| Background:Lung cancer（LC）is the most commonly diagnosed malignancy with the highest mortality worldwide,threatening human health.Its incidence rate is constantly increasing in china.In Yunnan province,especially in Xuanwei municipality,the incidence of NSCLC is ranked the first place in our country.Up to now,the development and progression of LC are identified to correlate with expression and regulation mechanism of oncogenes.O-glycan synthesis enzyme core 2 beta 1,6 N-acetylglucosaminyltransferase（GCNT3/C2Gn T-2）,as a kind of enzyme catalyzing the formation of mucin,has been reported to be correlated with tumorigenesis and tumor progression in hepatocellular carcinoma,pancreatic cancer,colorectal cancer and pancreatic cancer,and it plays a role in cell proliferation,migration and invasion.However,little is known about the role of GCNT3 in NSCLC.Thus,m RNA microarray between 15 paired NSCLC tumor tissues and adjacent normal tissues from the patients in Yunnan Xuanwei was conducted in our previous study to find that GCNT3 could act as a cancer-promoting gene in NSCLC.The biological function and clinical significance of GCNT3 in NSCLC has not yet been established.Therefore,we aimed to investigated the role of GCNT3 in the tumorigenesis and progression of NSCLC,which could be a potential biomarker for NSCLC diagnosis and therapy.Methods:1.Through m RNA microarray between 15 paired NSCLC tumor tissues and adjacent normal tissues from the patients in Yunnan Xuanwei,GCNT3 was found to express higher in NSCLC tissues than adjacent normal tissues;We determined the m RNA and protein levels of GCNT3 in paired NSCLC tissues and normal lung tissues by q RT-PCR and immunohistochemistry（IHC）staining,and analyzed the relationship between GCNT3 expression and clinicopathological parameters of patients with NSCLC;2.We investigate the biological effects of GCNT3 knockdown or overexpression on NSCLC cells（cell proliferation,migration,invasion,cell cycle and apoptosis）;3.GCNT3 was identified to be a target of mi R-302b-3p evidenced by mi RIP assay and micro RNA microarray;Then we investigate the biological effects of mi R-302b-3p mimic transfection on NSCLC cells（cell proliferation,migration,invasion）;The rescue experiment was conducted to identify whether GCNT3 overexpression could reverse the phenotype of mi R-302b-3p overexpression on the condition of mi R-302b-3p and GCNT3co-overexpression;The expression of GCNT3,EMT proteins and Erk proteins were also detected by western blot.Results:1.m RNA microarray(P=8.4×10-7,fold change=4.382),q RT-PCR（P<0.001）,immunohistochemistry staining（IHC,P<0.001）and data analysis from the Cancer Genome Atlas（TCGA,P<0.001）were conducted to demonstrate that GCNT3 expression was significantly up-regulated in NSCLC tissue;Up-regulation of GCNT3 was related to TNM stage（P=0.0078）,lymph node metastasis（P=0.0016）and patients’overall survival（P=0.0033）.Nevertheless,there was no correlation between GCNT3 expression level and the age,gender and tumor size（all P>0.05）;2.The result of q RT-PCR（P<0.001）and Western blot（P<0.001）showed that the expression level of GCNT3 in NCI-H292 and XWLC were significantly upregulated;Knockdown of GCNT3 in XWLC cell lines（XWLC and NCI-H292）could significantly inhibit cell proliferation(PNCI-H292<0.01,PXWLC<0.001),migration（PNCI-H292<0.001,PXWLC<0.01）and invasion(PNCI-H292<0.01,proliferation（PNCI-H292<0.05,PXWLC<0.01）,migration（PNCI-H292<0.05,PXWLC<0.01）and invasion(PNCI-H292<0.05,PXWLC<0.05)ability;3.Mi RIP method and Multiplex mi RNA array was conducted to find out that the mi R-302b-3p would target GCNT3,and mi R-302b-3p was interacted with GCNT3 m RNA most robustly;The result of q RT-PCR showed that the expression level of mi R-302b-3p in NCI-H292 and XWLC were significantly downregulated（P<0.001）;Mi R-302b-3p overexpression significantly suppressed both NCI-H292 and XWLC cell proliferation(PNCI-H292<0.001,PXWLC<0.05),migration(PNCI-H292<0.001,PXWLC<0.01)and invasion(PNCI-H292<0.01,PXWLC<0.01),and the protein levels of N-cadherin and Vimentin were downregulated,and the protein level of E-caderherin was markedly increased;Mi R-302b-3p and GCNT3 co-overexpression reversed the mi R-302b-3p overexpression induced NCI-H292 and XWLC cell proliferation（PNCI-H292<0.05,PXWLC<0.05）,migration（PNCI-H292<0.01,PXWLC<0.01）and invasion(PNCI-H292<0.001,PXWLC<0.01)abilities decline,and abrogated the effects of mi R-302b-3p on the levels of these proteins;Overexpression of mi R-302b-3p downregulated the protein level of p-Erk,the co-transfection with mi R-302b-3p and GCNT3 abrogated the effects of mi R-302b-3p on the levels of p-Erk.Conclusion:GCNT3 m RNA expression was significantly upregulated in NSCLC Tissues and NSCLC cell lines（NCI-H292 and XWLC）,and the expression of GCNT3 was correlated with TNM stage,lymph node metastasis and patients’overall survival;GCNT3,as a cancer-promoting gene in NSCLC,negatively controlled by mi R-302b-3p,regulated NSCLC proliferation,migration and invasion through facilitating activation of ERK signaling pathway. |
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