Bladder cancer tissue and cells

Clinicopathological data and tissue specimens were obtained from 30 patients who underwent surgical resection for bladder cancer at the Department of Urology, Nanjing Drum Tower Hospital from September 2019 to September 2020. All patients were confirmed to have bladder cancer through pathological examination, and they had not received any prior radiotherapy, chemotherapy, or other cancer-related treatments. Written informed consent was obtained from all patients, and the study was approved by the ethics committee of Nanjing Drum Tower Hospital.

Human bladder cancer cell lines (T24, 253J, J82, 5637, and RT-4) and human normal bladder epithelial cells (SV-HUC-1) were obtained from the ATCC Culture Collection. The cells were cultured in 1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Beyotime, Shanghai, China). The cells were maintained in a cell incubator (Thermo) at 37 °C with 5% CO2.

Isolation and culture of cancer stem-like cells

The side population cells, representing the cancer stem cells, were isolated from bladder cancer cell lines (T24 and 253J) using the ALDEFLUOR® assay (Stemcell Technologies, Vancouver, BC, Canada), as previously described [38, 39]. The sorted side population cells were confirmed to possess cancer stem-like cell properties by tumorigenesis in vivo and in vitro, as well as OCT4 expression detection (Additional file 1: Fig. S1). The isolated cancer stem cells were cultured in DMEM/F12 medium supplemented with 2% B27, 40 ng/mL basic fibroblast growth factor (bFGF), and 20 ng/mL epidermal growth factor (EGF).

Plasmid construction

Wild-type MARCH7 (MARCH7-WT), mutant MARCH7 (MARCH7-Mut), and NOD1 were amplified using Premix-PrimeSTAR-HS (TAKARA, Dalian, China). The DNA fragments were extracted using a MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (TAKARA) and cloned into a pcDNA3.1 (+) empty vector using BamHI and XhoI enzymes (Thermo Fisher Scientific). The MARCH7 (NM_001282805.2) and NOD1 (NM_006092.4) sequences were obtained from the National Center for Biotechnology Information (NCBI). The MARCH7-WT and MARCH7-Mut constructs were tagged with a Flag tag. The plasmids were designed and constructed by Shanghai Yuanmu Biotechnology Co., Ltd. (Shanghai, China). The plasmids were used for stable transfection of cancer cells.

Cell transfection and construction of stable transfected cell lines

The cancer cells were seeded in 6-well plates at a density of 2 × 106 cells per well. When the cells reached 70% confluence, the culture medium was aspirated, and 1 μg MARCH7-pcDNA3.1 or pcDNA3.1 (empty vector) were mixed with DMEM, incubated for 5 min, mixed with LipofectamineTM2000 (Invitrogen), and incubated for 20 min. The mixture (400 μl) was added to the cell medium, and 1 ml of cell culture medium was added after 6 h. The cells were further cultured for 1 day, added G418 (Sigma) into medium with 800 μg/ml concentration. After 30 days selection, the transfected stem cells with MARCH7-vector and the empty vector were referred to as the MARCH7 group and the vector group, respectively.

Same as above procedure, cells were transfected with different concentrations (1 and 1.5 μg) of Flag-MARCH7-WT or MARCH7-Mut, the transfection efficiency was determined by western blotting. Neomycin was added to the screen to ensure stable transfection. The proteasome inhibitor, MG132 (1 µM), was added to the control and experimental groups. After 10 h, the cells were washed. After trypsinization, the cells were centrifuged at 800 rpm for 5 min, and the complete medium was added. The cells were resuspended in a cryopreservation tube placed in a − 80 °C freezer overnight. Flag-MARCH7-WT and Flag-MARCH-Mut were constructed to verify the binding of MARCH7 and NOD1.

Spheroid formation assay

The effect of MARCH7 on spheroid formation was investigated in bladder cancer stem cells. The cells were seeded in ultra-low attachment 96-well plates (Corning, NY, USA) at a density of 200 cells per well. The plates were gently swirled to distribute the cells evenly and incubated for 7 days to form spheroids. The number and size of spheroids were observed and measured under a microscope.

Bioinformatics analysis

The indicated genes expression were analyzed by “Gene Differential Express” in the “Pan-Cancer” module of the STARBASE [40] (http://starbase.sysu.edu.cn/index.php). “MARCH7” was entered in the “Gene” option, and MARCH7 expression data for various cancers was obtained. Bladder urothelial carcinoma (BLCA) was selected to obtain mRNA expression data of MARCH7 mRNA in bladder cancer tissue. mRNA levels were obtained from RNA-seq data using the log2 algorithm (FPKM + 0.01). Additionally, we used UbiBroswer online tool (http://ubibrowser.bio-it.cn/ubibrowser/) to predict the potential substrates of MARCH7 following the instructions [41]. Afterwards, we detected the top 10 substrates of MARCH7 by conducting co-IP and western blot.

Quantitative real-time PCR (q-PCR)

The levels of MARCH family genes were detected using q-PCR. Total RNA was separated from tissues frozen in liquid nitrogen, as well as from bladder cancer cells and normal bladder epithelial cells, by lysis with TRIzol reagent. After the RNA concentration was determined, the total RNA was reverse-transcribed into cDNA. Using cDNA as a template, qPCR amplification was performed using the SYBR Premix Ex TaqTM qRT-PCR kit (TaKaRa, Dalian, China). The primers specific for MARCH family genes are listed in Table 1 and were purchased from Ribobio. (Gungzhou, China). The reaction conditions included pre-denaturation at 95 °C for 5 s, denaturation at 95 °C for 5 s, and annealing at 60 °C for 30 s, total 40 cycles. Using GAPDH as an internal reference, the relative expression of different genes in MARCH7-transfected and vector-transfected cells was computed using the 2−ΔΔCt method.

Table 1 Primers for q-PCRImmunoblotting and immunohistochemistry (IHC)

Protein expression levels of MARCH7 and NOD1 were detected using immunoblotting. Cells were lysed with RIPA buffer (Beyotime) supplemented with protease and phosphatase inhibitors. Protein concentrations were determined using a BCA Protein Assay Kit (Beyotime). Protein samples were separated by SDS-PAGE and transferred to PVDF membranes (Bio-Rad). The membranes were incubated with primary antibodies against -MARCH7 (Abcam, Cambridge, UK), NOD1 (Abcam), β-actin (Abcam), OCT4 (Abcam), CD44 (Thermo Fisher), CD133 (Thermo Fisher), PCNA (Novus Biologicals, Colorado, USA),Cyclin A1 (Novus Biologicals), N-cadherin (Novus Biologicals), E-cadherin (Novus Biologicals), Tubulin-α (Novus Biologicals), Flag (Novus Biologicals), HA (Novus Biologicals), NOD2 (Novus Biologicals), P65 (Novus Biologicals). They were followed by secondary antibodies conjugated to horseradish peroxidase (HRP) (Abcam). Protein bands were visualized using enhanced chemiluminescence (ECL) reagents (Millipore, Billerica, MA, USA). Protein bands were visualized using enhanced chemiluminescence (ECL) reagents (Millipore, Billerica, MA, USA). Band intensities were quantified using ImageJ software.

The expression of MARCH7 and NOD1 in bladder tumors and adjacent normal tissues was examined using IHC staining [42]. Paraffin-embedded tissue sections were deparaffinized and rehydrated. Antigen retrieval was performed by heating the sections in citrate buffer (pH 6.0) using a microwave. The sections were incubated with primary antibodies against MARCH7 (Abcam) and NOD1 (Abcam), followed by HRP-conjugated secondary antibodies (Abcam). The staining was visualized using 3,3′-diaminobenzidine (DAB) substrate (Beyotime). The sections were counterstained with hematoxylin, dehydrated, and mounted.

Flowing cytometry detecting CSCs marker CD44 and CD133

The stable transfected T24CSCs and 253 J CSCs were collected by centrifuging at 100g × 5 min. Then, they were washed with PBS gently and centrifuged for three times. Next, they were suspended anti-CD44-FITC- or anti-CD133-PE-contained buffer (140 mM NaCl, 2.5 mM CaCl2, 10 mM HEPES, pH7.2) for 30 min. Finally, the stained cells were objected to flowing cytometry analysis. The obtained data were analyzed and showed by using the Flow Jo V10 (Tree Star, California, Auckland, Fairbert Street, USA).

Cell viability assay

Cell viability was assessed using a Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan). The transfected cells were seeded in 96-well plates at a density of 5000 cells per well and incubated for 24, 48, 72, and 96 h. CCK-8 solution (10 μl) was added to each well, and the plates were incubated for 2 h at 37 °C. The absorbance at 450 nm was measured using a microplate reader (BioTek, Winooski, VT, USA).

Colony formation assay

Stem-like cells were seeded in 6-well plates at a density of 300 stably transfected cells per well. The medium was changed every two days. After incubation for 14 d, the colonies were stained with crystal violet. Briefly, the medium was aspirated, 1 ml methanol was added to each well, and the cells were fixed for 30 min. Then, methanol was discarded, and 0.5% crystal violet solution (Beyotime) for 1 h at room. Finally, the colonies were gently rinsed with crystal violet solution, dried, and the number of colonies (≥ 50 cells) was counted under a microscope (Olympus Corporation). All operations require caution to prevent cell detachment.

Transwell assay

The indicated cells were resuspended in DMEM without FBS as 1 × 105/ml. A total of 0.2 mL of cell suspension were added to the upper chamber of the transwell coated with matrigel (Corning, Shanghai, China), and the culture medium containing 10% FBS was added to the lower chamber for 24 h. Carefully wipe the cells in upper chamber with a cotton ball, fixed the cells which invaded into the lower chamber with methanol, and stained with 3% crystal violet. The number of transmembrane cells was counted in five fields. For the rescue assay, the cell concentration was 1 × 104/ml. All operations require caution to prevent cell detachment.

Dual luciferase assay

Majorly, 5000 bladder cancer cells (T24 with or without NOD1 overexpression) were seed into 24-well plates without antibiotics until. Cignal Finder 10-Pathway Reporter Arrays (Qiagen) were employed to identify the potential pathway(s) regulated by NOD1 as our previous reports [43, 44].

To validate NOD1 and MARCH7 effect on NF-κB pathway, NF-κB luciferase reporter which contains 3 NF-κB-binding sites and internal control pRL-TK plasmid, NOD1 and MARCH7 were transfected into T24 as indicated. 48 h later, dual luciferase reporter assay kit (Promega) detected the luciferase activities of the cells following the instruction of manual, with reporter luciferase activity normalized to renilla luciferase activity.

Immunoprecipitation assay

Immunoprecipitation was employed to detect the interaction between NOD1 and MARCH7, and determine the ubiquitination of HA-NOD1 or NOD1, as described previously [45]. Briefly, HA-NOD1 and Flag-MARCH7 were transfected into the cells. After 48 h of transfection, MG132 (10 μM) was added and incubated for another 24 h. Samples were collected at 2000 rpm for 5 min, suspended in 2% SDS buffer, boiled for 10 min, and sheared with a sonication device. Afterwards, the supernatant was rotated with anti-HA or anti-FLAG for 12 h, followed by protein A- or G-agarose beads for another 2 h. The bead-containing samples were washed thrice with pre-cooled PBS. Finally, the beads were removed by centrifugation at 20,000 ×g for 3 min. The residual supernatant was collected and boiled in 5X SDS loading buffer for 10 min. Besides, IgG was used as a negative control. These samples were loaded onto SDS-PAGE gels for immunoblotting with antibodies to determine the indicated proteins and protein ubiquitination.

In vitro ubiquitination detection

To determine MARCH7 may directly interact with NOD1, the Flag-MARCH7-WT, Flag-MARCH7-Mut, HA-NOD1 proteins were purified by Hangzhou Jingjie Biotechnology Co., Ltd.. Co-IP assay to determine the direct interaction between MARCH7 and NOD1 as described above. The ubiquitination detecting kit (Cat. no. VB2948, Viva Bioscience, Shanghai, China) was applied to examine the ubiquitination of NOD1. Briefly, the purified NOD1 protein was mixed with purified Flag-MARCH7-WT or Flag-MARCH7-Mut, and the mixed proteins were incubated in the tubs containing E1, E2, ATP, ubiquitin for 12 h. Afterwards, they samples were used for Co-IP using anti-HA to determine the effects of MARCH7-WT or -Mut on ubiquitination of HA-NOD1. Finally, these samples were added loading buffer for western blotting analysis.

Xenograft model

Male BALB/c nude mice aged 4–5 weeks were purchased from GemPharmatech (Chengdu, China) and housed in an SPF room. After 7 days adaptive phase, the mice were blindly and randomly divided as indicated groups (3 mice per group). The procedure for using the animals followed the Guidance Suggestions for the Care and Use of Laboratory Animals formulated by the Ministry of Science and Technology of China, and animal experiments were approved by the Animal Ethics Committee of West China Second University Hospital. The optimized experimental procedures minimized the number of experimental animals and alleviated their suffering. For xenograft tumor generation, 2 × 106 T24 cells and sorted stem cells were injected subcutaneously into the flank. The length and width of the tumors were measured with Vernier calipers every 4 days, and the tumor volume was calculated as length × width2/2. When the weight loss of an animal reaches 20–25%, or when the animal exhibits cachexia or wasting symptoms, or when the size of a solid tumor exceeds 10% of the animal’s body weight, the animal's life should be terminated. The nude mice were sacrificed at the indicated endpoint, and the tumors were excised and weighed.

Statistical analysis

Statistical analyses were performed using SPSS version 19.0. All the data were from three independent experiments. The measurement data are shown as \(\overline\pm S\). One-way ANOVA was applied for comparison between multiple groups, and the LSD-t test was performed for pairwise comparison between groups. Enumeration data were analyzed using the χ2 test. P < 0.05 was considered statistically significant at P < 0.05.