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Thyroid cancer (THCA) is a common malignancy of the endocrine system which threatens people's health and life quality. It is urgent to find the marker gene of THCA. BHLHE40 is a key gene involved in tumor malignant progression. However, the role of BHLHE40 in THCA remains unclear. In this study, 346 upregulated and 302 downregulated genes were found by analyzing the Gene Expression Omnibus database. BHLHE40 was upregulated in THCA. BHLHE40 and its related differentially expressed genes were involved in cell adhesion and differentiation in THCA. Moreover, BHLHE40 was also highly expressed in THCA cells and tissues. Downregulation of BHLHE40 inhibited cell growth and metastasis. Knockdown of BHLHE40 conditioned media retarded cell migration in M2 macrophages. In addition, knockdown of BHLHE40 inhibited CD206 and CD163 expression and decreased the secretion of interleukin-10 in M2 macrophage. Therefore, BHLHE40 has the potential to be used as a biomarker of immune infiltration and tumorigenesis in THCA.
Keywords: BHLHE40, cell migration, immune infiltration, poor survival, thyroid cancer
| Introduction | |  |
Thyroid cancer (THCA) is a common malignancy of the endocrine system and its incidence and mortality have continued to increase over the past decades.[1] THCA develops from two different cell types, including follicular cells and parafollicular (C) cells. More than 90% of THCAs originate from follicular cells, the epithelial cells responsible for iodine uptake and thyroid hormone synthesis.[2] Follicular THCA is the most diagnosed THCA and has been subdivided into follicular (follicular thyroid carcinoma, FTC), papillary thyroid cancer (PTC), partially differentiated (poorly differentiated thyroid cancer), or interstitial (anaplastic thyroid cancer) THCA. The FTC and PTC forms are characterized by differentiated cancers, with PTC alone accounting for approximately 80% of all thyroid tissue malignancies.[3],[4] It is crucial to clarify the underlying mechanisms of thyroid carcinogenesis and develop more effective diagnostic and therapeutic strategies.
Tumor-associated macrophages (TAMs) are the major component of leukocyte infiltration in many solid tumors. There are two recognized phenotypes of polarized macrophages, including classically activated macrophages (M1) and alternatively activated macrophages (M2).[5] Infiltration of M2 macrophages is positively correlated with tumor progression, and M2 macrophages promote the growth of various tumor cells[6] and promote tumor cell survival.[7] Tumor cells induce macrophage infiltration in tumor tissues and stimulate macrophage transition to M2 phenotype to promote their malignant progression. However, the underlying mechanisms by which cancer cells recruit and polarize macrophages to M2 type remain elusive.
BHLHE40 is a stress response transcription factor that is important for many cellular physiological responses.[8] The aberrant expression of BHLHE40 is associated with tumor malignant progression. It has been reported that knockdown of BHLHE40 significantly reduced primary tumor growth and lung metastasis in in situ xenografts and experimental metastasis models of breast cancer.[9] The expression of BHLHE40 is upregulated in peripheral blood mononuclear cells from patients with hepatocellular carcinoma (HCC), and BHLHE40 is associated with poor prognosis in patients with HCC, suggesting that it may be a promising diagnostic and prognostic biomarker for HBV-associated HCC.[10] BHLHE40 is highly expressed in pancreatic cancer and is closely associated with the migratory invasion and epithelial–mesenchymal transition (EMT) of pancreatic cancer cells.[11] However, the role of BHLHE40 in THCA remains unclear.
In this study, Gene Expression Omnibus (GEO) microarray data analysis revealed that BHLHE40 was highly expressed in THCA tissues, which was confirmed by TCGA data analysis. TIMER database analysis revealed that high BHLHE40 expression was associated with macrophage infiltration and was positively correlated with the expression of M2 type macrophages, TAM markers. Experimental studies revealed that BHLHE40 promoted THCA cell proliferation and recruited M2 macrophage infiltration, as well as promoted M2 type macrophage polarization.
| Materials and Methods | | |
Cell lines and tissue specimens
THCA cell lines (TPC-1 and BCPAP) and normal cells of human thyroid gland (Nthy-ori 3-1) were purchased from Procell and cultured with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and Pen-Strep. THCA tissue specimens were collected from Fujian Medical University Cancer Hospital. All the experiments were directed by manipulation instruction and approved by Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital (Approval No. SQ2018-108).
Microarray data, principal component analysis plot, volcano plot, and Kaplan–Meier analysis
GSE129562 gene expression profiling data were used in this study, which had not been previously simultaneously studied from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). The chip-based GPL10558 platform (Illumina HumanHT-12 V4.0 expression beadchip) was applied for the mRNA expression profiling of both databases. The GSE129562 dataset contains 8 THCA samples and 8 normal thyroid gland tissue samples.[12] The THCA differentially expressed genes (DEGs) in GEO microarray data (GSE129562) were analyzed by GEO2R (|logFC| >1 and P < 0.05),[13] principal component analysis (PCA) plot using FactoMineR package in R language, heatmap using Pheatmap, and heat volcano plot using ggplot2. The correlation between BHLHE40 expression and patient's survival rate was conducted by kmplot online platform (http://kmplot.com/analysis/).
TIMER and UALCAN analysis
The BHLHE40 expression level in THCA based on TCGA database was analyzed using TIMER online platform (https://cistrome.shinyapps.io/timer/) and UALCAN online platform (http://ualcan.path.uab.edu/index.html). The analysis of BHLHE40 expression in THCA tissues and normal tissues was performed by GEO microarray data.
Gene ontology-Kyoto Encyclopedia of Genes and Genomes enrichment analysis
Based on UALCAN database analysis of genes with relevance to BHLHE40 expression in THCA, genes with Pearson-CC ≥0.4 were selected for Gene ontology-Kyoto Encyclopedia of Genes and Genomes (GO-KEGG) in the Metascape analysis platform (http://metascape.org/gp/index.html#/main/step1) enrichment analysis.
Correlation analysis
TIMER data were used to analyze the relationship between BHLHE40 expression and the marker of six tumor-infiltrating immune cells, including B-cells, CD8+ T cells, CD4+ T cells, purity cells, macrophages, neutrophils, and dendritic cells. We analyzed the correlation between BHLHE40 expression with the abundance of immune infiltrates using the gene module, including monocytes, TAMs, M1 macrophages, and M2 macrophages.
Immunohistochemistry experiment
Thirty paired tissues were used in immunohistochemistry (IHC) analysis. All tissues were embedded, sliced, incubated with the indicated primary antibodies, and photographed by microscope as depicted by a previous study.[14] All tissues were manipulated with the approval of the ethics committee.
Real-time quantitative polymerase chain reaction, Western blotting, and ELISA assay
Total RNA was extracted as previously depicted, and then was reversely transcribed into cDNA which was used for the analysis of mRNA expression. The primers of BHLHE40 (forward primer: 5'-GACGGGGAATAAAGCGGAGC-3'; reverse primer: 5'-CCGGTCACGTCTCTTTTTCTC-3') were used in this experiment. For Western blotting, proteins were isolated by EBC buffer and then performed with electrophoresis, membrane transfer, blocking, incubation with antibodies, and imaging by ECL reagent. Membranes were incubated with anti-BHLHE40 (sc-101023, Santa Cruz Biotechnology, Texas, USA), CD206 (sc-376108, Santa Cruz Biotechnology, Texas, USA), and CD163 (sc-20066, Santa Cruz Biotechnology, Texas, USA) antibodies. For ELISA assay, cell lines were seeded into a 96-well plate, and the supernatant media were collected to analyze the concentration of interleukin-10 (IL-10) secreted by M2 cells.
Cell counting kit-8 assay and colony formation analysis
5000 THCA cells were seeded into a 96-well plate, and the cell growth rate at day 1, day 2, and day 3 were detected by cell counting kit-8 (CCK8) reagent. For the colony formation assay, 500 THCA cell lines were seeded into 60 mm dishes. After 2 weeks, cells were washed with phosphate-buffered saline, fixed with fixation buffer, and stained by crystal violet.
Transwell assay
M2 macrophage preparation
THP-1 cells were allowed to differentiate to M0 intermediate stage with paramethoxyamphetamine stimulation, and then, M0 macrophages were polarized to M2 macrophages with IL-4 and IL-13 stimulation, and cultured with the following media from tumor cells: (1) control (medium without tumor cells), (2) shNC conditioned medium (CM), and (3) shBHLHE40-2 CM. For Transwell assay, cells were seeded into upper chamber, and after 24 h, the count of migrated cells was analyzed.
Statistical analysis
The Student's t-test was used in statistical analysis. Data were presented as means ± standard error of the mean of three independent experiments. P < 0.05 was considered a statistically significant difference.
| Results | | |
BHLHE40 expression was increased in thyroid cancer
First, to screen the DEGs in THCA, we analyzed the GEO database by GEO2R, and the results showed that there was a big difference in gene expression between THCA tissues and normal tissues from GSE129562 [Figure 1]a, [Figure 1]b, [Figure 1]c. Next, 346 upregulated and 302 downregulated genes were selected by volcano plot [Figure 1]d. BHLHE40 was one of the upregulated DEGs in THCA, and we further used the TIMER database and UALCAN database to confirm the high expression of BHLHE40 in THCA [Figure 2]a and [Figure 2]b. Moreover, the expression of BHLHE40 was increased in tumor tissues based on sample type, cancer stages, patient's race, and gender compared with normal group [Figure 2]b. We also found that BHLHE40 was overexpressed in THCA tissues compared to normal tissues from GEO dataset [Figure 2]c. | Figure 1: Analysis of DEGs in THCA and normal tissues based on GEO data. (a) The DEGs between THCA tissues and normal tissues from GSE129562 were analyzed by batch correction. (b) FactoMineR package of R language was used to conduct PCA plot. (c) Heatmap of DEGs in THCA was used to analyze the expression of genes. (d) gplot was used to analyze the volcano plot of DEGs (fold change >2, P adjust <0.05). DEGs: Differentially expressed genes, THCA: Thyroid cancer, GEO: Gene Expression Omnibus, PCA: Principal component analysis.
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 | Figure 2: BHLHE40 was highly expressed in THCA. (a) The expression of BHLHE40 in THCA from TCGA dataset was analyzed by TIMER (https://cistrome.shinyapps.io/timer/). (b) The expression of BHLHE40 in THCA based on sample types, individual cancer stages, patient's race, gender, age, tumor history, and nodal metastasis status was analyzed by UALCAN (http://ualcan.path.uab.edu/analysis.html). (c) GSE129562 was used to analyze the expression of BHLHE40 in THCA tissues. ***P < 0.001. THCA: Thyroid cancer.
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Collectively, our data indicated that BHLHE is highly expressed in THCA and is associated with poor survival.
BHLHE40 was associated with immune cell and macrophage infiltration
Next, to clarify the function of BHLHE40 in THCA, we used GO-KEGG enrichment analysis to find the key genes that have a correlation with BHLHE40 from the UALCAN database. As shown in [Figure 3]a, [Figure 3]b and [Figure 3]c, BHLHE40 and related genes were enriched in regulating cell adhesion and duct formation. | Figure 3: GO-KEGG enrichment of BHLHE40 and related genes. (a-c) Based on UALCAN database analysis of genes with relevance to BHLHE40 expression in THCA, genes with Pearson-CC ≥0.4 were selected for GO-KEGG in the Metascape analysis platform (http://metascape.org/gp/index.html#/main/step1) enrichment analysis. Data were shown with column chart (a) and network plot (b and c). GO-KEGG: Gene ontology-Kyoto Encyclopedia of Genes and Genomes, THCA: Thyroid cancer.
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To figure out the role of BHLHE40 in immune cell infiltration in THCA, we analyzed the relationship between BHLHE40 and marker genes involved in immune cell and macrophage infiltration. As shown in [Figure 4]a, the expression of BHLHE40 was positively correlated with infiltration of B-cell (r = 0.47, P = 7.92e-28), CD4+ cell (r = 0.471, P = 2.96e-28), macrophage (r = 0.368, P = 4.01e-17), neutrophil (r = 0.535, P = 1.84e-35), and dendritic cell (r = 0.498, P = 9.60e-32). Furthermore, there was a positive correlation between BHLHE40 and macrophage markers, including monocyte, M1, M2, and TAM [Figure 4]b. Taken together, these data suggested that BHLHE40 may be positively associated with immune cell and macrophage infiltration. | Figure 4: Correlation analysis of BHLHE40 with markers of immune cell and macrophage infiltration. (a) Dot plot was used to analyze the correlation between BHLHE40 and immune infiltrates in THCA. (b) Correlation analysis between BHLHE40 and macrophage markers, including monocyte, M1, M2, and TAM. THCA: Thyroid cancer, TAM: Tumor-associated macrophage.
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BHLHE40 was overexpressed in thyroid cancer tissues and cells
In addition to the analysis of BHLHE40 expression in GEO dataset and online platform, we also measured the expression of BHLHE40 in patient tissues. As shown in [Figure 5]a, the mRNA expression of BHLHE40 was increased in tumor specimens compared to normal group. Moreover, the expression of BHLHE40 was also upregulated in tumor group compared to normal group at protein level, as detected by Western blotting and IHC [Figure 5]b and [Figure 5]c. CD206, a M2 macrophage marker, was overexpressed in tumor specimens compared to normal tissues [Figure 5]c. Collectively, our data suggested that BHLHE40 was highly expressed in THCA tissues. | Figure 5: BHLHE40 was highly expressed in THCA tissues and cells. (a and b) Thirty pairs of THCA specimens and matched adjacent normal tissues were collected to analyze the expression of BHLHE40 by RT-qPCR (a) and Western blotting (b). ***P < 0.001. (c) IHC assay was used to analyze the expression of BHLHE40 and CD206 in THCA specimens. THCA: Thyroid cancer, RT-qPCR: Real-time reverse transcription–quantitative polymerase chain reaction.
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BHLHE40 facilitated thyroid cancer cell growth and migration
Based on the analysis from GEO, BHLHE40 was associated with cell adhesion and differentiation. We next clarified the role of BHLHE40 in cell proliferation and migration. First, the expression of BHLHE40 was measured by RT-qPCR and Western blotting. As shown in [Figure 6]a and [Figure 6]b, BHLHE40 was highly expressed in TPC-1 and BCPAP cells compared to Nthy-ori 3-1 cells. Then, we used TPC-1 and BCPAP cells for indicated plasmid transfection [Figure 6]c. Knockdown of BHLHE40 suppressed cell proliferation of TPC-1 and BCPAP cells transfected with shBHLHE40 compared to cells transfected with shNC [Figure 6]d. Knockdown of BHLHE40 also decreased colony formation of TPC-1 and BCPAP cells [Figure 6]e. In addition, knockdown of BHLHE40 inhibited cell migration and invasion of TPC-1 and BCPAP cells [Figure 6]f. Collectively, these data indicated that BHLHE40 promoted cell growth and cell migration in THCA cells. | Figure 6: BHLHE40 promoted THCA cell proliferation and migration. (a and b) The expression of BHLHE40 in THCA cell lines (TPC-1 and BCPAP) and normal cells of human thyroid gland (Nthy-ori 3-1) was analyzed by RT-qPCR (a) and Western blotting (b). (c) TPC-1 and BCPAP cells were transfected with shBHLHE40 plasmids, and Western blotting assay was used to analyze the expression of BHLHE40. (d) Cell viability in BHLHE40 deficiency cell lines was conducted by CCK8 assay. (e) Crystal violet staining assay was used to analyze cell colony formation in THCA cell lines with BHLHE40 inhibition. (f) Cell migration and invasion were analyzed by Transwell assay. **P < 0.01. ***P < 0.001. THCA: Thyroid cancer, RT-qPCR: Real-time reverse transcription–quantitative polymerase chain reaction, CCK8: Cell counting kit-8.
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Next, we analyzed the role of BHLHE40 in M2 macrophage infiltration. As shown in [Figure 7]a, M2 macrophage with shNC CM treatment exerted higher migration compared to the control group, while M2 macrophage treated with shBHLHE40-2# CM retarded cell migration compared to shNC CM-treated cells. Next, we analyzed the expression of CD163 and CD206 (markers of M2 macrophage) in the cell lines. As shown in [Figure 7]b, BHLHE40 deficiency inhibited CD206 and CD163 expression in M2 macrophage. IL-10 is a marker of M2 macrophages, then the concentration of IL-10 was further analyzed by ELISA. As shown in [Figure 7]c, M2 macrophage treated with shNC CM exerted a higher concentration of IL-10 compared to control group, while knockdown of BHLHE40 decreased the concentration of IL-10 compared to shNC group. Taken together, our data indicated that BHLHE40 facilitated the recruitment of M2 macrophages and its polarization. | Figure 7: BHLHE40 promoted the recruitment of M2 macrophages and the polarization of macrophages toward M2. (a) M2 macrophages were cultured with three media including control group (medium without tumor cells), shNC conditioned media, and shBHLHE40-2# conditioned media. Cell migration in M2 macrophage was analyzed by Transwell assay. (b) The expression of CD163 and CD206 in M2 macrophage with BHLHE40 knockdown was analyzed by Western blotting. (c) ELISA assay was used to measure the concentration of IL-10 in M2 macrophage. **P < 0.01, ***P < 0.001, *compared to control group. ^P < 0.05, ^^P < 0.01, ^^^P < 0.001, ^compared with shNC group. IL-10: Interleukin-10.
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| Discussion | | |
THCA, as one of the three glandular tumors, has become a common cancer that threatens patient's health and life quality.[15] It is of great importance to clarify the underlying molecular mechanism of the development of THCA and explore biomarkers for diagnosing and therapy of THCA. In this study, BHLHE40, as one of the upregulated expression genes in THCA tissues and cell lines, promoted cell growth, colony formation, cell migration, and invasion of THCA cells. Moreover, GO-KEGG enrichment analysis showed that BHLHE40 was correlated with immune cell infiltration and cell differentiation. BHLHE40 promoted the recruitment and polarization of M2 macrophages.
BHLHE40, as a transcriptional repressor, was involved in the regulation of cell cycle arrest, senescence, differentiation, and apoptosis by binding to class B E-box (CACGTG).[16],[17] BHLHE40 mainly played a role in transcriptional activation and promoting of cell survival.[17] For example, BHLHE40 activated the transcription of pro-survival factors such as BIRC5 and DeltaNp63 in tumor cells.[18],[19] BHLHE40 negatively regulated the expression of genes encoding transcription factors c-Maf and Mafb, increased the expression of transcripts encoding cell cycle-related proteins, and promoted the proliferation of large peritoneal macrophages.[20] Here, we also demonstrated that knockdown of BHLHE40 reduced cell growth and migration, and high expression of BHLHE40 was associated with poor survival. However, the factor that determined the selectivity of BHLHE40 to repress or activate transcription remains unclear.
DEC1 (BHLHE40/Stra13/Sharp2), as a basic helix-loop-helix (bHLH) transcription factor, was involved in the regulation of apoptosis and cell proliferation and response to hypoxia.[8],[11],[17],[18],[19],[21] EMT was an important step leading to the invasion and migration of various tumor cells. A previous study reported that BHLHE40-mediated upregulation of INHBA promoted colon cancer cell migration.[22] Knockdown of BHLHE40 in preeclampsia placenta restored cell viability, migration, and invasion through upregulating SNX16 by transcriptional repression of miR-196a-5p.[23] BHLHE40 induced cell survival and migration by blocking DNA binding of histone deacetylase (HDAC) 1 and HDAC2 and inducing HBEGF transcription.[9] BHLHE40 was involved in the Notch signaling pathway in tumors and regulated aggressive tumor regeneration in a THCA model.[24] BHLHE40 promoted cell growth and invasiveness and sustained THCA progression. Abnormal signaling pathways in THCA cells enabled BHLHE40 to cooperate with the Notch signaling pathway to promote target gene transcription and tumor invasiveness. In both satellite cells and THCA, BHLHE40 increased the expression of the Notch ligand Jagged. Regarding the mechanism in regulation of BHLHE40 in THCA, we speculated that Notch signaling pathway may exert an important role in regulating cell proliferation and cell migration, which will be explored in future studies.
A previous study demonstrated that BHLHE40 can activate the transcription of a series of cytokines required to activate mouse CD4+ T cells.[25] In in vitro generated memory CD8+ T cells, BHLHE40 overexpression led to attenuated recall responses, but the physiological role of BHLHE40 in regulating CD8+ Teff and/or Tmem responses is unclear.[26] BHLHE40 was specifically required for Trm cells and TILs for development, fitness, and versatility.[26] In addition, depletion of DEC1 protected the heart from perivascular fibrosis, affected M1/M2 macrophage polarization, and reduced apoptosis.[21] We also demonstrated that BHLHE40 deficiency decreased cell migration of M2 macrophage and polarization of M2 macrophage.
| Conclusion | | |
BHLHE40 was highly expressed in THCA, and knockdown of BHLHE40 decreased cell growth, migration, and invasion. There was a correlation between BHLHE40 and immune cell infiltration and polarization of macrophages toward M2. BHLHE40 has the potential to be used as a new biomarker in tumorigenesis of THCA.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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Correspondence Address:
Huaying Li,
Department of Oncology Rehabilitation, Rehabilitation Hospital Affiliated to Fujian University of Traditional Chinese Medicine, No. 13, Hudong Branch Road, Gulou, Fuzhou, Fujian
China
 Source of Support: None, Conflict of Interest: None DOI: 10.4103/cjop.CJOP-D-22-00076
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7] |
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