MiRNA signatures of intracellular and extracellular fractions from cancer cell lines
Ontology highlight
ABSTRACT: For the purpose of finding a novel tumor marker, we conducted microRNA profiling analysis in fractions of cells, exosomes, and culture medium from seven cancer cell lines. Ranking data of the intracellular and extracellular microRNAs obtained by microarray analysis, we found that let-7 microRNA family are rich in all fractions from AZ-P7a cells, a metastatic gastric cancer cell line.
Project description:For the purpose of finding a novel tumor marker, we conducted microRNA profiling analysis in fractions of cells, exosomes, and culture medium from seven cancer cell lines. Ranking data of the intracellular and extracellular microRNAs obtained by microarray analysis, we found that let-7 microRNA family are rich in all fractions from AZ-P7a cells, a metastatic gastric cancer cell line. miRNA profiling in fractions of cells, exosomes, and culture medium were conducted by miRNA microarray analysis. Two replicates of intra-samples were performed.
Project description:Both disseminated tumor cells and noncancerous host cells contributed to cancer progression cooperatively in bone marrow. Bone marrow samples were obtained from 4 gastric cancer patients, and were separated into 3 fractions (CD45 positive, CD45 negative/EpCAM positive, and CD14 positive fractions) by the automagnetic-activated cell separation (AutoMACS) system using CD45, EpCAM, and CD14 microbeads (Miltenyi Biotec, Germany). microRNA expression profiles in each fractions were evaluated in order to identify candidate prognostic markers for gastric cancer patients.
Project description:Both disseminated tumor cells and noncancerous host cells contributed to cancer progression cooperatively in bone marrow. Bone marrow samples were obtained from 4 gastric cancer patients, and were separated into 3 fractions (CD45 positive, CD45 negative/EpCAM positive, and CD14 positive fractions) by the automagnetic-activated cell separation (AutoMACS) system using CD45, EpCAM, and CD14 microbeads (Miltenyi Biotec, Germany). microRNA expression profiles in each fractions were evaluated in order to identify candidate prognostic markers for gastric cancer patients. In 4 patients with gastric cancer, bone marrow samples (40 mL) were obtained from iliac bones. Nucleated cells were collected by gradient centrifugation using Ficoll-Paque PREMIUM (GE Healthcare Life Science, USA) and Leucosep (Greiner Bio-One, Germany) according to the manufacturer’s instructions. Next, we separated bone marrow cells into 3 fractions using MACS: CD45 positive (CD45+), CD45 negative/EpCAM positive (CD45-/EpCAM+), and CD14 positive (CD14+). microRNA expression levels of whole bone marrow cells and each fractions were measured by the miRCURY™ LNA array microarray (6th gen-hsa, mmu & rno#208402, Exiqon, Vedbaek, Denmark). The miRCURY™ LNA array microarray slides were scanned using the Agilent G2505C Microarray Scanner System (Agilent Technologies, Inc., USA) and the data analysis was carried out using the Feature Extraction 10.7.3.1 (Agilent Technologies, Inc., USA).
Project description:Circulating extracellular RNAs (exRNAs) have great promise as novel clinically plasma-based biomarkers for cancer diagnosis and prognosis. knowledge of the difference or association between these different sources of exRNAs is very limited. We performed a sequential physical and biochemical precipitation to receive 4 plasma fractions (platelets and cell debris, Thrombin-treated fraction, extracellular vesicles and supernatant) from each plasma sample. After total RNA extraction, we made ligation-free libraries and performed RNA-seq to evaluate full spectrum of RNA abundance in each fraction. All RNAs were included without size selection during the library preparation. We utilized a successive stepwise alignment strategy to map the RNA sequences to different RNA categories. We found that each plasma fraction had its own unique distribution of RNA species. Hierarchical cluster analysis showed similarity in samples with same fraction and significant differences between different fractions. In addition, we also observed abundance difference of unique transcript. Furthermore, a considerable proportion of exogenous RNAs and novel predicted miRNAs in all plasma fractions were detected. These results demonstrate that thorough inspection of all plasma fractions is necessary for exRNA-based biomarker study and appropriate sampling processes is needed for illustrating the complexity of plasma-based EVs study.
Project description:Most cancer-related deaths are caused by distant metastases, which are tumour cells that have escaped from a primary tumour and passed into the bloodstream to colonize a new organ. In this context, communication between tumour and stromal cells is essential. Indeed, tumor cells interact with cells in the tumor microenvironment and are able to modify them to their advantage. Both extracellular vesicles (EVs) and exosomes are heterogeneous populations of small vesicles present in the tumor microenvironment and in body fluids that have recently emerged as powerful mediators involved in this communication and their transport in fluids. Tumor cells release large quantities of exosomes containing tumor markers, which can then spread to distant locations.
The exosomes are of endosomal origin. They are composed of proteins, lipids, RNA and DNA, and they circulate in the bloodstream. They can be internalized by specific distant cells and thus deliver a functional message. It has recently been shown that tumor exosomes containing pro-metastatic factors form pre-metastatic niches, before the tumor cells actually arrive, while determining the metastatic organotropism of tumors. These properties are now opening up new avenues of research in tumor biomarkers. In recent years, several studies have highlighted different markers contained specifically in exosomes derived from cancer cells. Consequently, exosomes are considered as potential reservoirs of tumor biomarkers that could be clinically useful for the non-invasive diagnosis of cancer, with the advantage of being performed by liquid biopsy. The study of microRNA (miRNA) is of particular interest. Indeed, miRNAs are small non-coding RNAs (between 21 and 25 nucleotides) involved in the regulation of gene expression and which are frequently deregulated in cancer. Several studies underline that the variation of free miRNAs in the blood is correlated with the progression of the disease, particularly in colon cancer. However, the stability of free miRNAs is controversial. Therefore, exosomes represent a very advantageous means of transporting miRNAs in the blood, as they are able to protect miRNAs from degradation by RNAase.
The hypothesis of the project is that circulating exosomes derived from tumours contain markers including specific miRNAs that could be used as biomarkers of early prognosis (survival and progression), easily measured in blood samples from patients with colon cancer. But other molecules contained in exosomes could also be of interest.
Project description:Exosomes are nano-sized (30-150 nm), membrane encapsulated vesicles that are released by cells into the extracellular space and function as intercellular signaling vectors through the horizontal transfer of biologic molecules, including microRNA (miRNA). Although secreted by both normal and malignant cells, there is evidence that cancer-derived exosomes enable the tumor to manipulate its microenvironment, contributing to the capacity of the tumor for immune evasion, growth, invasion, and metastatic spread. The purpose of this study was to apply miRNA-sequencing to exosomes isolated from conditioned culture media to comprehensively characterize miRNA secreted in exosomes from 4 distinct head and neck squamous cell carcinoma (HNSCC) cell lines and normal oral epithelial cells.
Project description:In order to screen miRNAs related to cachexia, we collected plasma from patients with gastric cancer cachexia or without cachexia, and extracted exosomes from plasma. Then, we analyzed the gene expression profile of 3 gastric cancer patients with cachexia and 2 gastric cancer patients with non cachexia.