Antibodies were used in the study: anti-Fbw7 (for western blots) (ab109617, Abcam, Cambridge, UK), anti-Fbw7 (for IHC) (H00055294-M02, Abnova, Taipei City, Taiwan), anti-LDHA, 3682; anti-phospho-LDHATyr10, 8176; anti-HK2, 2867; anti-Glut1, 12939s; anti-PDK1, 5662; anti-Ubiquitin, 3936; anti-Flag Tag, 14793; anti-His Tag, 9991 (Cell Signaling Technology, Beverly, MA, USA), anti-β-actin, 60008-2-Ig (Proteintech, Rosemont, IL, USA). And other chemicals were purchased from Amresco (Dallas, TX, USA) and Sigma-Aldrich (St. Louis, MO, USA).
The ABC-DLBCL cell lines SU-DHL-2, OCI-LY-3, OCI-LY-10, U2932 and HEK293 were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and maintained in a humidified incubator with 5% CO2 at 37°C. Cell lines were confirmed by short tandem repeats-polymerase chain reaction (STR-PCR) genotyping and compared with American Type Culture Collection (ATCC) database.
Plasmids encoding Flag-Fbw7, His-LDHA and HA-Ubiquitin were constructed by cloning PCR amplified into pcDNA3.1. LDHA siRNA target sequence was 5’-GUUCAUCAUUCCCAACAUUTT-3’ (siRNA1) and 5’- AAUGUUGGGAAUGAUGAACTT-3’ (siRNA2). LDHA siRNA and siRNA-control (siRNA-NC), miR-223 inhibitor and mimic were purchased from RIBOBIO (Guangzhou, China). The constructs were transfected using Lipofectamine 2000 and 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacture.
The Lactate dehydrogenase (LDH) assay kit (C0016, Beyotime Biotechnology) and L-Lactate Assay Kit II (1200051002, Eton Bioscience) were used to measure intracellular LDH and lactate levels, respectively, in DLBCL cells according to the manufacturer's protocols. ATP generation was detected using an ATP detection assay kit (S0026, Beyotime Biotechnology).
Tissue samples and IHC
Thirty-two human DLBCL patients were collected at the Guangdong Provincial People’s Hospital. And written informed consent was obtained from all of the patients and this study was approved by the Biomedical Research Ethics Committee of Guangdong Provincial People’s Hospital. And patients with detail clinicopathological characteristics are rendered in Supplementary Table S1. For immunohistochemistry (IHC) detection, Paraffin embedded tissues were sectioned at 4-µm thickness, microwaved in Na citrate buffer, pH 6, for 10min and following primary antibody (anti-Fbw7 or anti-LDHA). Secondary antibody was biotinylated, and then sections were developed with diaminobenzidine. Staining results were estimated by two pathologists and the scoring criteria was: 0 (no staining), 1 (weak staining), 2 (intermediate staining), and 3 (strong staining).
Western blot analysis
Cells were homogenized in RIPA lysis buffer on ice for 30 min and then centrifuged at 14000 ×g for 30 min. Whole protein extract was measured using the BCA Assay (Pierce, Rockford, IL, USA), 30 µg of whole protein was added onto 10% SDS-PAGE gels and transferred onto PVDF membranes (Millipore, Billerica,MA, USA).
Immunoprecipitation and ubiquitination assay
For protein immunoprecipitation analysis, cells were lysed and 1μg of antibodies (anti-Fbw7 or anti-LDHA) was added, which was incubated with protein A/G beads (Millipore) and eluted in SDS buffer at 95°C for 4 min. The beads were then washed and boiled in SDS loading buffer and immunoprecipitation complexes were detected by western blotting analysis.
For ubiquitylation assay, cells were transfected with indicated plasmids and/or siRNA and then treated with MG132 (20 μM). And then cells were harvested, lysed and followed by immunoprecipitation of His-tag LDHA and western blotting to detect His-LDHA ubiquitination.
Quantification PCR analysis
For mRNA quantification, total RNAs were isolated using RNAiso (Takara, Shiga, Japan) Plus and 1 µg of total RNA used for cDNA synthesis using PrimeScript RT Master Mix (Takara). Real-time PCR reactions were carried out using SYBR Premix Ex Taq (Takara) and run on ABI 7500 PCR system (Applied Biosystems, Carlsbad, CA, USA). The PCR program consisted of 40 cycles at 95°C for 5 s and 60°C for 30 s. Gene expression was determined relative to β-actin (for mRNA) or U6 (for miRNA) and calculated using the 2−ΔΔCT method. The miDETECT A TrackTM miRNA qPCR Kit (RIBOBIO, Guangzhou, China) was used to examine the expression levels of mature miR-223. The gene-specific primers used for the PCR reaction is listed in Supplementary Table S2.
NOD/SCID mice (4 to 6 weeks) were purchased from the Experimental Animal Tech Co. of Weitonglihua (Beijing, China). Mice were injected with 1 ×107 cells in 100 μl PBS in the left flank. The size of the subcutaneous tumor was measured using calipers twice a week. The tumor volume was calculated using the following formula: tumor volume (mm3) = (width × height × depth)/2. These experiments were assessed and approved by the Committee of Care and Use of Laboratory Animals of Guangdong Provincial People’s Hospital.
To investigate whether the production of miR-223 could promote DLBCL tumor growth in vivo, hsa-miR-223 agomir (RIBOBIO, Guangzhou, China) (1 nmol in 100 μl PBS, twice a week for 4 weeks) was administered intraperitoneally at 1 week following SU-DHL-2 cell injection.
Dual luciferase reporter assays
The targeting sites for miR-223 and the Fbw7 3’-UTR were predicted using the TargetScan algorithm (www.targetscan.org). To validate miR-223-predicted targets, a dual luciferase reporter assay was performed according to the manufacturer (Promega, WI, USA). Cells were transiently transfected with Renilla luciferase vector for 3’-UTR of Fbw7(WT) or mutated miR-223 binding sites. The reporter constructs were co-transfected with negative control or miR-223 mimic into the indicated cells for 24 hours. Firefly luciferase was used as an internal control. Cell lysates were prepared and luciferase activity was detected using a Spark multimode microplate reader (TECAN, Mannedorf, Switzerland).
SPSS 23.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for Statistical analyses. All data are showed as the mean ± SD. Experiments were repeated at least three times. One-way analysis of variance or a two-tailed unpaired Student t test was applied to evaluate the data. Differences were known as significant at *, P < 0.05; **, P < 0.01; and ***, P < 0.001. The relationship between Fbw7 level and LDHA were assessed using the Pearson χ2 test. Each assay was performed in triplicate.