The prognostic genes were identified as follows: The home page of the Human Protein Atlas database (https://www.proteinatlas.org/humanproteome/pathology) was opened and ‘urothelial cancer’ under the ‘correlation analysis and prognostic genes’ section was selected. There are 1,092 prognostic genes in the online table. A gene is considered prognostic by the Pathology Atlas if correlation analysis of gene expression (FPKM) and prognosis (survival) resulted in Kaplan-Meier plots with high significance (P<0.001) (4). Here, the P-values were calculated with the best expression cut-offs. Based on the cut-off value of each gene, patients were classified into two groups (high expression group and low expression group) and the association between survival and gene expression was assessed. The best expression cut-off refers to the FPKM value that yields maximal difference with regards to survival between the two groups at the lowest log-rank P-value (4).

In addition to the P-values calculated with the best expression cut-offs, the Pathology Atlas also provides the P-values with the median expression cut-offs. Median expression refers to the median FPKM value calculated based on the gene expression data from all patients in this dataset (4). This information is attained as follows: i) The web page of each gene in the table is opened, ii) the median expression value under the ‘urothelial cancer-interactive survival scatter plot & survival analysis’ section is selected and the Kaplan-Meier curve and P-value are adjusted to provide results based on the median expression.

The candidate genes for investigation were screened by initially selecting 29 genes with P<1×10⁻⁶ (best expression cut-off). A total of six genes with P≥0.01 (median expression cut-off) were excluded from the final analysis. Among the remaining 23 genes, five genes [ZNF224 (8), CCNL1 (9), APOBEC3H (10), ANAPC4 (11) and IKBKB (12)] were further excluded as they have previously been reported to be associated with tumor development. Cloning primers were designed for the remaining 18 genes (Table SI). Following PCR amplification, restriction digestion ligation and transformation, 12 genes (ABRACL, MITD1, ZNF524, EMP1, HSPB6, CXorf38, TRIM38, ZNF182, ZNF195, SPRN, PTPN6 and LIPT1) were successfully cloned into the pcDNA3.1+ eukaryotic expression vector (Invitrogen; Thermo Fisher Scientific, Inc.).

A total of 10 patients with bladder cancer from the Affiliated Hospital of Qingdao University were enrolled in the present study between December 2016 and June 2018. The patients had not received radiotherapy or chemotherapy prior to surgery and the pathological type is urothelial cancer. The primary tumors, adjacent normal tissues (the distance between the tumor tissues and normal tissues >3 cm) and metastatic lymph nodes were surgically removed by radical cystectomy and pelvic lymphadenectomy, and immediately fixed in 10% formalin at room temperature for 24 h and embedded in paraffin. The present study was approved by the Human Ethics Committee of The Affiliated Hospital of Qingdao University (Qingdao, China; approval no. QYFYWZLL25948). The present study was performed in accordance with the latest version of the Declaration of Helsinki (13). Written informed consent was provided by all patients prior to the study start. The detailed clinical information of each patient is listed in Table I.

The gene coding sequences of ABRACL, MITD1, ZNF524, EMP1, HSPB6, CXorf38, TRIM38, ZNF182, ZNF195, SPRN, PTPN6 and LIPT1 were amplified via PCR. PCR thermocycling conditions were as follows: 98°C for 1 min and 30 cycles of 98°C for 10 sec, 55°C for 15 sec, and 72°C for 30 sec. PrimeSTAR MAX polymerase (cat. no. R045A; Takara Biomedical Technology (Beijing), Co., Ltd.) was used and the DNA of T24 cell was the template. The products were subsequently cloned into a pcDNA3.1+ vector (Invitrogen; Thermo Fisher Scientific, Inc.) with double digestion and ligation. Following transformation of the ligation products into DH5α competent cells (Takara Biomedical Technology, Co., Ltd.), respectively, the bacteria were cultured using the spread plate method (14) and 10 bacterial colonies for each gene were validated via PCR. The PCR products of positive colonies were sent to Sanger sequencing (BGI, Inc.) and the sequence compare was performed using Chromas software v2.4.1 (http://technelysium.com.au/wp/chromas) for the sequence correctness. The primer sequences used for cloning are listed in Table SI.

Transfection was performed at room temperature for 6 h. The T24 and 5637 bladder tumor cell lines (cat. no. SCSP-536 and TCHu 1; Chinese Academy of Sciences Cell Bank) were seeded into 12-well plates at a density of 100,000 cells/well the day before transfection. For transfection with the gene expression constructs, 1 µg of the gene expression vector (constructed by ourselves) and 2 µl p3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) were diluted into Opti-MEM solution (Invitrogen; Thermo Fisher Scientific, Inc.) and mixed with equal volumes of Opti-MEM, which contained 1.5 µl Lipofectamine^® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Following incubation at room temperature for 5 min, the 12 gene mixtures were added into the appropriate cell cultures, respectively. With regards to the siRNA transfection of genes ABACL, CXof38, LIPT1, MITD1, ZNF524, TRIM38 and EHBP1, 30 pmol siRNA or negative control (NC) oligonucleotide was diluted into Opti-MEM solution and mixed with equal volumes Opti-MEM, which contained 3 µl Lipofectamine^® 3000 reagent. Cells were collected by trypsinization for the following experiments (15), 48 h post-transfection. The siRNAs and NC oligonucleotide (cat. no. siN0000002-1-5) were produced by Guangzhou RiboBio, Co., Ltd., and the siRNA targets of the assessed genes are presented in Table SII.

Cells that underwent transfection were reseeded into 96-well culture plates at a density of 2,000 cells/well, and maintained in 100 µl RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc.) at 37°C in 5% CO₂. The wells in the borders of the plates were filled with phosphate buffered saline (PBS; Beyotime Institute of Biotechnology) to protect against the influence of culture medium volatilization. Cells were incubated with 10 µl MTT reagent (Beyotime Institute of Biotechnology) at 37°C for 4 h, 72 h post-transfection. Following the MTT incubation, the purple formazan crystals were dissolved using 100 µl dimethyl sulfoxide (Beyotime Biotechnology) and viability was subsequently analyzed at a wavelength of 490 nm using a spectrophotometer (Thermo Fisher Scientific, Inc.) (16,17). All experiments were performed in triplicate.

The Transwell assay was performed as previously described (18). Cells that underwent transfection were washed twice with RPMI-1640 medium without FBS. A total of 1×10⁵ cells were resuspended in RPMI-1640 medium without FBS and plated in the upper chambers of Transwell plates (cat. no. 353097; BD Biosciences), which had transparent polyester membranes and a pore size of 8 µm. RPMI-1640 medium supplemented with 10% FBS was plated in the lower chambers. Following incubation at 37°C for 12 h, the upper chambers were fixed with 100% methyl alcohol at room temperature for 10 min and subsequently stained with 0.1% crystal violet at room temperature for 10 min, and the excess dye was washed off with water. The unmigrated cells were removed using cotton swabs. Stained cells were counted in five randomly selected fields using a light microscope (magnification, ×100) (18).

T24 cells were seeded into 6-well culture plates at a density of 1×10⁶ cells/well the day before transfection. Cells transfected with the negative control and MITD1 siRNAs were further cultured in PRMI-1640 medium supplemented with 10% FBS at 37°C for 48 h. Cells were scratched with 200 µl pipette tips, washed once with PBS, and further incubated in serum-free medium at 37°C cell incubator for 48 h. The scratches were observed at 0, 24 and 48 h, respectively, using an inverted light microscope (magnification, ×100; IX73; Olympus Corporation). The results were quantified using ImageJ software (version 1.50i; National Institutes of Health) and analyzed using GraphPad Prism software (version 6; GraphPad Software, Inc.). All experiments were performed in triplicate.

IHC analysis was performed as previously described (19). Briefly, the surgically removed tissues were fixed in 10% formalin at room temperature for 24 h and embedded in paraffin. Paraffin-embedded tissue samples were cut into 4-µm-thick sections and heated overnight at 50°C. Prior to immunostaining, deparaffinization and hydration were performed in xylene and a descending ethanol series (100, 95 and 75%). Heat Induced Epitope Retrieval was performed by heating the slides immersed in retrieval solution: 10 mM sodium citrate buffer, pH 6.0, with 1 mM EDTA, at 125°C for 4 min in a pressure boiler. Tissue sections were incubated with 0.3% (v/v) H₂O₂ in 95% ethanol at room temperature for 5 min to inhibit endogenous peroxidase activity. Tissue sections were washed twice with PBS. Immunohistochemical staining was performed with Polymer HRP Detection System for Rabbit Primary Antibody (cat. no. PV-6001; ZSGB-BIO, Inc.). All incubations were performed at room temperature and reagents were applied at a volume of 200 µl per slide. According to the instruction manual, blocking was not necessary due to the special design of the primary antibody dilution buffer. The slides were incubated with MITD1 antibody (cat. no. HPA036162; Atlas Antibodies AB; 1:50) at room temperature for 30 min. The slides were rinsed three times with PBS, and subsequently incubated with peroxidase labeled polymer conjugated to goat anti-rabbit IgG antibody working solution (cat. no. PV-6001; ZSGB-BIO, Inc.) at room temperature for 30 min. The slides were re-washed twice in wash buffer and developed at room temperature for 5 min using DAB (cat. no. PV-6001; ZSGB-BIO, Inc.) as the substrate. Counterstaining was performed using hematoxylin solution (cat. no. C0107; Beyotime Institute of Biotechnology) for 3 min at room temperature. Cells were observed under a light microscope (magnification ×400).

RNA was extracted from cells that underwent transfection using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The library construction, sequencing and data analysis were performed by Annoroad Gene Technology Corporation (http://en.annoroad.com), with an Illumina Sequencing platform (HiSeq2500; Illumina, Inc.). Sequencing libraries were generated using the NEBNext^® Ultra™ RNA Library Prep kit for Illumina^® (cat. no. E7530L; NEB, Inc. http://www.neb.com), according to the manufacturer's instructions. RNA integrity and concentration were assessed using the RNA Nano 6000 Assay kit of the Bioanalyzer 2100 system (cat. no. 5067-1511; Agilent Technologies, Inc.). The concentration of library was measured using the Qubit^® RNA Assay kit (cat. no. Q32852; Thermo Fisher Scientific, Inc.) to preliminarily quantify and subsequently dilute to 1 ng/µl. The accurate concentration was measured using a StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, Inc.). The valid concentrations of the libraries are greater than 10 nM. The clustering of the index-coded samples was performed on a cBot cluster generation system using the HiSeq PE Cluster kit v4-cBot-HS (cat. no. PE-401-4001; Illumina, Inc.), according to the manufacturer's instructions. Following cluster generation, the libraries were sequenced on an Illumina platform (HiSeq2500; Illumina, Inc.) and 150-bp paired-end raw reads were generated.

The Perl script was used to filter the original data to guarantee the data quality. The steps were as follows: i) Trim Smart-seq2 public primer sequence from the reads; ii) remove the contaminated reads of adapters; iii) remove the low quality reads and iv) remove the reads with N base >5% for total bases. The reference genomes and the annotation file were downloaded from ENSEMBL database (http://www.ensembl.org/index.html). Bowtie2 v2.2.3 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) was used to build the genome index, and Clean Data was subsequently aligned to the reference genome using HISAT2 v2.1.0 (http://ccb.jhu.edu/software/hisat2/faq.shtml). Reads Count for each gene in each sample was counted using HTSeq v0.6.0 (https://htseq.readthedocs.io), and Fragments Per Kilobase Millon Mapped Reads (FPKM) was subsequently calculated to estimate the expression level of genes in each sample. DESeq2 (http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html) was used to compare differences in gene expression levels between groups. Genes with P≤0.05 and fold change (FC)≥1.5 were classified as differentially expressed genes (DEGs).

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Database for Annotation, Visualization and Integration Discovery (DAVID; version 6.8; http://david.ncifcrf.gov/home.jsp). DEGs between the control and MITD1 knockdown groups were uploaded onto DAVID. The enriched KEGG pathways of the genes were assessed using the ‘functional annotation tool’ within DAVID. Modified fisher exact P-value was calculated to determine the enriched extent. Gene count=2 was used as the threshold to include the items in the table.

Transfection efficiency was demonstrated via RT-qPCR. Total RNA were extracted from T24 and 5637 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed at 37°C for 15 min using the PrimeScript™ RT reagent kit with gDNA Eraser (cat. no. RR047; Takara Biomedical Technology, Inc.). qPCR was subsequently performed using Platinum™ SYBR™ Green qPCR SuperMix-UDG (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. GAPDH was used as the reference gene. The qPCR primers are listed in Table SIII. qPCR thermocycling conditions were as follows: 95°C for 1 min and 40 cycles of 95°C for 15 sec, 60°C for 30 sec, and 72°C for 15 sec. All data were analyzed using the 2^−ΔΔCq method (20).

Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, Inc.). Experiments were performed at least in triplicate, and data are presented as the mean ± standard deviation. Unpaired Student's t-test was used to compare differences between two groups, while one-way ANOVA followed by Tukey's post hoc test was used to compare differences between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

In order to measure the function of the prognostic genes on cell proliferation and migration, the present study focused on the no bias system-level analysis of the Pathology Atlas, which is based on the genome-wide transcriptomics analysis of TCGA project and the Human Protein Atlas project (4) (http://www.proteinatlas.org). The present study selected the top 18 genes significantly associated with survival in patients with urothelial cancer in the Pathology Atlas project, which met the following criteria: i) The prognostic P<1×10⁻⁶ (best cut-off); ii) the prognostic P<1×10⁻² (median expression cut-off) and iii) has rarely been reported to be associated with cancer progression. Following PCR amplification and restriction digestion, 12 genes (ABRACL, MITD1, ZNF524, EMP1, HSPB6, CXorf38, TRIM38, ZNF182, ZNF195, SPRN, PTPN6 and LIPT1) were successfully cloned into the pcDNA3.1+ eukaryotic expression vector (Fig. 1A). The process of screening the 12 genes is presented in Fig. 1B and detailed information of the 12 genes is presented in Table II. All 12 constructs were validated via Sanger sequencing.

Recombinant gene transfection may induce cell death or change cell proliferation rate (21). Thus, the present study assessed cell viability via the MTT assay 72 h post-transfection (Fig. 2A). The results demonstrated that in the in vitro experiments, there was no significant change in cell viability induced by transfection of the 12 gene expression constructors (Fig. 2B).

The 12 gene expression vectors were subsequently transfected into T24 bladder cancer cells, respectively. The migratory ability of cells was assessed via the Transwell assay, 48 h post-transfection. The results demonstrated that the migratory rate of cells transfected with CXorf38, EMP1, TRIM38, ZNF182 and ZNF524 did not changed compared with the mean value of the 12 overexpression groups. However, ABRACL, PTPN6, SPRN and ZNF195 transfection increased the cell migratory ability. The three genes, including two favorable-survival associated genes (MITD1 and LIPT1) and one unfavorable-survival associated gene, HSPB6, inhibited cell migration at a certain level (Fig. 3).

A siRNA library of 7 genes, in which each gene had two siRNAs, was used to screen the genes regulating cell migration with a loss-of-function strategy (Fig. 4A). MITD1 knockdown markedly increased cell migration (Fig. 4B and C). The wound healing assay was performed to validate the role of MITD1 on cell migration and the results demonstrated that MITD1 knockdown significantly promoted cell migration (Fig. 5).

As MITD1 inhibited cell migration in vitro, downstream genes were investigated with RNA sequencing. T24 cells were transfected with MITD1 siRNAs or NC oligonucleotide, while the untreated cells were cultured as the BLANK group. Cell RNA was extracted using TRIzol^® reagent 48 h post-transfection, and transcriptome sequencing was subsequently performed. The results demonstrated that cells transfected with MITD1 siRNA exhibited dysregulation of the migration associated genes, KISS1, SPANXB1, SPINT1, PIWIL2, SNAI1, APLN, EDN1 and CTHRC1 compared with the NC and BLANK groups (Fig. 6). The enriched KEGG pathways were analyzed using DAVID Bioinformatics Resources 6.8. The results demonstrated that ‘transcriptional dysregulation in cancer’ was significantly enriched (P=0.019) (Table SIV).

Lymphatic metastasis is one of the main characteristics underlying bladder cancer progression, and a key predictor of cancer-specific survival (22). To identify the associations between MITD1 and lymphatic metastasis, the present study assessed MITD1 protein expression in the primary tumors and paired metastasis in lymph nodes. IHC analysis demonstrated that there was a significant decrease in MITD1 protein expression in the metastatic tumors compared with the primary tumors (mean IHC score, 6.875 vs. 2.800; P<0.001; Fig. 7).