Effect Of NeuroD1 On The Expression Of Cell Cycle Regulation Factor P21 In Colon Cancer Cells

Colorectal cancer is the third most common cancer and is the second cancer that causes mortality.There are about 1.2 million new cases of colorectal cancer,and about700,000 mortality caused by colorectal cancer each year.The occurrence of colorectal cancer is due to the accumulation of genetic mutations and epigenetic instability in colonic mucosal cells,which ultimately leads to cell proliferation and metastasis.Therefore,elucidating the molecular mechanism underlying the formation of colorectal cancer will definitely help us to develop strategies for tumor prevention and therapy.p21 is a cell cycle-dependent kinase inhibitor 1A（CDKN1A）,which mediates p53-dependent cell cycle arrest.p21 protein binds to and inhibits the activities of cyclin dependent kinases（CDKs）/cyclins complexes,thereby negatively regulates cell cycle progression and subsequently inhibits tumor cell proliferation.p21 is a major cell cycle checkpoint inhibitor,and downregulation of p21 results in the growth of many tumors such as breast cancer,nasopharyngeal carcinoma,colorectal cancer and liver cancer.However,unlike p53,whose mutations could be found in about 50%of tumor patients,p21 is rarely mutated in human tumors.NeuroD1（neuronal differentiation 1）is a basic helix-loop-helix（bHLH）type transcription factor that mediates transcriptional activation by binding to the E-box in the promoter of its target genes.NeuroD1 has been known to play critical roles in neurogenesis.Previous study has shown that in Xenopus embryo,ectopic expression of NeuroD1 could lead to the coversion of epithelial cells into neuron.In term of tumorigenesis,some studies have found that NeuroD1 is involved in the tumorigenesis of neuroblastoma.NeuroD1 is also involved in regulating the formation of tumor sphere from neuroblastoma originated from MYCN transgenic mice.However,the roles of NeuroD1 in colorectal cancer,as well as the molecular mechanism underlying them,have not been fully elucidated.In this study,we found that NeuroD1 is highly expressed in colon cancer patients,and knockdown of NeuroD1 inhibits the proliferation of colon cancer cells.In colon cancer cells with wild-type p53 gene,reporter assay showed that the activity of p21luciferase reporter significantly increased when NeuroD1 was knocked down.Furthermore,quantitative RT-PCR and western blotting results showed that knocking down NeuroD1 resulted in significant increase of p21 mRNA and protein expression levels.Concomitantly,overexpression of NeuroD1 grossly suppressed the expression levels of p21 mRNA and protein.Subsequently,we examined the regulatory effect of NeuroD1 on tumor cells proliferation,and found that knocking down NeuroD1 robustly suppressed the proliferation and colony formation potentials of tumor cells.We also performed cell cycle analysis using propidium iodide staining followed by flow cyctometry.The results showed that while NeuroD1 knockdown did not affect the apoptotic rate,it induced G₂/M cell cycle arrest and suppressed the expression levels of Cyclin-B and Cdc2.These results indicate that NeuroD1 could negatively regulate p21expression,and thus regulates cell cycle progression and cell proliferation.p53 is an critical tumor suppressor gene whose expression could be induced by DNA damage.In this study,our results showed that knocking down NeuroD1 induced the expression levels of p53 mRNA and protein significantly.By using dual luciferase reporter assay,we found that knocking down NeuroD1 robustly increased the activity of p53 reporter gene,indicating that NeuroD1 might regulate the expression of p53 and thereby induced the expression level of p21.We then performed chromatin immunoprecipitation（ChIP）assay to confirm the direct binding of NeuroD1 on the promoter region of p53.Furthermore,by predicting NeuroD1 binding region on p53promoter and mutating the predicted binding site,we determined the NeuroD1 binding site on p53 promoter,CAGATT.Taken together,our study describes for the first time that NeuroD1 suppresses the expression of p21 by directly regulating the transcriptional activity of p53,thereby enhances cell cycle progression and subsequently,tumor cell proliferation and colony formation potential.This study not only provides new perspective regarding the novel function of NeuroD1,but also provides a basis for further study for elucidating the roles of NeuroD1 in the progression of colorectal cancer.