Development of microRNA expression signatures for gastric cancer
ABSTRACT: To further investigate of microRNA expression profile of gastric cancer, we have employed whole microRNA gene chip as a discovery platform to identify gene differential expression between gastric cancer tissues with mormal gastric tissues.Expression of four microRNAs (miR-125b,miR-451,miR-192b,miR-200b) from this signature was quantified and verified in the same RNA samples by real-time PCR. Overall design: A total of 4 consecutive patients with gastric cancer undergoing elective surgery were entered into this study. After obtaining informed consent from the patients and after receiving the approval of the ethics committee, the following tissue samples were collected: tumor (3 × 3 × 5 mm) and adjacent normal tissues (3 × 3 × 5 mm).
INSTRUMENT(S): Agilent-021827 Human miRNA Microarray [miRNA_107_Sep09_2_105]
Project description:To further investigate of microRNA expression profile of gastric cancer, we have employed whole microRNA gene chip as a discovery platform to identify gene differential expression between gastric cancer tissues with mormal gastric tissues.Expression of four microRNAs (miR-125b,miR-451,miR-192b,miR-200b) from this signature was quantified and verified in the same RNA samples by real-time PCR. A total of 4 consecutive patients with gastric cancer undergoing elective surgery were entered into this study. After obtaining informed consent from the patients and after receiving the approval of the ethics committee, the following tissue samples were collected: tumor (3 × 3 × 5 mm) and adjacent normal tissues (3 × 3 × 5 mm).
Project description:MicroRNA (miRNA) deregulation has long been associated with various cancers. Among an expanding list of cancer related miRNAs, deregulation of miR-125b has been well documented in various cancers including breast. Based on current knowledge, miR-125b is considered to be a tumor suppressor in breast cancers. While important mRNA targets have been defined for miR-125b, here, we aimed to further investigate direct/indirect consequences of miR-125b expression in breast cancer cells by using a transcriptome approach. Upon miR-125b expression, a total of 140 cancer related genes were found to be differentially expressed in breast cancer cells. Overall design: MCF7 cells were stably transfected with pSUPER-mir-125b and pSUPER empty vector controls. Polyclones were selected by G418, molecular confirmation of mir-125b expression was completed by TaqMan RT-qPCR assays.
Project description:The oncomir microRNA-125b (miR-125b) is up-regulated in a variety of human neoplastic blood disorders and constitutive up-regulation of miR-125b in mice can promote myeloid and B cell leukemia. We found that miR-125b promotes myeloid and B cell neoplasm by inducing tumorigenesis in hematopoietic progenitor cells. Our study demonstrates that miR-125b induces myeloid leukemia by enhancing myeloid progenitor output from stem cells as well as inducing immortality, self-renewal, and tumorigenesis in myeloid progenitors. Through functional and genetic analyses, we demonstrated that miR-125b induces myeloid and B cell leukemia by inhibiting IRF4 but through distinct mechanisms; it induces myeloid leukemia through repressing IRF4 at the mRNA level without altering the genomic DNA and induces B cell leukemia via genetic deletion of the gene encoding IRF4. The cancer myeloid (Cd11b+ sorted) and B cells (CD19+ sorted) were harvested from mice that over-express miR-125b. The genomic DNA was extracted from these cells. A total of 4 cancer samples (Two myeloid cancer samples and two B cell cancer samples) were analyzed. As control, genomic DNA from cells harvested from healthy C57bl/6 mice were harvested.
Project description:microRNA miR-144/451 is highly expressed during erythropoiesis. We deleted the miR-144/451 gene locus in mice and compared the transcriptomes of miR-144/451-null bone marrow erythroid precursors to stage-matched wild-type control cells. Overall design: Ter119+/CD71+/FSC-high bone marrow erythroblasts were sorted directly into Trizol LS reagent. Total RNAs extracted from three miR-144/451 knock-out and three wide type mice were analyzed using Affymetrix Mouse Genome 430 2.0 Arrays.
Project description:To investigate the role and mechanism of miR-125b on NCCIT cells without bias, we analyzed differentially expression microRNA profile among miR-125b antagomir-, miR-125b agomir-, and negative control-transfected NCCIT tumor cells by microRNA-seq. Overall design: Total RNA (1 μg for each samples) was extracted from miR-125b antagomir-, miR-125b agomir-, and control oligonucleotide (GenePharma, Shanghai, China)-transfected NCCIT (ATCC® CRL-2073) cells using miRcute miRNA Isolation Kit (Tiangen, Beijing, China). Three duplicate samples for each treatment were included. Then, equal amounts of total RNA were analysed using BGISEQ-500 platform and the sequencing procedure was performed by BGI (Shenzhen, China)
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.
Project description:Colorectal cancer cell lines were transduced with lentiviral antisense miRNA inhibitors (miRZIPs) targeting miR-429, miR-200b, miR-194 and miR-203. Global mRNA expression profiles were generated with Illumina arrays on transduced and control cells to identify mRNAs whose expression is regulated by the above microRNAs. Overall design: Colorectal cancer cell lines transduced with control vectors or with microRNA targeting vectors were profiled, together with wild-type cells, using two independent RNA extractions.
Project description:microRNA miR-144/451 is highly expressed during erythropoiesis. We deleted the miR-144/451 gene locus in mice and compared the transcriptomes of miR-144/451-null bone marrow erythroid precursors to stage-matched wild-type control cells. Ter119+/CD71+/FSC-high bone marrow erythroblasts were sorted directly into Trizol LS reagent. Total RNAs extracted from three miR-144/451 knock-out and three wide type mice were analyzed using Affymetrix Mouse Genome 430 2.0 Arrays.
Project description:Increasing evidence suggests that microRNAs may play important roles in regulating self-renewal and differentiation in mammalian stem cells (SCs). Here, we explore this issue in skin. We first characterize microRNA expression profiles of skin SCs versus their committed proliferative progenies and identify a microRNA subset associating with “stemness”. Of these, miR-125b is dramatically downregulated in early SC-progeny. We engineer an inducible mice system and show that when miR-125b is sustained in SC-progenies, tissue balance is reversibly skewed towards stemness at the expense of epidermal, oil-gland and HF differentiation. Using gain-and-loss of function in vitro, we further implicate miR-125b as a repressor of SC differentiation. In vivo, transcripts repressed upon miR-125b induction are enriched >700% for predicted miR-125b targets normally downregulated upon SC-lineage commitment. We verify some of these miR-125b targets, and show that Blimp1 and VDR in particular can account for many tissue imbalances we see when miR-125b is deregulated. We used microarrays to compare the global miRNA expression profile of P4 stage hair follicle ORS cells from DTG (K14-rtTA,TRE-miR-125b) and control littermates. Hair follicle cells were isolated from P4 Backskin (Dox since P3 for 24hrs) of DTG (K14-rtTA/TRE-miR-125b/K14-H2BGFP), TRE (TRE-miR-125b/K14-H2BGFP), KrtA (K14-rtTA/K14-H2BGFP) as following: interfollicular epidermis sheet was pealed from hair follicle & dermis after dispase treatment.The hair follicle & dermis were first digested by collagenase (Sigma). Intact hair follicles were separated from dermal cells by low speed spinning (20g). The hair follicles were then digested by Trypsin and filtered by 40 µm cell strainers. The isolated hair follicle cells were FACS sorted. During FACS, cells were first gated against CD34 (endothelial cells), CD45 (immune cells), CD114 (melanocytes) and DAPI (dead cells). ORS cells were sorted from the remaining cells as α6HiGFPHi.
Project description:Transcriptional profiling of breast cancer cells comparing pre-control transfected cells with cells transfected with pre-miR-125b. We searched for miR-125b targets by systematic screening of mRNA profiling of pre-miR-125b transfected MCF-7 cells and MDA-MB-435 cells. Two-condition experiment, pre-miR-125b Transfected vs. pre-control Transfected MCF-7 cells. One replicate per array.