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PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Transfect 293T cells using Lipofectamine 2000, and replace the medium with fresh medium after 6h.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Check the transfection efficiency using the fluorescence microscope after 48h of transfection, and collect the supernatant after 72h.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The GIST-882 or GIST-T1 cells were added with the corresponding volume of polybrene 0.5h before the addition of virus infection.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The cells were replaced after 12h of infection, after 48h the infection efficiency was observed under a fluorescence microscope.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The bottom of the Trans-well chambers (BD) was evenly spread with substrate gel and placed in an incubator at 37 °C for 0.5h to allow solidification.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
After cell counting, the cells were mixed with serum-free DMEM and added directly to the chambers, and the appropriate volume of complete medium was added outside the chambers.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The cells were fixed with methanol, stained crystal violet, and photographed under a microscope.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The average of five random fields of view per chamber was taken to compare each group's migration and invasion ability.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
In this experiment, 3–5 replicate wells were set up to test various cells.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The viability of cells was measured in vitro using the CCK-8 kit.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
2 × 10 cells per well were inoculated in a 96-well plate and incubated for 48h.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The 10 % CCK-8 solution was added to each well and incubated for approximately 2h.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The absorbance of each well was measured at 450 nm.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
For cell digestion and counting, the suspension was added to a six-well plate for incubation with 1 × 10 cells per well.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Clones were observed after 9–14 days.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Wash twice with PBS, add 1 ml of methanol to each well for fixation, and leave for 10min at room temperature.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Stain with 0.1 mg/ml crystal violet and leave for 10min at room temperature.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Dry and count at room temperature.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Cell membrane components were isolated from GIST-882 cells of imatinib-resistant strain and tissues of GIST-resistant patients (Beyotime Membrane Extraction Kit and Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Total RNA and cell membrane RNA were extracted with TRIZOL reagent, and PIGR endogenous antibody for RIP-Seq experiments.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Validation was performed using RIP-RT-PCR.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
3′ RACE and 5′ RACE experiments were performed using kits from SMARTer RACE cDNA Amplification Kit (Clontech).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Northern-blot assays were performed using the Northern Max® Kit from Thermo Fisher Scientific.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The online website STRING (https://cn.string-db.org/) was used to predict proteins with potential interactions with PIGR.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Based on the Coding Potential Calculator (CPC, http://cpc.cbi.pku.edu.cn/), the Coding-Potential Assessment Tool (CPAT, http://cpc.cbi.pku.edu.cn/), and Phylogenetic Codon Substitution Frequencies (PhyloCSF score), the LINC00870 sequence was analyzed for protein-coding ability.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The original data of Sarcoma second-generation sequencing were collected from the TCGA database.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
All statistical analyses of bioinformatics were performed with Rstudio software (version 4.0.2; http://www.rstudio.com/products/rstudio).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The Limma package was used to analyze the differences in gene expression between tumor samples and healthy samples, and a volcano map and heat map were drawn.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
R package clusterProfiler (https://guangchuangyu.github.io/software/clusterProfiler) was used to process the GSEA analysis.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The correlation analysis and visualization of the gene expression result were processed using ggplot2 in R software.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Statistical analyses were performed using GraphPad Prism 9 (GraphPad, San Diego, California).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Statistical analysis was done using the paired Student's t-test, the Pearson correlation, or the Log-rank survival analysis.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The p-values less than 0.05 were considered statistically different.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
In this study, we performed RNA sequencing analysis on five GIST patients (cancer and paracancerous tissues) who were resistant to imatinib therapy.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
We combined these results with RNA sequencing data obtained from 263 TCGA-Sarcoma tumor tissues and two normal tissues.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Using filtering conditions of log2Fold change (Tumor/Normal) > 4 and Pearson correlation coefficient (P < 0.001), we identified three genes (PIGR, AKR7A3, PLS1) that were highly expressed in GIST patients who were resistant to imatinib therapy (Fig. S1 A-C).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The differential gene expression profiles analysis between the two data sets indicated that the PIGR gene was significantly highly expressed in GIST imatinib-resistant patients (Fig. 1A and B).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Further analysis of the signaling pathways closely regulated by PIGR using Gene Set Enrichment Analysis (GSEA) showed that PIGR played an inhibitory role in multiple immune activation pathways, such as immune receptor activation and organ- or tissue-specific immune responses (Fig. 1C).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
We utilized the STRING online prediction tool to identify potential molecular targets of PIGR and examined their correlation at the mRNA level.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Our analysis revealed a significant association between PIGR and VNN1 as well as CEACAM6 (Fig. 1D and E).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Vanin-1 (VNN1) has pantetheinase activity and plays a crucial role in tumorigenesis and immune regulation [, , , , ].
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
CEACAM6, a carcinoembryonic antigen immunoglobulin family member, is a cell surface adhesion factor that plays a vital role in tumorigenesis, proliferation and metastasis .
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Based on the results obtained in this study, it is reasonable to infer that PIGR may have a pivotal role in regulating immune responses and promoting tumor progression.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Fig. 1PIGR as a candidate gene for the study.(A–B) Hot-map showed the expression profiles of apparently differential genes. (
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The analysis of the signaling pathways that regulate PIGR using GSEA. (
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
D–E) The online website STRING was used to predict proteins with potential interactions with PIGR and these proteins were analyzed for relevance at the mRNA level using bioinformatics.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Fig. 1 PIGR as a candidate gene for the study. (
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
A–B) Hot-map showed the expression profiles of apparently differential genes. (
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The analysis of the signaling pathways that regulate PIGR using GSEA. (
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
D–E) The online website STRING was used to predict proteins with potential interactions with PIGR and these proteins were analyzed for relevance at the mRNA level using bioinformatics.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
In light of the crucial role of PIGR in GIST imatinib resistance, we investigated the expression levels of PIGR in imatinib non-resistant cancer samples and their corresponding paracancerous controls, as well as in imatinib-resistant cancer samples and their paracancerous controls.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The results revealed significantly higher mRNA levels of PIGR in GIST tumor samples compared to their paracancerous controls (Fig. 2A, P < 0.001).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Moreover, the mRNA levels of PIGR were markedly higher in imatinib-resistant GIST samples than in non-resistant samples (Fig. 2A, P < 0.001).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
We further categorized the GIST patients into two groups based on PIGR expression levels and examined the correlation between PIGR expression and overall survival time.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The data showed that patients with high PIGR expression levels had a poorer prognosis (Fig. 2B).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Taken together, these results suggest that PIGR is significantly upregulated in imatinib-resistant GIST patients, and higher PIGR expression levels are associated with poorer prognosis.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Fig. 2Expression and prognosis of PIGR in GIST tissues and imatinib-resistant tissues.(A) The mRNA expression of PIGR in different tissues of GIST. (
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Using the log-rank test, the figure on the left shows the survival analysis of PIGR expression differences in GIST patients.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The figure on the right shows GIST patients divided into positive and negative groups of PIGR expression by RNA-FISH.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
∗∗∗P < 0.001.Fig.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
2 Expression and prognosis of PIGR in GIST tissues and imatinib-resistant tissues. (
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The mRNA expression of PIGR in different tissues of GIST. (
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Using the log-rank test, the figure on the left shows the survival analysis of PIGR expression differences in GIST patients.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The figure on the right shows GIST patients divided into positive and negative groups of PIGR expression by RNA-FISH.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
∗∗∗P < 0.001.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
PIGR plays a crucial role in bridging innate and adaptive immunity, suggesting a potential link between imatinib resistance and immune dysregulation [, , ].
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
To further investigate the specific function of PIGR in GIST, we designed sgRNAs against PIGR sequences and performed endogenous interference using CRISPRdCas9 in GIST-T1 and GIST-882 cell lines, followed by relevant cell function experiments.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Our results showed that interfering with PIGR significantly suppressed cell invasion capability, while overexpression significantly promoted it in vitro (Fig. 3A and B).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Furthermore, in vivo experiments showed that overexpression of PIGR significantly promoted lung colonization and liver metastasis of GIST cells (Fig. 3C and D).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
These findings suggest that PIGR acts as an oncogene that promotes the malignant phenotype of GIST.Fig.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
3PIGR functions as an oncogene in GIST.(A–B) The effect of interfering or overexpressing PIGR on the migration and invasion ability of GIST-T1 and GIST-882 cells. (
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
C–D) The effect of PIGR overexpression on the invasive ability of GIST-T1 and GIST-882 cells was analyzed via the tail vein model (C) and GIST in situ model (D).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Mean ± SEM of 3 independent experiments.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
∗∗∗P < 0.001 versus control.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Fig. 3 PIGR functions as an oncogene in GIST. (
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
A–B) The effect of interfering or overexpressing PIGR on the migration and invasion ability of GIST-T1 and GIST-882 cells. (
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
C–D) The effect of PIGR overexpression on the invasive ability of GIST-T1 and GIST-882 cells was analyzed via the tail vein model (C) and GIST in situ model (D).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Mean ± SEM of 3 independent experiments.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
∗∗∗P < 0.001 versus control.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
To facilitate subsequent investigations, we established an imatinib-resistant cell line, GIST-882 (IR-GIST-882), selected based on the SDHB marker as imatinib-resistant .
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Our results confirmed the successful construction of the stable resistant cell line (Figs. S2A and B).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
To investigate the molecular mechanisms underlying the role of PIGR in drug-resistant GIST, we extracted RNA from cell membrane fractions of tissues from drug-resistant patients, as well as from cells of drug-resistant strains, for RIP-seq analysis.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
This analysis aimed to identify RNA molecules specifically bound to PIGR (Fig. 4A).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Our study revealed a specific interaction between PIGR and LINC00870 (Fig. 4B), indicating that PIGR may mediate imatinib resistance in GIST through this interaction.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Fig. 4Exploration of specific mechanisms for PIGR.(A) The RIP-seq workflow. (
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The RNA molecules interacting with PIGR were verified using RIP-PCR.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The workflow was created with BioRender.com.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Fig. 4 Exploration of specific mechanisms for PIGR. (
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The RIP-seq workflow. (
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The RNA molecules interacting with PIGR were verified using RIP-PCR.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The workflow was created with BioRender.com.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
To clarify the transcript sequence information of LINC00870 in GIST cells, we conducted RACE assay and Northern-blot assay, which led us to determine the primary transcript length of 2700bp (Fig. 5A and B).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Furthermore, we isolated nucleus and cytoplasmic RNA using extraction kits and discovered that LINC00870 was predominantly localized in the cytoplasmic region (Fig. 5C).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Using bioinformatics tools such as CPC, CPAT, and phyloCSF sites, we predicted that LINC00870 is a non-coding RNA (Fig. 5D).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
To confirm the non-coding nature of LINC00870, we assessed overexpressing LINC00870 cells at both RNA and protein levels.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
Agarose gel electrophoresis confirmed effective RNA expression for both sense and antisense strands of LINC00870 (Fig. 5E).
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
However, Western blot analysis revealed no protein expression, indicating that LINC00870 RNA does not encode proteins (Fig. 5F).Fig.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
5Basic characteristics and biological functions of LINC00870.(A) LINC00870 sequence information and length were analyzed using 3′RACE and 5′RACE experiments.
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The uncropped gel image can be found in Supplementary Figure 3. (
PMC11834037
LINC00870 promotes imatinib resistance in gastrointestinal stromal tumor via inhibiting PIGR glycosylation modifications
The number and length of LINC00870 transcripts in GIST cell lines and tissues were determined by a Northern blot assay.