The specific reagents induced antibodies against Hexokinase II (ab209847), PDK1 (ab110025), IDH3B (ab247089), TRPM7 (ab109438), UQCRC1 (ab223746), H3 (ab1791), rabbit anti-PKM2 (ab85555), goat anti-rabbit IgG (ab6721) and goat anti-mouse IgG (ab6728, Abcam, Cambridge, USA), phospho-AMPKα (#2535, Cell Signaling Technology, Saint Louis, USA), Alexa Fluor488-conjugated goat anti-rabbit IgG (#35552), Alexa Fluor594-conjugated goat anti-mouse IgG (#35560, Invitrogen, Waltham, USA), compounds of Dorsomorphin (Compound C) 2HCl (S7306) and metformin HCl (S1950, Selleckchem, Houston, USA) and DAPI staining solution (C1005, Beyotime, Beijing, China).
We recruited 60 ovarian cancer patients without prior radiotherapy and chemotherapy in the Department of Gynecology, Cancer Hospital, Xiangya Medical College of Central South University of China. We collected surgical ovarian specimens when they underwent a surgery for removal of the tumor. Individual patients singed the written informed consent. The experiments were approved by the Joint Ethics Committee of Hunan Cancer Hospital and Affiliated Tumor Hospital of Xiangya Medical College of Central South University of China (approval number: KYJJ-2019-001).
Cells and culture
Human ovarian cancer SKOV3 and HO8910 cells were provided by the Cancer Institute, Central South University and they were identified by STR. SKOV3 and HO8910 cells were cultured in RPMI1640 medium containing 10% fetal bowel serum (FBS, Life Technologies, Carlsbad, USA) at 37°C in 5% CO2. Some cells were cultured in a hypoxic incubator of 1% O2, 5% CO2, and 94% N2 at 37°C.
Sequencing of mRNAs
Total RNA was extracted from different groups of SKOV3 sh-control and SKOV3 sh-TRPM7 cells and the samples (1–2 µg/each) were used for generation of libraries using the KAPA Stranded RNA-Seq Library Prep Kit (Illumina), according to the manufacturer’s protocol by KangChen BioTech, China. The barcoded libraries were purified and quantified using an Agilent 2100 bioanalyzer. The mixed libraries of different samples were denatured with 0.1M NAOH, diluted to 8 pM and amplified in situ on TruSeq SR Cluster Kit v3-cBot-HS (#GD-401-3001, Illumina, USA). The samples were sequenced at 2 × 150 bp in an Illumina X-ten/NovaSeq sequencer. The levels of gene expression were measured as fragments per kilobase per million reads (FPKM) and the differentially expressed genes (DEGs) were defined when a fold change ≥ 2.0 and a P-value of < 0.05.
We analyzed the DEGs between the sh-control (C) and sh-TRPM7 (T) groups using Gene Set Enrichment Analysis (GSEA, GSEA v4.0.3 for windows) (http://software.broadinstitute.org/gsea/index.jsp) . First, we analyzed the DEGs in the biological signaling using the MSigDB (Molecular Signatures Database) (http://software.broadinstitute.org/gsea/msigdb) with 1000-rounds. The identified DEGs were defined with a false discovery rate (FDR) < 0.25 and a family-wise error rate (FWR) < 0.05. Second, the DEGs were subjected to the Gene Ontology (GO) analysis organism (http://www.geneontology.org), particularly in Biological Process, Cellular Component and Molecular Function. The DEGs in the top GO lists were analyzed by Fisher’s exact test with a P-value of ≤ 0.05. Finally, the DEGs were analyzed for their pathways using the KEGG with a P-value of < 0.05.
The expression levels of specific proteins in ovarian cancer tissues were characterized by IHC using a kit (CW2069, CoWin Biosciences). Briefly, individual paraffin-embedded tissue sections (3 µm) were treated with 3% bowel serum albumin (BSA) and underwent routine-antigen retrieval. The sections were probed with mouse anti-Hexokinase II (1: 500), anti-PDK1 (1: 600), anti-IDH3B (1: 2000), anti-phospho-AMPKα (1: 100), anti-TRPM7 (1: 700), UQCRC1 (1: 400) or control mouse or rabbit IgG at 4°C overnight. The sections were cultured with horseradish peroxidase (HRP)-conjugated detection antibodies (1:5000) and reacted with DAB. The sections were counter-stained with hematoxylin. The immune staining signals were photoimaged under a light microscope and semi-quantitatively evaluated by two pathologists in a blinded manner, according to the percentages of positively stained cells and the intensity of IHC signals. The percentages of positively stained cells were scored as 0: <5%; 1: 6%-25%; 2: 26 %-50%; 3: >50%. The intensity of IHC signals was quantified as 0: colorless; 1: light yellow; 2: brown yellow; 3: dark brown. A final score in each image was achieved as the intensity score × percentage score and the levels of protein expression were defined as 0: no expression; 1–4: low levels of its expression; 5–9: high levels of its expression.
Transduction and transfection
The lentiviruses for TRPM7 knockdown, and HIF-1α overexpression or knockdown were purchased from Genepharma (shanghai, China). SKOV3 and HO8910 cells (5×105/well) were cultured overnight and transduced with lentivirus at a MOI of 5 for expression of Con-sh, TRPM7-sh1, TRPM7-sh2, TRPM7-sh3, TRPM7-sh4, HIF-1α-sh or HIF-1a. The target sequences are shown in the Supplementary Tables S2. After 48 hours of incubation, their total RNAs and proteins were extracted for verification of gene silencing or over-expression. Some cells were cultured in the presence of G418 (500 µg/ml, Biofrox, Germany) for 14 days to generate stable gene silencing or over-expressing cells. Subsequently, we characterized the cell clones by quantitative real-time PCR (qRT-PCR) and Western blot.
SKOV3-Con-sh, SKOV3-TRPM7-sh, HO8910-Con-sh and HO8910-TRPM7-sh cells were stained with rabbit anti-Hexokinase II (1: 100), mouse anti-PDK1(1: 100), rabbit anti-IDH3B (1: 100), mouse anti-UQCRC1 (1: 70), rabbit anti-PKM2 (1: 50). Subsequently, they were reacted with Alexa Fluor488-goat anti-rabbit IgG and Alexa Fluor594-goat anti-mouse IgG, and nuclear-stained with DAPI. The cells were examined under a fluorescent microscope.
We extracted total RNAs from each specimen or group of cells using Trizol reagent, and reversely transcribed them into cDNA using Revert Aid First Strand cDNA Synthesis Kit (K1622, Thermoscientific, USA). We quantified the relative levels of target gene to Tubulin mRNA transcripts by qRT-PCR using the FastStart Essential DNA Green Master kit (06924204001, Lifescience, Roche, Mannheim, Germany) and specific primers in the RocheLightCycler® 96 instrument and software (05815916001, Lifescience). The sequences of primers are shown in the Supplementary Tables S3. We performed the PCR reactions in triplicate and analyzed the data using the 2−ΔΔCt method.
We performed Western blot assays to quantify the relative levels of target protein expression in different groups of cells . Briefly, we performed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the cell lysate proteins (30–50 µg/lane) using 10% gels and transferred them onto polyvinylidene difluoride (PVDF) membranes. We treated the membranes with 5% BSA in TBST and probed the membranes overnight at 4 ° C with antibodies against HK2, PDK1, IDH3B, UQCRC1, AMPK, phosphor-AMPK, TRPM7 and α-Tubulin. Subsequently, we incubated the membranes with HRP-labeled second antibodies and developed the blots with the ECL substrate (32109, ThermoScientific). Finally, we quantified the relative levels of individual proteins to α-Tubulin using the ImageJ software (Madison, USA).
Animal experiments and PET/CT study
We obtained female BALB/c nude mice (6-week-old, ~ 20 g) from SLA Laboratory Animal (Changsha, China) and maintained them in a specific pathogen-free room. In the first set of experiments, we injected each mouse subcutaneously with five millions of SKOV3-Con-sh or SKOV3-TRPM7-sh cells in 0.15 mL of saline. Four weeks later, we dissected, weighed, photoimaged the tumors. In another set of experiments, we injected each mouse subcutaneously with 5x106 SKOV3 cells. Three weeks later, the experimental group of mice were intraperitoneally injected 50 mg/kg carvacrol (an inhibitor of TRPM7, Sigma-Aldrich, 282197) in DMSO vehicle daily for 7 consecutive days while the control mice received vehicle alone. At the end of treatment, we used PET/CT to measure the tumors in mice and euthanized them. We dissected, weighed and frozen tumors. Subsequently, we fixed some tumors from each group of mice in 10% formalin and paraffin-embedded them as well as froze some tumors in liquid nitrogen for subsequent experiments (Figure S5). To perform the PET/CT imaging, we fasted the mice overnight and injected individual mice intravenously with approximately 200 ± 10 µCi 18-fluoro-6-deoxy-glucose (FDG, Wuhan Union Hospital PET Center, Wuhan, China). One hour later, we anesthetized the mice with 2% isoflurane and imaged them using PET with a static mode of 10 min and using CT scan of normal mode in the TransPET Discoverist 180 system (Raycan Technology, Suzhou, China). We reconstructed the PET images using the three-dimensional (3D) OSEM method with a voxel size of 0.5×0.5×0.5 mm3. We reconstructed the CT images using FDK algorithm with 256×256×256 matrix. We displayed the images with software Carimas (Turku PET Center, Turku, Finland). We calculated the mean standardized uptake value (SUV) using the following formula: mean pixel value with the decay-corrected region-of-interest activity (µCi /kg)/(injected dose [µCi]/weight [kg]). The experimental protocols were approved by the Animal Research and Care Committee of our hospital (approval number: 2019-014).
The cell proliferation was tested by EdU assay, using the BeyoClick ™ EdU-594 Cell Proliferation Assay Kit (Byotime), per the manufacturer’s protocol. Briefly, the different groups of cells (3×105 cells/well) were cultured in 6-well plates for 48 h and labeled in triplicate with EdU and DAPI. The fluorescent signals were captured under a fluorescence microscope (Zeiss, Germany).
The different groups of cells were stained in duplicate using the Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme, Nanjing, China). The frequency of apoptotic cells was mwasured by flow cytometry in the BD Flow Cytometry System.
Intracellular ROS levels
The contents of intracellular ROS in each group of cells were quantified using Reactive Oxygen Species Assay Kit (Byotime), per the manufacturer’s protocol. The frequency of fluorescence positive cells was quantified by flow cytometry using CellQuest software.
Measurement of glucose uptake and lactate production
The impact of TRPM7 silencing on glucose uptake and lactate production in ieach group of ovarian cancer cells was tested using the glucose uptake colorimetric assay kit and lactate colorimetric assay kit (Biovision, USA), following the manufacturer’s protocols.
We performed seahorse assays to quantitatively measure ECAR and OCR of individual groups of cells using Seahorse XF Glycolysis Stress Test Kit and Seahorse XF Cell Mito Stress Test Kit in the Seahorse XFe 96 Extracellular Flux Analyzer (Seahorse Bioscience, USA), following the manufacturer’s protocols. Briefly, individual groups of cells (1 × 104 cells/well) were cultured into a Seahorse XF 96-well microplate and their baseline measures were determined. Subsequently, the cells were treated sequentially with glucose, oligomycin (the oxidative phosphorylation inhibitor), and 2-DG (the glycolytic inhibitor) at indicated dose and time points for measurement of ECAR. Similarly, the cells were treated sequentially with oligomycin, oxidative phosphorylation FCCP (p-trifluoromethoxy carbonyl cyanide phenylhydrazone, the reversible inhibitor), and rotenone/antimycin A (Rote/AA, the mitochondrial complex I inhibitor and the mitochondrial complex III inhibitor, respectively). Data were analyzed by Seahorse XF-96 Wave software and expressed as pmols/min for OCR and mpH/min for ECAR, respectively.
The levels of ATP production in each group of cells were determined by ATP assays using ATP Assay Kit (Byotime). Briefly, individual groups of cells were suspended in an ice-cold ATP-releasing buffer, and centrifuged. Individual supernatants or standard (100 µL each) were mixed with 100 µL ATP detection solution and analyzed using the Dual-Luciferase Reporter Assay System (Promega). After normalized with protein concentrations, the luminescent signals were used to quantify the levels of ATP, according to the standard curve prepared with known concentrations (1 nM − 1 mM) of ATP.
In vivo ubiquitination assays
The impact of TRPM7 silencing on the stability of HIF-1α in ovarian cancer cells was examined by in vivo ubiquitination assays. Briefly, the different groups of cells were treated in triplicate with CC (20 µM) under a normoxic or hypoxic condition for one day. The cells were treated with MG132 (10 µM) for 8 h, and the levels of HIF-1α ubiquitination were determined by IP with anti-HIF-1α antibody, followed by Western blot using anti-Ub antibody (10201-1-AP, Porteintech, 1:1000).
We expressed the data as representative images or the mean ± SD of each group from three independent experiments. We compared the data from multiple groups using ANOVA and post hoc Bonferroni analysis and the data from two groups by Student’s T-test using the SPSS version 18.0 (SPSS, Chicago, IL, USA). We analysed the potential correlation of two variables by Spearman’s rank test. We defined statistical significant difference when a P-value of < 0.05 and labelled the significant levels as *P < 0.05, **P < 0.01, ***P < 0.001 in the figures.