ABSTRACT
BACKGROUND: Gastric cancer is the fourth most common cancer in the world and the second most prevalent cause of cancer related death. The development of gastric cancer is mainly associated with H. Pylori infection leading to a focus in pathology studies on bacterial and environmental factors, and to a lesser extent on the mechanistic development of the tumour. MicroRNAs are small non-coding RNA molecules involved in post-transcriptional gene regulation. They are found to regulate genes involved in diverse biological functions and alterations in microRNA expression have been linked to the pathogenesis of many malignancies. The current study is focused on identifying microRNAs involved in gastric carcinogenesis and to explore their mechanistic relevance by characterizing their targets. RESULTS: Invitrogen NCode miRNA microarrays identified miR-449 to be decreased in 1-year-old Gastrin KO mice and in H. Pylori infected gastric tissues compared to tissues from wild type animals. Growth rate of gastric cell lines over-expressing miR-449 was inhibited by 60% compared to controls. FACS cell cycle analysis of miR-449 over-expressing cells showed a significant increase in the sub-G1 fraction indicative of apoptosis. ß-Gal assays indicated a senescent phenotype of gastric cell lines over-expressing miR-449. Affymetrix 133v2 arrays identified GMNN, MET, CCNE2, SIRT1 and CDK6 as miR-449 targets. Luciferase assays were used to confirm GMNN, MET, CCNE2 and SIRT1 as direct targets. We also show that miR-449 over-expression activated p53 and its downstream target p21 as well as the apoptosis markers cleaved CASP3 and PARP. Importantly, qPCR analyses showed a loss of miR-449 expression in human clinical gastric tumours compared to normal tissues. CONCLUSIONS: In this study, we document a diminished expression of miR-449 in Gastrin KO mice and further confirmed its loss in human gastric tumours. We investigated the function of miR-449 by identifying its direct targets. Furthermore we show that miR-449 induces senescence and apoptosis by activating the p53 pathway.
Subject(s)
Down-Regulation/genetics , Gene Expression Regulation, Neoplastic , MicroRNAs/genetics , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Adenoma/pathology , Animals , Base Sequence , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Cellular Senescence , Gastrins/deficiency , Gastrins/metabolism , Helicobacter Infections/complications , Helicobacter Infections/genetics , Helicobacter pylori/physiology , Humans , Mice , Mice, Knockout , MicroRNAs/metabolism , Molecular Sequence Data , Pyloric Antrum/metabolism , Pyloric Antrum/pathology , Signal Transduction/genetics , Stomach Neoplasms/complications , Tumor Suppressor Protein p53/metabolismABSTRACT
MicroRNAs (miRNAs) are small non-coding RNAs, which regulate gene expression through base pairing with mRNA and which are crucially involved in carcinogenesis (the so-called oncomiRs). We compared the miRNA signature between acquired melanocytic nevi showing clinical atypia (atypic nevi, AN) and common acquired nevi (common nevi, CN). We obtained miRNA profiles from 41 biopsies (22 AN and 19 CN) and showed that AN could be differentiated from CN on the basis of the expression of 36 miRNAs (false discovery rate <0.05). OncomiRs were present in this group, and we further confirmed the differential expression of miR-125b and let-7c by qRT-PCR. Our data suggest that miRNAs are functionally involved in the pathogenesis of nevi and possibly malignant melanoma.
Subject(s)
MicroRNAs/metabolism , Nevus, Pigmented/diagnosis , Nevus, Pigmented/metabolism , Adult , Cluster Analysis , Down-Regulation/genetics , Female , Gene Expression/genetics , Gene Expression Profiling , Humans , Male , Middle Aged , Nevus, Pigmented/etiology , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Up-Regulation/genetics , Young AdultABSTRACT
CONTEXT: microRNA (miRNA) expression profiling and classification of tissue obtained from fine-needle aspirates (FNA) could be a major improvement of the preoperative diagnosis of thyroid nodules. OBJECTIVE: Before this can be clinically implemented, a robust and non-toxic method for obtaining sufficient quantity and quality of RNA from single in vivo FNA has to be established. RNAlater is a non-toxic RNA-stabilizing agent. However, due to the high density of RNAlater, pelleting of the tissue samples is difficult, and results in low recovery of RNA that is insufficient for subsequent miRNA array expression analysis. We therefore developed a simple centrifugation method for capturing tissue stored in RNAlater on a 0.45-µm filter. DESIGN: FNA from 24 patients with a solitary cold thyroid nodule was stored in Trizol, liquid nitrogen, or RNAlater. The tissue stored in RNAlater was either pelleted by centrifugation or captured on the 0.45-µm filters. RNA was extracted using the Trizol method. To validate results, FNA from additional 30 patients were analyzed based on the modified RNAlater protocol. MAIN OUTCOME: Capturing FNA tissue samples on the filters increased the RNA yield 10 fold and maintained RNA purity, permitting miRNA array expression profiling and allowing comparable levels of known miRNA-clusters regardless of preservation technique. Results were confirmed in an additional 30 patients. CONCLUSION: The modified RNAlater protocol is well suited for isolating RNA from single thyroid in vivo FNA in a clinical setting. Furthermore this permits shipping of FNA samples at room temperature from peripheral centers to a centralized array core facility.
