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Identification of circRNA-interacting proteins in pancreatic ductal adenocarcinoma

ABSTRACT: Many studies have demonstrated the importance of circRNA in regulating gene expression through functioning as microRNA sponges. However, the roles of circRNA-protein interaction are not fully understood. Importantly, how circRNA-protein interaction contributes the progression of pancreatic ductal adenocarcinoma is largely unexplored. Therefore, RNA Pull down assay for investigating RNA-protein interaction was performed in PANC-1 cells.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Pancreatic Ductal Carcinoma

SUBMITTER: Chi Hin Wong

LAB HEAD: Chi Hin Wong

PROVIDER: PXD015048 | Pride | 2022-02-15

REPOSITORIES: Pride

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			Action	DRS
	C_R.raw	Raw
	FX_R.raw	Raw
	F_R.raw	Raw
	GX_R.raw	Raw
	G_R.raw	Raw

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1 - 5 of 45

Publications

CircRTN4 promotes pancreatic cancer progression through a novel CircRNA-miRNA-lncRNA pathway and stabilizing epithelial-mesenchymal transition protein.

Wong Chi Hin CH Lou Ut Kei UK Fung Frederic Khe-Cheong FK Tong Joanna H M JHM Zhang Chang-Hua CH To Ka-Fai KF Chan Stephen Lam SL Chen Yangchao Y

Molecular cancer 20220104 1

<h4>Background</h4>Circular RNAs (circRNAs) play important roles in many biological processes. However, the detailed mechanism underlying the critical roles of circRNAs in cancer remains largely unexplored. We aim to explore the molecular mechanisms of circRTN4 with critical roles in pancreatic ductal adenocarcinoma (PDAC).<h4>Methods</h4>CircRTN4 expression level was examined in PDAC primary tumors. The oncogenic roles of circRTN4 in PDAC tumor growth and metastasis were studied in mouse tumor ...[more]

PMID: 34983537

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Synthetic circular RNA functions as a miR-21 sponge to suppress gastric carcinoma cell proliferation

Project description:MicroRNA (miRNA) sponges containing miRNA complementary binding sites constitute a potentially useful strategy for miRNA-inhibition therapeutics in cancer patients. Recently, naturally occurring circular RNAs (circRNAs) have been revealed to function as efficient microRNA sponges. We hypothesized that synthetic circRNA sponges targeting oncomiRs could be constructed and used to achieve potentially therapeutic microRNA loss of function. In this study, linear RNA molecules containing five miR-21 binding sites were transcribed in vitro. After dephosphorylation by calf intestinal phosphatase and phosphorylation by T4 polynucleotide kinase, circRNA sponges were circularized using 5’-3’ end ligation by T4 RNA ligase 1. Synthetic circular sponge stability was assayed in the presence of RNase R or fetal bovine serum. Luciferase reporter and cell proliferation assays were performed to assess competitive inhibition of miR-21 activity by circRNA sponges in NCI-N87 gastric cancer cells. Tandem Mass Tag (TMT) labeling proteomics analysis and Western blotting were performed to delineate effects of circRNA sponges on miR-21 downstream targeted proteins. Our experiments revealed that artificial circRNA sponges can be synthesized using enzymatic ligation. These synthetic circRNA sponges are more resistant than their linear RNA counterparts to nuclease degradation in vitro. They effectively suppress the activity of miR-21 on its downstream protein targets, including the important cancer protein DAXX. Finally, they also inhibit gastric cancer cell proliferation. Our results suggest that synthetic circRNA sponges represent a rapid, effective, convenient strategy to achieve loss of miRNA function in vitro, with potential future therapeutic application in vivo.

2018-10-22 | PXD008584 | Pride

CircHIPK3 and FMRP compete for direct binding to BRCA1 mRNA controlling its protein levels and DNA damage response [miRNA_pulldown]

Project description:Circular RNAs (circRNAs) are covalently closed RNA molecules widely expressed in eukaryotes and regulated in several pathological processes, including cancer. Many studies point to their mechanism of action as miRNA and protein sponges; however, we propose a new functionality based on circRNA-mRNA interaction to regulate mRNA fate. We show that the widely expressed circHIPK3 directly interacts in vivo with the BRCA1 mRNA through the back-splicing region. This interaction favoured the translation of BRCA1 by competing for the binding of the FMRP1 RNA-binding protein, which acts as a repressor of BRCA1 translation. CircHIPK3 depletion or disruption of the CircRNA-mRNA interaction decreased BRCA1 levels and increased DNA damage, sensitizing several cancer cell lines to DNA-damage-inducing agents and rendering them susceptible to synthetic lethality. Additionally, mimicking circHIPK3 interaction with lock-nucleic acid (LNA) oligonucleotides restores BRCA1 levels in hereditary breast cancer model cells, underscoring circRNA-mRNA interaction’s importance in regulating cell homeostasis and drug response.

2024-09-13 | GSE246225 | GEO

CircHIPK3 and FMRP compete for direct binding to BRCA1 mRNA controlling its protein levels and DNA damage response [RNA_pulldown]

2024-09-13 | GSE246224 | GEO

The Regulatory Network and Role of the circRNA-miRNA-mRNA ceRNA Network in the Progression and the Immune Response of Wilms Tumor based on RNA-seq

Project description:Circular RNA (circRNA), which is a newly discovered non-coding RNA, has been documented to play important roles in miRNA sponges, and the dysregulation of which is involved in cancer development. However, circRNA expression profiles and their role in initiation and progression of Wilms tumor (WT) remain largely unclear at present. Here, we used paired WT samples and high-throughput RNA sequencing to identify differentially expressed circRNAs.

2022-05-25 | GSE197046 | GEO

Structured elements drive circular RNA translation and expand the human proteome, part1

Project description:The human genome encodes tens of thousands circular RNAs (circRNAs) whose levels correlate with many disease states. While studies have focused on the non-coding functions of circRNAs, emerging evidence suggests that a handful of circRNAs encode proteins. Translation canonically starts by recognition of mRNA 5’cap and scanning to the start codon; how circRNA translation initiates remains unclear. Here, we developed a high-throughput screen to systematically identify and quantify RNA sequences that can direct circRNA translation. We identify and validate over 17,000 circRNA internal ribosome entry sites (IRES) and reveal that 18S rRNA complementarity and a structured RNA element on the IRES are important for facilitating circRNA cap-independent translation. With genomic and peptidomic analyses of the IRES, we identified nearly 1,000 putative endogenous protein-coding circRNAs and hundreds of translational units encoded by these circRNAs. We further characterized circFGFR1p, a protein encoded by circFGFR1, functions as a negative regulator of FGFR1 to suppress cell growth under stress conditions. The circRNA proteome may be important links among circRNA, biological control, and disease.

2021-08-23 | PXD024993 | Pride

circITGB6-pulled down proteins in A549 cells

Project description:A549 cells were transfected with 100 pmol DNA probes complementary to the back-splice site of circITGB6 or PDPN 3’UTR region. 24 h later, cells were washed with PBS and lysed with the lysis buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1.5 mM MgCl2, 0.5% NP-40, 0.5% Triton X-100, 10% glycerol, RNase inhibitors (Thermo Scientific), protease and phosphatase inhibitor cocktails). Pre-treated streptavidin magnetic beads were incubated with cell lysates at 4°C for 1 h, followed by washing five times with the washing buffer (20 mM Tri-HCl, pH7.5, 1 mM EDTA and 450 mM NaCl) and elution with Laemmli sample buffer. The circITGB6- or PDPN mRNA- interacting proteins were subjected to SDS-PAGE and visualized by coomassie blue staining or immunoblot analysis. Protein bands were excised and identified by LC-MS/MS mass spectrometry and Proteome Discoverer software (version 1.4; Thermo Scientific).

2023-10-13 | PXD043341 | Pride

Profiling of protein interactions of the circRNA cZNF292 in endothelial cells

Project description:Circular RNAs are a class of non-coding RNAs mostly generated by back-splicing RNA transcripts of protein-coding gene loci. In difference to canoncial splicing, back-splicing ligates downstream splice donor site with an upstream splice acceptor generating a covalently closed circular RNA. CircRNAs have been reported to influence cellular functions by acting as miRNA sponges, regulation of host gene transcription or by protein interactions. The circRNA cZNF292 has been previously reported to be regulated under hypoxia and to influence endothelial cell angiogenesis and functions. The present data set aims to identfy protein interactions partners of cZNF292 in endothelial cells to further characterize the mechanism of action of cZNF292. Dataset comprises 8 replicates of proteins identified after biotin-based 2'O-Me RNA antisense affinity purification of the circRNA cZNF292 from HUVEC (human umbilical vein endothelial cell) lysates compared to a control probe dataset. Purification was performed without cross-linking under low salt and mild detergent conditions.

2022-11-29 | PXD029794 | Pride

Transcriptomic profiles of two sponge species upon exposure to microbial-associated molecular patterns (lipopolysaccharide and peptidoglycan) compared against control (sterile artificial seawater)

Project description:The interaction of animals with microbes relies on the specific recognition of microbial-derived molecules by receptors of the immune system. Sponges (phylum Porifera), as sister group of the Eumetazoa, provide insights into conserved mechanisms for animal-microbe crosstalk, but empirical data is limited. Here we aimed to characterize the immune response of sponges upon microbial stimuli by RNA-Seq. Two sponges species from the Mediterranean Sea, Aplysina aerophoba and Dysidea avara, were challenged with microbial-associated molecular patterns (lipopolysaccharide and peptidoglycan) or sterile artificial seawater (control) in aquarium experiments. Sponge tissue samples were collected 1h, 3h, and 5h after treatment. The response of the sponges to the treatments was assessed by differential gene expression analysis of RNA-Seq data. For each species, we compared the transcriptomic profiles of the samples in MAMP treatment to control within each time point.

2018-11-26 | E-MTAB-6757 | biostudies-arrayexpress

Expression profiles of circRNA in HCT-8 cells infected with Cryptosporidium parvum IId subtype

Project description:Cryptosporidium parvum is one of the most important opportunistic enteric parasites, causing severe diarrhea in immunocomprised human and animals. However, few effective control agents were available for this parasite. circular RNA (circRNA) was discovered to play key roles in many diseases, and the well-known regulatory mechanism for circRNAs is that act as miRNA sponges competitively binding to miRNAs to block miRNA-mRNA interaction. Here, using microarray assay, we investigated the expression profiles of circRNAs in HCT-8 cells after the infection of C. parvum IId subtype, the prevalent subtype of China. A total of 178 circRNAs were dysregulated expressed in HCT-8 cells at 24 h post infection (pi) of C. parvum IId subtype.

2022-12-15 | GSE162990 | GEO

Negative regulation of Type I IFN signaling by phosphorylation of STAT2 on T387

Project description:The samples were derived from STAT2 IP experiments from human fibrosarcoma cells. This experiment involved the transfection of cells with a flag tagged STAT2 and the goal of these experiments is to identify STAT2 binding partners.

2018-10-26 | PXD005008 | Pride

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