Protein-coding genes account for only a small part of the human genome, whereas the vast majority of transcripts make up the non-coding RNAs including long non-coding RNAs (lncRNAs). Accumulating evidence indicates that lncRNAs could play a critical role in regulation of cellular processes such as cell growth and apoptosis as well as cancer progression and metastasis. LncRNA loc285194 was previously shown to be within a tumor suppressor unit in osteosarcoma and to suppress tumor cell growth. However, it is unknown regarding the regulation of loc285194. Moreover, the underlying mechanism by which loc285194 functions as a potential tumor suppressor is elusive. In this study, we show that loc285194 is a p53 transcription target; ectopic expression of loc285194 inhibits tumor cell growth both in vitro and in vivo. Through deletion analysis, we identify an active region responsible for tumor cell growth inhibition within exon 4, which harbors two miR-211 binding sites. Importantly, this loc285194-mediated growth inhibition is in part due to specific suppression of miR-211. We further demonstrate a reciprocal repression between loc285194 and miR-211; in contrast to loc285194, miR-211 promotes cell growth. Finally, we detect downregulation of loc285194 in colon cancer specimens by quantitative PCR arrays and in situ hybridization of tissue microarrays. Together, these results suggest that loc285194 is a p53-regulated tumor suppressor, which acts in part through repression of miR-211.
Abstract:Exosomes are small membrane-bound vesicles secreted by most cell types. Exosomes contain various functional proteins, mRNAs and microRNAs (miRNAs) that could be used for diagnostic and therapeutic purposes. How we should store the samples before RNA isolation and whether those long term stored samples could be used for circulating RNA investigation because of RNase is unknown. The aim of the study was to determine the stability of circulating miRNA in exosomes and plasma. Exosomes were isolated from plasma samples by using ExoQuick Precipitation methods. RNA was extracted from exosomes and the corresponding plasma samples with a Qiagen miRNeasy Mini kit. The concentration of RNA was measured by a Qubit ® RNA HS Assay Kit, and quantitative PCR was used for individual miRNA expression level detection. Results showed that exosomal miRNA showed extra stability under different storage conditions and no significant influence on plasma miRNA, except for short term storage at 4 °C. It is thus indicated that exosome miRNAs can be good biomarkers based on their stability under various storage conditions.
Therapeutic applications of microRNA (miRNA) in KRAS-driven pancreatic cancers might be valuable, but few studies have explored this area. Here, we report that miR-96 directly targets the KRAS oncogene and functions as a tumor-suppressing miRNA in pancreatic cancer cells. Ectopic expression of miR-96 through a synthetic miRNA precursor inhibited KRAS, dampened Akt signaling, and triggered apoptosis in cells. In human clinical specimens, miR-96 was downregulated or deleted where an association with KRAS elevations was observed. In vitro and in vivo assays established that miR-96 decreased cancer cell invasion and migration and slowed tumor growth in a manner associated with KRAS downregulation. Our findings identify miR-96 as a potent regulator of KRAS, which may provide a novel therapeutic strategy for treatment of pancreatic cancer and other KRAS-driven cancers.
It is well known that upon stress, the level of the tumor suppressor p53 is remarkably elevated. However, despite extensive studies, the underlying mechanism involving important inter-players for stress-induced p53 regulation is still not fully understood. We present evidence that the human lincRNA-RoR (RoR) is a strong negative regulator of p53. Unlike MDM2 that causes p53 degradation through the ubiquitin-proteasome pathway, RoR suppresses p53 translation through direct interaction with the heterogeneous nuclear ribonucleoprotein I (hnRNP I). Importantly, a 28-base RoR sequence carrying hnRNP I binding motifs is essential and sufficient for p53 repression. We further show that RoR inhibits p53-mediated cell cycle arrest and apoptosis. Finally, we demonstrate a RoR-p53 autoregulatory feedback loop where p53 transcriptionally induces RoR expression. Together, these results suggest that the RoR-hnRNP I-p53 axis may constitute an additional surveillance network for the cell to better respond to various stresses.
IntroductionExosomes are 30-100 nm membrane vesicles of endocytic origin, mediating diverse biological functions including tumor cell invasion, cell-cell communication and antigen presentation through transfer of proteins, mRNAs and microRNAs. Recent evidence suggests that microRNAs can be released through ceramide-dependent secretory machinery regulated by neutral sphingomyelinase 2 (nSMase2) enzyme encoded by the smpd3 gene that triggers exosome secretion. However, whether exosome-mediated microRNA transfer plays any role in cell invasion remains poorly understood. Thus, the aim of this study was to identify the exosomal microRNAs involved in breast cancer invasion.MethodsThe expression level of endogenous and exosomal miRNAs were examined by real time PCR and the expression level of target proteins were detected by western blot. Scanning electron and confocal microscopy were used to characterize exosomes and to study its uptake and transfer. Luciferase reporter plasmids and its mutant were used to confirm direct targeting. Furthermore, the functional significance of exosomal miR-10b was estimated by invasion assay.ResultsIn this study, we demonstrate that microRNA carrying exosomes can be transferred among different cell lines through direct uptake. miR-10b is highly expressed in metastatic breast cancer MDA-MB-231 cells as compared to non-metastatic breast cancer cells or non-malignant breast cells; it is actively secreted into medium via exosomes. In particular, nSMase2 or ceramide promotes the exosome-mediated miR-10b secretion whereas ceramide inhibitor suppresses this secretion. Moreover, upon uptake, miR-10b can suppress the protein level of its target genes such as HOXD10 and KLF4, indicating its functional significance. Finally, treatment with exosomes derived from MDA-MB-231 cells could induce the invasion ability of non-malignant HMLE cells.ConclusionTogether, our results suggest that a set of specific microRNAs may play an important role in modulating tumor microenvironment through exosomes. Thus, a better understanding of this process may aid in the development of novel therapeutic agents.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-256) contains supplementary material, which is available to authorized users.
SummaryThe Duffy antigen/receptor for chemokines (DARC), first identified on erythrocytes, functions not only as a promiscuous chemokine receptor but also as a receptor for the malarial parasite, Plasmodium vivax. The recent finding that DARC is ubiquitously expressed by endothelial cells lining postcapillary venules provides a possible insight into the function of this receptor because this anatomic site is an active interface for leukocyte trafficking. However, the biological significance of DARC is questionable since it has not yet been determined whether individuals lacking the expression of this protein on their erythrocytes (Duffy negative individuals), who are apparently immunologicaUy normal, express the receptor on endothelial cells. However, we report here that DARC is indeed expressed in endothelial cells lining postcapillary venules and splenic sinusoids in individuals who lack the erythrocyte receptor. These findings are based on immunohistochemical, biochemical, and molecular biological analysis of tissues from Duff-y negative individuals. We also present data showing that, in contrast to erythrocyte DAILC, cells transfected with DARC internalize radiolabeled ligand. We conclude that the DARC may play a critical role in mediating the effects of proinflammatory chemokines on the interactions between leukocyte and endothelial cells since the molecular pathology of the Duffy negative genotype maintains expression on the latter cell type.
Aberrantly expressed microRNA (miRNA) is frequently associated with a variety of cancers, including pancreatic ductal adenocarcinoma (PDAC). In this study, we investigated the expression and possible role of miR-217 in PDAC. Data obtained by locked nucleic acid in situ hybridization and real-time quantitative polymerase chain reaction showed that miR-217 was downregulated in 76.2% (16/21) of PDAC tissues and in all tested PDAC cell lines when compared with the corresponding normal pancreatic tissue. Overexpression of miR-217 in PDAC cells inhibited tumor cell growth and anchorage-independent colony formation and miR-217 decreased tumor cell growth in nude mouse xenografts in vivo. Using in silico predictions, KRAS was defined as a potential direct target of miR-217. Data from the dual-luciferase reporter gene assay showed that KRAS was a direct target of miR-217. Upregulation of miR-217 could decrease KRAS protein levels and reduce the constitutive phosphorylation of downstream AKT. Downregulation of miR-217 expression in PDAC cells could increase cell anchorage-independent colony formation and KRAS protein levels. Furthermore, miR-217 expression was observed to be negatively correlated with KRAS protein expression in PDAC cell lines. We conclude that the frequently downregulated miR-217 can regulate KRAS and function as a tumor suppressor in PDAC. Therefore, miR-217 may serve as a useful therapeutic agent for miRNA-based PDAC therapy.
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