Significance Of GCNT3 Expression And Identificantion Of Ascl2 Auto-regulation In Colorectal Cancer

Background and ObjectiveColorectal cancer(CRC)is a common fatal malignant tumor,and the dominating reason of death among CRC patients is unmanageable recurrence and metastasis.With the increasing trend,colorectal cancer is gradually becoming a financial burden on the government.Therefore,the identification of biomarkers may help to detecte those patients who have higher risk of recurrence in order to be achieved an early diagnosis and further therapy.Newly synthesized proteins are assembled within the endoplasmic reticulum and transported to the Golgi apparatus,where they are posttranslationally modified by glycosylation.Epithelial mucins classified into secretory and membrane-bound members possess common structural characteristics and at least some common functions,such as cell adhesion,motility,and development.Glycosylation of membrane-tethered mucin at the cell surface play a crucial role in carcinogenesis.It is recognized that the aberrant glycosylation occurs during the development and further progression of colorectal cancer.C2GnT-2,encoded by GCNT3 gene,catalyzes the adding of N-acetyl-D-galactosamine(GalNAc)residue to the Ser/Thr residues on the folded proteins to form Core 2 mucin-type O-glycan,Core 4 O-glycan and I branches.C2GnT-2 expression expressed in normal pancreas,trachea,kidney,stomach and intestine is altered in the tumour development and progression.The potential role of GCNT3 in CRC cells was previously reported and the enforced expression of GCNT3 in CRC cells suppressed cell adhesion,motility,and invasion as well as colony formation ability.The low GCNT3 expression is a promising prognostic biomarker for colon cancer that could be used to identify early-stage colon cancer patients at high risk of relapse by the quantitation of GCNT3 mRNA in 119 cases of CRC patients.In this study,we used a TCGA data in order to confirm the association between GCNT3 expression in CRC tissues and the prognosis of these 442 patients who were pathologically diagnosed.Ascl2,a basic helix-loop-helix transcription factor,is a downstream target of Wnt signaling pathway and plays an important role in intestinal stem cells and CRC progenitor cells.Ascl2 overexpressed in colorectal cancer affects the clinical behavior of the CRC cells.However,the molecular mechanism of Ascl2 overexpression in CRC cells remains unclear.In this study,we reported an Ascl2 auto-regulation in CRC cells,including Ascl2 binding to its own promoter and the auto-transcriptinal activation of Ascl2 gene.Methods1.Correlation between GCNT3 expression levels and prognosis of CRC patients,and its effect on the ability of proliferation.1.1 GCNT3 expression data and clinical information of the 442 patients were obtained from The Cancer Genome Atlas databases(TCGA)(https://tcga-data.nci.nih.gov/tcga).For survival analysis,the optimal cutoffs for the TCGA cohorts were calculated for the low and high groups with the the X-tile Software(Yale University,New Haven,CT,USA)using the Kaplan–Meier method.The Cox regression analysis is used for evaluating the prognostic significance of GCNT3 expression levels and determining the statistical significance of the curve comparisons.The relationship between GCNT3 expression level and patient clinical information was analyzed using chi-square test.1.2 Effect of GCNT3 on the proliferation ability of colorectal cancer cells was assessed by MTT assay.The shRNA-GCNT3/HT-29 and shRNA-Ctr/HT-29 cells were described previously and maintained in our lab.2.Identification of auto-regulation of Ascl22.1 Western blot and Real-time-PCR were used to confirm whether endogenous Ascl2 expression could be activated by exogenous Ascl2.2.2 Luciferase assays and ChIP assays were used to confirm whether Ascl2 bound to its own promoter and further transcriptional regulation by itself,namely auto-regulation activation.Results1.Correlation between GCNT3 expression levels and prognosis of CRC patients,and its effect on the ability of proliferation.We found that GCNT3 expression was significantly decreased in CRC tissues compared with adjacent normal colorectal tissues.Patients of high GCNT3 group had a higher disease-free survival rate than low GCNT3 group.The proliferation ability of shRNA-GCNT3/HT-29 cells from days 3 to 4 after seeding was significantly higher compared with the control cells.2.Identification of auto-regulation of Ascl2Endogenous Ascl2 expression can be directly activated by exogenous Ascl2.Ascl2 can bind to its own promoter and exerted Ascl2 transcription.Conclusions1.GCNT3,reduced in cancerous tissues,might be an independent disease-free survival indicator in CRC patients.2.Ascl2 transcription in colon cancer cells can be activated by Ascl2 itself,including Ascl2 binding with its own promoter and initiating its transcription.