BHLHE41 suppresses MCF‐7 cell invasion via MAPK/JNK pathway

Abstract Deregulation of the basic helix‐loop‐helix family member e41 (BHLHE41) has been characterized as a marker of progression of several cancers. In this study, we aimed to explore the mechanism by which BHLHE41 regulates the invasion of breast cancer cells. BHLHE41 suppresses, whereas the silencing of BHLHE41 promotes tumour invasion of both MCF‐7 and MDA‐MB‐231 cells. Meanwhile, BHLHE41 down‐regulated the transcription and translation of SNAI1, SNAI2, VIM and CDH2, and up‐regulated those of CLDN1, CLDN4 and CDH1. Reporter assay indicated that silencing of BHLHE41 dramatically activated the MAPK/JNK signalling pathway in MCF‐7 cell line and the hypoxia signalling pathway in MDA‐MB‐231 cell line. Furthermore, silencing of BHLHE41 activated the MAPK/JNK signalling pathway by up‐regulating phosphorylated JNK and failed to affect the expression of HIF‐1 alpha in MCF‐7 cells. After blocking the MAPK/JNK signalling pathway by specific inhibitor SP600125, silencing of BHLHE41 failed to promote tumour cell invasion. These results suggest that BHLHE41 facilitates MCF‐7 cell invasion mainly via the activation of MAPK/JNK signalling pathway. In conclusion, although BHLHE41 suppresses tumour invasion in MCF‐7 and MDA‐MB‐231 cell lines, the specific regulatory mechanisms may be different.

Recent studies indicate that BHLHE41 is involved in regulating tumour migration and invasion. 5,6,19,24 Asanoma et al 19 demonstrated that BHLHE41 suppressed tumour invasion and metastasis in endometrial cancer by inhibiting TWIST1 transcription. Li et al 6 pointed out that BHLHE41 suppressed tumour proliferation and metastasis by regulating ERK/NF-kappaB pathway in gastric cancer. BHLHE41 was demonstrated to be a crucial regulator of the invasive and metastatic phenotype in triple-negative breast cancer and suppressed metastasis by promoting the degradation of hypoxia-inducible factors in MDA-MB-231 cells. 8 Thus far, most researchers believe that BHLHE41 plays a role in suppressing cell invasion and metastasis in tumour progression. The detailed mechanisms, however, were not always consistently demonstrated in the above studies. 6,8,19 Therefore, further work is needed to understand the mechanism by which BHLHE41 regulates cell invasion and metastasis.

| Cell culture
The MCF-7 and MDA-MB-231 cell line was obtained from the Shanghai Cell Bank (Shanghai, China). Both cell lines were authenticated by DNA profiling by short tandem repeat (STR). Following that, the cell samples were frozen and individual aliquots were cultured typically for analysis within ten passages. The cells were routinely grown in DMEM (Invitrogen) plus 10% foetal bovine serum (FBS; Invitrogen), 100 μg/mL streptomycin (Sigma) and 100 IU/ mL penicillin (Sigma), and passaged every other day using trypsin (0.25%, Invitrogen).

| Matrigel invasion assay
Cell invasion assays were performed using 24-well Transwell chambers (8-μm pore-sized; Costar). The inserts were coated with 20 μL Matrigel (1:3 dilution; BD Bioscience). Forty-eight hours after transfection, the cells were trypsinized and resuspended in 100 μL of a serum-free medium at a concentration of 3 × 10 5 cells. The cells were then added to the upper chamber of Transwell plates, and 10% FBS was placed in the lower chamber as a chemoattractant. After an 18-hour incubation period, the cells that had migrated through the Matrigel-coated filter were fixed with 4% paraformaldehyde, stained with haematoxylin.
The cells on the lower surface of the filters were counted under a light microscope in 10 randomly selected fields at 40× magnification.

| qRT-PCR
The total RNA was extracted from the cells and isolated using an RNeasy RNA isolation kit (QIAGEN). First-strand cDNA samples were synthesized from the total RNA (1 µg) by using ReverTra Ace (TOYOBO). RT-PCR was performed using an aliquot of the firststrand cDNA as a template under standard conditions with Taq DNA polymerase (QIAGEN). Real-time PCR was performed using the SYBR Green Master Mix (Applied Biosystems). The relative expression levels of the target genes were calculated using a △△CT method after normalizing with the housekeeping gene, 18 S. The sequence information used is shown in Table 1.

| Western blot (WB)
The total protein extraction was performed using a lysis buffer (Pierce). The total protein concentration in the extract was estimated by the Bradford method. 12  ProteinTech Group, Inc). The membranes were washed and were treated for 2 hours at 37°C with peroxidase-conjugated antimouse or anti-rabbit IgG (Santa Cruz Biotechnology). The bound proteins were visualized using electrochemiluminescence (Pierce) and detected with a bio-imaging system (DNR Bio-Imaging Systems).

| Co-immunoprecipitation (Co-IP)
The whole-cell lysates from the MCF-7 cells expressing MYC-SP1 or interacting with either Flag-BHLHE40 or His-BHLHE41 were used for immunoprecipitation. The lysates were immunoprecipitated with either anti-SP1 or -His antibody bound to protein G PLUS-agarose (Santa Cruz Biotechnology) overnight at 4°C and then separated by SDS-PAGE. The immunoprecipitated proteins were visualized by WB with an anti-SP1, -Flag or -His antibody.
After 24 hours, the cells were transfected with BHLHE40 or

| Luciferase reporter assay
Cignal Finder Reporter Assay Kit (10-pathway) (Product no. 336821) was purchased from QIAGEN (China), and the procedure was performed according to the manufacturer's instructions.

TA B L E 1 Primers used for the qRT-PCR analysis
The sequences for the sense and antisense BHLHE41 siRNAs

| Statistical analyses
All analyses were performed using SPSS version 22.0 for Windows (SPSS). Mann-Whitney U test was used to analyse the differences in expression levels and results of invasion assay. The data of the reporter assay and ChIP were analysed using Student's t test. The statistical differences between the treatment groups were determined using Super ANOVA and Scheffé's test. A P-value of <.05 was considered statistically significant.

| BHLHE41 expression suppressed tumour cell migration and invasion of MCF-7 cells and MDA-MB-231 cells
We also detect the role of exogenous BHLHE41 in cell invasion.

| BHLHE41 did not affect the cellular location of CLDN1 and CLDN4
We next performed immunofluorescence staining, to investigate whether BHLHE41 affects their intracellular localization of CLDN1 and CLDN4. As shown in Figure  F I G U R E 1 BHLHE41 silencing by siRNA promoted tumour cell migration and invasion of MCF-7 and MDA-MB-231 cells. A, B, Three different siRNAs targeting BHLHE41 were used for the knockdown experiments. The validity of these siRNAs was confirmed by qRT-PCR. si-BHLHE41#3 was used in the following knockdown experiments because of the optimal effects. C, D, Transwell assay for assessing cell migration and invasion. As measured by the transwell assay with or without gel cover, BHLHE41 silencing led to a significant increase in the number of migrated cells. E, F, Transwell assays were repeated in triplicate. Ten views were selected for each trial, and the migrated cells were counted for the final statistical analysis. Each value represents the mean ± SE (bars) of three independent experiments, ***P < .001. G, H, Real-time PCR analyses of BHLHE41, CLDN1, CLDN4, SNAI1,  Transwell assays were repeated in triplicate. Ten views were selected for each trial, and the migrated or invasion cells were counted for the final statistical analysis. Each value represents the mean ± SE (bars) of three independent experiments, ***P < .001

| BHLHE41 suppressed MCF-7 cell invasion via MAPK/JNK signalling pathway
We previously reported that BHLHE40, another member of the DEC subfamily, regulated MCF-7 cell invasion through interaction with SP1 and therefore negatively regulated the transcription of CLDN1. In the current study, we performed Co-IP to detect whether BHLHE41 could interact with SP1 and play a similar role in regulating CLDN1 transcription. As shown in Figure 4, BHLHE41 failed to interact with SP1 and BHLHE40 was used here as a positive control.
As BHLHE41 was reported to be involved in the regulation of many signalling pathways, we further screened multi-signalling pathways by reporter assay in MCF-7 and MDA-MB-231 cells.
We silenced BHLHE41 by siRNA to investigate the effects of endogenous BHLHE41 on these signalling pathways. As shown in  Figure 5F). and MDA-MB-231). In fact, this discrepancy between the performance of BHLHE40 and BHLHE41 is not uncommon. 9,[28][29][30] This paradox was also observed in the regulation of apoptosis and cell proliferation. A possible explanation is that BHLHE40 may negatively regulate the transcription of BHLHE41 and vice versa. 30 Another explanation is the diversity in protein structure, that is, BHLHE41 has a Gla/Gla-rich region in its C-terminal. 5 In addition, we found that BHLHE41 failed to interact with sp1 and therefore could not form a transcriptional complex as BHLHE40 did. This result indicates that BHLHE41 regulates cell invasion in a different manner as compared to that of BHLHE40.

| D ISCUSS I ON
We screened multiple pathways by reporter assay to find the underlying mechanism of BHLHE41. Interestingly, the activated signalling pathway induced by BHLHE41 is not the same as that

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

Yang Liu and Enhua Wang conceived and designed the experiments;
Di Zhang, Qin Zheng, Chen Wang and Na Zhao performed the experiments; Di Zhang, Yang Liu and Enhua wrote the paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.