Motor neuron and pancreas homeobox 1/HLXB9 promotes sustained proliferation in bladder cancer by upregulating CCNE1/2

SV-HUC-1, T24, MGH-U4, 253 J, 639 V, 5637, RT4, and 575A cells were purchased from American Type Culture Collection and cultured in DMEM with 10% foetal bovine serum (FBS, HyClone, Logan, UT, USA). All cells were incubated at 37 °C in 5% CO₂ atmosphere.

This study was conducted on 167 paraffin-embedded, archived bladder cancer samples that had been histopathologically and clinically diagnosed at the Sun Yat-sen University Cancer Center from 2005 to 2011. Additional file 1: Table S1 summarizes the clinicopathological characteristics. Ethical approval from the Institutional Research Ethics Committee and prior patient consent had been obtained for the use of the clinical specimens for research purposes. Freshly collected bladder cancer tissues were frozen and stored in liquid nitrogen until used.

Total RNA was extracted from cultured cells using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. 2 μg extracted RNA from each sample was used for cDNA synthesis with M-MLV Reverse Transcriptase (Promega, Madison, US). cDNAs were amplified and quantified by SYBR-Green in CFX96 Real Time System C1000 Cycler (Bio-Rad Laboratories, Singapore). Expression data were normalized to the housekeeping gene GAPDH and calculated as 2^{-[(Ct of gene) - (Ct of GAPDH)]}, where Ct represents the threshold cycle for each transcript. Primers used in the PCR reactions are listed in the Additional file 2: Table S4.

Cells were harvested and equal quantities of denatured protein samples were resolved on SDS-polyacrylamide gels, and then transferred onto polyvinylidene difluoride membranes. The membrane was blocked and incubated with primary antibodies, followed by the horseradixh peroxidase-conjugated secondary antibody. Proteins were visualized using ECL reagents. An anti-MNX1 rabbit polyclonal antibody (1:500 dilution; Sigma Aldrich), an anti-CCNE1 Rabbit polyclonal antibody (1:1000 dilution;Proteintech), an anti-CCNE2 Rabbit polyclonal antibody (1:1000 dilution;Proteintech), an anti-α-tubulin mouse monoclonal antibody (1:4000 dilution; Sigma-Aldrich), an anti-Rb rabbit polyclonal antibody (1:1000 dilution; Cell Signaling Technology), an anti-p-Rb Rabbit polyclonal antibody (1:1000 dilution; Cell Signaling Technology), were used in this study.

Immunohistochemical analysis was done to study altered protein expression in 167 human bladder cancer tissues. In brief, paraffin-embedded specimens were cut into 4-μm sections and baked at 65 °C for 30 min. The sections were deparaffinized with xylenes and rehydrated. Sections were submerged into EDTA antigenic retrieval buffer and microwaved for antigenic retrieval. The sections were treated with 3% hydrogen peroxide in methanol to quench the endogenous peroxidase activity, followed by incubation with 1% bovine serum albumin to block nonspecific binding. Rabbit anti-MNX1 (1:500 dilution; Sigma Aldrich) was incubated with the sections overnight at 4 °C. For negative controls, the rabbit anti-MNX1 antibody was replaced with normal goat serum, or the rabbit anti-MNX1 antibody was blocked with a recombinant MNX1 polypeptide by coincubation at 4 °C overnight preceding the immunohistochemical staining procedure. After washing, the tissue sections were treated with biotinylated anti-rabbit secondary antibody (Zymed), followed by further incubation with streptavidin-horseradish peroxidase complex (Zymed). The tissue sections were immersed in 3-amino-9-ethyl carbazole and counterstained with 10% Mayer’s hematoxylin, dehydrated, and mounted in Crystal Mount.

Two independent pathologists blinded to the clinical outcome scored and evaluated the staining results. The scores were determined by combining the proportion of positively-stained cells and the intensity of staining. Cell proportions were scored as follows: 0, no positive cells; 1, < 10% positive cells; 2, 10–35% positive cells; 3, 35–75% positive cells; 4, > 75% positive cells. Staining intensity was graded according to the following standard: 0, no staining; 1, weak staining (light yellow); 2, moderate staining (yellow brown); 3, strong staining (brown). The staining index (SI) was calculated as the product of the staining intensity score and the proportion of positive cells. Using this method of assessment, we evaluated protein expression by determining the SI, with possible scores of 0, 1, 2, 3, 4, 6, 8, 9, and 12. Samples with a SI ≥ 6 were defined as high expression, and samples with a SI < 6 were defined as low expression. The cutoff value was determined on the basis of a measure of heterogeneity using the log-rank test with respect to 5-year overall and relapse-free survival.

The human MNX1 cDNAs were PCR-amplified and cloned into the pMSCV-puro-retro vector (Clontech). Two shRNAs against MNX1 in pLKO.1-puro vector were purchased (Transheep Bio). Transfection of these plasmids was performed using the Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s instructions. Cells (2 × 10⁵) were seeded and infected by retrovirus generated by pMSCV-puro-cDNAs or pLKO.1-puro-shRNAs for 3 days. The stable cell lines were selected with 0.5 μg/ml puromycin for 7 days. The sequences of primers are provided in the Additional file 2: Table S5.

Cells (0.2 × 10⁴ per well) were seeded in 96-well plates. At each time point, the cells were stained with 100 μl sterile 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl- 2H-tetrazolium bromide (MTT) dye (0.5 mg/ml; Sigma) for 4 h at 37C, followed by removal of the culture medium and addition of 150 μl of dimethyl sulphoxide (Sigma). The absorbance was measured at 570 nm, with 655 nm as the reference wavelength. All experiments were performed in triplicate.

Cells were plated on 6 well-plates (0.5 × 10³ cells per plate) and cultured for 10 days. The colonies were stained with 1% crystal violet for 30 s after fixation with 10% formaldehyde for 5 min.

The bladder cancer cells were grown on cover slips. BrdU was added to the cells and incubated for 2 h. The cells were then fixed and stained with anti-BrdU antibody (Upstate, Temecula, CA, USA) according to the manufacturer’s instructions. The percentage of BrdU-positive cells in five random low-power fields was counted and reported as the mean ± SD.

CCNE1 promoter from − 1750 to + 350 and CCNE2 promoter from − 2450 to + 350 were amplified by PCR, and then cloned into the pGL3 plasmid using the Sac II and Xho1 restriction enzymes. Primers for promoter amplification were: CCNE1-promoter-F: gccCCGCGGcctgttactggtgattcctaacg; R: gccCTCGAGgtgtcccctccacccca; CCNE2-promoter-F: gccAGATCTgaaaggggagactgggctg; R: gccGTCGACaaaaaaaggcacagaataaagaaat.

Luciferase assays were performed in stable cell lines with MNX1 overexpression or knockdown. Briefly, 3 × 10⁴ stably transfected cells were cultured in triplicate in 48-well plates for 24 h. Then, 100 ng luciferase reporter plasmids or the control-luciferase plasmid, plus 1 ng pRL-TK Renilla plasmid (Promega), were transiently transfected into the indicated stable cell lines using the Lipofectamine 3000 reagent (Invitrogen), according to the manufacturer’s recommendations. Luciferase and Renilla signals were measured 24 h after transfection, using the Dual Luciferase Reporter Assay Kit (Promega). Primers of the promoters were presented in the Additional file 2: Table S5.

Cells (4 × 10⁶) in a 100 mm culture dish were treated with 1% final concentration of formaldehyde to cross-link proteins to DNA, and the reaction was stopped by addition of glycine. The cell lysates were sonicated to shear DNA to sizes of 300–1000 bp. Equal aliquots of chromatin supernatants were incubated with 1 μg of anti-MNX1, or anti-immunoglobulinG antibodies (Millipore, Billerica, MA, USA) overnight at 4 °C with rotation. After reverse cross-link of protein/DNA complexes to free DNA, PCR was performed. Specific primers for ChIP were presented in the Additional file 2: Table S6.

Male BALB/c-nu mice (5–6 weeks old, 18–20 g) were purchased from the Slac-Jingda Animal Laboratory (Hunan, China), and housed in barrier facilities on a 12-h light/dark cycle. The Institutional Animal Care and Use Committee of Sun Yat-sen University approved all experimental procedures. The mice were randomly assigned to groups (n = 8 per group) and their dorsal flanks were subcutaneously injected with 1 × 10⁶ T24 cells. After 7 days, tumor formation kinetics were estimated by measuring tumor size at 3-day intervals. Tumor volume was calculated using the eq. (L*W²)/2. The animals were euthanized on day 42, and the tumors were excised, weighed, and paraffin-embedded. Serial 6.0-μm sections were obtained and stained with anti–Ki-67 (Dako, Glostrup, Denmark) and anti-BrdU.

Statistical analyses were performed using the SPSS version 19.0 (SPSS Inc.) statistical software package. The log-rank test, χ² test, Spearman rank correlation test, and Student t-test (two-tailed) were used. Multivariate statistical analysis was performed using a Cox regression model. Data are the mean ± SD. P < 0.05 was considered statistically significant.

To investigate the spectrum of MNX1 expression in bladder cancer, we first analyzed MNX1 expression in publicly available human bladder cancer datasets from The Cancer Genome Atlas (TCGA) [24] and Gene Expression Omnibus (GEO) [25]. MNX1 mRNA levels were obviously upregulated in bladder cancer samples compared with normal bladder tissues (Fig. 1a, b). We then detected MNX1 expression in bladder cancer cell lines and tissues. Western blotting and real-time PCR showed that MNX1 mRNA and protein expression, respectively, were markedly upregulated in all bladder cancer cell lines compared to primary normal urethral epithelial cells (Fig. 1c, d). Consistently, MNX1 expression was significantly higher in eight human bladder cancer tissues than in the paired adjacent non-tumor tissues (Fig. 1e, f). These findings demonstrate that MNX1 is upregulated in bladder cancer.

To evaluate the relationship between MNX1 expression and the clinicopathological features of bladder cancer, we analyzed 167 human bladder cancer tissue samples by IHC. Similarly, IHC indicated that MNX1 was greatly overexpressed in bladder cancer samples (Fig. 2a, b). The potential correlation between the expression status of MNX1 and clinical pathologic parameters of patients with bladder cancer was further analyzed (Additional file 1: Table S2). Furthermore, Kaplan–Meier survival curves and log-rank testing showed that patients with high MNX1 expression had shorter 5-year overall survival and relapse-free survival than those with low MNX1 expression (Fig. 2c). In addition, univariate Cox regression analysis indicated that the MNX1 expression and T stage status were each recognized as independent prognostic factors for the 5-year overall survival in bladder cancer (Fig. 2d and Additional file 1: Table S3). Taken together, these results show that MNX1 is upregulated in bladder cancer cell lines and tissues, and that it might lead to poor clinical outcome.

To further investigate the effect of MNX1 in bladder cancer progression, we first established MNX1 stable overexpression and knockdown in T24 and 5637 cell lines (Fig. 3a). Strikingly, we found that MNX1 overexpression enhanced bladder cancer cell proliferation. The tetrazolium (MTT) assay showed that MNX1 upregulation increased the proliferation rate of bladder cancer cells; MNX1 depletion reduced it (Fig. 3b). The subsequent colony formation assay showed that MNX1 promoted bladder cancer cell colony numbers significantly, while silencing MNX1 had the opposite effects, and the colony numbers were counted and shown in histogram (Fig. 3c and d). These data suggest that MNX1 plays an important role in bladder cancer cell proliferation.

To further evaluate whether this MNX1-induced promotion of cell proliferation was due to the inhibition of cell cycle arrest, we examined the effect of MNX1 on cell cycle of bladder cancer cells. Cell lines stably overexpressing MNX1 had significantly increased proportions of cells in the S phase, but reduced proportions of cells in the G1 phase. In contrast, silencing MNX1 had the opposite effects (Fig. 4a). The BrdU incorporation assay showed that the percentages of BrdU-incorporating cells were significantly enhanced in MNX1-overexpressing cells and were reduced in MNX1-silenced cells (Fig. 4b). These results indicate that MNX1 promotes cell cycle progression of bladder cancer cells, confirming that MNX1 promotes bladder cancer cell proliferation.

To further explore the effects of MNX1 in bladder cancer tumorigenesis in vivo, we established MNX1 stable overexpression and knockdown in T24 cells (Fig. 5a). MNX1-overexpressing, MNX1 knockdown, or vector cells were subcutaneously injected into BALB/c-nu mice, and the tumor growth was measured. The MNX1-overexpressing group had prominently increased tumor growth, whereas it was obviously suppressed in the MNX1 knockdown group, both as compared with the vector group (Fig. 5b, c). Moreover, tumors from the MNX1 upregulation group exhibited high expression of the proliferation marker Ki-67 compared to the vector group. In contrast, MNX1 downregulation was associated with low Ki-67 expression (Fig. 5d). These results indicate that MNX1 promotes bladder cancer cell growth in vivo.

As MNX1 is involved in the cell cycle regulation of bladder cancer cells, we examined the expression of cell cycle regulators. Real-time PCR and western blotting revealed that multiple cell cycle regulators, especially CCNE1 and CCNE2, were robustly increased in MNX1-overexpressing cells, but were reduced in MNX1-silenced cells compared to the control cells (Fig. 6a, b). Moreover, phosphorylation of Rb, the downstream target protein of the CCNE–CDK2 complex, was induced in MNX1-overexpressing cells, but was suppressed in the MNX1-silenced cells (Fig. 6b). Consistently, using the cBioportal program (http://​www.​cbioportal.​org), we found that most of these regulators were positively correlated with MNX1 expression (Additional file 3: Figure S1). Intriguingly, among these cyclin genes, CCNE1 and CCNE2 showed the highest correlation with MNX1 (r = 0.32 and 0.26), suggesting that MNX1 might play a role in CCNE1/2 transcription (Fig. 6c). As expected, luciferase reporter assays revealed that MNX1 overexpression activated the luciferase activity of CCNE1 and CCNE2 promoters in the bladder cancer cells, whereas MNX1 downregulation attenuated it (Fig. 6d, e). We then examined whether MNX1 upregulates CCNE1 and CCNE2 transcriptionally using chromatin immunoprecipitation (ChIP). The ChIP showed that MNX1 can bind to different regions within the CCNE1 and CCNE2 promoters (Fig. 6d, e). The above data indicate that MNX1 regulates CCNE1 and CCNE2 by directly targeting their promoter elements.