The Mechanism Of BHLHE41 Suppressing MCF-7 Cell Invasion

Objective:As one member of the DEC subfamily(BHLHE40 / DEC1 and BHLHE41 / DEC2),Basic helix-loop-helix family member e41(BHLHE41)plays a pivotal role in regulating circadian rhythm,whose function is achieved by BHLHE41 protein entering the nucleus and competing with CLOCK-BMAL1 heterodimer for E-Box element binding.Moreover,other pathways like PI3K/Akt,Notch,ERK/NF-kappaB,BCR and hypoxia are employed by BHLHE41 to regulate non-circadianis.It is shown in current studies that BHLHE41 is entailed in regulating tumor migration and invasion(such as endometrial cancer and gastric cancer),whereas BHLHE41 may suppress cell invasion and metastasis in tumor progression,and the mechanism is yet to be known.Previous report has shown that invasion and metastasis were suppressed by BHLHE41 through promoting the degradation of HIF-1α in triple-negative breast cancer cell line MDA-MB-231.Whether and how BHLHE41 suppresses invasion and metastasis in MCF-7 cells(luminal A phenotype)is the research objective of this study.Methods:GEPIA online was used to analyze the expression of BHLHE41 gene in breast cancer tissues and normal breast tissues.WB was used to detect the expression of BHLHE41 protein in breast cancer and adjacent normal breast tissue.Obtained MCF-7 and MDA-MB-231 cell line was authenticated by DNA profiling using short tandem repeat(STR),with the cell samples being cultured in DMEM.After obtaining the Plasmids pCMV,pCMV-Myc-SP1,pcDNA3.1-BHLHE40,pcDNA3.1-BHLHE41 and pcDNA3.1,plasmid pcDNA3.1-BHLHE40 was inserted with N-His-Flag-tag,and the plasmid pcDNA3.1-BHLHE41 was inserted with N-His-Myc-tag.MCF-7 and MDA-MB-231 cells were transfected with the plasmids using Lipofectamine LTX(Invitrogen).Having designed three kinds of BHLHE41 siRNAs,we transfected BHLHE41 siRNA into MCF-7 and MDA-MB-231 cell lines by Lipofectamine 3000reagent(Invitrogen).After silencing BHLHE41 in MCF-7 and MDA-MB-231 cells,the mRNA and protein levels of VIM,CDH1,CDH2,SNAI1,SNAI2,CLDN1 and CLDN4 were detected by using qRT-PCR and WB in order to investigate the relationship between BHLHE41 and epithelial-mesenchymal transition(EMT)and tight junction(TJ).For the purpose of detecting the role of exogenous BHLHE41 in cell invasion,MCF-7 and MDA-MB-231 cell lines were transfected with His-Myc-BHLHE41,using the vector(pcDNA)as a control,thus the mRNA and protein expression of EMT and TJ by qRT-PCR and WB were detect as well.We performed immunofluorescence to locate CLDN1,CLDN4,and BHLHE41 in MCF-7 and MDA-MB-231 cells,and to find out whether BHLHE41 affects their intracellular localization of CLDN1 and CLDN4.We performed Co-IP to detect whether BHLHE41 could interact with SP1 and play a similar role as BHLHE40.siRNA in MCF-7 and MDA-MB-231 cells silencing BHLHE41,my team investigated the effects of endogenous BHLHE41 on these signaling pathways.At the meantime,we performed WB to detect the status of MAPK/ERK,hypoxia,MAPK/JNK,NFκB etc.signaling pathway after silencing BHLHE41 by siRNA.Further more,we blocked MAPK/JNK signaling pathway with a specific inhibitor SP600125 in MCF-7 cells,then treated with BHLHE41 siRNA,for the purpose of further investigating whether BHLHE41 suppressed the invasion of MCF-7 cell line via MAPK/JNK signaling pathway.Results:1.TCGA database was used to analyze the expression of BHLHE41 gene in 1085 breast cancer tissues and 112 normal breast tissues,with the result showing that the expression of BHLHE41 in breast cancer tissues was lower than that in normal breast tissues.After detecting expression of BHLHE41 protein in paired specimens of breast cancer and normal breast tissue from 16 patients by WB,we found that the expression of BHLHE41 protein in breast cancer tissue was lower than that in adjacent normal breast tissue.2.BHLHE41 silenced by siRNA promotes tumor cell migration and invasion of MCF-7cells and MDA-MB-231 cells.Both were transfected with BHLHE41 siRNA.The si RNA with optimal effect(siBHLHE41#3)was selected by using qRT-PCR analysis.The number of cells that migrated and traversed the gel were less in the control group than that in the BHLHE41 siRNA group in MCF-7 and MDA-MB-231 cell line.We further analyzed the variations of EMT and TJ-associated genes by qRT-PCR and WB analysis.BHLHE41 siRNA increased the mRNA and protein levels of CLDN1 and CLDN4,while decreased those of SNAI1,SNAI2,VIM and CDH2 in both MCF-7 and MDA-MB-231 cells.The mRNA and protein levels of CDH1 were also downregulated in MCF-7 cells.3.Exogenous BHLHE41 expression suppressed tumor cell migration and invasion.After transfecting MCF-7 and MDA-MB-231 cell lines with His-Myc-BHLHE41,the number of cells that migrated and traversed the gel were more in the control group than that in the BHLHE41 transfected group.qRT-PCR and WB were used to detect the mRNA and protein expression of EMT and TJ.Having been transfected with BHLHE41 in MCF-7 cells,the m RNA and protein levels of CLDN1,CLDN4,and CDH1 were upregulated,and by contrast those of SNAI1,SNAI2,VIM,and CDH2 were downregulated.The results were similar to MCF-7 cell line except for CDH1 in MDA-MB-231 cell line.4.BHLHE41 did not affect the cellular location of CLDN1 and CLDN4.Immunofluorescence confirmed that BHLHE41 is normally expressed in the nuclear of MCF-7 and MDA-MB-231 cells.BHLHE41 did not affect the cellular location of CLDN1 and CLDN4,both of which exhibited a cytoplasmic expression pattern regardless of the BHLHE41 expression level.5.We performed Co-IP to confirmed that BHLHE41 failed to interact with SP1 in MCF-7 cells,and BHLHE41 regulates cell invasion in a different manner as compared to that of BHLHE40.6.BHLHE41 suppressed MCF-7 cell invasion via MAPK/JNK signaling pathway.Multi-signaling pathways were screened by reporter assay in MCF-7 and MDA-MB-231 cells.MAPK/ERK,NFκB,MAPK/JNK and hypoxia signaling pathways were activated after silencing BHLHE41 by using siRNA in MCF-7 cell line,among which the variations of the MAPK/JNK signaling pathway was the most apparent.The variations of hypoxia and p53 signaling pathway were the most apparent in MDA-MB-231 cell line,while MAPK/JNK signaling pathway was slightlyactivated.After treatment with BHLHE41 siRNA in MCF-7 cells,phosphorylated JNK was obviously upregulated,which indicated the activated state of MAPK/JNK signaling pathway.After blocking MAPK/JNK signaling pathway with a specific inhibitor SP600125,the induction of phosphorylated JNK by BHLHE41 si RNA was reversed.Conclusion:BHLHE41 expression in breast cancer tissues is lower than that in normal breast tissues.BHLHE41 suppressed invasion in breast cancer cells MCF-7 and MDA-MB-231 cells.BHLHE41 failed to interact with SP1 and BHLHE41 regulates cell invasion in a different manner as compared to that of BHLHE40.Silencing of BHLHE41 by siRNA activated MAPK/JNK signaling pathway in MCF-7cells,or activate hypoxia signaling pathway in MDA-MB-231 cells.BHLHE41 suppresses invasion by inhibiting EMT via MAPK/JNK signaling pathway in MCF-7 cells.