Interaction With SP1 Is Responsible For BHLHE40-mediated Transcriptional Suppression Of The CLDN1 And Facilitated Cell Invasion In MCF-7 Cells

Objective: Basic helix-loop-helix family member e40 is located in 3p26.1,acts as transcriptional repressor involved in the regulation of the circadian rhythm by negatively regulating the activity of the clock genes and clock-controlled genes.Recent research demonstrated that BHLHE40 may involve in regulating the progression of tumor cells.However,the mechanism by which BHLHE40 regulates the invasion and metastasis of tumor cells,is still unclear.In recent years,the incidence of breast cancer in our country is increasing,therefore,to further explore the mechanism by which BHLHE40 regulates the invasion and metastasis of tumor cells is of great significance and may provide a new target for the treatment of breast cancer.Methods: In this study,breast cancer cell MCF-7 was selected as the research object,we induced the exogenous expression of BHLHE40 or silenced the BHLHE40 by si RNA,then explored the expression level of EMT related molecules by Western blotting and Real-time PCR,and explored the invasion and migration of tumor cells by Matrigel invasion assay.The molecular mechanism of suppressing the transcription of CLDN1 was further explored by immunofluorescence,Co-immunoprecipitation,Luciferase reporter assay and Ch IP assay.Results: 1.In our vitro assays show that BHLHE40 expression promoted tumor cell invasion and migration in MCF-7 cells.While silence of BHLHE40 by si RNA suppressed the cell invasion and migration in MCF-7 cells.The m RNA and protein levels of CLDN1,CLDN4,CDH1 were downregulated,while those of SNAI1,SNAI2 and VIM were upregulated by BHLHE40 overexpression.We got a consistent result by BHLHE40 si RNA.2.BHLHE40 suppressed CLDN1 transcription not through directly binding with the E-box motif of the CLDN1 promoter regions.Further studies indicated that BHLHE40 inhibits the transcription of CLDN1 by a physical interaction between BHLHE40 and SP1 which attenuated the latter's transcriptional activation.3.By constructing the sliced variants(BHLHE40?BHLH and BHLHE40?Orange),we found that the BHLH and Orange regions are essential for the binding of BHLHE40 to SP1.Conclusion: 1.BHLHE40 expression promoted tumor cell invasion and migration in MCF-7 cells.2.BHLHE40 suppressed CLDN1 transcription by binding with SP1,instead of directly binding with E-box motif in MCF-7 cells.3.The BHLH and Orange regions of BHLHE40 are essential for the binding of BHLHE40 to SP1.