GATA binding protein 1 recruits histone deacetylase 2 to the promoter region of nuclear receptor binding protein 2 to affect the tumor microenvironment and malignancy of thyroid carcinoma

ABSTRACT The tumor microenvironment (TME) and activated angiogenesis in thyroid carcinoma (TC) are critical for tumor growth and metastasis. Nuclear receptor binding protein 2 (NRBP2) has been suggested as a tumor suppressor. This study examines the function of NRBP2 in the progression of TC and the regulatory mechanism. By analyzing bioinformatic tools including GSE165724 dataset and the Cancer Genome Atlas system, we predicted NRBP2 as a poorly expressed gene in TC. Decreased NRBP2 expression was detected in TC tumor tissues and cells. Poor expression of NRBP2 was linked to unfavorable prognosis of patients. GATA binding protein 1 (GATA1) was found as a negative regulator of NRBP2. It recruited histone deacetylase2 (HDAC2) to the NRBP2 promoter to trigger histone deacetylation. NRBP2 overexpression suppressed growth of TC cells, and it reduced expression of TME markers, M2 polarization of macrophages, and angiogenesis in TC. Similar results were reproduced in vivo in nude mice. However, the anti-oncogenic roles of NRBP2 were blocked after further overexpression of GATA1 or HDAC2. In summary, this study demonstrates that GATA1 recruits HDAC2 to the NRBP2 promoter and enhances the TME and angiogenesis in TC cells.


Introduction
Thyroid carcinoma (TC or THCA) represents the ninth most common cancer among all cancer types [1] and the most prevalent endocrine cancer as a neoplasm of the thyroid epithelium [2]. Ionizing radiation, especially in childhood, is the only wellrecognized risk factor of TC [1]. Papillary TC (PTC), arising from the follicular cells, is the most frequent type that makes up about 80% of all cases [2]. The prognosis of patients with well-differentiated PTC was favorable with the 5-year survival rate reaching 97.5%, but the poorly differentiated types and anaplastic carcinomas are aggressive and lethal [3][4][5]. Tumor microenvironment (TME) plays a critical role in tumor migration and invasion [6]. Identifying novel molecules involved in TME maintenance and TC development may provide new biomarkers for improved risk prediction and help develop therapeutic options.
Advanced bioinformatic analytical systems have provided considerable benefits for researchers to identify crucial molecules implicated in the progression of human diseases including cancers [7,8]. In the present study, the microarray analysis using the TCrelated gene expressing dataset GSE165724 from Gene Expression Omnibus (GEO; https://www.ncbi. nlm.nih.gov/geo/) suggested that nuclear receptor binding protein 2 (NRBP2) is significantly downregulated in TC tissues. The NRBP family initially participate in the transport between the endoplasmic reticulum and Golgi [9]. Moreover, the NRBPs are suggested to play tumor suppressive role consistently [10]. However, the function of NRBP2 in the progression of TC remains not clear.
Gene alterations by genetical and epigenetic regulations are frequently involved in almost every stage of cancers including TC [11]. In this work, the bioinformatics analysis using JASPAR (http://jas par.genereg.net/) suggested GATA binding protein 1 (GATA1) as a candidate negative regulator of NRBP2. GATA1 is a master transcription factor in erythropoiesis which exert key functions in regulating proliferation, differentiation, and death of erythroid cells [12]. Recent evidence suggested the oncogenic role of GATA1 in human malignancies [13,14]. Epigenetic mechanisms such as acetylation and methylation modifications that control transcriptional dysregulation in cancer development have aroused increasing concerns [15]. Overexpression of GATA1 has been observed to interact with the histone methyltransferase SET7 and to augment vascular endothelial growth factor (VEGF)-induced angiogenesis [16]. Interestingly, data in the UCSC browser (https://genome.ucsc. edu/index.html) demonstrated that there is significant acetylation of histone H3 lysine 9 (H3K9ac) in the promoter region of NRBP2. The histone H3 lysine 9 (H3K9) is a widely studied acetylation site producing H3K9ac, which is essentially correlated with transcriptional activation in cells [17]. However, as mentioned above, NRBP2 was predicted to be poorly expressed in TC. Could this be caused by reduced H3K9ac level in the promoter of NRBP2? We therefore hypothesized that GATA1 possibly interacts with specific histone deacetylases (HDACs) to reduce transcriptional activity of NRBP2, which participates in the progression of TC.

Clinical samples
From February 2016 to October 2017, 71 patients with TC (27-81 years old, male: female = 2:5) admitted into the First Affiliated Hospital of Henan University of Science and Technology were enrolled into this research. The patients had low echogenicity, calcification and unclear border on the interior of the nodule by transthyroid ultrasound. According to the ACR Thyroid Imaging, Reporting and Data System (TI-RADS) scores [18], all patients were categorized in the TR3-TR5 grade, and they were diagnosed as TC according to the pathologic diagnosis. The fresh TC tumor tissues and the para-tumorous tissues were harvested during surgery and instantly frozen in liquid nitrogen. The research was approved by the Ethical Committee of the First Affiliated Hospital of Henan University of Science and Technology (Approval No. 2015.12.15) and adhered to the Declaration of Helsinki. Each participant signed the informed consent form.

Bioinformatic analysis
The TC-related GEO dataset GSE165724 comprises 16 TC tissues and 12 healthy control tissues. Genes with differential expression between tumor and normal tissues were identified with Log Fold Change < −2 and adjusted p value < 0.01 as the screening thresholds. The data were analyzed using an R limma Package, and the heatmap was produced using the R Volcano package.

Cell transfection
Overexpression vectors of NRBP2, HDAC2 and GATA1 (oe-NRBP2, oe-HDAC2 and oe-GATA1) , the short hairpin (sh) RNA of NRBP2 and GATA1 (sh-NRBP2 and sh-GATA1), and the negative control (NC) vectors (oe-NC and sh-NC) were respectively transfected into TPC-1 and CAL62 cells using the pCMV6-AC-GFP vector (FH1215, Fenghui Biotechnology, Hunan, China). The overexpression plasmids and shRNA fragments were procured from Sigma-Aldrich Chemical Company (Merck KGaA, Darmstadt, Germany). In short, the target fragments and vectors were digested overnight at 37°C and then ligated with T4 DNA lignase. After that, 10 μL ligation product was added into 100 μL DH5α competent cells (TianGen Biotech Co., Ltd., Beijing, China). After concentration and centrifugation, the positive colony was identified by colony PCR. The vectors were extracted using a GenElute™ plasmid miniprep kit (PLN350, Sigma-Aldrich). Cell transfection was performed utilizing a Lipofectamine 2000 kit (11668019; Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA). In brief, 1 × 10 5 cells were incubated in 24-well plates for 24 h before transfection. Thereafter, 1 μL Lipofectamine 2000 was added to the centrifugation tube with serum-free RPMI 1640 and mixed to prepare 25 μL diluted transfection reagent at a concentration of 25 nM. The vector solution for transfection and the dilution were loaded into centrifugation tubes and allowed to stand for 15 min until complete mixing. After that, the transfection compounds were loaded into cells in 0.45 mL medium for 6 h, and the cells were cultured in RPMI-1640 for 48 h. The transient transfection efficiency was identified by reverse transcription quantitative polymerase-chain reaction (RT-qPCR). Stably transfected cells were screened after culture with 1 μg/mL puromycin for 48 h. Later, the cells were harvested and the transfection efficacy was determined.

RT-qPCR
Total RNA was extracted using the TRIzol reagent (15596018, Invitrogen). The RNA concentration was examined by a NanoDrop TM Lite Spectrophotometer (Thermo Fisher Scientific), and a TaqMan PrimeScript RT kit (RR047A, Takara Holdings Inc., Kyoto, Japan) was used for RNA reverse transcription. After that, qPCR was conducted on an ABI 7500 qPCR system (Applied Biosystems, Inc., Carlsbad, CA, USA). Table 1 lists the sequence information of primers. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal loading. Gene expression value was analyzed using the 2 −ΔΔCt method [19].

5-ethynyl-2'-deoxyuridine (EdU) labeling assay
Cell proliferation was analyzed using the EdU kit (C10310-2, RiboBio Co., Ltd., Guangzhou, Guangdong China). In short, transfected cells were cultured in 12-well plates for 36 h. After that, the cells were cultured in serum-free medium containing EdU (50 μM) for 2 h, washed with phosphate-buffered saline, and fixed for 30 min. After that, the cells were stained with Apollo and Hoechst 33,342 in the dark for 30 min. The staining images were captured under a microscope (Olympus, Tokyo, Japan) with five random fields of views included [21].

Colony formation assay
Exponentially growing TPC-1 or CAL-62 cells were adjusted to 10 3 cells/mL in the culture medium. After that, 1.5 mL cell suspension was seeded in culture dishes mixed with 5% agar and culture medium at a ratio of 1:9. The cells were cultured at 37°C with 5% CO 2 for 2 weeks. After that, the cells were fixed for 30 min and stained with 0.1% crystal violet for 3 min. The colonies were observed and counted under microscopy [22].

Co-culture with macrophages
Transfected TPC-1 and CAL62 cells were loaded into the Transwell upper wells. Wells filled with RPMI-1640 were set as controls. THP-1 cells (American Type Culture Collection, Manassas, VA, USA) were induced with phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich) to obtain the phenotype of M0 macrophages, which were loaded into the basolateral chambers. After 48 h, relative mRNA expression of CD206 and CD163 (macrophage biomarkers) in the macrophages was examined using RT-qPCR. The percentage of CD206/CD163-positive cells was examined using flow cytometry [23].

Tube formation assay
Human umbilical vein endothelial cells (HUVECs) (Yiyuan Biotechnology Corporation, Guangzhou, Guangdong, China) were cultured into Matrigelcoated 24-well plates at 2 × 10 4 cells per well and cocultured with the supernatant of TPC-1 and CAL62 cells. After 24 h, the number of vascular branches was examined under a phase-contrast microscope (Olympus) with 5 random fields included [24].

CRISPR-cas9 system
The single-guide RNA (sgRNA) of GATA1 and the NC were obtained from Sigma-Aldrich. The sgRNA was cloned to the Cas-9 plasmid using the restriction endonuclease. The ligation products were transformed into competent cells, which were cultured for 12 h for plasmid monoclonal amplification. Thereafter, the plasmids were screened and extracted, and then transfected into TPC1 and CAL26 cells to construct GATA1-deleted cells and the wild-type (WT) control cells. The primer sequences are as follows: GATA1-promoter-1#: Guide Sequence: GCCCCCATAAGCACTATTG, protospacer adjacent motif: GGG; GATA1-promoter-2#: Guide Sequence: CGCTTCTTGGGCCGGATGA, protospacer adjacent motif: GGG.

Animal experiments
Twenty-four female BALB/c nude mice (4-5 weeks old, 15-18 g) were acquired from SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The mice were allocated into four groups, n = 6 in each. TPC-1 or CAL62 cells transfected with oe-NRBP2 or oe-NC were injected into the mice subcutaneously. Thereafter, the volume (V) of xenograft tumors was examined once per week. On the 36 th day, the mice were sacrificed via intraperitoneal injection of overdosed barbiturate (120 mg/kg). The tumors were weighed and used for further analysis [27]. All animal experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital, and College of Clinical Medicine of Henan University of Science and Technology (Approval No. 2019. 1.19) and adhered to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, Maryland, USA). Significant efforts were made to reduce the usage and suffering of conscious animals.

Chromatin immunoprecipitation (ChIP)-qPCR
A ChIP analysis kit (Cat#53008, Active Motif, Carlsbad, CA, USA) was used. The TC cells were crosslinked in 1% methanol for 10 min and neutralized with glycine for 5 min. After that, the cells were resuspended in SDS lysis buffer, ultrasonicated and centrifuged. The supernatant was diluted in IP dilution buffer. Anti-GATA1 (1:50, #3535, CST), anti-H3K9ac (1:100, GTX128944, Genetex) and IgG (1:1,000, Cat#2729S, CST) were used for IP reaction. Thereafter, the samples were added with Protein A-agarose for 1 h of incubation. The precipitates were washed and de-crosslinked, and the purified DNA was determined by qPCR [29]. The primer sequences are presented in Table 2.

Luciferase assay
The promoter sequence of NRBP2 was obtained from UCSC and sub-cloned to the pGL3 luciferase vector (E1751, Promega, Fitchburg, WI, USA). To confirm the binding between NRBP2 promoter and GATA1 or HDAC2, the WT GATA1 or HDAC2 sequence was cloned into the pGL3 vector to construct pGL3-WT luciferase vectors. Sequence products were gene fragments containing motif-binding sequences, and product lengths were controlled to range from 500 to 600 bp. The putative-binding sequence between NRBP2 promoter and GATA1 was obtained from JASPAR, whereas its H3K9ac binding site with HDAC2 was obtained for UCSC. After that, different doses of pGL3-GATA1-WT or pGL3-HDAC2-WT vectors were transfected with pCMV6-AC-GFP-based oe-GATA1 or oe-HDAC2 into 293 T cells. After 24 h, the cells were collected. The luciferase activity in cells was examined using a dual-luciferase reporter gene system (Promega) [30].

Statistical analysis
Prism 8.01 (GraphPad, La Jolla, CA, USA) and SPSS20.0 (IBM Corp. Armonk, NY, USA) was used for data analysis. Data are presented as the mean ± standard deviation from three repetitions. Differences analyzed by t test, or by one-or twoway analysis of variance followed by Tukey's posthoc comparison. Correlations between gene expression and the clinical characteristics of patients with TC were analyzed by the Fisher's exact test or the Chi-square test. *p < 0.05 represents significant difference.

Starting paragraph
We obtained via bioinformatic analyses that NRBP2 is a downregulated in gene TC tissues and GATA1 a candidate negative regulator of NRBP2. With significant H3K9ac modification predicted in NRBP2

Poor expression of NRBP2 in TC cells is linked to poor prognosis in patients
The TC-related gene expression dataset GSE165724 comprising 16 TC tissues and 12 healthy control tissues was analyzed. By using Log Fold Change < −2 and adjusted p value < 0.01 as the screening thresholds, 834 genes with differential expression in TC tissues were screened (Figure 1(a)). These genes were compared with the data in The Cancer Genome Atlas-Thyroid Carcinoma (TCGA-THCA) (https:// www.cancer.gov/types/thyroid), which suggested that NRBP2 was significantly reduced in TC tissues (Figure 1(b)). Moreover, we obtained the IHC data of NRBP2 in normal thyroid tissues and TC tissues from the HUMAN PROTEIN ATLAS (HPA, https:// www.proteinatlas.org/). It was suggested that the NRBP2 expression is lower in tumor samples (moderate) than that in healthy samples (high) (Figure 1 (c)). After that, we examined the expression of NRBP2 mRNA in the 71 included patients using RT-qPCR and confirmed that NRBP2 was expressed at low levels in the tumor tissues versus the adjacent tissues (adjacent tissue vs. tumor: 9.55 vs. 3.44 (all mean values, the same below); p < 0.0001) (Figure 1 (d)  (Figure 1(g)).

Overexpression of NRBP2 reduces activity of TC cells in vitro
To examine the effect of NRBP2 on TC cell growth, TPC-1 and CAL62 cells which showed the lowest expression of NRBP2 were transfected with oe-NRBP2. The successful upregulation was confirmed by RT-qPCR (oe-NC vs. oe-NRBP2 TPC-1: 1.00 vs.  (Figure 2(e)).

NRBP2 reduces expression of TME markers and angiogenesis in TC cells
To examine the correlation between NRBP2 and the TME in TC, we first examined the levels of TME markers IL-6 and VEGFA in the supernatant of TPC-1 and CAL62 cells. It was observed that NRBP2  (Figure 3(a)). After that, the M0 macrophages were co-cultured with TPC-1 and CAL62 cells, and then the polarization of the macrophages was examined by flow cytometry.  (Figure 3(b)). Moreover,  (Figure 3(d)).

NRBP2 reduces TC tumorigenesis and M2 macrophage infiltration in vivo
The role of NRBP2 in the tumorigenesis of TC in vivo was then explored.  (Figure 4(c)). In addition, the IHC results also suggested that the staining intensity of Arg1 (M2macrophage marker) and CD31 (angiogenesis marker) in cells was reduced after NRBP2 overexpression    (Figure 4(d,e)). These results suggest that NRBP2 reduces TC tumorigenesis and M2 macrophage infiltration in vivo.

GATA1 recruits HDAC2 to suppress NRBP2 expression
The results above preliminarily suggested that GATA1 could transcriptionally suppress NRBP2 expression to induce growth of TC cells and the changes in TME. We then explored whether there are other epigenetic regulations such as DNA methylations and histone modifications that affect NRBP2 expression. Data in the UCSC browser suggested that a histone modification marker H3K9ac is enriched at the NRBP2 promoter ( Figure 6(a)). Thereafter, we examined the H3K9ac level in three pairs of collected tumor tissues and adjacent tissues using anti-H3K9ac. It was found that the H3K9ac level, namely the histone acetylation level, was significantly reduced in tumor tissues (adjacent tissue vs. tumor: Paired 1: 3.34 vs. 0.41, p < 0.0001; Paired 2: 5.16 vs. 0.24. p < 0.0001; Paired 3: 3.97 vs. 0.57, p < 0.0001) (Figure 6(b)). We then speculated that under pathological conditions of TC, aberrant deacetylation modification might reduce the expression of NRBP2 and therefore promote the onset and development of TC. To validate this, a HDACsspecific inhibitor Tacedinaline was administrated into TPC-1 and CAL62 cells. It was observed that after the HDAC inhibition, the NRBP2 mRNA and protein levels in cells were significantly enhanced (mRNA: DMSO vs. Tacedinaline (Figure 6(c,d)). Linking this to the results in Figure 5, we hypothesized that GATA1 possibly recruits certain HDACs to the promoter region of NRBP2 to suppress its expression. Therefore, a Co-IP assay was performed where anti-GATA1 was introduced into TPC-1 and CAL62 cells for IP. After that, the western blot analysis identified HDAC2 expression in the precipitated compounds pulled down by anti-GATA1, whereas no expression of HDAC1 or HDAC3 was detected ( Figure 6(e)). To examine the regulation of HDAC2 on NRBP2 expression, the luciferase vector containing NRBP2 promoter sequence was co-transfected with oe-HDAC2 into 293 T cells. Importantly, it was found that oe-HDAC2 also reduced the luciferase activity in cells (0 μg: 1.00; 1 μg: 0.74; 2 μg: 0.52; 3 μg: 0.37; 4 μg: 0.16; 5 μg: 0.07; 0 μg vs. 1 μg: p = 0.0001; 0 μg vs. 2 μg: p < 0.0001; 0 μg vs. 3 μg: p < 0.0001; 0 μg vs. 4 μg: p < 0.0001; 0 μg vs. 5 μg: p < 0.0001) (Figure 6(f)). Thereafter, the oe-GATA1-transfected TPC-1 and CAL62 cells were further treated with a HDAC2specific inhibitor CAY10683, after which we found that the expression of NRBP2 was significantly restored (mRNA: oe-GATA1 + DMSO vs. oe-    (Figure 6(g,h)). These results further indicate that HDAC2 is required for GATA1-mediated NRBP2 downregulation. We also induced shRNA silencing of GATA1 in TPC-1 and CAL62 cells. In this setting, it was observed that the HDAC2 mRNA in cells was not significantly changed, but the NRBP2 expression was significantly elevated (TPC-1: sh-NC vs. sh-GATA1  (Figure 6(i)). These results, collectively, suggest that GATA1 recruits HDAC2 to the promoter region of NRBP2 to reduce its expression.

Discussion
Angiogenesis is an essential process for the supply of nutrients and oxygen to the rapid growth and dissemination of tumor cells [31]. Aberrant TME plays a critical role in tumor angiogenesis, invasion and metastasis [6,32]. This study reports that GATA1-and HDAC2-mediated NRBP2 downregulation induced TME and angiogenesis in TC  Bioinformatics tools including GEO datasets and TCGA-THCA have offered great convenience in the (h), angiogenesis ability of HUVECs co-cultured with TPC-1 and CAL62 cells determined by tube formation assay. Three repetitions were performed. **p < 0.01. fast identification of hub genes implicated in TC progression [33]. In this work, by analyzing the GSE165724 dataset and TCGA-THCA, we obtained NRBP2 as a significantly downregulated gene in TC. The poor expression of NRBP2 was validated in the clinical tissues and TC cell lines. Low NRBP2 expression was linked to tumor infiltration, lymph node metastasis, and the consequent poor prognosis of patients. NRBP2 shows a 59% amino acid similarity to NRBP1 which has been identified to regulate intestinal progenitor cell homeostasis and suppress tumor formation [34]. As for NRBP2 itself, it has recently been demonstrated as a tumor inhibitor in breast cancer since its poor expression was related to poor prognosis of patients and its high expression limited tumor metastasis [35]. Likewise, NRBP2 was poorly expressed in medulloblastoma and it reduced survival and growth of tumor cells upon overexpression [36]. In addition, NRBP2 showed a chemosensitizing effect in hepatocellular carcinoma cells [37]. Our experimental results first showed that NRBP2 weakened proliferation and colony formation abilities of TC cells and increased cell apoptosis.
There is not always a direct correlation between gene alteration and the invasiveness of TC, whereas the altered gene expression may affect the TME to influence the tumor angiogenesis and dissemination [6]. It has been well-recognized that the TME is beneficial for many stages of tumor development from occurrence to metastasis, and it largely affects tumor treatment and the clinical outcome [38]. However, current studies concerning TME of TC are contradictory. For instance, tumor-infiltrating lymphocytes (TILs) were reported to be linked to extra-thyroidal extension [39], but the following researches of TILs did not have a significant positive role in TC [40]. Elevated CD8 + T cell tumor infiltration in patients with differentiated PTC was correlated with increased disease-free survival [41]. However, infiltration of CD8 + T cell has also been observed to predict relapse in PTC [42]. Among the immune regulators, tumor-associated macrophages (TAMs), mostly the immunosuppressive M2 types, are generally playing tumor enhancing roles in PTC and are present in more aggressive tumor types [41,43,44]. The M2 macrophages are closely correlated with angiogenesis and lymph angiogenesis in cancer [45]. Importantly, our experiments suggested that NRBP2 overexpression significantly decreased the expression of TME biomarkers IL-6 and VEGFA [46] in the TPC-1 and CAL62 cells and, and overexpression of NRBP2 in cells reduced the M2 Figure 8. Graphical abstract. In TC, GATA1 recruits HDAC2 to the promoter region of NRBP2 to induce NRBP2 transcriptional suppression by deacetylation, which leads to increased M2 polarization of macrophages as well as increased angiogenesis, proliferation, and tumor development.
polarization of the co-cultured macrophages. In addition, it was found that the angiogenesis ability of HUVECs was reduced when cultured in an NRBP2-overexpressing condition. In vivo, overexpression of NRBP2 also reduced weight and volume of and the TAM infiltration in the xenograft tumors. These results revealed a tumor-suppressing role of NRBP2 in TC by weakening TME and angiogenesis.
Our subsequent integrated bioinformatic analyses and ChIP-qPCR assays suggested that GATA1 negatively regulated NRBP2 transcription. The tumorpromoting role of GATA1 has been witnessed in human malignancies such as ovarian cancer [13] and colorectal cancer [14] by promoting cell proliferation and invasiveness. GATA1 is a critical factor in erythropoiesis, while little has been concerned about its involvement in TME. As for angiogenesis, GATA1 has been reported to interact with the histone methyltransferase SET7 to trigger VEGF-induced angiogenesis in breast cancer [16]. Importantly, by using the UCSC browser concerning the potential epigenetic regulations, we predicated that there is a significant H3K9ac modification near the NRBP2 promoter. However, the H3K9ac modification, which is usually correlated with gene activation, was reduced in the NRBP2 promoter in TC tissues. In addition, we found that treatment with a HDAC inhibitor Tacedinaline restored the H3K9ac level in the NRBP2 promoter in TPC-1 and CAL62 cells, and treatment with the HDAC2-specific inhibitor CAY10683 restored the NRBP2 levels in cells suppressed by oe-GATA1, indicating that HDAC2 is required for GATA1-mediated NRBP2 downregulation. The transcriptional co-factor friend of GATA1 was found to recruit histone deacetylase NuRD to the mast cell gene promoter [47]. We then surmised that GATA1 may recruit specific HDACs to the NRBP2 promoter to induce transcriptional repression via deacetylation of H3K9ac. Importantly, the Co-IP, western blot, and dual immunofluorescence staining assays confirmed a binding relationship between GATA1 and HDAC2 in the nucleus of TC cells. HDAC2 is a widely investigated HDAC which locates in nucleus and can exert functions alone [48]. HDAC2 regulates gene transcription by deacetylating the N-terminal tails of the core histones, leading to more condensed chromatin state and reduced transcriptional activity [49]. A study by Li et al. suggested that a T-box transcription factor TBX3 recruits HDAC1 and HDAC2 to transcriptionally suppress expression of p57 to promote proliferation of PTC cells [50]. Here, we confirmed that overexpression of GATA1 or HDAC2 blocked the roles of NRBP2 and restored proliferation of PTC cells. Inhibition of HDACs has been reported to induce M2 polarization of macrophages in nitrogen mustardinduced lung injury [51]. Here, we confirmed that overexpression of GATA1 or HDAC1 strengthened the TME, induced M2 polarizations of macrophages and induced angiogenesis of HUVECs co-cultured with the PTC cells.

Conclusion
In conclusion, this study reports that GATA1 can recruit HDAC2 to the NRBP2 promoter to induce its transcriptional suppression, which leads to M2 polarization of macrophages and tumor angiogenesis and development ( Figure 8). GATA1 and HDAC2 may serve as potential therapeutic targets for TC, though more intensive pre-clinical researches are required. Hopefully we will see more new findings in TME in TC as this field is developing rapidly.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Availability of data and materials
All the data generated or analyzed during this study are included in this published article.