Clinical sample collection and cell culture
Fresh BC tissue tissues and corresponding non-tumour normal tissues were collected from patients who underwent radical cystectomy. The samples were immediately cleaned with normal saline and snap-frozen in liquid nitrogen after resection. All samples were confirmed independently by two pathologists. The patients were informed of the study contents and signed informed consent forms. This study was approved and supported by the Ethics Committee of the Second Affiliated Hospital of Kunming Medical University.
Bladder cancer cell lines and cell culture
The BC cell lines T24 and 5637 and the normal bladder uroepithelium cell lines SV-HUC-1 and HEK-293T were purchased from the Institute of Cell Research, Chinese Academy of Sciences (Shanghai, China). T24 and SV-HUC-1 cells were cultured in DMEM (Gibco, USA) with 10% foetal bovine serum (Gibco, USA), and 5637 cells were cultured with RPMI-1640 medium (Gibco, USA) and 10% foetal bovine serum. All cells were cultured at 37 °C in a humidified 5% CO2 atmosphere.
RNA extraction and quantitative real-time PCR (qRT–PCR)
Total RNA was extracted from bladder tissue and cells using TRIzol reagent (Thermo Fisher, USA) according to the manufacturer's instructions. The primers for GAPDH were 5′-AAAGGGTCATCATCTCTG-3′ (forward) and 5′-GCTGTT GTCATACTTCTC-3′ (reverse). The primers for U6 were 5′-CTCGCTTCGGCAGC ACA-3′ (forward) and 5′-AACGCTTCACGAATTTGCGT--3′ (reverse). The primers for SPRR1B were 5′-CAAGGTTCCAGAGCCATG-3′ (forward) and 5′-TACTTCTGCTTGGTCTTCT-3′ (reverse). The primers for HAGLROS were 5′-GACGAATACACCTCTGAA-3′ (forward) and 5′-AGTCTTAGCCTACTTCCT-3′ (reverse). The miRNA primers were designed and synthesized by RiboBio Biotechnology (Guangzhou, China). qRT–PCR assays were performed using a qRT–PCR Starter Kit (RiboBio, Guangzhou, China) and a LightCycle 96 Fluorescent Quantitative PCR System (Roche, Switzerland) according to the manufacturer’s instructions. Expression was normalized by GAPDH and U6. Each experiment was repeated at least thrice.
RNA fluorescent in situ hybridization (FISH) and subcellular fractionation assay
FISH was performed to detect the subcellular colocalization of HAGLROS, miR-330-5p and SPRR1B. A FISH kit was purchased from Focofish Biotechnology (Guangzhou, China). The U6 probes were purchased from Sangon (Shanghai, China). Cell nuclei were counterstained with DAPI (Sangon, Shanghai, China). The cytoplasmic and nuclear fractions of T24 and 5637 cells were extracted using nuclear and cytoplasmic extraction reagents (Thermo Fisher, USA) according to the manufacturer’s protocol. Then, the RNA was extracted, and qRT–PCR was performed as previously described. Each experiment was repeated at least thrice.
Briefly, total RNA was extracted from three pairs of fresh bladder cancer tissues and corresponding adjacent tissues. The eligible RNAs were sent to the sequencing company for quality control, library construction and sequencing.
Plasmids overexpressing HAGLROS (OE-HAGLROS), short hairpin (sh) HAGLROS and sh-SPRR1B were designed and synthesized by GenoMeditech Biotechnology (Shanghai, China). T24 and 5637 cells were transfected. The cells were harvested 48-72 hours. Purinomycin (Solarbio, Beijing, China) was used to screen stably transfected cells. The transfection efficiency was evaluated by qRT–PCR. The miR-330-5p mimics/inhibitors were purchased from RiboBio Biotechnology (Guangzhou, China). Each experiment was repeated at least thrice.
Western blot assay
Western blot assay was used to detect protein expression. The tissues and cells were lysed with ice-cold RIPA lysis buffer (Beyotime, Shanghai, China). The protein was quantified using a Bradford Protein Assay Kit (Beyotime, Beijing, China), separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Sigma, USA), transferred to a polyvinylidene fluoride (PVDF) membrane, blocked with bovine serum albumin (Solarbio, Beijing, China), hybridized with SPRR1B (Abcam, MA, USA) and GAPDH (Abmart, Shanghai, China), and incubated with a secondary antibody. The PVDF (Millipore, USA) membrane was treated with BeyoECL Plus reagent (Beyotime, China). Then, the bands were detected using a gel imager system (Bio–Rad, USA) and analysed by ImageJ software (NIH, Bethesda, MD, USA). Each experiment was repeated at least thrice.
Cell counting Kit-8 (CCK-8) assay
BC cell proliferation was observed using an Enhanced Cell Counting Kit-8 (Beyotime, Beijing, China) according to the manufacturer’s instructions. Briefly, 5× 103 cells were seeded onto 96–well plates per well. After 24 hours, 10 µl CCK-8 solution was added to each well, and the cells were incubated for 1 hour. Finally, the absorbance was measured at 450 nm using a microplate reader (Thermo Fisher, USA). Each experiment was repeated at least thrice.
Wounding healing assay
The migration capability of cells was tested by wound healing assay. The wounds were scratched using a 200-μl pipette tip in approximately 90% confluent cells. The migration of cells was acquired using a microscope (Olympus, Tokyo, Japan). Finally, wound healing areas were measured by ImageJ software (NIH, Bethesda, MD, USA). Each experiment was repeated at least thrice.
The invasive capability of cells was observed using a Transwell assay. Briefly, 100 µl diluted Matrigel (Corning, USA) was added to the upper chamber of the Transwell (Corning, USA), and the Transwells were incubated at 37 °C for 1 hour. Then, 200 μl serum-free medium with 5×104 cells was seeded into each upper chamber. Culture medium containing 10% FBS was added to the lower chamber. The cells in the upper chamber were wiped using cotton after 48 hours of incubation, fixed with paraformaldehyde, and stained with crystal violet. The number of invaded cells were recorded under a microscope. Each experiment was repeated at least thrice.
Flow cytometry assay
The cell cycle was determined by using a Cell cycle Analysis Kit (Beyotime, Shanghai, China) according to the manufacturer's instructions. The cell cycle was assessed by flow cytometry. Each experiment was repeated at least thrice.
Protein expression levels were detected by immunohistochemical staining. The bladder tissues were prepared into paraffin sections. Then, these sections were deparaffinized, used for antigen retrieval, and incubated with a primary antibody against SPRR1B (Proteintech, Wuhan, China, 1:150) overnight at 4 °C. A universal two-step assay kit (Zsboi, Beijing, China) was used for IHC. The immunostaining images were detected using an optical microscope. Each experiment was repeated at least thrice.
Dual-luciferase reporter assay
Luciferase reporter plasmids were designed and synthesized by GenoMeditech (Shanghai, China). HEK-293T cells were cotransfected with luciferase reporter plasmid and microRNAs. A dual-luciferase reporter assay system (Promega, USA) was used to determine the luciferase activity 48 h later according to the instructions. Each experiment was repeated at least thrice.
Tumour xenograft implantation in nude mice
T24 cells (2 × 106 cells) were subcutaneously inoculated into BALB/c nude mice (18-22 g). Then, tumour volumes were measured every 3 days. The animal experiments were performed at the Department of Experimental Animals, Kunming Medical University and approved and supported by the Ethics Committee of the Second Affiliated Hospital of Kunming Medical University. Each experiment was repeated at least thrice.
The DEGseq2 R package was used to identify differentially expressed lncRNAs and mRNAs (DE-lncRNAs and DE-mRNAs) according to the thresholds |log2 (FC) |>1.0 and P value < 0.05. A total of 408 BC samples and 19 normal samples were downloaded from TCGA database (https://portal.gdc.cancer.gov/). The DE-lncRNAs and DE-mRNAs overlapped between the RNA-sequencing data and TCGA data. The differential expression of candidate genes and the relationship of their expression levels were evaluated in data retrieved from the starBase database (https://starbase.sysu.edu.cn/), the GEIPIA database (http://gepia.cancer-pku.cn/), the Lnc2Cancer database (http://bio-bigdata.hrbmu.edu.cn/lnc2cancer/) and the LncBase database (https://diana.e-ce.uth.gr/lncbase/).
All data were obtained from three independent experiments performed at least thrice. All results were presented as the mean ± SD. Statistical analysis was performed with one-way ANOVA and Student’s t test using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). In this study, P < 0.05 was considered statistically significant.