Delphinidin upregulates microRNA-let-7b expression through family with sequence similarity 222 member B

Abstract

Delphinidin is a major anthocyanidin belonging to the flavonoid family with pharmacological activities against cancer, inflammation, and muscle atrophy. However, the mechanism regulating its activity is yet to be completely elucidated. The present study aimed to identify the molecules responsible for the effects of delphinidin. We investigated the effects of delphinidin on melanoma cells and observed that delphinidin upregulates the expression of let-7b, a microRNA (miRNA) that inhibits the proliferation of melanoma cells. Delphinidin-induced upregulation of let-7b led to the suppression of cyclin D1, a target gene associated with cell cycle progression. Using genetic functional screening, we identified that family with sequence similarity 222 member B (Fam222B) is an important factor behind the inhibitory effects of delphinidin against melanoma cell proliferation and that the transfection with small interfering RNA specifically targeting Fam222B altered miRNAs expression in B16 cells. In summary, delphinidin exerts antimelanoma effects by increasing let-7b expression through Fam222B, and Fam222B is involved in regulating miRNA expression.

Introduction

Anthocyanins are red, purple, and blue natural plant pigments found in flowers, fruits, and vegetables. Over 600 types of anthocyanins have been identified, including six major anthocyanidins with complex glycosylation patterns. Owing to their beneficial health effects, anthocyanidins have been widely investigated¹. Delphinidin is a major anthocyanidin that exhibits antioxidative, anticancer, anti-inflammatory, and other biological activities^{2, 3–4}. However, the molecules responsible for its biological activity remain unknown.

MicroRNAs (miRNAs), which are endogenously encoded small RNAs, downregulate gene expression via mRNA cleavage or translational inhibition. They are expressed in animals, plants, and bacteria and have physiological functions, including cell growth⁵apoptosis⁶metabolism⁷and inflammation⁸. Moreover, changes in miRNAs expression are being investigated in terms of human diseases, such as cancer, cardiovascular diseases, and skeletal muscle diseases^{9, 10–11}. For example, the expression of let-7 family members is reduced in many cancer types¹²and let-7b has been shown to effectively inhibit the growth of lung and breast cancer cells^13,14. Regulating miRNA expression could be an effective strategy to improve physical condition, and the health effects of polyphenols have been reported to be associated with miRNAs¹⁵. In our previous studies, we demonstrated that delphinidin alters miRNA expression^16,17. Thus, we hypothesized that miRNAs may play a key role behind the biological effects of delphinidin.

Biomolecules mediate the bioactivity of food factors, including amino acids, fatty acids, vitamins, and polyphenols. For example, (−)-epigallocatechin-3-O-gallate (EGCG), a major catechin in green tea, binds to the cell surface protein 67-kDa laminin receptor (67LR)¹⁸ and exerts various effects, including anticancer^19,20anti-inflammatory²¹and anti-allergic effects²² through 67LR. Previously, we used the genetic suppressor element (GSE) method to identify molecules that are responsible for the effects of EGCG^23,24 and soy isoflavone metabolite equol²⁵. GSE libraries are complementary DNA (cDNA) fragments encoding dominant negative peptides that act as protein function inhibitors or antisense RNAs that suppress the expression of target genes²⁶. GSE method can be used to identify molecules that are related to the function of various food ingredients.

The present study aimed to identify the molecules involved in the biological activity of delphinidin and evaluate the involvement of miRNAs in the effect of delphinidin. Moreover, we attempted to elucidate the molecular basis of delphinidin’s function using the GSE method.

Results

Effects of delphinidin on the proliferation of melanoma cells

In our previous study, we used B16 melanoma cell lines to evaluate miRNAs involvement in the function of EGCG²⁷ and perform the GSE method^{23, 24–25}. To study the effects of delphinidin on melanoma cells, we first determined whether delphinidin at the indicated treatment concentrations could influence the number of B16 cells. Our data demonstrated that delphinidin exerted inhibitory effects on B16 cell proliferation (Fig. 1a). There was no difference between the 0 and 10 µM delphinidin groups at 24 h; however, the cell numbers were significantly suppressed in the 10 µM delphinidin group at 48 and 72 h (Fig. 1b). Delphinidin inhibited the proliferation of human melanoma cell line Hs294t (Fig. 1c). The cyclin D1 protein encoded by the CCND1 gene is a key molecule of physiological cell cycle regulation²⁸. As the transcription of cyclin D1 depends on upstream pathways that are essential for cancer development, it has been identified as a therapeutic target. On the basis of this hypothesis, we examined the effects of delphinidin on cyclin D1 expression. We observed that delphinidin suppressed cyclin D1 protein levels in B16 cells (Fig. 1d).

Fig. 1 [Images not available. See PDF.]

Effects of delphinidin on the proliferation of melanoma cells. (a) B16 cells were treated with delphinidin for 48 h, and the cell numbers were measured. (b) B16 cells were treated with 10 µM delphinidin for the indicated time, and the cell numbers were measured. (c) Hs294t cells were treated with delphinidin for 48 h, and the cell numbers were measured. (d) B16 cells were treated with delphinidin for 48 h, and then cyclin D1 levels were assessed by Western blot analysis. Data are presented as mean ± SEM (n = 3). Statistical analysis was conducted using Student’s t-test in (b) and using one-way ANOVA followed by Dunnett’s multiple comparison test in (a) and (c). *P < 0.05, **P < 0.01, and ***P < 0.001.

Delphinidin exerts antimelanoma effects by increasing let-7b expression

MiRNA-let-7b is a member of the let-7 family, which targets essential oncogenes and suppresses tumor progression. Let-7b has numerous target genes associated with tumor development, including cyclin D1²⁹. Our previous study demonstrated that delphinidin upregulated let-7b expression in mouse plasma¹⁷and we hypothesized that delphinidin may increase let-7b expression in melanoma. We analyzed the effects of delphinidin on let-7b expression using RT-qPCR and found that delphinidin significantly upregulated let-7b expression in B16 cells (Fig. 2a and b) and Hs294t cells (Fig. 2c). In this experiment, although there was no change in tumor weight (Supplementary Fig. 1), the oral administration of delphinidin increased let-7b expression in melanoma tumors (Fig. 2d). Additionally, we used a let-7b inhibitor to determine whether the delphinidin-induced upregulation of let-7b was associated with the growth inhibitory activity in B16 cells. Delphinidin suppressed B16 cell proliferation and decreased cyclin D1 protein levels in control inhibitor-transfected cells, whereas the let-7b inhibitor eliminated the effects of delphinidin (Fig. 2e and f). These results indicate that delphinidin exerts antimelanoma effects by upregulating let-7b expression.

Fig. 2 [Images not available. See PDF.]

Delphinidin exerts antimelanoma effects by increasing let-7b expression. (a) B16 cells were treated with different delphinidin concentrations for 48 h, and then let-7b expression was analyzed using RT-qPCR (n = 3). (b) B16 cells were treated with 10 µM delphinidin at different time intervals, and then let-7b expression was measured using RT-qPCR (n = 3). (c) Hs294t cells were treated with delphinidin for 48 h, and let-7b expression was measured using RT-qPCR (n = 3). (d) Fifteen days after B16 cell injection, the mice were orally administered with delphinidin (20 mg/kg of body weight) or dH₂O. After 48 h of administration, let-7b expression in the tumor was measured using RT-qPCR (n = 5). (e and f) B16 cells were transfected with 50 nM let-7b inhibitor for 48 h and then treated with 10 µM delphinidin for 48 h, after which cell number and cyclin D1 protein levels were assessed (n = 3). Data are presented as mean ± SEM. Statistical analysis was conducted using Student’s t-test in (b-e) and using one-way ANOVA followed by Dunnett’s multiple comparison test in (a). *P < 0.05, **P < 0.01, and ***P < 0.001.

Identification of molecules that regulate the growth inhibitory effects of melanoma cells by delphinidin

Inhibition of specific genes is an approach to elucidate gene function, and selection by the GSE method makes it possible to identify molecules involved in the function of food components. In the present study, the GSE method was used to elucidate the molecular mechanism behind the beneficial effect of delphinidin (Fig. 3a). Before culturing with delphinidin for 4 weeks, B16 cells were transfected with the GSE library. The GSE conferring resistance to delphinidin was recovered from B16 cells and a sequence corresponding to family with sequence similarity 222 member B (Fam222B) was identified. Considering there is little information with regard to Fam222B, its expression levels were examined in B16 cells, human melanoma cell lines (A375, Hs294t, and MeWo), and NHEMs. The protein levels of Fam222B were detected in all cells (Fig. 3b). B16 cells were stimulated with delphinidin, and its mRNA expression and protein levels were assessed to elucidate the effects of delphinidin on Fam222B. The results showed that delphinidin did not change Fam222B mRNA expression (Fig. 3c) and protein levels (Fig. 3d) in B16 cells. These data suggest that delphinidin may affect Fam222B function without changing its expression.

Fig. 3 [Images not available. See PDF.]

Identification of molecules that regulate the growth inhibitory effects of melanoma cells by delphinidin. (a) Schematic of genetic screening for molecules that mediate the inhibition of melanoma cell proliferation by delphinidin. (b) Fam222B protein levels in melanoma cell lines and NHEMs were assessed using Western blot analysis (n = 1). (c and d) Fam222B mRNA expression and protein levels were evaluated in B16 cells treated with delphinidin for 48 h (n = 3). Data are presented as mean ± SEM. Statistical analysis was conducted using one-way ANOVA followed by Dunnett’s multiple comparison test.

Fam222B is involved in delphinidin’s antimelanoma activity in B16 cells

To assess the role of Fam222B in the effects of delphinidin, targeted small interfering RNA (siRNA) was introduced to silence its expression in B16 cells (Fig. 4a). Delphinidin suppressed B16 cell proliferation in control-siRNA-transfected cells, whereas the inhibitory effects of delphinidin were lost in Fam222B-siRNA-transfected B16 cells (Fig. 4b). Furthermore, delphinidin had no effects on cyclin D1 protein levels and let-7b expression in Fam222B-siRNA-transfected B16 cells (Fig. 4c and d).

Fig. 4 [Images not available. See PDF.]

Fam222B is involved in the antimelanoma activity of delphinidin in B16 cells. (a) Fam222B mRNA expression in B16 cells transfected with 10 nM Fam222B-siRNA for 48 h. (b) B16 cells were transfected with 10 nM Fam222B-siRNA for 48 h and then treated with 10 µM delphinidin for 48 h. The cell numbers were measured. (c) B16 cells transfected with Fam222B-siRNA were treated with 10 µM delphinidin for 48 h, and cyclin D1 protein levels were measured using Western blot analysis. (d) B16 cells transfected with Fam222B-siRNA were treated with 10 µM delphinidin for 48 h, and let-7b expression was measured by RT-qPCR. Data are presented as mean ± SEM (n = 3). Statistical analysis was conducted using Student’s t-test. *P < 0.05 and **P < 0.01.

Fam222B suppression inhibits MiRNA expression

We previously found that delphinidin alters miRNA expression in mouse skeletal muscle and blood^16,17. Our data showed that delphinidin increases let-7b expression through Fam222B, and we speculate that Fam222B may be involved in changing miRNA expression. To determine whether Fam222B regulates miRNA expression in B16 cells, we performed microarray analysis to compare the miRNA expression profiles between control and Fam222B-siRNA-transfected B16 cells. Fam222B silencing tended to decrease the expression of 337 miRNAs as shown in Fig. 5a. Through Gene ontology (GO) analysis, it was demonstrated that miRNAs down-regulated by Fam222B-siRNA were mainly enriched in the GO terms ‘transcription’, ‘protein ubiquitination’, ‘protein transport’ and ‘cell cycle process’ for biological processes (Fig. 5b). Pathway analysis based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database showed that the miRNAs with decreased expression were associated with cancer, protein processing, autophagy and other pathways (Fig. 5c). Using RT-qPCR analysis, we confirmed that Fam222B-siRNA decreased the expression of miR-10a, let-7b, and let-7c in B16 cells (Fig. 5d). The blood levels of these miRNAs, which exert anticancer effects^{30, 31–32}are increased due to delphinidin intake¹⁷. MiR-23a, whose expression is increased in skeletal muscle due to delphinidin intake¹⁶was also suppressed by Fam222B-siRNA (Fig. 5d).

Fig. 5 [Images not available. See PDF.]

Fam222B suppression inhibits miRNA expression. (a) MiRNA microarray analysis was performed to compare the miRNA expression profile in control and Fam222B-silenced B16 cells. The red diagonal line marks the 1:1 ratio of control-siRNA and Fam222B-siRNA samples. (b) GO analysis of down-regulated miRNAs. (c) KEGG pathway analysis of down-regulated miRNAs. (d) B16 cells were transfected with Fam222B-siRNA, and miRNA expression was measured using RT-qPCR after 48 h (n = 3). Statistical analysis was conducted using Student’s t-test. Data are presented as mean ± SEM. *P < 0.05 and **P < 0.01.

Discussion

The purpose of this study was to identify the underlying molecular factor involved in the mechanism of delphinidin activity. Results showed that delphinidin could upregulate the expression of let-7b in melanoma cell lines and melanoma tumors, with let-7b inhibition effectively eliminating the antimelanoma effects of delphinidin. Using selection by GSEs, a B16 cell clone was generated, in which the introduced genetic element conferred protecting against delphinidin, enabling the identification of Fam222B molecule, associated with delphinidin activity. Subsequently, the role of Fam222B in the antimelanoma cell proliferation activity of delphinidin was confirmed in B16 cells.

Cell growth inhibition is associated with the progression of numerous cell signaling pathways that lead to cell cycle arrest. Cell cycle regulation by plant polyphenols has received considerable attention. Currently, a large number of flavonoids have been shown to prevent the cell cycle activation of cancer cells³³. For example, delphinidin could induce cell cycle arrest at the G2/M phase in breast³⁴ and colon cancer cell lines³⁵. In this study, we observed that delphinidin inhibited B16 cells proliferation and reduced cyclin D1 protein levels (Fig. 1c). Thus, delphinidin may prevent cell growth by inducing the G2/M arrest of melanoma cells. We also revealed that let-7b is involved in the mechanism by which delphinidin suppresses cyclin D1 expression (Fig. 2e). The expression of let-7 family members is essential for tumor suppression, and the regulatory process involves numerous factors³⁶. The RNA-binding protein LIN28 is a key factor that regulates the expression of let-7 family members by suppressing their maturation process³⁷. Furthermore, let-7 precursors bound to LIN28 can be oligouridylated and degraded by terminal uridyltransferases³⁸. Delphinidin may affect the expression of let-7b by acting on these upstream factors.

Our data suggest that Fam222B is essential not only for the inhibitory effects of delphinidin in melanoma cell proliferation but also for the modulation of let-7b expression. Although the specific function of Fam222B remains unknown, Fam222B may be an important molecule involved in the biological activity of delphinidin. Many studies have demonstrated the relationship between oxidative stress and melanoma development. In melanoma cells, signaling pathways that are involved in various cellular processes are regulated by high reactive oxygen species (ROS) levels³⁹. Anthocyanidins have antioxidant properties⁴⁰and delphinidin is a ROS scavenger in various cells^41,42. However, ROS have been reported to downregulate cyclin D1 expression and induce cell cycle arrest⁴³. Because delphinidin suppressed cyclin D1 protein levels through Fam222B, we predict that the Fam222B-mediated anticancer effect of delphinidin is separate from its antioxidant activity. Since delphinidin may regulate let-7b expression without altering Fam222B expression, it is possible that it affects the activity or localization of Fam222B or that additional molecules are involved. Further studies using in vivo models are needed to elucidate the Fam222B-mediated effects of delphinidin. Moreover, in vivo studies should be performed using Fam222B-knockout mice to clarify the direct relationship between Fam222B and delphinidin bioactivities.

Typically, the amount of miRNA expression is the result of transcription and processing. Several molecules that bind to miRNA promoter regions and regulate miRNA expression have also been identified. c-Myc has been reported to directly regulate the expression of miR-9, miR-15a/16 − 1, and the miR-17–92 cluster⁴⁴and p53 can bind to the promoter element of specific miRNAs and upregulate their expression⁴⁵. In the nucleus, miRNAs are transcribed as primary miRNAs from DNA, and these primary miRNAs are processed into precursor miRNAs by the RNase Drosha⁴⁶. In the cytoplasm, precursor miRNAs are further cleaved into mature miRNAs by another RNase Dicer⁴⁷ and finally assembled into miRNA-induced silencing complexes. RNA-binding proteins are important factors that determine the functions of miRNAs because they control the biogenesis, localization, and stability of miRNAs. RNA-binding proteins, including p68 and p72, have been found to interact with miRNA processing complexes and control miRNA maturation^48,49. Fam222B silencing altered the expression of miRNAs (Up: 25, Down: 337), indicating that Fam222B may be involved in the transcription, processing, or stability of miRNAs, including let-7b. Moreover, knockdown of Fam222B suppressed miR-23a expression (Fig. 5b), indicating that Fam222B may be involved in delphinidin’s effects in regulating miRNA expression and suppressing muscle atrophy. The protein structure is closely linked to its function. Thus, it may be possible to understand the relationship between Fam222B and miRNA by analyzing the structure of the Fam222B protein. Several techniques are needed to determine the structure of the Fam222B protein, including X-ray crystallography and nuclear magnetic resonance.

In summary, these findings indicate that Fam222B and let-7b are important efficacy determinants of the anticancer activity of delphinidin. We hope that our study will help in understanding the biological mechanism behind the activity of delphinidin.

Materials and methods

Cell culture

Mouse melanoma B16 cells, human melanoma cell lines (A375, Hs294t, and MeWo), and normal human epidermal melanocytes (NHEMs) were obtained from the American Type Culture Collection (Manassas, VA, USA). B16 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) supplemented with 5% fetal bovine serum (FBS, Gibco, Waltham, MA, USA), whereas other cells were cultured in DMEM with 10% FBS. All cells were maintained at 37 °C in a humidified atmosphere of 5% CO₂.

Reagents

Delphinidin (≥ 97% pure) was purchased from Extrasynthese (Genay, France). Anti-β-actin antibody, hsa-let-7b-5p inhibitor, negative control inhibitor, mmu-Fam222B-siRNA, and negative control-siRNA were obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-cyclin D1 and anti-Fam222B antibodies were procured from Santa Cruz (Heidelberg, Germany) and Abcam (Cambridge, UK), respectively.

Cell proliferation assay

B16 cells were seeded in a 24-well plate at a density of 1 × 10⁴ cells/mL. After preculture, the cells were treated with the indicated concentrations of delphinidin in a medium containing 5% FBS. Subsequently, the cells were harvested from the wells using trypsin and then counted using a hematocytometer.

Western blot analysis

After delphinidin treatment, the total protein was isolated from B16 cells using a cell lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM ethylenediaminetetraacetic acid, 50 mM NaF, 30 mM Na₄P₂O₇, 1 mM phenylmethanesulfonyl fluoride, 2 µg/mL aprotinin, and 1 mM pervanadate. Western blot analysis was performed as described in a previous study¹⁶.

Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR)

Total RNA was isolated from cell or tumor samples using the TRI Reagent (Cosmo Bio Co., Tokyo, Japan). Complementary DNA (cDNA) was prepared from total RNA using the PrimeScript® RT Reagent Kit (Takara Bio Inc., Shiga, Japan) to analyze mRNA expression. RT-qPCR was performed on a CFX96 real-time PCR analysis system (Bio-Rad Laboratories, Hercules, CA, USA) using the SsoAdvanced™ Universal SYBR Green Supermix. The following specific primers were used: mouse Fam222B, forward 5′- CCAGCAATAAGGGTCCAAGCA-3′ and reverse 5′-TGCCCATAAACCACCACAGGTA-3′; mouse β-actin, forward 5′-CATCCGTAAAGACCTCTATGCCAAC-3′ and reverse 5′-ATGGAGCCACCGATCCACA-3′. Fam222B mRNA expression was normalized to β-actin.

For miRNA detection, total RNA was reverse transcribed to synthesize cDNA using the miRCURY LNA^™ RT Lit (QIAGEN, Hilden, Germany). Subsequently, RT-qPCR was performed using the miRCURY LNA^™ SYBR Green PCR Kit (QIAGEN) on a CFX96 real-time PCR analysis system (Bio-Rad). The LNA™ PCR primer mix, miR-10a-5p (#YP00204778), miR-23a-3p (#YP00204772), let-7b-5p (#YP00204750), and let-7c-5p (#YP00204767) were purchased from QIAGEN. In this study, the expression of let-7b was normalized with that of U6 (QIAGEN, #YP00203907).

Animal experiment

Four-week-old C57BL/6J male mice were obtained from Kyudo Co., Ltd. (Saga, Japan) and then acclimated for 1 week. The mice were subcutaneously injected on the back with 5 × 10⁵ B16 cells suspended in 100 µL of phosphate-buffered saline (PBS). Subsequently, vehicle (dH₂O) alone or in combination with delphinidin (20 mg/kg of body weight) was orally administered 15 days after injection. Then, the RNAs of melanoma tumors were extracted from samples 48 h after administration. The animal experiment was conducted in accordance with the Law (No. 105) and Notification (No. 6) of the Japanese government and the Animal Research: Reporting of In Vivo Experiments guidelines (https://arriveguidelines.org). The study protocol was approved by the Animal Care and Use Committee of Kyushu University (approval number A30-041).

GSE library transduction and delphinidin selection

A library was prepared as described in a previous study²³. In brief, AmphoPack-293 and EcoPack2-293 cells (Clontech, Palo Alto, CA, USA) were transfected with the GSE library using FuGene 6 (Roche Diagnostics, Mannheim, Germany). The retrovirus-containing medium was harvested from AmphoPack-293 and EcoPack2-293 cells after 12, 24, 36, and 48 h. The culture medium was filtered through a 0.45-µm membrane, and B16 cells were incubated with retrovirus particle-packaged GSEs with 8 µg/mL of Polybrene (Sigma-Aldrich). Infection was performed four times at 12-h intervals. Subsequently, B16 cells were cultured and incubated with 50 µM delphinidin for 4 weeks. The cells grown at delphinidin concentrations that inhibited cell proliferation were harvested. cDNA was synthesized from total RNA using Moloney mouse leukemia virus-reverse transcriptase (Amersham Biosciences, Uppsala, Sweden) with oligo(dT)20 and reverse pLPCX primers. The product was amplified by PCR using TaKaRa Ex Taq polymerase (Takara Bio). The pLPCX primer sequences were as follows: forward 5′-GATCCGCTAGCGCTACCGGACTCAGAT-3′ and reverse 5′-CTTTCATTCCCCCCTTTTTCTGGAGAC-3′. Subsequently, the PCR fragments were subcloned into the pTARGET (Promega, Madison, WI, USA) vector and identified by DNA sequencing.

RNA transfection

MiRNA inhibitor or siRNA was introduced into B16 cells using the Lipofectamine™ RNAiMAX Kit (Invitrogen, Carlsbad, CA, USA). Before being added to the cells, DMEM, RNA reagents, and RNAiMAX were mixed with a pipette and then set for 10 min at room temperature.

MiRNA microarray

Microarray analysis on the 3D-Gene® Mouse miRNA Oligo Chip was outsourced to Toray Industries, Inc. (Tokyo, Japan). Briefly, the RNA samples were extracted from B16 cells using TRI Reagent. The RNA concentration was measured using the NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and RNA fluorescence labeling was performed using the 3D-Gene® miRNA labeling Kit (Toray Industries Inc.). The 3D-Gene® Scanner (Toray Industries Inc.) was used for scanning.

GO and pathway analysis

The GO and pathway annotation of the downregulated miRNA proceeded through miRPath v4.0 (http://62.217.122.229:3838/app/miRPathv4). Figure 5B and C display the top 10 outcomes from the GO biological process and KEGG pathway⁵⁰ analyses.

Statistical analyses

Data were presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism software (version 7). We used Student’s t-test or one-way ANOVA followed by Dunnett’s test, according to the design of each experiment (shown in figure legends). Statistical comparisons at P < 0.05 indicated with asterisks were considered significant.

Acknowledgements

We thank Enago (www.enago.jp) for the English language editing.

Author contributions

M.M., Y.F. and H.T. contributed to the study conception and design. M.M., Y.M., S.Y., I.L., S.Y. and M.K. performed data collection and analysis. M.M., Y.M. and H.T. prepared the manuscript. All authors read and approved the final manuscript.

Funding

This study was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI grant number JP20H05683 and JP 25H00923 (to H. Tachibana) and JP17H06936 (to M. Murata).

Data availability

The microarray data presented in this study have been deposited in the Gene Expression Omnibus (GEO) repository, accession number GSE289508. Other datasets are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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