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PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Our findings suggest that PIGR plays a crucial role in imatinib resistance in GIST, as evidenced by the significantly higher mRNA expression of PIGR in imatinib-resistant GIST samples than in non-resistant samples.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Furthermore, we identified a specific binding between PIGR and LINC00870 through the RIP-seq assay.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
In recent years, there has been increasing research interest in the regulatory role of lncRNA in tumor development and progression [, , , ].
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The significance of lncRNA in various pathological and physiological processes is well established , although no studies have reported on the regulatory mechanisms of LINC00870 in GIST drug resistance and immunity.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
We established an imatinib-resistant cell line, IR-GIST-882, through a high-dose gap-action approach, which provided an effective tool for studying imatinib resistance.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Furthermore, by overexpressing LINC00870 in GIST-882 and GIST-T1 cell lines, we observed increased invasion, migration, and cladogenesis of GIST cells, suggesting that LINC00870 functions as an oncogene in GIST.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
LncRNAs are pivotal as regulatory and predictive factors in cancer treatment resistance and can be used as adjunctive therapeutic agents to enhance the efficacy of existing therapies, such as chemotherapy, radiotherapy, immune checkpoint inhibitors, and targeted therapy .
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Their substantial dysregulation across various cancer types, coupled with their heterogeneous expression, positions them as ideal biomarkers and therapeutic targets for personalized medicine .
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Despite their potential, the clinical application of LncRNAs-based therapies faces challenges, including issues of tolerance, toxicity, and off-target effects, which necessitate further investigation.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The cell lines and drug-resistant variants used in our study are derived from human sources.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
However, due to interspecies differences, in vivo studies often utilize immunodeficient mouse models, which have significant limitations, particularly the absence of immune cells that may influence assessments of immune dysregulation.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Current cellular models in tumor immunology include human peripheral blood mononuclear cell (Hu-PBMC) and human CD34 (Hu-CD34) humanized mouse models, which effectively reconstruct the immune system within immunodeficient mice, thereby enabling the examination of tumor dynamics within an immune-enriched microenvironment .
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
To investigate the roles of LINC00870 and PIGR within the immune cell microenvironment, employing humanized mouse models would provide more reliable results.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
While this study proposes a potential strategy to overcome imatinib resistance through the inhibition of PIGR or LINC00870, it is essential to draw insights from other research to further validate its feasibility and safety in clinical settings [, , ].
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Additionally, the pharmacokinetics and pharmacodynamics of these strategies should be investigated to optimize dosing regimens, enhance therapeutic efficacy, and minimize side effects .
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
We hope that the GIST-imatinib resistance-related targets identified in this study will provide valuable references and assistance for the treatment of GIST patients with imatinib resistance.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
In summary, our study investigated the mechanisms underlying resistance to targeted therapies in GIST patients and identified the PIGR-LINC00870 complex as a potential key factor in inducing drug resistance.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
These findings suggest that targeting this complex could offer a promising approach to the comprehensive treatment of GIST by combining targeted therapy, immunotherapy, and molecular therapy.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
This study provides valuable insights into the clinical management of GIST and offers potential clinical applications for improving patient outcomes.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Further studies are warranted to validate the clinical significance of this complex and explore its potential as a therapeutic target for GIST.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Yuan Li: Writing – original draft, Formal analysis, Data curation.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Zhiqiang Dai: Formal analysis, Data curation.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Zewei Cheng: Formal analysis, Data curation.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Junyi He: Software, Methodology.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Yirui Yin: Software, Methodology.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Xinyou Liu: Software, Formal analysis.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Jiwei Zhang: Writing – review & editing.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Guohua Hu: Data curation.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Yueda Chen: Writing – review & editing, Supervision, Funding acquisition.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Xuefei Wang: Writing – review & editing, Supervision, Funding acquisition.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Yebo Shao: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Not applicable.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
All datasets involved in this study can be viewed in the Gene Expression Omnibus (GEO number: GSE155800) or data availability part of the corresponding articles.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
All data pertinent to this study, whether generated or analyzed, are comprehensively presented in this manuscript and its supplementary information.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
For any additional inquiries or requests, interested parties are encouraged to contact the corresponding authors.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The animal research was approved by the Institutional Animal Care and Use Committee of Shanghai Immunocan Biotechnology Co., Ltd (Approval Number: YMNK-IACUC-F006, Approval Date: November 14, 2022).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The human research was approved by the Clinical Research Ethics Committee of Fudan University Zhongshan Hospital (Approval Number: B2023-199, Approval Date: November 19, 2023).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Additionally, it should be noted that at our teaching hospital, all patients admitted for surgical procedures are required to sign consent forms allowing for the use of their surgical specimens for research purposes.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Therefore, research involving surgical specimens is exempt from the requirement for explicit informed consent.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
This work was supported by the Natural Science Foundation of Fujian Province (supported by 10.13039/501100005270Fujian Provincial Department of Science and Technology) under grant number 2022J011413, Natural Science Foundation of Xiamen under grant number 3502Z20227276, 2021 Funding support for high-level medical and healthcare talents in Xiamen — Funding support for high potential talents, Project of Science and Technology of Xiamen City under grant number 3502Z20224015, Key Clinical Specialty Discipline Construction Program of Fujian, the Clinical Medical Research Center Project of Xiamen City, Xiamen medical and health guidance project (supported by Xiamen Science and Technology Bureau) under grant number 3502Z20209041, Science and Technology Project of Fujian Provincial Health Commission under grant number 2020QNB063, Construction of gastric cancer organoid biobank and the application in personalized screening of chemotherapy and targeted drugs and Clinical Research Center Project for Precision medicine of abdominal tumor of Fujian Province.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Recent evidence has demonstrated that abnormal expression and regulation of circular RNA (circRNAs) are implicated in the development and progression of various tumors.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
The aim of this study was to investigate the effects of circ_SMA4 in Gastrointestinal Stromal Tumors (GISTs) malignant progression.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Human circRNAs microarray analysis was conducted to identify differentially expressed (DE) circRNAs in GISTs.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
The effect of circ_SMA4 on cell proliferation, invasion, migration, and apoptosis was assessed in both in vitro and in vivo settings.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Dual-luciferase reporter assay, RT-qPCR, Western-blot, and rescue assay were employed to confirm the interaction between circ_SMA4/miR-494-3p/ KIT axis.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
The results revealed that circ_SMA4 was significantly upregulated in GISTs, and exhibited high diagnostic efficiency with an AUC of 0.9824 (P < 0.01).
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
circ_SMA4 promoted cell proliferation, invasion, migration, while inhibiting apoptosis in GISTs cells, both in vitro and in vivo.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Silencing circ_SMA4 partially inhibited GISTs malignant progression.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Additionally, circ_SMA4 acted as a competing endogenous RNA (ceRNA) by targeting miR-494-3p, and KIT was identified as a functional gene for miR-494-3p in GISTs.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Furthermore, the results confirmed that circ_SMA4/miR-494-3p/ KIT axis plays a role in activating the JAK/STAT signaling pathway in GISTs.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Therefore, for the first time, we have identified and emphasized that circ_SMA4 is significantly upregulated and plays an oncogenic role in GISTs by sponging miR-494-3p to activate the KIT/JAK/STAT pathway.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
These findings underscore circ_SMA4 may serve as a novel diagnostic biomarker and therapeutic target for GISTs.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Gastrointestinal stromal tumors (GISTs) are infrequently encountered among gastrointestinal carcinomas, originating from mesenchymal tissue.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
These tumors are identified by the presence of the CD117 receptor in cells and exhibit diverse biological phenotypes, ranging from benign to highly malignant.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Among the most prevalent non-epithelial neoplasms, GISTs are predominantly situated in the stomach (55.6%) and small intestine (31.8%).
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
The preferred approach for treatment is radical surgery, and molecular targeted therapies, such as imatinib, have demonstrated efficacy in enhancing the survival rates of advanced patients harboring c-kit and/or PDGFRα mutations.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Despite this, the availability of effective tumor biomarkers for GISTs diagnosis and prognostication remains limited.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Circular RNA (circRNA) represents an emerging class of endogenous non-coding RNA characterized by a distinctive closed-loop structure, wherein the 3′ and 5′ ends are covalently linked.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
This unique structure imparts resistance to exonucleases, rendering circRNAs more stable compared to traditional linear RNA, including lncRNA and miRNA.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Categorically, circRNAs can be classified into four types based on their source: exonic circRNAs (ecircRNA), intronic circRNA (ciRNA), exonic- intronic circRNA (EIciRNA), and intergenic circRNAs.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Notably, 80% of circRNAs belong to the ecircRNA category.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
The functional roles of circRNAs are diverse and impactful.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
They can function as microRNA (miRNA/miR) sponges, competitively binding to miRNA response elements to modulate downstream target gene expression.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
This concept gained support with the identification of cerebellar degeneration-related protein (CDR1as), also known as CiRS-7, which unveiled circRNAs’ ability to function as competing endogenous RNAs (ceRNA) with over 60 miRNA binding sites.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Simultaneously, circRNAs exert influence at the post-translational level, impacting gene function.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Intriguingly, a single circRNA can regulate multiple miRNAs, and reciprocally, the same miRNAs can regulate multiple mRNA genes, forming an extensive circRNA-miRNA-mRNA competitive network that significantly influences tumor development.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Additionally, certain circRNAs exhibit activities through interactions with proteins, and some ecircRNAs may participate in assembly and protein ribosome translation processes.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Reports indicate that circRNAs are implicated in diverse biological processes, including signal transduction, transcription, cell cycle regulation, RNA-binding protein interactions, stress responses, protein metabolism, cellular immunity, and cell structure.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Recent studies underscore the involvement of abnormal circRNA expression and regulation in the occurrence and progression of various tumors.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Consequently, circRNAs hold significant value as biomarkers for cancer diagnosis, prediction, and treatment response evaluation.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Furthermore, they may serve as potential targets for innovative cancer treatments.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Our preliminary study revealed that circ_SMA4 (hsa_circRNA_103870) was significantly up-regulated in GISTs (n = 20), and circ_SMA4 also showed high diagnostic values, and significantly associated with tumor size, mitotic figure, and malignant degrees in GISTs.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
We hypothesized that circ_SMA4 might be critical circRNA and may present as potential diagnostic biomarkers for GISTs.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
To validate our hypothesis, we established cell models with circ_SMA4 overexpression and knockdown to investigate the effect of circ_SMA4 on GISTs malignant progression.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Its mechanism of action is also being explored in depth.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Twenty pairs of GISTs and adjacent tissues were collected from The Second Xiangya Hospital of Central South University.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
All pathological specimens were confirmed by experienced pathologists and did not undergo pre-operative radiotherapy, chemotherapy, or imatinib targeted therapy.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
The clinicopathological features are detailed in Supplemental Table SI.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Tissues were collected during surgical operations and promptly stored in liquid nitrogen.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
The present study was approved by the ethics committee of The Second Xiangya Hospital of Central South University, and informed consents were obtained from all participants.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Total RNAs from each sample were extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany).
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Differences in circular RNAs (circRNA) between the two sample groups were analyzed using the Arraystar Human circRNA Array V2 (8 × 15 K; Arraystar).
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
The GISTs cell lines GIST-T1 and GIST-882 and Human Stomach smooth muscle cells (HGSMC) were obtained from the American Type Culture Collection (ATCC).
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
These cell lines underwent regular authentication through Short Tandem Repeat (STR) analysis and were routinely screened for mycoplasma contamination to ensure their integrity.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
The cell lines were cultured in Dulbecco’s minimum essential medium supplemented with 10% fetal calf serum at 37 °C in a humidified atmosphere containing 5% CO2.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Total RNA extraction was conducted utilizing TRIzol reagent.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Subsequently, the synthesis of complementary DNA (cDNA) was performed using the PrimeScript™ RT-PCR Kit following the manufacturer’s protocol.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
The primer sequences for circular RNAs (circRNAs), microRNA (miRNA), and messenger RNA (mRNA) were custom-synthesized by Shanghai GeneChem Co., Ltd. GAPDH served as the internal reference for circRNAs and mRNA, while U6 was employed as the internal reference for miRNA.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
The thermocycling conditions involved an initial step at 95 °C for 15 min, followed by 40 cycles at 94 °C for 15 s, 55 °C for 30 s, and 70 °C for 30 s. Following the PCR procedure, the relative expression levels were determined using the 2 method.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Cells were plated at a density of 2000 cells per well in a 96-well plate and exposed to Pembrolizumab (0.0–1.0 µM) for a duration of 24 h. Assessment of cell viability was conducted by measuring absorbance at 492 nm following incubation with CCK-8 solution.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
The formula employed for calculating Cell Viability was:[12pt] $$ = - } -)} 100\%$$ This experiment was repeated three times for robustness and reliability.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
For the experiment, a Transwell system featuring an 8-µm pore size from Corning was employed.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
In this setup, the upper chamber of the Transwell was seeded with 2 × 10 cells cultured in 500 µL of serum-free medium, while the bottom well was supplemented with 1 mL of complete culture medium containing 10% serum.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
After undergoing a 48-hour incubation period in a dedicated incubator, the cells were fixed using 4% paraformaldehyde and subsequently stained with crystal violet solution.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
To eliminate any remaining cells that had not migrated, a cotton swab was utilized.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
The remaining cells were then observed and imaged under a microscope to ensure thorough analysis and documentation.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
This process was conducted with attention to detail and precision.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
To evaluate cell migration, a wound healing assay was conducted.
PMC11422497
circ_SMA4 promotes gastrointestinal stromal tumors malignant progression by sponging miR-494-3p/KIT axis and activating JAK/STAT pathway
Following trypsinization and cell counting, 5 × 10 cells were seeded into each well of a pre-marked 6-well plate with evenly spaced horizontal lines.