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PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Seven-week-old male BALB/c-nude mice were purchased from Orient Bio (Gyeonggi, South Korea).
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
A total of 1 × 10 GIST430 or GIST430-V654A cells were mixed with an equal volume of Matrigel (BD Biosciences), and each mouse was subcutaneously injected with 0.1 ml of the cell-Matrigel mixture into the flank.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Tumor volume was measured using a digital Vernier caliper (Mitutoyo; Kanagawa, Japan) three times a week after implantation and was calculated as 1/2 × length × width .
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
When the tumor volume had reached 200–300 mm about 3 weeks, the mice were randomized into five groups (n = 4–6/group) according to cell line and administered an intraperitoneal injection of 2% DMSO in PBS (vehicle), 50 mg kg imatinib, 30 mg kg PF429242, 1 mg kg bortezomib, 30 mg kg PF429242, and 1 mg kg bortezomib.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Eight injections were performed at three-day intervals, for a total of eight injections.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Body weight and tumor volume were measured three times a week after drug treatment.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
At the end of the experiments, the animals were sacrificed, and the tumors were isolated, weighed, and stored at −80 °C until further analyses.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Tumors were paraffin-embedded and 4 μm sections were used for immunohistochemistry.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
The sections were deparaffinized and treated with BLOXALL endogenous blocking solution (Vector Laboratories, CA, USA) to inhibit endogenous peroxidase activity.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
The epitope retrieval was conducted with Retrieve-All Antigen 1 (pH 8), followed by blocking with PBS supplemented with 2% normal horse serum and 3% BSA.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Sections were incubated with individual primary antibodies at 4 °C overnight and with the appropriate secondary antibody at room temperature for 1 h. Peroxidase activity was revealed using 3,3-diaminobenzidine (Vector Laboratories), and sections were counterstained with hematoxylin (Vector Laboratories).
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
After dehydrogenation with alcohol and xylene, sections were viewed and photographed with an Olympus BX51 microscope (Olympus, Tokyo, Japan) and Mosaic2.1 software.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
The following antibodies were used: Ki-67 (1:100, Abcam, Cambridge, UK), cleaved caspase-3 (1:100, Cell Signaling Technology, MA, USA), and KIT (1:200, Abcam).
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Ki-67, cleaved caspase-3, and KIT.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Positive staining in xenografted tumors were quantified using ImageJ software (NIH).
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Three HPF (high-power fields, at ×400 magnification) were used for each tumor .
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
All data were analyzed using GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA, USA) and expressed as mean ± SEM.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
A comparison of the means among groups was performed using Mann-Whitney U test for body weight, tumor weight, and tumor volume data, or one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test for IHC data.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Fisher’s exact test, Student’s t test, and Kaplan–Meier survival analysis were used to analyze the relationship between nuclear ATF6 expression and clinicopathological parameters (SPSS software, SPSS, Inc., Chicago, IL, USA).
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Statistical significance was set at P < 0.05.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
•Knockdown of circAPP inhibits GIST proliferation and the Warburg effect.•miR-6838-5p induces GIST proliferation and the Warburg effect.•CircAPP activates GIST proliferation and the Warburg effect by regulating the miR-6838-5p/CDV3 axis.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Knockdown of circAPP inhibits GIST proliferation and the Warburg effect.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
miR-6838-5p induces GIST proliferation and the Warburg effect.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
CircAPP activates GIST proliferation and the Warburg effect by regulating the miR-6838-5p/CDV3 axis.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Gastrointestinal Stromal Tumor (GIST) occurs mainly in the stomach and small intestine but also in the colon and rectum, esophagus, and other organs.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Mutations that activate Tyrosine Kinase (KIT) or platelet-derived growth factor receptor α are reported to be the main molecular mechanism of GIST.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Currently, KIT inhibitor (imatinib) is the first-line chemotherapeutic drug for GIST treatment, but most GIST patients develop imatinib resistance.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Although key aspects of the pathogenesis of GIST have been elucidated, the mechanism of oncogene overexpression and gene expression regulators in GIST remains unclear.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Metabolic reprogramming is the earliest known cancer hallmark, which achieves tumor cell proliferation and survival by altering bioenergetic and biosynthetic pathways, and tumor cells exhibit a high rate of glycolysis, also known as the Warburg effect.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Therefore, exploring the regulatory mechanism of the Warburg effect is likely to develop new strategies for GIST treatment.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
CircRNAs are endogenous non-coding RNAs that differ from traditional linear RNAs in that they have covalently closed structures.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
CircRNA/microRNAs (miRNAs) interaction can regulate gene expression and participate in various physiological and pathological processes.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Furthermore, due to the high stability and conservation of circRNAs and their high abundance in body fluids, circRNAs have been considered potential biomarkers for GIST.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
CircRNA Amyloid Precursor Protein (circAPP) is an abnormally highly expressed circRNA in GIST.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
In the preliminary experiments, it was also determined that circAPP was abnormally highly expressed in GIST.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Therefore, this study focused on exploring the function and potential downstream molecular mechanisms of circAPP in GIST.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
CircRNAs expressed in the cytoplasm contain miRNA binding sites to act as miRNA sponges, preventing miRNAs from interacting with mRNAs in the 3′Untranslated Region (UTR), thereby modifying miRNA downstream mRNAs, thereby affecting a series of cellular activities.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
miRNAs are capable of modifying post-transcriptional gene expression by recognizing and binding to the 3′-UTR of target gene mRNAs.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Dysregulated miRNAs are key to many cancers including GIST, where they act as tumor suppressors or oncogenes.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
For example, miR-4510 promotes GIST cell invasion and metastasis whereas miR-494 suppresses GIST development.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
miR-6838-5p has been confirmed to be involved in osteosarcoma, gastric cancer, and renal cell carcinoma.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
However, no studies have demonstrated its actions in GIST.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Targeting to unearth the mechanism of circAPP in GIST, various studies were included, ultimately discovering the mediatory impacts of circAPP on proliferation, migration, and Warburg effect through miR-6838-5p targeting CDV3 in GIST, and hopefully replenishing the options for molecule-targeted therapy in GIST.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Between April 2013 and July 2015, 52 pairs of GIST tissue samples and adjacent normal tissue (≥ 5 cm from cancer tissue) were obtained from the Affiliated Hospital of Inner Mongolia Minzu University.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Inclusion criteria: 1) All the included subjects met the diagnostic criteria of GIST according to the criteria of “Chinese Expert Consensus on Diagnosis and Treatment of Gastrointestinal Mesenchymal Tumor (2017 Edition)” and “NIH2008 Modified Chinese Consensus 2017 Revised”; 2) Patients with GIST observed by abdominal CT or ultrasound endoscopy with tumor located in the stomach, and those who were diagnosed by pathology; 3) Clinical data of all the study subjects must be accurate and complete; 4) Patients not undergoing any radiotherapy or chemotherapy; 5) Patients with no history of other concomitant malignant tumors.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
With written informed consent, tissue sampling was done with the approval of the Ethics Committee of the Affiliated Hospital of Inner Mongolia Minzu University (n° 201211Y62).
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
After diagnosis by a pathologist, tissue specimens were frozen in liquid nitrogen and then stored at -80 °C to determine gene expression.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
All methods in the study were carried out in accordance with the ARRIVE guidelines.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
The human gastric mucosal epithelial cell line GES-1 and the human GIST cell lines GIST-882 and GIST-T1 (Cosmo, Tokyo, Japan) were serially passaged for no more than 6 months.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Cells were cultured in DMEM (Gibco; Thermo Fisher Scientific) in a humidified incubator at 37 °C with 5 % CO2.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
10 % heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific), 100 U/mL penicillin, and 100 mg/mL streptomycin were supplementary to the culture medium.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
The cell lines were all validated by STR profiles and tested negative for mycoplasma contamination.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Under the guidelines of TRIzol (Invitrogen), total RNA was extracted.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
For reverse transcription, the PrimeScript RT kit (Takara, Shiga, Japan) was employed.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Taking cDNA as a template, RNA quantification was done using specific primers (Table 1) and TB Green qPCR Master Mix (Takara) on a 480 II instrument (Roche Diagnostics, Basel, Switzerland).
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Expression levels of circRNA, mRNA, and miRNA were calculated by the 2 method and standardized to GAPDH or U6.Table 1Primers.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Table 1PrimerscircAPPForward: 5′-TGACTGAAGGGAAGTGTGCC-3′Reverse: 5′-CAAACTCACCAATGGCGCTG-3′miR-6838-5pForward: 5′-GCAAGCAGCAGTGGCAAG-3′Reverse: 5′-GCAGGGTCCGAGGTATTC-3′GAPDHForward: 5′- CACCCACTCCTCCACCTTTG-3′Reverse: 5′-CCACCACCCTGTTGCTGTAG-3′U6Forward: 5′-CTCGCTTCGGCAGCACA-3′Reverse: 5′-AACGCTTCACGAATTTGCGT-3′Note: circAPP, circular RNA APP; miR-6838-5p, microRNA-6838-5p; GAPDH, Glyceraldehyde-3-Phosphate Dehydrogenase.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Primers.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Note: circAPP, circular RNA APP; miR-6838-5p, microRNA-6838-5p; GAPDH, Glyceraldehyde-3-Phosphate Dehydrogenase.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
For RNase R treatment, total RNA (2 μg) was cultured with 3 U/μg RNase R (Geneseed Biotech, Guangzhou, China) at 37°C for 10 min.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Then, RNA purification was done with the RNeasy MinElute Cleanup kit (Qiagen, Hilden, Germany).
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
For actinomycin D treatment, RNA was treated with 2 mg/mL actinomycin D (Sigma-Aldrich).
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Dimethyl sulfoxide was taken as a control.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Finally, RNA expression levels were quantitatively analyzed.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Subcellular localization of circAPP in GIST-882 cells was analyzed.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Nuclear and cytoplasmic fractions were isolated using the PARIS™ kit (Invitrogen), with U6 and 18S rRNA as positive references, respectively.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Small interfering RNA targeting circAPP and CDV3, small interfering RNA negative control (si-circAPP: 5’-ATGGATGTTTGCGAAACTCATCT-3’, si-CDV3, si-NC), overexpression plasmid targeting circAPP and CDV3 and negative control (pcDNA 3.1-circAPP/CDV3, pcDNA 3.1) were supplied by RiboBio (Guangzhou, China), whereas miR-6838-5p mimic (5’-AAGCAGCAGTGGCAAGACTCCT-3’)/inhibitor and mimic/inhibitor NC were by Genepharma (Shanghai, China).
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Lipofectamine 2000 (Invitrogen) served for the transfection of cells seeded at 2×10/well on 6-well plates.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Briefly, when cells were 70-80 % confluent, oligonucleotides or plasmids were diluted in Opti-MEM® Medium.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Lipofectamine 2000 was then diluted with Opti-MEM® Medium.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Lipofectamine 2000 was mixed with the diluted oligonucleotide or plasmid and added to each well at 100 μL/well.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
After 48h, the transfection efficiency was determined by RT-qPCR or western blot.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Cell samples from log phase cultures were transferred to 6-well plates at 500 cells/well and cultured for 14 days.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Afterward, samples were fixed in 4 % paraformaldehyde and counted after 0.1 % crystal violet staining.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
GIST-882 and GES-1 cells were incubated with 30 μM EdU (KeyGen Biotech, Jiangsu, China) for 2h and treated with DAPI (Sigma-Aldrich) to stain the nuclear.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Fluorescent images were taken to assess the EdU-positive rate.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Apoptotic cells were assessed with an annexin V-FITC/PI kit (Vazyme, Nanjing, China).
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Cells were resuspended and stained with Annexin V-FITC and PI solutions (5 μL each solution) in the dark.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Apoptotic cells were detected on a FACSCanto II flow cytometer (BD Biosciences) to calculate the apoptosis rate.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Glucose consumption, lactate level, and ATP were determined by glucose assay kits (Sigma), lactate colorimetric/fluorometric kits (BioVision, CA, USA), and ATP assay kits (Thermo Fisher Scientific), respectively.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
ATP/ADP was measured using the ApoSENSOR ADP/ATP Ratio Assay Kit (K255-200, BioVision, CA, USA) and luminescence was measured by spectroscopy (Molecular Devices, CA USA).
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Cells (10 cells) were seeded onto luminometer plates, incubated with nucleotide release buffer, and added with 1 µL of ATP monitoring enzyme.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Data were recorded at 1 min (Data A) and 10 min (Data B).
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
ADP convertase was then added, and values were read (Data C). .
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
NAD/NADH was tested using the EnzyChrom™ NAD/NADH Ratio Assay Kit (E2ND-100, Bioassay Systems, CA, USA).
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
A total of 10 cells were homogenized and cultured with 100 mL NAD extraction buffer for NAD assay or 100 mL NADH extraction buffer for NADH assay.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Next, 20 µL of assay buffer was added to and 100 mL of back-extraction buffer was then supplemented.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
The mixture was then centrifuged, and the supernatant was transferred to the working reagent.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
OD565 nm was read at 0 and 15 min.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Total protein from cells and tissues was isolated using Lysis Buffer (Thermo Fisher Scientific) and analyzed for concentration using the BCA Assay Kit (Bio-Rad).
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Proteins (30 μg) were separated by 10 % SDS-PAGE and then transferred to PVFD membranes (Millipore, MA, USA).
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Membranes were blocked with 5 % skim milk, mixed with primary antibodies GAPDH (60004-1-Ig, Proteintech), CDV3 (LS-C205853, LSBio), HK2 (22029-1-AP, Proteintech), PKM2 (15822-1-AP, Proteintech), followed by the secondary antibody (Sigma-Aldrich).
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
To visualize protein bands, a Western blot substrate (Thermo Fisher Scientific) was added, and Image Lab analysis software (Bio-Rad) was employed for reading protein band intensity.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
CircAPP and CDV3-3′UTR wild-type sequences (WT-circAPP, WT-CDV3) or mutant sequences (MUT-circAPP, MUT-CDV3) containing the miR-6838-5p binding site were cloned into the vector psiCHECK-2.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
The above-mentioned reporter gene vector, along with miR-6838-5p mimic or mimic NC was co-transfected into GIST-882 cells (5×10) in the 24-well plates using Lipofectamine 2000 (Invitrogen).
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Cells were lysed after 48h, and luciferase activity was evaluated using Dual-Luciferase Assay kit (Promega).
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
The RIP assay was performed using the Imprint® RIP Kit (Sigma-Aldrich).
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
GIST-882 cells (1×10) were lysed with RIP buffers and interacted with antibody-coated magnetic beads at 4°C overnight.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
Anti-IgG was the control for Anti-Ago2.
PMC11269792
CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor
After RNA isolation from the magnetic beads, analysis of RNA expression was carried out by RT-Qpcr.