SLC12A8 plays a key role in bladder cancer progression and EMT

2.1 Bioinformatics analysis

The RNASeq data from TCGA (https://cancergenome.nih.gov/) [20], including 414 samples of bladder cancer patients and 19 normal samples, were used for the SLC12A8 expression analysis. Among the 414 tumor samples, 405 cases with relatively complete clinical data were used to study the relationship between SLC12A8 expression and clinical factors. Moreover, the data obtained from TCGA and GTEx (tumor = 411, normal = 28) were processed by UCSC (https://genome.ucsc.edu/) and used for the SLC12A8 expression analysis. In addition, the SLC12A8 expression in infiltrating bladder urothelial carcinoma and normal tissues was analyzed using Oncomine database (https://www.oncomine.org). The median expression of the gene was used as a boundary to classify the samples into high and low expression groups. Gene expression profiling interactive analysis (GEPIA; http://gepia.cancer-pku.cn/) website was applied to detect the relationship between SLC12A8 and E-cadherin/β-catenin/vimentin/snail/slug in bladder cancer by the Pearson correlation coefficient.

2.2 Cell culture

The human bladder cancer cell lines (5637, J82, and T24) and normal control cells SV40-immortalized human uroepithelial cells (SV-HUC-1) were obtained from the Chinese Academy of Medical Sciences (Shanghai, Chinese). All cells were cultured in RPMI-1640 mixed with 100 units/mL penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum. Then, they were maintained under 5% CO₂ at 37°C for subsequent experiments.

2.3 Transfection

The T24 cell line underwent transfection with si-SLC12A8#1 (5′-GCCATGTATATCACCGGCTT-3′) or si-SLC12A8#2 (5′-CCTACAAGATAGCTTCCTCT-3′). The control siRNA sequence (si-con) was 5′-CGAACUCACUGGUCUGACC-3′. For upregulation of SLC12A8, the plasmid vector pcDNA3.1-SLC12A8 was transfected into 5637 cell line, and pcDNA3.1 empty vector was used as a control. Transfection was performed as described in the Lipofectamine 2000 Transfection Kit. When the cell concentration reached 80% in the six-well plate, the antibiotic-free medium was replaced 2 h before the transfection, and then, cell transfection was performed. The expression of the SLC12A8 could be observed after 24 h.

2.4 RNA isolation and qRT-PCR assays

The total RNA was extracted by Trizol reagent. After reverse transcription to form cDNA, the real-time PCR system was performed to assess mRNA-SLC12A8 expression. The qRT-PCR condition was as follows: initial denaturation (5 min, 95°C), 40 cycles of denaturation (30 s, 95°C), annealing (45 s, 60°C), and extension (30 s, 72°C). Each experiment was assessed in triplicate. Quantification was done using the 2^−ΔΔCT method to calculate the mRNA expression level of SLC12A8. The sequences of primers in this assay were listed as follows: SLC12A8: F: 5′- TGGCGTCTACTCCATGATCTCC-3′, R: 5′-CCGAGATGGATTCAGCAAAGCC-3′; GAPDH: F: 5′-TGTGTCCGTCGTGGATCTGA-3′, R: 5′-CCTGCTTCACCACCTTCTTGA-3′. GAPDH served as the internal reference.

2.5 Western blotting assays

After transfection for 24 h, cells in the six-well plate were placed on ice. After extracting by RIPA lysate (with protease inhibitor), the concentrations of protein were determined by the BCA method. Then, proteins were heated at 95°C for 5 min before isolating by SDS–PAGE, and they were transferred onto a PVDF membrane subsequently. The membrane was then blocked for 1 h using 5% skim milk at an ambient temperature. Following that, the membrane was incubated with primary antibodies at 4°C overnight. The primary antibodies used in this study were as follows: SLC12A8 (ab122152, Abcam), E-cadherin (ab40772, Abcam), β-catenin (ab227499, Abcam), vimentin (ab45939, Abcam), snail (ab82846, Abcam), and slug (ab27568, Abcam). After incubation, the membrane was taken out and washed using TBST (tris buffered saline with Tween) for 3 × 5 min. Subsequently, bands were finally examined by ECL (enhanced chemiluminescence) after incubating the membrane with the secondary antibody at an ambient temperature for 1 h. The gray values were scanned by Quantity One software, and the ratio of target protein/GAPDH was calculated.

2.6 Cell viability assays

Cell viability was detected by the CCK8 kit (Lianke Bio, China). Briefly, the cells were digested after 24 h transfection to prepare cell suspension. Then, cell suspension was plated in 96-well plates, and the number of cells in each well was about 1 × 10³. The 96-well plates were incubated under standard conditions, and cell viability was examined every 24 h. A total of 10 µL CCK8 was added into each well at the indicated time points and incubated for 1.5 h at 37°C. Finally, the optical density (OD) value at 450 nm was detected, and the proliferation curve was drawn.

2.7 Transwell invasion and migration assays

After transfection, cells were treated to prepare cell suspension with serum-free culture. Then, cells were inoculated in the upper chamber at the density of 1 × 10⁵ cells per well. The upper layer membranes were either coated with or without 100 µL Matrigel (serum-free medium diluted 1:6), which used to evaluate the migration and the invasion capacity respectively. About 500 µL culture medium was placed in the bottom chambers. Cells were grown in six-well plates overnight, and then, the chambers were taken out. The cells on the upper layers were wiped by swab, and the cells that invaded and migrated to the bottom layer were fixed in 4% paraformaldehyde for 0.5 h and stained using 0.1% crystal violet for 20 min after washing with PBS. Cells in five different fields were selected for calculation, and images were captured under a microscope.

2.8 Plate clone formation assay

Exponentially growing cells were trypsinized to individual cells to make cell suspensions. Then, cells were seeded in 60 mm Petri dish at 400 cells/Petri dish density with 5 mL culture solution prewarmed at 37°C. After that, cells were incubated under 5% CO₂ at 37°C for 1–2 weeks to form colonies. The cell culture was terminated when there is visible clone formation. Subsequently, cells were stained using 0.1% crystal violet for 0.5 h after being fixed with 5 mL 4% paraformaldehyde for 0.5 h. The Petri dish was air dried after the staining solution was slowly washed with the running water. Then, the number of clones was counted and the size of clones were compared.

2.9 Data analysis and statistics

SPSS 22.0 and Graphpad Prism 5.0 software were performed for all the statistical analyses, and all experiments data were represented as means ± SD. The comparison between two composition ratios of the data was based on Student’s t-test, and comparison among multiple samples was conducted by one-way ANOVA with post hoc Dunnett’s test [21]. Kaplan–Meier analysis was conducted in SPSS 22.0 software to evaluate the association between SLC12A8 expression and survival of bladder cancer patients. Furthermore, the log-rank test was adopted to evaluate the statistical significance [22]. To investigate whether SLC12A8 can be used as an independent predictor of bladder cancer prognosis, Cox regression was used. The association between gene expression and clinical features was assessed using the chi-square test. p < 0.05 was regarded as significance, and all experiments were repeated for three times.

3.1 SLC12A8 was upregulated in bladder cancer

Rely on the TCGA database, the SLC12A8 expression level was demonstrated notably higher in bladder cancer tissue compared with normal tissue (p = 0.0005; Figure 1a). Moreover, data from TCGA and GTEx presented the similar result, and SLC12A8 was highly expressed in bladder cancer compared with the normal samples (p = 1.07 × 10⁻¹²; Figure 1b). Besides, the results from Sanchez-Carbayo Bladder 2 statistics from Oncomine database indicated that SLC12A8 expression was significantly enhanced in infiltrating bladder urothelial carcinoma compared with the normal bladder tissues, which was consistent with the aforementioned results (p < 0.0001, Figure 1c).

Figure 1

Expression of SLC12A8 in bladder cancer and its prognostic value. (a) SLC12A8 expression level was remarkably higher in bladder cancer tissues (n = 414) than that in normal tissues (n = 19) based on data from the TCGA database, p = 0.0005. (b) Data from TCGA and GTE _X showed that SLC12A8 was significantly upregulated in bladder cancer samples (n = 411) than that in normal samples (n = 28), p = 1.07 × 10⁻¹². (c) SLC12A8 expression was meaningfully higher in infiltrating bladder urothelial carcinoma (n = 81) compared with normal bladder (n = 48) in the Oncomine dataset, p < 0.0001. (d) SLC12A8 expression in (5637, J82, and T24) cell lines was higher than normal control cells (SV-HUC-1), **p < 0.01. (e) Kaplan–Meier analysis showed the bladder cancer patients’ survival with unequal SLC12A8 expression level, p = 0.043. (f) Kaplan–Meier analysis showed that high expression of SLC12A8 led to the worse disease-free survival in patients with bladder cancer, p = 0.003.

Afterward, the SLC12A8 expression levels in 5637, J82, and T24 cell lines and SV-HUC-1 cell line have been examined by applying qRT-PCR (Figure 1d). The outcomes suggested that the mRNA levels of SLC12A8 in 5637, J82, and T24 cell lines were notably higher compared with that in SV-HUC-1 cells (p < 0.01). From the results, the mRNA expression of SLC12A8 in T24 cells was the highest, while the expression value in 5637 cells was the lowest. Therefore, the T24 and 5637 cells were selected to carry out the subsequent SLC12A8 knockdown and upregulation experiments, respectively.

3.2 High SLC12A8 expression correlated with poor prognosis of bladder cancer

The correlation between SLC12A8 expression and clinicopathological features in bladder cancer patients is presented in Table 1. The bladder cancer patients were divided into SLC12A8 high expression group (n = 202) and SLC12A8 low expression group (n = 203) according to the median value of SLC12A8 expression. Table 1 demonstrates that the SLC12A8 expression was correlated with age (p ≤ 0.001), pathological stage (p ≤ 0.001), T stage (p ≤ 0.001), and N stage (p ≤ 0.001) of bladder cancer patients. However, the correlations between SLC12A8 expression and the gender and M stage were not significant.

In addition, to explore whether SLC12A8 expression levels were associated with prognosis of bladder cancer, the Kaplan–Maier analysis and log-rank comparison were carried out. The results demonstrated that higher SLC12A8 expression level was correlated with shorter OS in bladder cancer patients (p = 0.043, Figure 1e). Moreover, higher SLC12A8 expression level led to the worse disease-free survival (p = 0.003, Figure 1f).

The Cox regression analysis results showed that the SLC12A8 expression, Clinical stage, T stage, lymph node, metastasis, and age were notably correlated with OS of bladder cancer patients. Besides, pathologic-N could be used as an independent prognostic factor of bladder cancer by the multivariate analysis (Table 2; p = 0.013, HR = 2.034, 95% CI: 1.165–3.552).

3.3 SLC12A8 knockdown/upregulation efficiency determined by qRT-PCR and western blot analysis

In brief, human bladder cancer T24 cells were transfected with si-SLC12A8#1 and si-SLC12A8#2, while 5637 cells were transfected with pcDNA3.1-SLC12A8. The SLC12A8 mRNA and protein levels in T24 and 5637 cells were detected by qRT-PCR and western blot. The results of qRT-PCR suggested that mRNA-SLC12A8 level in T24 cells with depleted SLC12A8 was evidently lower than that in the control group (p < 0.01) (Figure 2a). The inhibitory effect was best in the si-SLC12A8#1 group, and the relative expression of mRNA was 17.84% of the control group. The outcomes of western blot revealed that the lower SLC12A8 protein expression was presented in si-SLC12A8#1 group (p < 0.01) (Figure 2b). For the accuracy of the inference experiment, the following loss of function assays was performed using both si-SLC12A8#1 and si-SLC12A8#2. However, the mRNA and protein expression levels of SLC12A8 were notably promoted in 5637 cells transfected with pcDNA3.1-SLC12A8 (Figure 2c and d).

Figure 2

Expression of SLC12A8 mRNA and protein in bladder cancer cells transfected with si-SLC12A8 or pcDNA3.1-SLC12A8. (a) The SLC12A8 mRNA expression levels were decreased in T24 cells after transfection with si-SLC12A8#1 or si-SLC12A8#2, **p < 0.01. (b) The SLC12A8 protein expression was decreased in T24 cells with depleted SLC12A8, **p < 0.01. (c) The mRNA-SLC12A8 expression level was remarkably increased in 5,637 cells with upregulated SLC12A8, **p < 0.01. (d) The SLC12A8 protein expression was notably higher in 5,637 cells with upregulated SLC12A8, **p < 0.01.

3.4 Knockdown/upregulation of SLC12A8 affected the growth, invasion, and migration of bladder cancer cells

To further explore the effect of SLC12A8 on the proliferation and motility of bladder cancer cells, CCK8 and colony formation assays were carried out in T24 and 5673 cells, which transfected with si-SLC12A8#1 and si-SLC12A8#2 or pcDNA3.1-SLC12A8, respectively. The results of the CCK8 assay indicated that the proliferative ability of T24 cells with silenced SLC12A8 by si-SLC12A8#1 or si-SLC12A8#2 was notably decreased (p < 0.01) (Figure 3a and b). However, it was showed that the viability of 5,637 cells was significantly promoted after upregulation of SLC12A8 (p < 0.01) (Figure 3c). The outcomes of plate cloning experiment showed that compared with the control group, the proliferation rate of the T24 cells was significantly reduced after knockdown of SLC12A8 by si-SLC12A8#1 or si-SLC12A8#2 (Figure 3d–e) (p < 0.01). Figure 3f showed that the results of plate cloning experiments indicated that the number of colonies in 5,637 cells group (36.33 ± 7.09) increased compared with the control group (11.33 ± 3.51) (p < 0.01). The above experimental data demonstrated that SLC12A8 upregulation could notably promote the proliferative capacity of bladder cancer cells.

Figure 3

SLC12A8 knockdown or upregulation affected the growth of bladder cancer cells. (a) The proliferation of T24 cells with downregulated SLC12A8 detected by CCK8, **p < 0.01. (b) The proliferation of T24 cells was examined by CCK8 after knockdown of SLC12A8 by si-SLC12A8#2, **p < 0.01. (c) The proliferation of 5,637 cells with upregulation of SLC12A8 detected by CCK8, **p < 0.01. (d) The plate cloning experiments showed that the proliferation rate of T24 cells in the si-SLC12A8#1 group was remarkably reduced compared with the si-con group, **p < 0.01. (e) Knockdown of SLC12A8 by si-SLC12A8#2 reduced the colony formation ability of T24 cells, **p < 0.01. (f) The plate cloning experiments showed that the proliferation rate of 5,637 cells in the pcDNA3.1-SLC12A8 group was notably promoted compared with vector group, **p < 0.01. (g) Knockdown of SLC12A8 by si-SLC12A8#1 repressed the invasion and migration of T24 cells, **p < 0.01. (h) The invasion and migration abilities of T24 cells were inhibited by si-SLC12A8#2 treatment, **p < 0.01. (i) Upregulation of SLC12A8 enhanced the invasion and migration of 5,637 cells, **p < 0.01.

Next, the Transwell assays were used to detect the association between SLC12A8 expression and bladder cancer metastasis. The outcomes showed that the invasive ability of the si-SLC12A8#1 group was 48.38% of the control group (p < 0.01), while the migratory ability was 52.26% of the control group (p < 0.01, Figure 3g). Similarly, the invasive (40.73%) and migratory (51.27%) abilities of T24 cells were reduced after knockdown of SLC12A8 by si-SLC12A8#2 treatment (p < 0.01, Figure 3h). The aforementioned results indicated that the knockdown of SLC12A8 can reduce the invasive and migratory capacities of bladder cancer cells. On the contrast, the number of transmembrane cells in pcDNA3.1-SLC12A8 group was notably increased. The invasive ability was 2.37 times of the control group (p < 0.01), while the migratory ability was 1.91 times of the control group (p < 0.01) (Figure 3i), illustrating that the invasive and migratory capacities of bladder cancer cells were enhanced by SLC12A8 upregulation.

3.5 EMT mediated the function of SLC12A8 in regulating bladder cancer

The correlation between SLC12A8 and EMT marker including CDH1 (E-cadherin), CTNNB1 (β-catenin), VIM (vimentin), SNAIL1 (snail), and SNAIL2 (slug) was analyzed in GEPIA website by the Pearson correlation coefficient (Figure 4a). The data showed that SLC12A8 expression was negatively correlated with E-cadherin and positively correlated with β-catenin, vimentin, snail, and slug. The western blot evidenced the aforementioned results: β-catenin, vimentin, snail, and slug protein were decreased, and E-cadherin protein was increased when SLC12A8 was knocked down by si-SLC12A8#1 or si-SLC12A8#2 in T24 cells (Figure 4b and c), while the result was opposite when SLC12A8 was upregulated in 5,637 cells (Figure 4d). The aforementioned outcomes revealed that SLC12A8 upregulation could remarkably promote the EMT process of 5,637 cells, while the EMT in T24 cells was notably inhibited when SLC12A8 was depleted.

Figure 4

EMT mediated the function of SLC12A8 in regulating bladder cancer. (a) The correlation between SLC12A8 and EMT markers was analyzed in GEPIA website by Pearson correlation coefficient. (b and c) The expression of EMT protein markers in T24 cells was detected by western blotting after depletion of SLC12A8 by si-SLC12A8#1 or si-SLC12A8#2, **p < 0.01. (d) The expression of EMT protein markers in 5637 cells was examined by western blotting with upregulation of SLC12A8, **p < 0.01.