (miRIP) method was used to identify the miRNAs targeting GCNT3. The protein were measured using western blot assay, and the mRNAs were measured by quantitative real-time PCR (qRT-PCR) assay. Cell proliferation was measured using Cell Counting Kit-8 (CCK-8) and a colony forming assays; cell migration and invasion assays were performed using 24-well Transwell chambers with 8-μm pores filter, and analyses of the cell cycle and apoptosis were performed via flow cytometric analysis. The dual luciferase reporter assay was performed to confirm whether GCNT3 gene was a direct target of miR-302b-3p. Results: GCNT3 was found to be highly expressed in both NSCLC tissues and cell lines, and higher expression correlated significantly with advanced tumor-node-metastasis (TNM) stage, positive lymph node metastasis, and poor overall survival. Knockdown of GCNT3 inhibited the proliferation, migration and invasion ability of NSCLC cells, while overexpression facilitated these activities. Further mechanistic experiments using miRIP and dual luciferase reporter assays revealed that GCNT3 was a direct target of miR-302b-3p. Low expression of miR-302b-3p was found in NSCLC cells and negatively correlated with GCNT3 levels, while miR-302b-3p overexpression inhibited the proliferation, migration and invasion of NSCLC cells. Co-transfection with miR-302b-3p and the expression vector of GCNT3 abrogated the effects of mir-302b-3p, confirming that miR-302b-3p inhibited NSCLC progression by targeting GCNT3. Western blotting revealed that E-cadherin, N-cadherin, vimentin, p-Erk and cyclin D1 were downstream molecules of miR-302b-3p/GCNT3 pathway. Conclusion: miR-302b-3p/GCNT3 axis regulated cell proliferation, migration, and invasion by activating the Erk signaling pathway and epithelial-mesenchymal transition (EMT), which was identified as a potential therapeutic target for NSCLC.