Histone deacetylase HDAC2 regulates microRNA‐125a expression in neuroblastoma

Abstract Background Neuroblastoma (NB) is an infrequent childhood malignancy of the peripheral sympathetic nervous system and is accountable for about 10% of pediatric tumors. microRNA (miR)‐125a has been implicated to serve as a tumor suppressor in various cancers. Herein, we set out to ascertain whether miR‐125a exerts antitumor effects in NB. Methods Downregulated miRNAs were identified by miRNA microarray analysis of NB tissues and paracancerous tissues. The expression of miR‐125a in NB tissues and cells was detected by reverse transcription‐quantitative (RT‐q) PCR, followed by prognostic analysis. Gene Ontology (GO) enrichment analysis was performed on target genes of differentially expressed miRNAs. Cell proliferation, apoptosis, and differentiation were detected by cell counting kit‐8 (CCK‐8), Hoechst staining, immunofluorescence, and western blot. NB cells were injected into nude mice to detect tumorigenic, apoptotic, and differentiation activities in vivo. Dual‐luciferase assay and chromatin immunoprecipitation (ChIP) were carried out to verify the binding relationship between miR‐125a and PHOX2B or histone deacetylases 2 (HDAC2), respectively. Finally, rescue experiments were conducted. Results miR‐125a was downregulated in NB tissues and cells, which was associated with poor prognosis. miR‐125a reduced NB cell proliferation and augmented apoptosis and differentiation. NB cells with miR‐125a overexpression decreased cell tumorigenesis and increased apoptosis and differentiation in xenograft tumor tissues. miR‐125a targeted PHOX2B, which was highly expressed in NB tissues and cells. HDAC2, highly expressed in NB tissues and cells, repressed miR‐125a transcription through histone deacetylation. Overexpression of HDAC2 or PHOX2B rescued the effects of miR‐125a on NB cell proliferation, apoptosis, and differentiation. Conclusion HDAC2 inhibited miR‐125a transcription through deacetylation, and miR‐125a suppressed NB development through binding to PHOX2B.


INTRODUCTION
Neuroblastoma (NB), the most common extracranial malignancy of childhood and the most common malignancy in infants, is an embryonal tumor of the sympathetic nervous system, developing during fetal or early postnatal time from sympathetic cells derived from the neural crest (Davidoff, 2012). NB is the major cause of death of children between the ages of 1 and 5 years and is responsible for about 13% of all mortality for pediatric cancers (Louis & Shohet, 2015). The most well-known prognostic factors for NB are age, stage, and MYCN amplification, and more than half of children diagnosed with high-risk NB either do not respond to conventional therapies or relapse after treatment (Berlanga et al., 2017). Therefore, understanding the molecular mechanism underlying NB is helpful to improve the prognosis of patients.
MicroRNAs (miRNAs) are small single-stranded RNAs that target messenger RNAs (mRNAs) at the posttranscriptional level by inhibiting translation within all facets of human physiology, and miRNAs are currently used as biomarkers for prognosis and tumor characterization in multiple cancers, including NB (Zammit et al., 2018). In mammalians, miR-125a is present in most adult organs and tissues where it targets proteins related to the mitogenic response, and the antiproliferative properties of miR-125a, together with the fact that this miRNA is downregulated in many cancers, give a substantial support to the notion that miR-125a exerts an onco-suppressive effect (Russo & Potenza, 2019). Moreover, epigenetic mechanisms, mainly including DNA methylation, posttranslational histone modifications, as well as noncoding RNAs, tightly control gene expression, embryogenesis, and tumorigenesis, thus playing a vital role in both physio-and pathological settings (Vogelstein et al., 2013). Acetylation is one of the main posttranslational protein modifications with various effects on the protein level and the metabolome level, and the interplay between acetylation and deacetylation is of great importance for cellular processes (Drazic et al., 2016). The role of histone deacetylases (HDACs) has been defined by shedding light on the possible mechanisms involved in NB tumorigenesis, acting as potential markers and novel therapeutic options (Fetahu & Taschner-Mandl, 2021). For one of them, HDAC2 was enhanced by MYCN, which in turn elevates stability and protein expression of MYCN, thereby describing a positive feedback loop (Kim & Carroll, 2004). Interestingly, HDAC2 has been reported to promote the development of tamoxifen resistance in breast cancer cells by downregulating miR-125a-5p . However, the possible correlation between HDAC2 and miR-125a remains unclear in NB.
In this study, we sought to identify and functionally characterize the role of miR-125a in NB, linking its downregulation to HDAC2.

Subjects
The study was approved by the ethical committee of the Hunan Children's Hospital and was in accordance with 2011 Declaration

miRNA microarray analysis
Tumor and adjacent tissues from three patients were randomly selected for gene expression analysis, and RNA was extracted using

Bioinformatics analysis
Differentially expressed genes-mediated biological processes in NB were analyzed using by Gene Ontology (GO) enrichment analysis.
GO enrichment analysis of miRNA target genes was performed in

Western blot
Cells were lysed in radio immunoprecipitation assay buffer ( Plus-ECL (Perkin Elmer) and analyzed using LAS-3000 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) to assess relative bands and molecular weights relative to standard molecular weight markers.

Immunohistochemistry
The mouse xenograft tumors were fixed in paraformaldehyde for 1 h, followed by conventional paraffin-embedding and sectioning (4 μm), and drying at 60°C. Paraffin sections were dewaxed with xylene and graded ethanol, and rinsed with distilled water. The slides were boiled in sodium citrate buffer (pH = 6.0, Solarbio, Beijing, China) for 10 min for antigen recovery. The sections were incubated in 3% hydrogen per-

Chromatin immunoprecipitation
The promoter of miR-125a was obtained from UCSC (https://genome.

Statistics
The

miR-125a is significantly poorly expressed in NB
Microarray analysis was performed using miRNA probes to identify miRNAs with tumor suppressor functions in NB, and miR-125a was identified to be significantly reduced in NB (Figure 1a). To probe the function of miR-125a in NB, we tested its expression in NB tissues.
The data presented that miR-125a was remarkably downregulated in NB tissues (n = 43) compared with adjacent tissues, which was the same as the microarray results (Figure 1b). Further analysis revealed that miR-125a was significantly reduced in NB cell lines SK-N-BE(2),  (Figure 1e), demonstrating that low miR-125a expression was closely linked to poor patient prognosis. The GO enrichment analysis of the differentially expressed miRNAs revealed that the differentially expressed miRNAs in NB were mainly enriched in apoptosis and cell differentiation processes, and miR-125a was highly enriched in these processes (Figure 1f), indicating that miR-125a mainly regulates NB cell activity through these pathways.

miR-125a reduces activity in NB cell lines
The effect of miR-125a on NB cell activity was examined according

miR-125a reduces the proliferation of NB cells in vivo
We verified the ability of miR-125a to reduce tumor growth in vivo by subcutaneous injection of SK-N-DZ and SK-N-AS cells previously transfected with miR-125a mimic, and tumor growth was monitored every 7 days. Overexpression of miR-125a delayed the growth of NB cells in vivo, resulting in a reduction of xenograft tumor volume and growth rate in mice (Figure 3a). Also, analysis of tumor weights confirmed the inhibition of xenograft tumors by miR-125a overexpression ( Figure 3b). The detection of cleaved-caspase-3 and β-Tubulin-III in tumors revealed that miR-125a increased apoptosis and differentiation (Figure 3c,d). Verification of miR-125a expression in tumors displayed that miR-125a was consistently expressed in tumors (Figure 3e), indicating that miR-125a overexpression reduced tumorigenic activity in NB cells and induced apoptosis and cell differentiation.

miR-125a targets PHOX2B
According to the gene transcription analysis between NB patients and healthy young donors in the GEO database GSE25624, 120 genes were upregulated and 21 genes were downregulated in NB (Figure 4a).
GO enrichment analysis of the differentially expressed genes revealed that 12 genes were mainly enriched in apoptosis and cell differentiation processes (Figure 4b). Since we obtained genes among miR-125a target genes enriched in apoptosis and cell differentiation process in Figure 1f, we compared these genes with those in Figure 4b and found that the overlapping gene was PHOX2B (Figure 4c). We then suggested that PHOX2B might be the gene mediated by miR-125a in NB.
To further prove this hypothesis, we first verified the binding sites between miR-125a and PHOX2B by a dual-luciferase assay and found that miR-125a mimic could indeed lead to a reduction in fluorescence intensity of PHOX2B-WT plasmid (Figure 4d). The expression of PHOX2B in NB tissues was found to be significantly elevated (Figure 4e) and negatively correlated with miR-125a expression (Figure 4f).
Detection of PHOX2B expression in cells revealed that PHOX2B was highly expressed in NB cell lines and significantly reduced by miR-125a induction (Figure 4g).

miR-125a expression is mediated by HDAC2
After probing the downstream gene of miR-125a, we revisited the results of Figure 4b and found an enrichment of HDACs in Figure 4c.
We were curious whether epigenetic modifications play a role in medi- miR-125a enrichment using antibodies to HDAC2 and H4ac was stronger than that using IgG ( Figure 5c). Meanwhile, HDAC2 enrichment was decreased in SK-N-DZ and SK-N-AS cells treated with BRD5298, while H4ac enrichment was increased (Figure 5d). The expression of HDAC2 was detected in NB tissues, and HDAC2 was found to be significantly elevated in NB tissues. Correlation analysis between HDAC2 and miR-125a showed a significant negative correlation ( Figure 5e). Detection of HDAC2 expression in NB cells revealed that it was significantly higher in NB cells (Figure 5f). Using HDAC2-OE to transfect NB cells, we observed that miR-125a was significantly reduced (Figure 5g), indicating the transcriptional repression of miR-125a by HDAC2 during the NB.

HDAC2/miR-125a/PHOX2B affects NB activity in vitro
To confirm whether PHOX2B is a key target of miR-125a and miR-125a is a downstream regulator of HDAC2, we tested whether ectopic expression of PHOX2B or HDAC2 could counteract the effect of miR-125a on NB cellular activity. We transfected NB cells with fragments expressing HDAC2 and PHOX2B in miR-125a overexpressing cells. Western blot assays of HDAC2 and PHOX2B protein expression in cells showed that both HDAC2-OE and PHOX2B-OE were successfully transfected (Figure 6a). The result of Hoechst staining showed that the pro-apoptotic effect of miR-125a on NB cells was partially neutralized by the overexpression of HDAC2 and PHOX2B (Figure 6b).

The observation of cell morphology and differentiation demonstrated
that the differentiation of cells under the influence of miR-125a was inhibited by HDAC2 and PHOX2B, and the expression of β-Tubulin-III in cells was reduced (Figure 6c). Finally, the quantification of protein levels in cells revealed that the pro-apoptotic protein expression was reduced under the influence of HDAC2, and the differentiationrelated protein expression was also diminished. Cells overexpressing PHOX2B showed the same trend as these overexpressing HDAC2 ( Figure 6d). It was demonstrated that both HDAC2 and PHOX2B were able to inhibit the effect of miR-125a to increase NB cell activity again.

HDAC2/miR-125a/PHOX2B affects NB activity in vivo
In the last part, we set out to evaluate the functional effects of HDAC2, miR-125a, and PHOX2B on the NB progression. The upregulation of

DISCUSSION
Significant progress has been achieved to understand the molecular mechanisms related to the etiology and pathogenesis of NB, and genome-wide association investigation, transcriptomics, genome sequencing, and high-throughput genome analysis have revealed genetic alterations and impaired pathways, which hold accountable for NB growth and development (Zafar et al., 2021). Moreover, the significance of miRNAs in the mediation of cell survival, proliferation and differentiation, and their involvement in the pathogenesis of NB has been appreciated in a recent review (Rezaei et al., 2021). In the present study, we found through microarray analysis that miR-125a was downregulated in NB, which was associated with short survival of NB patients and resistance to cell apoptosis and differentiation.
Specifically, miR-125a overexpression induced NB cell apoptosis and differentiation but suppressed tumor growth in vivo. We also found that miR-125a targeted and negatively regulated PHOX2B expression.
Further, the downregulation of miR-125a in NB cells was related to HDAC2 overexpression.
Consistently, we observed that miR-125a overexpression enhanced the expression of Bax, cleaved-caspase-3, GAP43, and NSE in NB cells, indicating the supporting role of miR-125a on NB cell apoptosis and differentiation. Cannabidiol was found to partially counteract the depletion of GAP43 and β-Tubulin-III, thus alleviating the injury of NB SH-SY5Y cells (Branca et al., 2019). Also, increased apoptosis and cleaved-caspase-3 were observed in mouse granulosa cells transfected with a miR-125a-5p mimic (Wang et al., 2016). This was largely in agreement with our in vitro and in vivo results regarding the assessment of cleaved-caspase-3 expression.
Integrated bioinformatics prediction and dual-luciferase assay revealed that PHOX2B is a putative target of miR-125a in NB, while HDAC2 binds to miR-125a to negatively regulate its expression. Thwin et al. (2020) revealed that PHOX2B was one of the core genes upregulated in bone marrow of patients with NB relative to the peripheral blood sample. Furthermore, PHOX2B expression in bone marrow aspirate could be a biomarker for NB patients at high risk and with poor response to treatment (El-Shazly et al., 2019). However, its functional role in NB needs to be clarified. Interestingly, Bachetti et al. (2015) found that miR-204 mediated downregulation of PHOX2B expression in NB cells posttranscriptionally. This suggests that PHOX2B, regulated by a certain miRNA, could involve in the apoptosis and differentiation in NB. Our rescue experiments validated this hypothesis.
Aberrant HDAC recruitment and expression and dysregulated H4ac have been described for tumor cells (Witt et al., 2009). In this study, it was noted that miR-125a expression only responded to HDAC2 inhibitor among three different HDAC inhibitors. The ChIP assay further substantiated that inhibition of HDAC2 expedited the H4ac, thus upregulating the expression of miR-125a. Overexpression of HDAC2 was highly correlated with high tumor grade, positive lymph node status, and dismal prognosis, and the HDAC inhibitor showed antitumor effects on breast cancer lines by mediation of miR-182 (Shan et al., 2017). Suppression of HDAC8 increased the doxorubicin sensitivity of NB cells via upregulation of miR-137 (Zhao et al., 2017), which highlights the involvement of HDAC in NB progression. Lodrini et al. (2013) reported that HDAC2 depletion enhanced promoter-associated histone H4ac and the following miR-183 expression, indicating epigenetic changes preceded transcriptional activation in NB. In addition, silencing of HDAC3 inhibited PAK6 expression by elevating miR-27a, ultimately inhibiting neuronal apoptosis and accelerating the recovery of spinal cord injury (Zhou et al., 2020). In the present study, we found that HDAC2 counteracted the stimulative effects of miR-125a on NB cell apoptosis and differentiation.

CONCLUSION
In conclusion, this study found that HDAC2 expression was significantly upregulated in NB cells, which inhibited miR-125a and upregulated PHOX2B expression in an H4ac-dependent manner. In addition, the study suggested that targeting miR-125a may be a therapeutic candidate for NB patients. Other pathways of miR-125a regulation will be explored in future studies.