The cell lines MDAMB231, BT549, 4 T1 and the HEK293T were purchased from the American Type Cell Culture Collection (ATCC). MDAMB231 and HEK293T cells were cultured with DMEM (41965, Life Technologies). BT549 and 4 T1 cells were cultured with RPMI-1640 (42401, Life Technologies). All cell lines were supplemented with 10% FBS (10270, Life Technologies) and maintained with 1% penicillin/streptomycin (C0222, Beyotime) with 5% CO2 at 37 °C, and were digested and passaged according to ATCC recommendations. The PowerPlex 21 system was used to profile all cell lines and validate their certification and authenticity.
Plasmids, siRNA and reagents
Plasmids were transfected with Lipo2000 (11,668,019, Thermo) based on manufacturer’s instructions. siRNA was transfected with Lipofectamine RNAiMAX (13,778,150, Thermo) based on manufacturer’s instructions. The Flag-RNF31, HA-Ub, HA-Ub-K48, HA-Ub-K63, HA-K48R and HA-Ub-KO plasmids were obtained from plasmid were used in our previous study . The RNF31 deletion constructs were purchased from HANBIO Biological (Shanghai, China). The Myc-YAP, YAP truncation mutants, lysine mutation YAP and TEAD-reporter plasmids were kindly gifted by Xiaofeng Zhou . The pLVX-RNF31 were constructed by cloning an RNF31 PCR fragment into the pLVX-EF1a-ZsGreen vector digested via EcoRI and NotI by T4 DNA ligase. We cloned an 800 bp fragment of human genomic DNA around the YAP–TEAD binding site in the 13-kb upstream from the PD-L1 gene into pGL4.26 luciferase vector (Promega). The human RNF31 target sequences (siRNA, GenePharma) were as follows: siRNA#1: GCG AUU AUA UGG CUA CAC A, UGU GUA GCC AUA UAA UCG C； siRNA#2: GGC GUG GUG UCA AGU UUA A, UUA AAC UUG ACA CCA CGC C；.
siRNA#3: UGAACAUCCUGGAGAAAUAUU, UAUUUCUCCAGGAUGUUCAUU.
The human YAP target sequence (siRNA, GenePharma) was as follows: GCU CAU UCC UCU CCA GCU UTT; AAG CUG GAG AGG AAU GAG CTT. The negative control (siRNA, GenePharma) was as follows: UUC UCC GAA CGU GUC ACG U; ACG UGA CAC GUU CGG AGA A. Some key reagents are provided below: Verteporfin were purchased from MCE (Cat. No. HY-B0146); Adezmapimod (SB 203580) were obtained from MCE (Cat. No. HY-10256); MG132 and Cycloheximide (CHX) were purchased from MCE (Cat. No. HY-13259) and MCE (Cat. No. HY-12320).
Quantitative real-time PCR
Quantitative real-time PCR were measured as described . Primers used were as follows: RNF31 5′-GAG CCC CG AAA CTA CCT CAA C′; 5′-CTT GAC ACC ACG CCA GTA CC-3′. CTGF: 5′-CAG CAT GGA CGT TCG TCT G-3′; 5′-AAC CAC GGT TTG GTC CTT GG-3′. CYR61: 5′-GGT CAA AGT TACC GGG CAG T-3′; 5′- GGA GGC ATC GAA TCC CAG C-3′. 36B4: 5′-CGA CCT GGA AGT CCA ACT AC-3′; 5′-ATC TGC TGC ATC TGC TTG-3′.
Coimmunoprecipitation (co-IP) assay and Western blot
Cells were lysed with Western and IP lysis buffer (P0013J, Beyotime) with a protease inhibitor cocktail (Roche). Total cell lysates were incubated with 30 μl of Protein Agarose (Beyotime, P2012) and IgG (Beyotime, 1:50) for preclearing 2 h at 4 °C, and immunoprecipitation was then performed with an indicated antibody for 4 h at 4 °C. As a negative control, Rabbit IgG (Beyotime, A7016, 1:50) or Moues IgG (Beyotime, A7028, 1:50) was used. Separation of proteins by SDS-polyacrylamide gel electrophoresis (PAGE) and transfer to nitrocellulose membranes (Millipore) were carried out. The antibodies were used as follows: anti-RNF31 (ab125189, Abcam, 1:2000); anti-HA (MMS-101R, COVANCE, 1:2000); anti-Flag (20543–1-AP, Proteintech, 1:2000); anti-YAP (14,074, Cell Signaling Technology, 1:2000; SC-101199, Santa Cruz, 1;500); PD-L1 (13,684, Cell Signaling Technology; 66,248–1-Ig, Proteintech); anti-Tubulin (11224–1-AP, Proteintech, 1:5000); anti-Histone-H3 (17168–1-AP, Proteintech, 1:1000) anti-Myc (Ab9106, Abcam, 1:2000); and anti-β-actin (8H10D10, Cell Signaling Technology, 1:10000). AffiniPure goat anti-mouse peroxidase-conjugated antibody IgG-HRP (A0216, Beyotime, 1:5000) or goat anti-rabbit IgG-HRP (A0208, Beyotime, 1；5000) secondary antibodies was incubated with the membranes after three washes with PBST. The signals were detected with the ECL Kit (Meilunbio, #MA0186).
Transwell, EdU and wound healing assay
MDAMB231 cells and BT549 cells were transfected with siRNF31 or sControl. The method of transwell assay, EdU assay and wound healing assay were measured as described [23, 24].
4% paraformaldehyde was used to fix MDAMB231 cells for 15 min, 0.25% Triton X-100 to permeabilize them for 15 min, and 3% BSA to block them for 1 h at room temperature. The rabbit anti-RNF31 antibody (ab125189, Abcam, 1:400), mouse anti-YAP antibody (SC-101199, 1:200) and mouse anti-PD-L1 antibody (1:400, 66,248–1-Ig, Proteintech) were used. The Alexa Flour 647 (Invitrogen) anti-mouse or Alexa Flour 488 anti-rabbit secondary antibodies (Invitrogen). The nuclei were stained with DAPI (Beyotime). Samples with only secondary antibodies and no primary antibodies were used as negative controls. Images were captured with a Nikon A+ laser scanning confocal system.
The streptavidin-peroxidase-biotin (SP) immunohistochemical method was used to measure RNF31 and YAP protein expression in 52 TNBC breast tissues. For primary antibodies, sections of tissues were incubated with RNF31 (ab125189, Abcam, 1:200) or YAP (14,074, Cell Signaling Technology, 1:200) antibodies overnight at 4 °C, while sections of xenografts from BALB/c mice with rat monoclonal anti-CD8 (eBioscience, 1:200). Immunoreactivity was measured as depicted in previous paper . This usage of clinical samples was reviewed and approved by the Ethical Board at Shandong University with written informed consent from all the patients.
Flow cytometric analyses
MDA-MB-231 and BT549 cells were transfected with siRNF31 or siControl. 48 hours after transfection. For apoptosis analysis, the cells were treated following the Apoptosis Detection Kit (A211–01, Vazyme). For CD44/CD24 analysis, the cells were digested and then washed with PBS (with 1% FBS) for 3 times, then resuspension in 100 μl PBS, and then stained with anti-CD44-PE (BD, 1:500) and anti-CD24-FITC (BD, 1:500). The samples were then washed by PBS 3 times and finally re-suspended in 400 μl PBS. For membrane PD-L1 analysis, the cells were digested and stained with PD-L1 antibody according to PD-L1 antibody (13,684, Cell Signaling Technology). The CD45 analysis was performed on tumor tissue samples from mice after they were washed with PBS, minced, and treated with a digestive solution containing 95% RPMI 1640, 2% FBS, 1% collagenase IV, and 1% DNase I and 1% Dispase II at 37 °C. Then, single cells were centrifuged (200 rpm for 30 min) and stained with anti-mouse CD45 eFluor 450 (B220, 48–0452-82). The data was analyzed using a Beckman FACS flow cytometer.
Specific pathogen-free (SPF) conditions were maintained, with an approximately 12 h light/12 h dark cycle, and free access to food and water were provided for all animals. 6-week-old female BALB/c nude mice and BALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. BALB/c nude mice (n = 6 for each group) were injected with 3 × 106 MDAMB231 cells in 100 μl PBS subcutaneously. 5 × 106 4 T1 cells in 100 μl PBS with 50 μl Matrixgel Basement Membrane Matrix (BD Biosciences) were injected into the BALB/c mammary fat fad. Tumor growth of 4 T1 cells in BALB/c mice treated with mPD-L1 antibody ((Bio X Cell, 10F.9G2)) or IgG ((Bio X Cell) as indicated. Tumors were measured at the indicated time points (n = 6 mice per group). Tumor volume was calculated by formula volume = length× (width2)/2. All animal protocols in this study were approved from the Animal Care and Use Committee of Xinxiang Medical University.
To directly detect K48 ubiquitinated and total ubiquitinated YAP enriched in cell extracts, HA-K48 ubiquitinated or HA-Ub plasmids, Myc-YAP and Flag-RNF31 or Flag-Vectors were transfected into HEK293T cells. After 24 hours, 10 μM MG132 was added to the cells for treating 8 hours. Then, cells were lysed with Western and IP lysis buffer (P0013J, Beyotime) with a protease inhibitor cocktail (Roche). Total cell lysates were incubated with 30 μl of Protein Agarose (Beyotime, P2012) for preclearing 2 h at 4 °C, and immunoprecipitation was then performed with anti-YAP antibody (14,074, Cell Signaling Technology) or Rabbit IgG (Beyotime, A7016) for 4 h at 4 °C. Western blot was performed with anti-HA antibody to detect YAP total polyubiquitinated or YAP K48 polyubiquitinated.
Chromatin immunoprecipitation (ChIP)
By reviewing Zanconato; et al. preserved ChIP-Seq data (GSE66081 ) and referring to Min Hwan Kim; et al. . We designed a pair of primers for ChIP analysis targeting the narrow peak of TEAD4 binding 13-kb upstream of PD-L1 transcription start site. Primers were used as follows: 5′-CAT CGG GAT TAC CAC GCT GA-3′ and 5′-TTC GTT CCA TTA GAG CGC GT-3′. ChIP assay was executed in indicated MDAMB231 cells according to the Magna ChIP A/G kit (17–10,085, Sigma-Aldrich) instructions. Sheared chromatin was immunoprecipitated by IgG or anti-Myc antibody at 4 °C for 12 h. YAP binding with the 13-kb upstream site was detected by quantitative PCR using SYBR Green qPCR assay (639,676, TaKaRa).
Dual-luciferase reporter assay
The Dual-Luciferase Reporter Assay System (E1910, Promega, United States) was used to detect Luciferase activity. The cells were transfected with the TEAD luciferase reporter or pGL4.26-YAP–TEAD binding site and the Renilla plasmid. After 24 hours, cells were lysis, and luciferase activity was detected. A GloMax-Multi Jr. (Promega-GloMax Promega, USA) was used to detect luciferase activity.
Publicly available clinical data analysis
Analysis of the expression of RNF31 was carried out with data for 1080 breast cancer samples from the TCGA database. Expression in ER positive, HER2-positive, and triple-negative breast cancer tissues and normal tissues were analyzed with GraphPad Prism 9. Analysis of RNF31 and YAP with clinical prognosis was analyzed by KMPLOT database (https://kmplot.com). RNF31 RNA-seq data were downloaded from the GEO datasets (GSE218406). For CORDENONSI YAP CONSERVED SIGNATURE gene sets were used and downloaded from the GSEA Molecular Signatures Database. GSEA was implemented by GSEA 4.3.2 software.
Statistical analyses were carried out and created using GraphPad Prism 9 software. All the data are showed with the mean ± standard deviation (SD) for at least 3 independent experiments. P value< 0.05 was considered to indicate statistically significant using two-tailed Student’s t test and Chi-square test. *P < 0.05, **P < 0.01, ***P < 0.001.