Expression Analysis of BIRC3 as One Target Gene of Transcription Factor NF-κB for Esophageal Cancer

Abstract

Esophageal cancer (ESCA) is one of the highest lethal malignancy tumors worldwide. Baculoviral IAP repeat-containing protein 3 (BIRC3) is the main inhibitor of apoptosis in many malignancies. The aim of this study was to clarify how BIRC3 acts in ESCA cells. Through TNMplot and GEPIA2 analysis, BIRC3 was found abundantly expressed in ESCA cells. The quantitative RT-PCR assay confirmed BIRC3 was pronouncedly induced in all used ESCA cell lines. In addition, proinflammatory cytokines TNFα and IL-1β were shown to have promotion effects on BIRC3 expression in ESCA cells. These promotive effects were blocked when the function of NF-κB was inhibited by bay 11-7082, which indicates the expression of the BIRC3 gene was regulated via the NF-κB transcription pathway in ESCA. Moreover, bioinformatics analysis showed that the BIRC3 gene had many NF-κB binding cis-elements. Chromatin immunoprecipitation was then performed and it was found that NF-κB directly interacts with cis-elements of the BIRC3 gene. In conclusion, our data proved that the high expression level of BIRC3 maintained the survival of ESCA cells. BIRC3 was up-regulated by proinflammatory cytokine TNFα and IL-1β through the NF-κB signaling pathway, and this may be helpful for esophageal cancer prevention and therapy.

1. Introduction

Due to high cancer-related deaths, esophageal cancer (ESCA) is the sixth most common malignancy tumor worldwide. Two major histological ESCA are squamous cell carcinoma (ESCC) and adenocarcinoma (EAC) [1]. Although the portion of ESCA in cancer patients has declined in recent years, the total number of ESCA patients still increased every year, especially in elderly patients over 70 years old [2]. China is one of the countries suffering from a rapidly increasing cancer burden. In 2017, China had the world’s highest number of esophageal cancer incident cases (235,000) and deaths (213,000) [3]. ESCA has become the fourth most common malignant cancer causing death in China [4]. The risk factors of ESCA in China are still not certain, but drinking beverages at high temperatures, low intake of fruit and vegetables, poor nutrition, and tobacco and alcohol use are considered as the main causes. Based on these strong risk factors, many precaution strategies have been taken to prevent ESCA. However, primary and secondary prevention of ESCA, such as changing daily routines and lifestyle, and the detection of precancerous and early cancerous lesions, are not as effective as expected [5]. Further information, particularly molecular mechanisms underlying ESCA progression, needs to be investigated for improving therapeutic outcomes and survival of patients.

Recently, thanks to high-throughput sequencing, a variety of gene-encoding proteins regulating cancer-related processes have been identified in relation to ESCA [6]. Genes involved in apoptosis, angiogenesis, or in oncogenes tumor suppressor processes were considered as important factors regulating ESCA forming. Among them, inhibitors of the apoptosis protein (IAP) family, regulating caspases, apoptosis, immunity, mitogenic kinase signaling, and cell proliferation, are the most studied molecular and therapeutic targets. Baculoviral IAP repeat containing 3 (BIRC3), also known as cellular IAP2 (cIAP2), which is a typical IAP protein, was a pro-oncogenic protein, associated with cancer evasion from apoptosis [7]. Previous studies have demonstrated that the high expression of BIRC3  correlated with many kinds of cancers, such as endometrial cancer, pancreatic cancer, and glioblastoma [8,9,10]; meanwhile, the overexpression of BIRC3 encourages tumor progression in different cancers [11]. The depletion of the BIRC3 gene can significantly suppress tumor initiation and progression [12]. Lower BIRC3 expression levels were associated with a favorable outcome and a BIRC3 level increase paralleled cancer recurrence and therapeutic resistance [13]. BIRC3  has become a potential therapeutic target in multiple cancers due to its multidirectional effects and complicated functions. However, the roles of BIRC3  in ESCA have only been investigated to a limited extent.

NF-κB (nuclear factor kappa B) signaling has been implicated in many hallmarks of cancer development, such as self-sufficiency in growth signals, insensitivity to growth inhibitors, and evasion of apoptosis [14]. In mammals, NF-κB contains five homologous subunits, including RelA/p65, c-Rel, RelB, p50/NF-κB1, and p52/NF-κB2. Previous studies suggest that both BIRC2 and BIRC3  contribute to the inhibition of apoptosis through an E3 ubiquitin-dependent manner [15]. The RING domain of BIRC3  is necessary for the interaction with the E3 ubiquitin ligase that leads to proteasomal degradation of MAP3K14, which is the central kinase of the NF-κB non-canonical pathway. Thus, BIRC3  is a biological negative regulator of non-canonical NF-κB signaling [16], which is alongside the canonical NF-κB signaling stimulated by tumor necrosis factor (TNF). On the other hand, BIRC3  was reportedly required for the canonical activation of NF-κB and MAPK signaling pathways by members of the TNF receptor (TNFR) superfamily [17]. NF-κB signaling is a crucial signaling axis, with a variety of key molecules aberrantly expressed or genetically altered in cancer patients [18]. It is still not clear whether BIRC3  regulates apoptosis in ESCA through the NF-κB pathway.

Meantime, BIRC3  is also involved in the control of inflammasome activation. Recent work has shown that BIRC3  can be induced by the inflammatory cytokine Interleukin-1β (IL-1β) in breast cancer cells, which resulted in a chemoresistance to doxorubicin [19]. It needs to be illustrated whether the functions of BIRC3  in esophageal cancer depend on the inflammatory cytokine. Here, the expression profiles of BIRC3 in five kinds of ESCA cells treated with TNF-α and IL-1β were analyzed to reveal the roles of BIRC3  in ESCA. Moreover, bioinformatics analyses of BIRC3  and chromatin immunoprecipitation (ChIP) in ESCA cells were performed to identify the BIRC3  acting manner in ESCA. The results provide proof that BIRC3  functions as a tumor stimulator in ESCA through the NF-κB pathway and it may be a therapeutic target for ESCA prognosis.

2. Materials and Methods

The expression profile of BIRC3 across various human cancer types was examined through TNMplot (https://tnmplot.com/analysis/ (accessed on 21 August 2022)) [9]. On the homepage, select ‘BIRC3′ from the gene list. The boxplots produced showed the expression level of BIRC3 with left bars representing normal samples and right bars representing tumor samples. Gene expression correlation analysis of BIRC3 in normal and esophageal cancer tissues was analyzed using the GEPIA2 web server (http://gepia2.cancer-pku.cn/#index (accessed on 21 August 2022)), setting the parameter as: “log scale: Yes”, “cancer name: ESCA”, “Matched Normal data: Match TCGA normal and GTEx data”. The prognostic value of the BIRC3 gene in esophageal cancer was analyzed using the Kaplan–Meier Plotter (http://kmplot.com/analysis/ (accessed on 21 August 2022)). The survival rates of patients with high and low levels of BIRC3 were shown using a Kaplan–Meier survival plot.

For BIRC3 gene expression analysis, the esophageal cancer cell lines KYSE150, KYSE510, KYSE450, TE3, and SHEEC cell lines from China ESCA patients were gifted by Professor Xu Yanli from Shantou University. The epithelial cell lines 293T and THLE3 purchased from BLUEFBIO Biotech (BLUEFBIO Biotech, Shanghai, China) were used as controls. Esophageal cancer cells were grown in RPMI1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% FBS (TransGen Biotech, Beijing, China), 1% penicillin and streptomycin (both from Beyotime Biotech, Shanghai, China) with 5% carbon dioxide at 37 °C. For NF-κB activation, 15 ng/mL, 30 ng/mL, and 60 ng/mL TNFα (PeproTech, Rocky Hill, NJ, USA) were added respectively and cultured for 1 h. For NF-κB inhibition, cells were pre-incubated with the downer 50 μmol/L Bay 11-7082 (Beyotime Biotech) for 30 min, then 30 ng/mL TNFα were added. After being cultured for 1 h, cells were collected for analysis. For the IL-1β experiment, cells were cultured with 10 ng/mL, 20 ng/mL, and 40 ng/mL human IL-1β (PeproTech), respectively, for 1 h. For the IL-1β effects but without NF-κB stimulation experiment, cells were incubated with 50 μmol/L Bay 11-7082 for 30 min and then cultured for 1 h with a medium containing 20 ng/mL IL-1β. Control cells were cultured in the same way but without TNFα, Bay 11-7082, or IL-1β. Cells were collected by centrifugation. The total RNA was extracted from collected cells using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) followed by isopropyl alcohol precipitation. RNA quality and concentration were assessed using NanoDrop (Applied Biosystem, Foster City, CA, USA). PrimeScript™ RT reagent Kit (Takara, Tokyo, Japan) was applied for cDNA synthesis following the manufacturer’s instructions. Gene relative expression was measured using TB Green Fast qPCR Mix (Takara) in a Real-Time Light Cycler 96 (Roche, Basel, Switzerland), using the PCR program with initial denaturing at 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s and 60 °C for 30 s. Relative mRNA expression levels were calculated according to the 2^−ΔΔCt equation. Cell lines 293T and THLE3 were used as the control group, respectively. Values for expression of each gene were normalized to the housekeeping gene GAPDH (GeneBank No: 2597). Specific primers used for PCR analysis were: GAPDH forward, 5′-ATTTGGTCGTATTGGGCG-3′, reverse, 5′-CTCGCTCCTGGAAGATGG-3′, and BIRC3 forward 5′-ATTTGATGAAAAGCGCCAACAC-3′, reverse, 5′-AACCCCAGCAGGAAAAGTGG-3′. Each PCR was carried out in triplicate and shown as mean ± SD.

The BIRC3-related genes were identified through GeneMANIA (http://genemania.org/ (accessed on 21 August 2022)). Setting parameter of GeneMANIA is: “organism: Homo sapiens”, “network: automatically selected weighting method”, all networks including physical interactions, co-expression, predicted, co-localization, pathway, genetic interactions, and shared protein domains were chosen. Further, the gene structure of BIRC3 was analyzed through UCSC software (http://genome.ucsc.edu/ (accessed on 21 August 2022)), and the cis-elements of the BIRC3 gene binding to NF-κB transcript factor (TF) were shown in a table.

To detect the relationship between the NF-κB transcript factor and the BIRC3 gene, ChIP was performed as described before [20]. Antibody for RelA TF (p65, Abcam, Cambridge, UK) was used for the affinity purification and complete rabbit IgG (Abcam) for the negative control. In addition, an antibody for acetylate histones H3K27Ac (Abcam) was applied to detect the acetylate histones signals. In brief, SHEEC cells treated with 30 ng/mL TNFα or 20 ng/mL IL-1β were applied for chromatin isolation, and were sheared on ice with a Branson 450 sonicator (Branson Ultrasonics, Danbury, CT, USA) for 3 cycles of 15 s pulses of half-maximal power with 30 s cooling time between pulses. Protein A-Agarose beads (Santa Cruz Bio-technology, Santa Cruz, CA, USA) were used for affinity purification, and samples were treated with proteinase K (Invitrogen). After precipitation, DNA was diluted in 100 μL water, and purified with a PCR products purification kit (Solarbio, Beijing, China). BIRC3 gene fragments were amplified with specific primers (BIRC3-84F, 5′-GCTGGAGTTCCCCTAAGTCC-3′, BIRC3-84R, 5′-TATCTGTACCAGGCGTTCCG-3′ and BIRC3-117F, 5′-GAACGCCTGGTACAGATAGGG-3′, BIRC3-117R, 5′-CCCAGTCTTTTCAAGCGACAC-3′), using the purified DNA as template. As input control, 8% volume of the supernatant from all pre-immune samples was set in parallel and re-suspended in 1000 μL of water for subsequent PCR.

All data in this study were calculated with IBM SPSS Statistics software (Version 13, IBM SPSS, Chicago, IL, USA). Significances between the groups were identified using t test or ANOVA followed by Dunnett’s multiple comparison test.

3. Results

3.1. In Silico Analysis of Gene Expression of BIRC3 in Tumors

The gene expression of BIRC3 in tumors and normal tissues was analyzed through the TNMplot web server. During 22 surveyed human cancers, the expression of BIRC3 is significantly higher in nine kinds of tumors (Figure 1A), including esophageal cancer. The further GEPIA2 analysis of the BIRC3 expression profile in esophageal cancer confirmed that BIRC3 had a much higher expression level than normal tissues (Figure 1B). Kaplan–Meier analysis revealed that a high expression level of BIRC3 was correlated with a good survival rate in esophageal squamous cell carcinoma cancer (p = 0.035; Figure 1C), but there is no significant correlation between the expression level of BIRC3 and the survival rate of patients for esophageal adenocarcinoma cancer (p = 0.2; Figure 1D). This observation suggests that BIRC3 acts in esophageal cancer, but its role seems to be comprehensive.

3.2. BIRC3-Induced Expression in Esophageal Cancer Cells

To analyze the BIRC3 gene expressions in esophageal cancer cells, quantitative real-time PCR analyses were performed. Five esophageal cancer cell lines were applied. Expressions of BIRC3 were detectable in all these cells. Compared with the control cells 293T and THLE3, BIRC3 was significantly induced to be expressed in all used esophageal cancer cells (Figure 2). Among the detected five kinds of esophageal cancer cell lines, KYSE150 cells have the lowest expression of BIRC3 with about 7.9 and 2.8 times more than the controls 293T and THLE3, respectively. While the gene inducement of BIRC3 in KYSE450 cells was most prominent, which was 1106.2 times and 6950.5 times more than the controls. The transcripts analysis confirmed the prediction of the GEPIA2 database (Figure 1B). The above finding suggests that BIRC3 is induced in esophageal cancer cells to maintain survival.

3.3. TNFα Encourages Expression of the BIRC3 Gene in Esophageal Cancer Cells

Previous reports showed that BIRC3 could negatively regulate NF-κB signaling activation [21]. TNFα is a common stimulator of NF-κB signaling. To validate whether the activation of NF-κB could affect the expression of BIRC3, mRNA levels in five kinds of esophageal cancer cells treated with three gradient concentrations of TNFα were measured by qPCR analysis. Figure 3A showed that after TNFα-stimulation, expressions of BIRC3 in five kinds of cells increased significantly. Among the three concentrations, 30 ng/mL TNFα had the best-induced effects on BIRC3 gene expression in five kinds of esophageal cancer cells. At the same time, the expression of BIRC3 in SHEEC cells treated with different TNFα had the highest increase, enhanced more than 9.0-fold.

Further, after NF-κB was inhibited with Bay 11-7082, the effects of TNFα stimulation on BIRC3 expression declined noticeably (Figure 3B). In KYSE510 and SHEEC cell lines, the increased degrees of mRNA of BIRC3 levels from 4.2-fold and 9.0-fold to 1.2-fold and 1.3-fold, respectively. While in KYSE150, KYSE450, and TE3, the expression of BIRC3 even decreased to 68.9%, 52.5%, and 70.6%, respectively. The results implicated that BIRC3  induced by TNFα was through the NF-κB pathway.

3.4. IL-1β Encourages the Expression of the BIRC3 Gene in Esophageal Cancer Cells

Relative expression of the BIRC3 gene in KYSE150, KYSE510, TE3, and SHEEC cells treated by IL-1β was determined by qPCR. As shown in Figure 4A, IL-1β up-regulated the expression of the BIRC3 gene in four kinds of esophageal cancer cells. However, various concentrations of IL-1β had different effects on BIRC3 expression in different esophageal cancer cell lines. In KYSE150, a higher concentration of IL-1β (40 ng/mL) did not affect the mRNA level of BIRC3 (not significantly, p > 0.05), while a lower concentration of IL-1β (10 ng/mL and 20 ng/mL) could promote BIRC3 expression to 1.44-fold and 1.64-fold, respectively. The effects of IL-1β on mRNA levels of BIRC3 in KYSE510 and TE3 were similar, whereby the low concentration of IL-1β (10 ng/mL) had no effect on BIRC3 expression, and medium concentration (20 ng/mL) of IL-1β increased mRNA of BIRC3 to its highest level, which was 4.27-fold and 2.64-fold, respectively. The BIRC3 expression in SHEEC was up-regulated by IL-1β, and encouraged mostly by 20 ng/mL IL-1β promoted to 19.28-fold more than the untreated control.

We also examined the mRNA levels of BIRC3 in KYSE150, KYSE510, TE3, and SHEEC cells treated with 50 μmol/L Bay 11-7082, and 20 ng/mL IL-1β combination. The results showed the inducing effect of IL-1β on BIRC3 gene expression was inhibited by Bay 11-7082 (Figure 4B). When NF-κB was suppressed, and even IL-1β was added, in KYSE150 and KYSE510, the mRNA level of BIRC3 had no changes, and in TE3 and SHEEC cell lines, the expression of BIRC3 declined from 3.1-fold and 19.3-fold to 1.1-fold and 3.8-fold, respectively. The results showed that the BIRC3 gene was up-regulated by IL-1β, but also depended on the NF-κB transcription factor.

3.5. BIRC3 Regulated by the NF-κB Transcription Factor

The related genes of BIRC3 were predicted via GeneMANIA. The twenty best-matched genes are listed in Table S1. Among them, four transcription factors (TFs) were predicted, including NF-κB TF. In addition, several genes involved in the NF-κB regulation pathway were found, such as TRAF1, TRAF2, TRAF3, and IKBKB (Figure S1). To further reveal the relationship between BIRC3 and NF-κB, the NF-κB/RelA binding sites at 1500 bp upstream of BIRC3 (chr11: 102,315,950–102,339,394, and GRCh38/hg38) were analyzed by UCSC (

Table 1. Analysis of NF-κB bound sites of BIRC3 promoter in UCSC database.

Factor Name	Position (Strand)	Core Match	Matrix Match	Sequence	Matrix Identifier
c-Rel	99 (−)	0.785	0.830	GTAAAtgccg	V$CREL_01
NF-κB (p65)	149 (−)	1.000	0.983	GGAAAtcccc	V$NFKAPPAB65_01
NF-κB (p65)	162 (+)	0.867	0.876	tgggtTTGCC	V$NFKAPPAB65_01
c-Rel	199 (+)	0.758	0.811	tggttATTAC	V$CREL_01
NF-κB (p65)	213 (−)	1.000	0.839	ggagtTCCCC	V$NFKAPPAB50_01
c-Rel	235 (−)	1.000	0.879	GGAAAgcacc	V$CREL_01
c-Rel	275 (−)	0.952	0.788	GGAACgcctg	V$CREL_01
c-Rel	353 (−)	1.000	0.849	GGAAAaggcc	V$CREL_01

). The results showed that there are eight NF-κB/RelA bound sites in the BIRC3 promoter region. Among them, two in the sense strand and six in the reverse strand. This indicates that BIRC3 is regulated through the NF-κB transcription pathway.

The special binding of the NF-κB to the BIRC3 gene was assessed via chromatin affinity purification. ChIP with a RelA TF (p65) antibody was performed on a protein extract derived from SHEEC cells (Figure 5A). The NF-κB transcript factor was shown to be able to bind to the promoter of the BIRC3 gene of both 84 bp and 117 bp intron fragments. At the same time, the acetylate histones H3K27Ac (H3) were also found interacting with the BIRC3 gene fragments 84 bp and 117 bp (Figure 5B). When SHEEC cells were treated with 30 ng/mL TNFα, the interaction between the NF-κB and BIRC3 gene 84 bp and 117 bp fragments increased 15.8- and 16.9-fold compared with negative controls, respectively (Figure 5C,D). In addition, the target 84 bp and 117 bp of the BIRC3 promoter DNA sequence was respectively 18.9- and 15.7-fold enriched after affinity purification with the p65 antibody in SHEEC cells after treatment by IL-1β (Figure 5E,F). These indicated that BIRC3 is regulated by NF-κB TF.

4. Discussion

ESCA has emerged as a major health issue in Asian countries, especially in China, due to its unclear pathogenesis and high death rate. BIRC3 (cIAP2) is a member of the inhibitor apoptosis (IAP) protein family, which controls survival and cell death through regulating caspases and apoptosis [22]. Commonly, BIRC3 acts as a pro-oncogenic protein, which helps cancer cells to avoid undergoing apoptosis. In different kinds of tumors, high expression of BIRC3 was detected to contribute to osteosarcoma growth and to reduce apoptosis [9]. In this study, TNMplot analysis showed BIRC3 increased in many kinds of cancers (Figure 1A), which implied that BIRC3 is an important ingredient in the process of cancer. GEPIA2 bioinformatics analysis (Figure 1B) also showed BIRC3 to be positively associated with esophageal cancer growth. As early as 2004, cIAP2 (BIRC3) was demonstrated to have a higher expression in esophageal cancer tissue than in normal mucosa; although, the differences were not significant [23]. Now, 18 years later, the esophageal cancer cell lines can be well classified. Here, in this study, the expression of BIRC3 was promoted obviously in all five kinds of esophageal cancer cells (Figure 2). It confirmed that BIRC3 plays a major role in ESCA pathogenesis. However, the high expression of BIRC3 was positively related to the survival rate of ESCA patients (Figure 1C,D). BIRC3 duplications were detected in (Chronic lymphocytic leukemia) CLL patients [24], and genetic inactivation or down-regulation of BIRC3  had unfavorable clinical contribution in cancer patients. All indicated that the genetic modulation of BIRC3  will have comprehensive effects when designing ESCA therapeutic approaches.

BIRC3  plays a role in protecting cells from apoptosis when exposed to proinflammatory cytokines. It has been demonstrated that BIRC3  protein expression increased noticeably in primary nasal epithelial cells when exposed to IFN-γ or TNF-α [25]. Our discovery also revealed that BIRC3  was up-regulated by cytokines such as TNFα and IL-1β (Figure 3 and Figure 4), which confirmed that BIRC3  was the key mediator in inflammatory and immunity processes. Previous studies have shown that the TNFα-initiated apoptosis in tumors was suppressed by NF-κB via activating a group of gene targets, such as inhibitor-of-apoptosis (IAP) proteins c-IAP1 and c-IAP2 (BIRC3) [26]. Proinflammatory cytokines TNFα and IL-1β both act in apoptosis promotion and activation of transcription factor NF-κB. Here, in all ESCA cell lines, BIRC3  was noticeably stimulated by TNFα, and suppressed by the NF-κB inhibitor bay 11-7082 (Figure 3). It is evidenced that TNFα-induced killing is somehow inhibited by the BIRC3  protein, which is activated through NF-κB activation.

The NF-κB signaling pathway was evidenced as an important regulatory mechanism in esophageal cancer. The ESCC could intervene by modulating genes involved in the NF-κB signaling pathway. As referred before, overexpressed FSTL1, which was an upstream stimulator of NF-κB, abolished the promoting effect of lncRNA on cell invasion, and further resulted in the low survival of ESCC patients [27]. BIRC3  was a downstream regulator in the NF-κB pathway. As referred previously, BIRC3  was one of the genes of the regulatory complex in the noncanonical NF-κB pathway [21]. Transcription factors regulated downstream genes often by occupying their promoter sites. Here, so many subunits of NF-κB transcription complexes, such as p65 (RelA) and c-Rel subunits, were found in the BIRC3  promoter (Table 1). Most importantly, as expected, NF-κB TF appeared to interact with BIRC3  gene fragments (including upstream and intron sequences). Finally, ChIP results confirm that BIRC3  is regulated by NF-κB (Figure 5). An enrichment of acetylated histones H3 was found around the BIRC3  promoter (Figure 5B), which suggests that NF-κB-enhanced expression of BIRC3  may occur by promoting histone acetylation in ESCA, just like that in breast cancer [28]. Activated NF-κB and up-regulated IAPs (BIRC2 and BIRC3) in ovarian cancer cells might mediate chemotherapy resistance [29]. Here, the stimulated NF-κB pathway could increase the expression of BIRC3  in ESCA and may provide a clinical therapeutic basement. Nevertheless, there must be other possible mutual accommodation between BIRC3  and NF-κB, and the increased NF-κB level partly resulted from the expression of the BIRC3  elevated level in ESCA. Further mechanisms of BIRC3/NF-κB signaling in ESCA will undoubtedly need to be revealed.

Based on the above findings, as well as the previous literature [30], a putative model of how the molecular mechanism of BIRC3  modulates esophageal tumor cell apoptosis via the NF-κB pathway was proposed (Figure 6). cIAPs are crucial regulators of canonical and non-canonical NF-κB activation pathways. There is a positive feedback loop between BIRC3 and NF-κB [31], the expression of BIRC3 was induced by NF-κB (Figure 3 and Figure 4), and BIRC3 promotes NF-κB activation. In the presence of proinflammatory cytokines, NF-κB was activated to suppress apoptosis through a canonical and non-canonical pathway [14]. Previous studies have shown that in the case of TNFα, TRAF2 and BIRC3 were activated via the NF-κB pathway to suppress apoptosis via inhibiting caspase-8 [26]. As one of the coding products of the canonical NF-κB pathway stimulated by both TNFα and IL-1β, the increased expression of BIRC3 could be inhibited when NF-κB was suppressed. The NF-κB inhibitor verified this notion. When the phosphorylation of IκBα was blocked by bay 11-7082, it caused the failure of NF-κB stimulation, and the gene expression of BIRC3 immediately declined to a constitutive expression normal level (Figure 3 and Figure 4). BIRC3 is also famous due to its function of proteasome degradation of MAP3K14, the major driver of activation of the non-canonical NF-κB pathway [32]. This study demonstrates that BIRC3 is a promoter of the NF-κB regulatory mechanism in ESCA to help cells withstand apoptosis. Therefore, further studies, in particular, to suppress both BIRC3 and NF-κB in ESCA patients, can yet be regarded as a new attempt in treatment.

5. Conclusions

In summary, our findings have shed some light on the regulation of BIRC3 in ESCA. BIRC3 was significantly induced in ESCA cell lines. Proinflammatory cytokine TNFα and IL-1β had promotive effects on BIRC3 expression via the NF-κB transcription pathway in ESCA. In particular, an in-depth disclosure of BIRC3 up-regulated through the NF-κB pathway in ESCA was uncovered. NF-κB directly interacts with CIS-elements of the BIRC3 gene to regulate its expression. Our data provided targeted BIRC3/NF-κB/as a relevant therapeutic agent of ESCA. Combined inhibition of BIRC3 and NF-κB may have clinical potential for ESCA treatment.