BHLHE41 Promotes The U87 And U251 Cell Proliferation Via ERK/cyclinD1 Signaling Pathway

Objective: Basic – helix-loop-helix transcriptional factor E41(BHLHE41),acts as a transcription factor involved in the regulation of a variety of biological functions,including circadian rhythm,development and differentiation,proliferation,apoptosis,inflammation and hypoxia response.Although the abnormal expression of BHLHE41 is associated with central nervous system diseases,such as major depression,its role in central nervous system tumors has rarely been reported.Methods: First,we induced the exogenous expression of BHLHE41 or silenced the BHLHE41 by si RNA.The CCK8 assay,colony formation assay,and flow cytometry were used to analyze the biological functions of BHLHE41 in U87 and U251 cells.Next,we used western blot to explore the expression of cyclins and signaling pathway-related proteins(PI3K/AKT,NF-? B,MAPK/ERK)in glioma cells after transfected and knockdown with BHLHE41.To further explore the specific mechanisms,we both examined the expression levels of P-ERK and cyclin D1 by Western blotting before and after treated with ERK signaling pathway inhibitor(SCH772984).Apoptosis was quantified at 20 h by Annexin V-FITC/PI double staining assay DMSO or cisplatin treatment.Meanwhile,WB analysis was performed to detect the variation of apoptosisrelated proteins.Then TCGA database(http://cancergenome.nig.gov)was used to analyze the associations between BHLHE41 expression with clinicopathological factors and the overall survival(OS)of glioma patients.Results: This study sought to elucidate the role of BHLHE41 in regulating the proliferation of glioblastoma cells.In the current study,forced BHLHE41 expression promoted tumor cell proliferation,while BHLHE41 depletion suppressed it in U87 and U251 cells.Functional study demonstrated that BHLHE41 upregulated the protein expression of cyclins(cyclin D1,cyclin D3,cyclin E1)and increased the proportion of cells in G2 and S phase compared to that in G1 phase.In other words,BHLHE41 has been shown to drive phase transition from G1 to S and G2 phase by upregulating these cyclins.The results of CCK8 and Colony Assay indicated that BHLHE41 had the ability to promote cell proliferation and colony formation.Furthermore,BHLHE41 promoted the phosphorylation of ERK and upregulated cyclin D1 expression.After blocking ERK signal pathway by specific inhibitor(SCH772984),the upregulation of cyclin D1 by BHLHE41 was reverse.Conclusion: Our finding indicated BHLHE41 upregulated cyclin D1 expression by activating MAPK/ERK signaling pathway,and therefore facilitated cell proliferation of U87 and U251 cells.Based on these studies,BHLHE41 may be a potential target for molecular therapy and drug design in glioblastoma.