Supplementary Material for: Downregulation of N-Acetylglucosaminyltransferase GCNT3 by miR-302b-3p Decreases Non-Small Cell Lung Cancer (NSCLC) Cell Proliferation, Migration and Invasion
datasetposted on 24.10.2018, 09:30 by Li Q., Ran P., Zhang X., Guo X., Yuan Y., Dong T., Zhu B., Zheng S., Xiao C.
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.