Cell culture

The uroepithelial cell line SV-HUC-1 (RRID: CVCL_3798) and bladder cancer cell lines UM-UC-3 (RRID: CVCL_1783), SW780 (RRID: CVCL_1728), RT4 (RRID: CVCL_0036), and 5637 (RRID: CVCL_0126) were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China), RT112 (RRID: CVCL_1670) was purchased from DSMZ, T24T (RRID: CVCL_M892) and EJ (RRID: CVCL_2893) were gifted by Dr. Guosong Jiang of Wuhan Union Hospital. All cell lines were cultured in appropriate complete medium and maintained at 37 °C in 5% CO2 condition.

All cell lines have been authenticated using short tandem repeat profiling within the last 3 years and tested for mycoplasma contamination by DAPI staining. All experiments were performed with mycoplasma-free cells.

Clinical tissue specimens

Fresh tumor specimens and adjacent non-tumorous tissues of BC patients were obtained from Wuhan Union Hospital. None of patients involved in this study received preoperative radiotherapy or chemotherapy. All procedures related to clinical specimens were approved by the ethics committee of the Union Hospital affiliated to Huazhong University of Science and Technology and undertaken in accordance with guidelines set forth by the Declaration of Helsinki.

Quantitative real-time PCR (qRT-PCR)

Total RNA that extracted from cell lines or fresh tissues with TRIzol reagent (Invitrogen, CA, USA) was reverse transcribed into cDNA using PrimeScript RT Reagent Kit (Takara, Kyoto, Japan). To quantify the expression level, qRT-PCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems, CA, USA). 2–ΔΔCT method was used to calculated the relative expression. GAPDH was used as the internal control. Primers used in this study were synthesized by Sangon Biotech (Shanghai, China) (Additional file 5: Table S2).

Western blot

Cellular or tissue protein was extracted with RIPA lysis buffer (Servicebio, Wuhan, China), and western blot was performed as previously described [27]. Antibodies use for western blot: anti-PABPN1 (66807-1-Ig, Proteintech, USA), anti-GAPDH (60004-1-Ig, Proteintech, USA), anti-ATP6AP2 (10926-1-AP, Proteintech, USA), anti-TMEM97 (26444-1-AP, Proteintech, USA), anti-ZNF777 (NBP1-03344, Novus Biologicals, USA), anti-CDCA2 (17701-1-AP, Proteintech, USA), anti-CCND3 (26755-1-AP, Proteintech, USA), anti-AACS (ab237802, Abcam, UK), anti-β-catenin (51067-2-AP, Proteintech, USA), anti-Histone H3 (17168-1-AP, Proteintech, USA), and anti-α-Tubulin (66031-1-Ig, Proteintech, USA).

Lentivirus construction and infection

Human PABPN1 cDNA was amplified and cloned into GV705 lentivirus vector (CMV enhancer-MCS-3FLAG-sv40-puromycin) (GeneChem, Shanghai, China). Oligos of PABPN1 shRNAs were synthesized and inserted into GV112 vector (hU6-MCS-CMV-Puromycin) (GeneChem, Shanghai, China). To establish stable cell lines, BC cells were infected with lentivirus vectors expressing PABPN1 (PABPN1) or PABPN1-shRNAs (PABPN1-Sh#1 and PABPN1-Sh#2), and were selected with 2 μg/ml puromycin for at least 30 days.

Cell counting kit-8 (CCK-8) assay

For CCK-8 assay, cells from different groups were seeded into a 96-well plate at 3 × 103 cells per well and incubated at 37 °C. To detect the proliferation ability, cells were incubated in 100 μL of complete medium adding with 10 μL of CCK-8 reagent (DOJINDO, Kumamoto, Japan) for 2 h at 37 °C. The absorbance, which represented the number of surviving cells, was measured at 450 nm using a microtiter plate reader.

Cell migration and invasion assays

Transwell system (Corning, NY, USA) was used to evaluated the migration or invasion capacity of BC cells. In brief, cells suspended in serum-free medium were seeded in upper chambers without (for migration assay) or with (for invasion assay) Matrigel, while the serum-containing medium was added into lower chambers. After 24 h, cells remaining on the top surface were gently removed, and cells migrated or invaded to the lower surface were fixed, stained, and photographed, under a light microscope.

Colony formation assay

BC cells were seeded in 6-well plates at the density of 500 cells per well, and incubated at 37 °C in 5% CO2 for 2 weeks. Cell colonies were immobilized, dyed and recorded using the digital single-lens reflex camera (D610, Nikon, Japan).

Cell cycle analysis

For cell cycle analysis, BC cells were collected and fixed in 70% ethanol at 4 °C. After 0.5 h, cells were washed with PBS and incubated with PI/RNase Staining Buffer (BD Biosciences, CA, USA) in the dark for 30 min. Cell cycle was analyzed by BD LSRFortessa X-20 (BD Biosciences, CA, USA).

Detection of neutral lipid, triglyceride, and phospholipid

Neutral lipid accumulation in BC cells was monitored by lipophilic fluorescence dye BODIPY 493/503 (Invitrogen, CA, USA). Briefly, cells were washed in Phosphate Buffered Saline (PBS), fixed with 4% paraformaldehyde, and stained with BODIPY 493/503 (1ug/mL) for 45 min at room temperature. Nuclei were counterstained with Hoechst (10 μg/mL) for 15 min. All fluorescence images were captured using a Nikon A1R-si Laser Scanning Confocal Microscope (Nikon, Tokyo, Japan). For quantitative estimation of phospholipid and triglyceride, the EnzyChrom Phospholipid Assay Kit and EnzyChrom Triglyceride Assay Kit (BioAssay Systems, CA, USA) were used according to manufacturer’s instructions.

In vivo tumor growth and metastasis assays

Animal experiments involved in this study were conducted in the light of the Guide for the Care and Use of Laboratory Animals [28], and approved by the ethics committee of Tongji Medical College of the Huazhong University of Science and Technology. BALB/c-nu mice (male, 3–5 weeks of age) were randomly divided into 4 groups. For the tumor growth assay, BC cells were injected subcutaneously into the right axilla of each mouse. After injection, tumor volumes were observed using an external caliper and recorded using the equation (L × W2)/2. On day 30, all of the mice were euthanized, and tumors were excised, weighed, and photographed. For the metastasis assay, cells were injected into the tail-vein of each mouse. On day 50, all animals were euthanized by cervical dislocation, and lungs were excised and imaged with the In Vivo Optical Imaging System (In Vivo FX PRO, Bruker, MA, USA).

Dual-luciferase reporter assay

Dual-luciferase reporters based on psiCHECK-2 vectors containing 3’UTRs of ATP6AP2, TMEM97, and ZNF777 were constructed, and then transfected into BC cells with corresponding miRNA mimics. After 48 h, the luciferase activities were measured with the Dual-Luciferase Reporter Assay System (Promega, WI, USA).

Rapid amplification of cDNA 3’ ends (3’RACE)

Total RNA was extracted and reversely transcribed into cDNA using the primer 5ʹ-CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTT-3ʹ. Forward primers designed specifically for PTCD1, AACS, ATP6AP2, PIGG, CDCA2, TMEM97, ZNF777, REPIN1, and CCND3 were designed (Additional file 5: Table S2) and mixed with the reverse primer 5ʹ-CCAGTGAGCAGAGTGACG-3ʹ to amplify the cDNA 3’ ends. Then products were electrophoresed on agarose gels and recorded under the ultraviolet light.

RNA binding protein immunoprecipitation (RIP) assay

For RIP assay, approximately 1 × 107 BC cells transfected with AACS, PIGG, and CDCA2 overexpressing vectors harboring wild-type or mutant 3’UTR were collected, and lysed in polysome lysis buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES–NaOH pH 7.0, 0.5% Nonidet P-40 supplemented with 1 mM dithiothreitol, 200 units/ml RNase OUT, and EDTA-free Protease Inhibitor Cocktail) on ice for 10 min. After the preparation of Protein A/G Magnetic Beads (MedChemExpress, NJ, USA) and immobilization of antibodies (anti-PABPN1, 66807-1-Ig, mouse anti-IgG1, 66360-1-Ig, Proteintech, USA), cell lysate was incubated with antibody-bead reaction overnight at 4 ℃. The next day, beads were washed with ice-cold NT-2 (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05% Nonidet P-40) six times, and incubated with Proteinase K digestion buffer (1 × NT-2 buffer supplemented with 1% sodium dodecyl sulfate, 1.2 mg/ml Proteinase K) at 55 °C for 30 min. RNA Purification was conducted and binding products were detected using qRT-PCR.

Heatmap analysis

We downloaded the Percentage of Distal polyA site Usage Index (PDUI) scores of every gene in The Cancer Genome Atlas Bladder Urothelial Carcinoma (TCGA-BLCA) samples from TC3A database (http://tc3a.org/) [29]. Genes favoring distal PAS usage (long 3’UTRs) have PDUI scores near 1, whereas genes favoring proximal PAS usage (short 3’UTRs) have PDUI scores near 0. Then, heatmap analysis and hierarchical clustering were conducted using MeV 4.9.0 application based on PDUI scores of genes in tumor samples. Three distinct subgroups were identified being characterized by different 3’UTR alteration patterns.

RNA sequencing (RNA-Seq)

RNA-seq was performed with the help of HaploX (Jiangxi, China). Total RNA from EJ cells with or without PABPN1 overexpressing was extracted, and RNA purity (OD260/280 and OD260/230), concentration (ng/μL), and integrity (RIN) was determined. Then, the mRNA library was constructed, and the sequencing was performed on an Illumina PE150 platform.

Dynamic analysis of alternative polyadenylation from RNA-Seq (DaPars)

To systematically investigate the impact of PABPN1 overexpressing on global APA landscape in BC cells, we utilized DaPars [16, 17], a well-established computational algorithm, to analysis the RNA-Seq files of PABPN1 overexpressing BC cell and the corresponding control cell. Briefly, DaPars uses a linear regression model to predict the proximal APA site and estimates the abundance of long form and short form of 3’ UTRs, then calculates the PDUI.

Statistical analyses

Statistical analyses were performed using the GraphPad Prism 7 (GraphPad, CA, USA). Data were presented as mean ± SD and from at least three independent experiments. Pearson correlation coefficient was used to analyze the expression correlation. Survival curves were calculated according to the Kaplan–Meier method. Differences between groups were analyzed by Student’s t test and p-value < 0.05 was considered statistically significant.