DACH1 antagonizes CXCL8 to repress tumorigenesis of lung adenocarcinoma and improve prognosis

Microarray analysis

CXCL8 mRNA expression datasets for lung cancer were downloaded from the ArrayExpress. GSE31210 expression profile consists of 226 primary ADC patients with pathological stage I–II. GSE68465 with 443 ADC cases includes histologic grade and tumor size. GSE32474 consists of nine lung cancer cell lines. Meta-analysis integrating 10 studies was performed as previously described [26], and the forest graphs were drawn by Stata 13.

Cell culture, establishment of cell lines stably expressing DACH1 and cell stimulation

The 293T and human bronchial epithelial (HBE) cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and human lung cancer cell lines (SKLU, A549, and H460) were cultured in 1640 medium supplemented with 10% FBS. Cells were maintained in an atmosphere of 5% carbon dioxide (CO₂) in a humidified 37 °C incubator. The expression vectors encoding DACH1 and DACH1 deficient in the DNA binding domain (ΔDS) were subcloned into lentivirus expression vector. Stable expressions of DACH1 in A549 and SKLU cells were confirmed by immunofluorescence. In the drug intervention group, 100 ng/ml 12-O-tetradecanoyl phorbol-13-acetate (TPA) and 10 ng/ml TNF-α were added into the supernatants, respectively, after 24 h of serum starvation. After 12 h of the stimulation, cells were subsequently used for further experiments.

ELISA

The blood samples were collected from about 100 patients who were diagnosed as ADC at various stages by April 2015 in Tongji hospital. The QuantiCyto Hu CXCL8 kit (H161125-008a) is a solid phase sandwich ELISA kit, used for the quantitative determination of CXCL8 in human serum, plasma, buffered solution, or cell culture medium. A monoclonal antibody specific for CXCL8 has been coated onto the wells of the microtiter strips provided by the manufacture. Diluted samples, including standards of known CXCL8 content, control specimens, and unknowns, were pipetted into these wells followed by the addition of a second biotinylated monoclonal antibody. Then, Streptavidin-Peroxidase (enzyme) was added to complete the four-member sandwich. After incubation and washing steps to rid the microplate of unbound substances, a substrate solution (TMB) was added and reacted with the enzyme-antibody-target complex to produce measurable signals. After stopping the reaction with the stop reagent, the values of optical densities (OD) were measured at 450 nm using Microplate Reader (BioRad). The intensity of this signal is directly proportional to the concentration of CXCL8 in original specimen.

Immunohistochemical staining

Commercially available tissue microarray slide (HLug-Ade150Sur-02, Shanghai Outdo Biotech Company) with 75 pairs of ADC samples and corresponding adjacent tissues was used to assess the correlation between the protein expression of CXCL8 and clinic-pathological variables and prognosis of ADC patients based on the detailed survival data. Specific primary antibody of CXCL8 (RLT2343, Ruiying Biology, 1:100) was utilized for immunohistochemistry (IHC) with a two-step protocol which was described formerly [27, 28]. Additionally, the protein expressions of DACH1, cyclin D1, Ki-67, and CXCL8 in A549-vector and A549-DACH1 xenograft mice models were examined by IHC. The specific primary antibodies were anti-DACH1-antibody (10914-1-AP, ProteinTech, 1:150), anti-Ki-67-antibody (ab15680, Abcam, 1:150), and anti-CXCL8-antibody (RLT2343, Ruiying Biology, 1:250). Ready to use rabbit anti-cyclin D1-antibody was from MAIXIN-Bio (RMA-0541). Slide images were captured by Mv Image software.

Analysis and quantification of staining

The immunohistochemical scores were graded by two people independently. Scores were dependent on the intensity and percentage of positive staining tumor cells in the whole tissue staining according to the Fromowitz standard as described above [29]. The staining intensity was scored as 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). The percentage of positive cells was divided into four levels: 1 (0–25% positive cells), 2 (26–50% positive cells), 3 (51–75% positive cells), and 4 (76–100% positive cells). The multiplication of the intensity and percentage was utilized to represent the final staining score. Score > 8 was defined as high CXCL8 staining, otherwise was defined as low CXCL8 staining. Moreover, at least five fields of × 200 magnification from each core were taken for quantification.

Human cytokine array

Human cytokine arrays spotted on nitrocellulose membranes were obtained from Raybiotech (Norcross, GA). Conditioned medium was prepared from SKLU-vector, SKLU-DACH1, A549-vector, and A549-DACH1 cells by culturing cells in serum free 1640 for 24–48 h. Cytokine array analysis was performed as described previously [21].

Western blot analysis

Cells were washed twice with cold phosphate buffered solution (PBS) and then lysed with RIPA lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF). Each sample was denatured in 100 °C boiling water in the presence of sodium dodecyl sulfate (SDS) and DL-Dithiothreitol (DTT). Twenty micrograms of protein from each sample was loaded on a 15% SDS-polyacrylamide gel, and the separated proteins were then transferred onto a polyvinylidene fluoride (PVDF) membrane. The primary antibodies were anti-CXCL8-antibody (60141-2-lg, ProteinTech, 1:1000), anti-DACH1-antibody (10914-1-AP, ProteinTech, 1:1000), and anti-β-actin-antibody (60008-1-lg, ProteinTech, 1:2000). Secondary staining and detection were conducted in accordance with standard protocols as previously described [30, 31].

Real-time PCR analysis

The primers of CXCL8 used for real-time PCR are as follows: forward primer, 5′- GACAGCAGAGCACACAAGC-3′; reverse primer, 5′- GGCAAAACTGCACCTTCAC-3′, which were applied in previous study [32]. Total mRNA was extracted from cells using Trizol on the basis of the manufacturer’s protocol (Takara Co, Japan). One microgram of total mRNA was subsequently reverse transcribed to complementary DNA (cDNA) with synthesis kit (TransStart). Quantification reactions were carried out using the ABI 7900 HT platform. Each reaction system was composed of 3 μl cDNA, 5 μl Tip Green qPCR SuperMix (TransStart), 2 μl 10 μM forward and reverse primer mix. Samples were amplified under the following cycling conditions: 95 °C for 30 s, and 45 cycles of 95 °C for 5 s, 59 °C for 15 s, and 72 °C for 10 s. Cycle thresholds were calculated as the expression fold changes through formula: 2^-△△CT.

Transwell migration assay

Transwell migration assays were conducted using an 8-μm pore size transwell filter insert (Corning Inc., Corning, NY, USA) coated with diluted Matrigel (BD Biosciences, Bedford, MA, USA). SKLU-vector, SKLU-DACH1, A549-vector, and A549-DACH1 cells were suspended in 1640 culture medium with 1% FBS, and 2.5 × 10⁴ cells in 100 μl serum-free medium were seeded on the upper chamber. Lower chamber was filled with 1640 culture medium supplemented with 2.5% FBS. In comparison group, 5 μl CXCL8 antibody (10 μg/ml) (MAB208, R&D systems) was added in the lower chamber to antagonize the chemotaxis effect of CXCL8, and 2 μl human CXCL8 factor (50 μg/ml) (200-08 M-5UG, PeProTech) was added in the lower chamber of the SKLU-DACH1 and A549-DACH1 group to restore the inhibitory action of DACH1. After incubation for 12 h at 37 °C with 5% CO₂, migratory cells were stained with 0.5% crystal violet solution and counted by light microscopy (200×).

Nude mice study

The animal experiment protocols were approved by the ethics committee of the Tongji Hospital of Huazhong University of Science and Technology. The methods used in this section were in accordance with the relevant guidelines and regulations. 1 × 10⁵ A549-vector, A549-DACH1, and A549-ΔDS cells were subcutaneously injected into 4-week-old athymic female nude mice purchased from Hunan SJA Laboratory Animal Co. The tumor volume was measured weekly for 6 weeks by using a digital caliper. Nude mice were sacrificed and subcutaneous tumor lumps were stripped on day 42 after implantation. Tumors were weighed by electronical scale.

Plasmid construction, transient transfection, and luciferase reporter gene assay

The 2000 bp sequence of CXCL8 promoter was cloned into pGL3-basic vector. The recombinant plasmid consisting of 163 bp sequence of CXCL8 promoter is a gift from Dr. Antonella Casola [33]. Cells were plated at a density of 1 × 10⁵ cells in a 24-well plate on the day prior to transfection with Superfect according to the manufacturer’s protocol (Qiagen, Valencia, CA). Transient transfection with the expression vectors encoding DACH1 and ΔDS were previously described [11, 21, 34]. One hundred fifty nanograms of expression vectors and 200 ng of promoter reporter plasmids were required in each well in accordance with the dose-response fashion. Luciferase reporter gene assay was conducted by the kit (TransDetect, FR101). Fluorescence intensity was quantified with luminometer (Biotech Synergy 2).

GIPZ Lentivirus shRNA vector expressing control or DACH1 was purchased from Open Biosystems. shDACH1-targeted sequences are CTGAAGCAATGAAGGTGAA and TGAGAATGTTTGTAAATGT. Stable cell line HBE was established by infecting with lentivirus expressing shDACH1 or shCTL. H460 cells were planted on an 8-well chamber and were transiently transfected with shDACH1 or shCTL using Lipofectamine 2000 following the standard protocol [19]. After 48 h culture, cells were routinely fixed, permeabalized by 0.5% Triton X-100 and blocked with 5% BSA, and then stained with antibody to DACH1 or CXCL8. Nuclei were count stained with Hoechst 33342.

Kaplan-Meier plotter

Kaplan-Meier survival curves with hazard ratio and log-rank p value were calculated and plotted with the analysis tool, which can be accessed online at http://kmplot.com/analysis/ [35]. The background database, downloaded from GEO (Affymetrix microarrays only), EGA, and TCGA, offers gene expression data, relapse free survival (RFS), and overall survival (OS) rates. The software is capable to assess the effect of 54,675 genes on survival using 10,188 cancer samples, among which includes 2437 cases of lung cancer. We analyzed the survival outcomes of NSCLC and ADC in different expression levels of DACH1 and CXCL8. The Kaplan-Meier survival curves downloaded from the website were resized in Adobe Illustrator CS6. The blend curves were obtained from IBM SPSS Statistics 19.0 using GSE31210 and resized in Adobe Illustrator CS6.

Statistical analysis

Statistical analyses between groups were calculated by Student’s t test and one-way ANOVA. The p value was set at 0.05. Mann-Whitney test was applied when the original data did not accord with normal distribution. Correlations between clinic-pathological features and IHC variables were calculated via Pearson chi-square test and continuity correction chi-square test. Based on the extracted survival data of IHC chip, the survival curve for OS and RFS were calculated using Kaplan-Meier method with the log-rank test, and univariate cumulative survival analyses were conducted with Cox regression model. Statistical analyses were conducted using SPSS 19.0 and GraphPad Prism 5.0. All data were presented as the mean ± SEM.

CXCL8 mRNA level positively correlates to the progression of ADC

Gene expression dataset (GSE68465) was extracted to estimate the mRNA level of CXCL8 in ADC. The results showed that the mRNA profile of CXCL8 was dramatically higher in tumor tissue than normal tissue (p < 0.0001) (Fig. 1a). ADC patients with poor and moderate differentiation expressed more CXCL8 than patients with well differentiation (p < 0.0001 and p = 0.0017) (Fig. 1b). Besides, CXCL8 level showed an upward trend with the increase of tumor size (p = 0.0128 and p = 0.0102) (Fig. 1c). We further integrated 10 studies consisting of 1070 patients with detailed clinic-pathological characteristics (Table 1). The meta-analysis indicated that increased CXCL8 mRNA level was significantly associated with advanced tumor stage in NSCLC patients (pooled OR = 1.52, 95% CI 1.13–2.05), especially in ADC patients (pooled OR = 1.71, 95% CI 1.12–2.61) (Fig. 1d, e). It was indicated that overexpression of CXCL8 mRNA occurred in ADC patients and was markedly correlated with the progression of ADC.

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Table 1

Characteristics of studies included for meta-analysis

First author	Year	Duration (months)	Histology	Stage	Patient number	Quality score	Detection	Platform
Yuan A [40]	2000	28	NSCLC	I–III	58	8	Microarray	Perkin-Elmer Applied Biosystems
Tang H [41]	2013	120	NSCLC	I–III	176	9	Microarray	IlluminaHumanWG-6v3.0
Raponi M [42]	2006	144	SQC	I–III	129	8	Microarray	AffymetrixHgu133plus2.0
Tomida S [43]	2009	109.8	ADC	I–III	117	9	Microarray	AgilentWholeHumanGenomeMicroarray 4x44K G4112F
Botling J [44]	2013	120	NSCLC	I–IV	196	8	Microarray	AffymetrixHgu133plus2.0
Beer DG [45]	2002	110.6	ADC	I–III	86	8	Microarray	AffymetrixHumanFullLength HuGeneFL
Landi MT [46]	2008	NR	ADC	I–IV	74	9	Microarray	AffymetrixHgu133a
Lu T [47]	2010	NR	ADC	I–IV	60	8	Microarray	AffymetrixHgu133plus2.0
Selamat SA [48]	2012	NR	ADC	I–III	58	8	Microarray	IlluminaHumanWG-6v3.0 expression
Meyerson M [49]	2015	NR	SQC	I–IV	135	8	Microarray	AffymetrixHgu133a

Cutoff value: median expression

NR not reporting, ADC lung adenocarcinoma, SQC lung squamous cell carcinoma

Protein abundance of CXCL8 correlates to the progression and prognosis of ADC

In order to determine the protein level of CXCL8, we conducted ELISA to analyze the serum level of CXCL8 in 20 normal samples and 48 ADC patients covering distinct pathological stages and histological grades. Of 48 ADC patients, 23 belong to operable stage I–IIIa, while the rest are unresectable advanced ADC (stage IIIb–IV). The results showed that ADC patients had much higher serum level of CXCL8 than normal samples (I–IIIa/normal: p < 0.0001; IIIb–IV/normal: p < 0.0001) (Fig. 2a). Moreover, patients with advanced ADC stages secreted more CXCL8 to peripheral circulation than patients with early stage of ADC (p = 0.0344) (Fig. 2a). We further analyzed the data based on distinct tumor grades. Patients with grade 3 had highest level of CXCL8 (grade 3/grade 1–2: p < 0.0001; grade 3/normal: p < 0.0001) (Fig. 2b), and patients with grade 1–2 expressed higher CXCL8 protein level than normal samples (grade 1–2/normal: p < 0.0001) (Fig. 2b). Correlation between CXCL8 expression and clinic-pathological features of 48 ADC patients was shown in Table 2.

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Table 2

Correlations between CXCL8 expression and clinic-pathological features of 68 ADC patients in ELISA

Variables	N	CXCL8 expression		P value
		Mean (pg/ml)	SEM (pg/ml)
Sample				< 0.0001b
Normal	20	7.45	0.15
Tumor	48	15.58	2.49
Age				0.3006a
≤ 55	20	11.94	0.77
> 55	28	18.18	4.20
Sex				0.4800a
Male	29	13.96	1.19
Female	19	18.06	6.08
Stage				0.0344a
I~IIIa	23	11.35	0.73
IIIb~IV	25	19.47	4.65
Grade				< 0.0001a
1~ 2	24	10.13	0.52
3	24	21.03	4.75

^aMann-Whitney

^bStudent’s t test

Then, we detected the CXCL8 expression on tumor tissues by IHC. Xenograft tumor tissue from A549 cell was used to test the specificity of anti-CXCL8 antibody (Fig. 2c). Immunohistochemical microarray including 75 pairs of ADC patients with survival data, clinic-pathological parameters, and corresponding adjacent tissues was stained and showed that the expression of CXCL8 was markedly higher in ADC tissues than in adjacent normal samples (p < 0.0001) (Fig. 2d). Patients with big tumor size (T3-T4) had upregulated CXCL8 expression compared with patients with small tumor size (T1-T2) (p = 0.006) (Fig. 2e). The same trend was also found in ADC patients with different TNM stages, that is samples with advanced stage (III) showed stronger staining than samples with early stages (I–II) (p = 0.0029) (Fig. 2f). Moreover, ADC patients with lymph node metastasis expressed more CXCL8 than those without lymph node metastasis (p = 0.0065) (Fig. 2g). However, there was no statistical difference of CXCL8 expression between tumors with distinct grades. Median OS times of patients with low CXCL8 and high CXCL8 were 51.5 ± 2.52 and 41 ± 2.81 months, respectively (p = 0.035) (Fig. 3a). By exploring the relationship between clinic-pathological parameters (age, sex, TNM stage, grade, lymph node metastasis, and tumor size) and CXCL8 expression, we found that the level of CXCL8 was relevant to tumor stage (Pearson chi-square test, p = 0.024) and lymph node metastasis (Pearson chi-square test, p = 0.004), nevertheless other parameters showed no statistical significance with CXCL8 (Table 3). Univariate Cox regression analysis was used to investigate the correlation between cumulative OS rates and clinic-pathological factors. As shown in Fig. 3b, four factors, including lymph node metastasis (hazard rate (HR) = 4.037; 95% CI 1.706–9.551; p = 0.001), tumor size (HR = 2.315; 95% CI 1.072–4.996; p = 0.033), TNM stage (HR = 5.965; 95% CI 2.588–13.753; p = 0.000), and CXCL8 expression (HR = 2.210; 95% CI 1.059–4.615; p = 0.035), were obviously associated with the clinical outcomes of ADC patients, while other parameters were not the direct prognostic factors. Our results suggested that increased CXCL8 protein occurred in ADC patients and was significantly correlated with tumor progression and poor prognosis.

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Table 3

Correlations between CXCL8 expression and clinic-pathological features of 75 ADC patients in immunohistochemistry chip (HLug-Ade150Sur-02)

Variables	N	CXCL8 expression		P value
		≤ 8 (low expression)	> 8 (high expression)
Age				0.865a
< 55	20	14	6
≥ 55	53	36	17
Sex				0.255a
Male	40	30	10
Female	35	22	13
Grade				0.249b
1~ 2	61	40	21
3	14	12	2
Stage				0.024a
I~II	40	30	10
III	18	8	10
Lymph node
N−	36	29	7	0.004a
N+	21	9	12
Tumor size
T1–T2	60	45	15	0.069b
T3–T4	15	7	8

^aPearson chi-square test

^bContinuity correction chi-square test

Negative correlation between CXCL8 and DACH1 in vitro

We analyzed mRNA expression of CXCL8 and DACH1 in early stage of ADC using the Arrayexpress database containing 226 ADC cases with stage I–II (GSE31210). The result showed that CXCL8 mRNA expression was positively correlated with tumor stage at mRNA level (p = 0.0009) (Fig. 4a). Meanwhile, DACH1 was negatively correlated with tumor stage in early stage of ADC (p = 0.0001) (Fig. 4b). Based on this observation, we conducted relevance analysis between CXCL8 and DACH1 at mRNA level in lung cancer tissues and cell lines. There was a negative correlation between CXCL8 and DACH1 (p = 0.001, p = 0.047) (Fig. 4c, d). Meanwhile, we also did the correlation analysis between CXCL8 and other known tumor suppressor genes, such as p53 and CDKN1B, and there were no significant negative relationship between CXCL8 and those genes in both lung cancer patients (p = 0.480, p = 0.264) and lung cancer cell lines (p = 0.063, p = 0.426) (Fig. 4e–h). It suggested the specificity of correlation between DACH1 and CXCL8 in lung cancer.

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Stable expression of DACH1 in A549 and SKLU cells was confirmed by immunofluorescence assay (Fig. 5a). Human cytokine array analysis intuitively showed that SKLU-DACH1 and A549-DACH1 cells secreted lower level of related cytokines, especially CXCL8, compared with the vector control group (Fig. 5b). Compared with the control group which was standardized as 1, the confidence intervals of CXCL8 expression level were 0.2–0.4 in SKLU-DACH1 cells (p < 0.0001) and 0.5–0.7 in A549-DACH1 cells (p < 0.0001). And the corresponding quantitative bar graph certified the trend (p < 0.0001) (Fig. 5c). Similarly, cell lines ELISA showed that lower protein levels of CXCL8 were detected in the conditional medium of SKLU-DACH1 and A549-DACH1 cells than in the SKLU-vector and A549-vector cells (p = 0.0039, p = 0.0055) (Fig. 5d). Western blot further suggested that SKLU-DACH1 cell line expressed decreased CXCL8 protein (Fig. 5e). We also conducted the real-time PCR to explore the mRNA level of CXCL8 using SKLU-vector, SKLU-DACH1, A549-vector, and A549-DACH1 cells. The results certified that CXCL8 mRNA expression was significantly downregulated under the condition of the existence of DACH1 regarding the corresponding vector group as the criterion (p = 0.0002, p = 0.004) (Fig. 5f). HBE cells steadily expressing shDACH1 and shCTL were established, as evidenced by GFP expression from vector (Fig. 6a). Real-time PCR analyses showed that the mRNA level of CXCL8 was upregulated in the loss of DACH1 (p = 0.0329) (Fig. 6b). Immunofluorescence assay exhibited the consistent trend that the inhibition of DACH1 could markedly stimulate the CXCL8 expression in H460 cell (Fig. 6c). Overall, we certified that DACH1 was a vital factor to inversely regulate the CXCL8 in vitro.

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DACH1 represses CXCL8-induced migration of lung cancer cells in vitro

To investigate whether overexpression of CXCL8 had a critical role in cell movement of ADC and whether DACH1 could interfere the CXCL8-induced migration, we performed transwell assay using three groups. Anti-CXCL8 antibody efficiently inhibit cell migration in both SKLU and A549 cells in comparison with the vector cells (p < 0.0001, p < 0.0001) (Fig. 7a, b). Meanwhile, the number of migratory cells was markedly reduced in SKLU and A549 cells expressing DACH1 compared with the vector group (p < 0.0001, p < 0.0001) (Fig. 7c, d). Based on this, we added the human CXCL8 factor into the SKLU-DACH1 and A549-DACH1 cells, and we found that CXCL8 factor restored the migratory ability of SKLU and A549 in spite of the presence of DACH1 (p < 0.0001, p < 0.0001) (Fig. 7e, f).

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DACH1 inhibits the ADC tumor growth and is negatively correlated with CXCL8, cyclin D1, and Ki-67 expression in vivo

The xenograft tumor models were established through subcutaneous injection of A549-vector, A549-DACH1, and A549-ΔDS cells in nude mice (Fig. 8c). A549 cells with sustained DACH1 expression possessed less tumorigenic potential and slower tumor growth rate than A549-vector and A549-ΔDS cells in vivo. Increased DACH1 expression inhibited the volume of tumor by 85% and decelerated the tumor growth (Fig. 8a). Besides, tumor weight was significantly reduced by 85% in mice implanted with A549-DACH1 cells in comparison with the control group (p < 0.0001) (Fig. 8b). Nevertheless, no statistical difference was found in the tumor growth rate and final tumor weight between the A549-vector tumor group and A549-ΔDS tumor group (Fig. 8a, b).

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Immunohistochemical staining of xenograft tumor tissues was conducted to evaluate the protein abundance of DACH1, cyclin D1, Ki-67, and CXCL8 in DACH1-overexpressed and control tumors. Images intuitively showed negative associations between DACH1 and the expressions of cyclin D1, Ki-67, and CXCL8 (Fig. 8d). And the semi-quantitative IHC scores exhibited the accordant trend that high level of DACH1 prominently downregulated the protein abundance of cyclin D1 (p < 0.0001), Ki-67 (p < 0.0001), and CXCL8 (p < 0.0001) (Fig. 8e–h). Our results suggested that besides inhibiting the CXCL8 expression, DACH1 could also reduce the expression of proliferation-related proteins, such as cyclin D1 and Ki-67, which explained the mechanism by which DACH1 restrained the tumorigenesis and growth processes in nude mice model.

DACH1 transcriptionally represses CXCL8 through AP-1 and NF-κB binding sites

To determine the mechanisms by which DACH1 suppressed the CXCL8 expression, several mutant DACH1 expression plasmids were examined to take effect on the CXCL8 promoter (Fig. 9a). We detected CXCL8 promoter activities under the influence of different DACH1 expression plasmids by using luciferase reporter gene assay. Before any treatment, we detected the basal fluorescence activity of empty vector, plasmids containing 2000 bp sequence of CXCL8 promoter and 163 bp sequence of CXCL8 promoter. As shown in Fig. 9b, plasmid containing 163 bp sequence of CXCL8 promoter exhibited higher basic activity (p < 0.0001); thus, we chose it for next experiments. The result suggested that DACH1 repressed the CXCL8 promoter mainly via its DS domain (DACH1/vector: p = 0.0016; DS/vector: p = 0.0056) (Fig. 9c). Meanwhile, activities of point mutants of CXCL8 promoter were detected under the effect of DACH1. DACH1 inhibited the activity of CXCL8 promoter by 90% (p = 0.0002). Mutations of the AP-1 site and NF-κB site on the CXCL8 promoter downregulated CXCL8 promoter activity by 50% and 80% respectively (p = 0.0019, p = 0.0003) and interfered the inhibitory function of DACH1 to some extent (Fig. 9d). DACH1 repressed ΔAP-1 and ΔNF-κB mutants by 75 and 50% (p = 0.0013, p = 0.0089) (Fig. 9d).

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CXCL8 stimulators, TPA and TNF-α, were supplemented to microenvironment of transfected 293T cells for 12 h. Luciferase reporter gene assay showed that TPA and TNF-α enhanced the CXCL8 promoter activity majorly through AP-1 and NF-κB sites, while the existence of DACH1 repressed the activation of CXCL8 promoter stimulated by TPA and TNF-α (Fig. 9d). Real-time PCR was conducted to evaluate the CXCL8 mRNA level in SKLU-vector, SKLU-DACH1, A549-vector, and A549-DACH1 cells under the influence of TPA and TNF-α. It was showed that TPA and TNF-α substantially stimulate the expression of CXCL8 (Fig. 9e, f). Overall, our results proved that DACH1 transcriptionally suppressed CXCL8 through AP-1 and NF-κB sites of CXCL8 promoter in the dependent of the DS domain of DACH1. TPA and TNF-α, acted as two major CXCL8 stimulators, efficiently upregulated the CXCL8 expression.

Relative expressions of CXCL8 and DACH1 predict survival in NSCLC

Kaplan-Meier curves indicated that patients with higher mRNA level of CXCL8 had shorter OS time and RFS time, which represent poor survival in NSCLC (Fig. 10a, c). And the subgroup analysis of ADC showed the same trend (Fig. 10b, d). On the contrary, high DACH1 predicted favorable OS and RFS in NSCLC, especially in ADC (Fig. 10e–h). The blend Kaplan-Meier curves showed that patients with high DACH1 and low CXCL8 had the longest OS and RFS time, whereas low DACH1 and high CXCL8 predicted poorest prognosis (Fig. 10i, j). Taken together, our study was consistent with a previous study [25] that DACH1 was negatively correlated with tumor progression, whereas high CXCL8 expression was associated with unfavorable prognosis of NSCLC patients.

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