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1.
Aging (Albany NY) ; 11(15): 5705-5725, 2019 08 10.
Article in English | MEDLINE | ID: mdl-31400752

ABSTRACT

Accumulating evidence reveals the principal role of long noncoding RNAs in the progression of clear cell renal cell carcinoma (ccRCC). However, little is known about the underlying mechanism of ADAM metallopeptidase with thrombospondin type 1 motif, 9 antisense RNA 2 (ADAMTS9-AS2) in ccRCC. Here, bioinformatics analyses verified ADAMTS9-AS2 is a long noncoding RNA and its high expression was associated with better prognosis of ccRCC. ADAMTS9-AS2 was clearly downregulated in ccRCC clinical samples and cell lines. Clinical data showed low-expressed ADAMTS9-AS2 was correlated with worse overall survival in ccRCC patients. Next, miR-27a-3p was identified as an inhibitory target of ADAMTS9-AS2 by dual-luciferase reporter and RNA immunoprecipitation assays. Both overexpressed ADAMTS9-AS2 and underexpressed miR-27a-3p in ccRCC cell lines led to the inhibition of cell proliferation and the reduction of chemoresistance. Additionally, Forkhead Box Protein O1 (FOXO1) was confirmed as the inhibitory target of miR-27a-3p. Induced by ADAMTS9-AS2 overexpression, cell proliferation and chemoresistance exhibited an obvious reduction, FOXO1 expression showed an evident increase, but all were reversed after miR-27a-3p was simultaneously overexpressed. Collectively, these results suggest ADAMTS9-AS2 inhibits the progression and impairs the chemoresistance of ccRCC via miR-27a-3p-mediated regulation of FOXO1 and may serve as a prognostic biomarker and therapeutic target for ccRCC.


Subject(s)
ADAMTS9 Protein/antagonists & inhibitors , ADAMTS9 Protein/genetics , Carcinoma, Renal Cell/genetics , Forkhead Box Protein O1/genetics , Kidney Neoplasms/genetics , MicroRNAs/genetics , RNA, Long Noncoding/genetics , Carcinoma, Renal Cell/metabolism , Carcinoma, Renal Cell/pathology , Cell Line, Tumor , Cell Proliferation/genetics , Computational Biology , Down-Regulation , Drug Resistance, Neoplasm/genetics , Female , Forkhead Box Protein O1/antagonists & inhibitors , Forkhead Box Protein O1/metabolism , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Kidney Neoplasms/metabolism , Kidney Neoplasms/pathology , Male , MicroRNAs/antagonists & inhibitors , MicroRNAs/metabolism , Middle Aged , Prognosis , RNA, Antisense/genetics , RNA, Antisense/metabolism , RNA, Long Noncoding/metabolism , Signal Transduction
2.
RNA ; 23(4): 483-492, 2017 04.
Article in English | MEDLINE | ID: mdl-28115596

ABSTRACT

Structured RNAs have a central role in cellular function. The capability of structured RNAs to adopt fixed architectural structures or undergo dynamic conformational changes contributes to their diverse role in the regulation of gene expression. Although numerous biophysical and biochemical tools have been developed to study structured RNAs, there is a continuing need for the development of new methods for the investigation of RNA structures, especially methods that allow RNA structure to be studied in solution close to its native cellular conditions. Here we use osmium tetroxide (OsO4) as a chemical probe of RNA structure. In this method, we have used fluorescence-based sequencing technologies to detect OsO4 modified RNA. We characterized the requirements for OsO4 modification of RNA by investigating three known structured RNAs: the M-box, glycine riboswitch RNAs, and tRNAasp Our results show that OsO4 predominantly modifies RNA at uracils that are conformationally exposed on the surface of the RNA. We also show that changes in OsO4 reactivity at flexible positions in the RNA correlate with ligand-driven conformational changes in the RNA structure. Osmium tetroxide modification of RNA will provide insights into the structural features of RNAs that are relevant to their underlying biological functions.


Subject(s)
Molecular Probes/chemistry , Osmium Tetroxide/chemistry , RNA, Transfer, Asp/chemistry , Riboswitch/genetics , Base Sequence , Nucleic Acid Conformation , RNA, Transfer, Asp/genetics , Staining and Labeling/methods , Uracil/chemistry
3.
Mol Med Rep ; 13(3): 2708-14, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26847233

ABSTRACT

Previous studies have suggested that lycopene has cytotoxic effects in a variety of types of human cancer. An improved understanding of the mechanisms underlying the anticancer effects of lycopene may provide novel therapeutic targets for cancer treatment. PC3 cells were treated with different concentrations of lycopene for 24 and 48 h, the level of protein kinase B (AKT2) was detected by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blotting. Additionally, the expression levels of microRNA (miR)­let­7f­1 were measured using RT­qPCR. miR­let­7f­1 function was analyzed using cell proliferation and apoptosis assays in gain­ and loss­of­function experiments. It was observed that lycopene downregulated the expression of AKT2 and upregulated the expression of miR­let­7f­1 in PC3 cells. Re­introduction of miR­let­7f­1 into PC3 cells was able to inhibit cell proliferation and induce apoptosis. Further investigation indicated that miR­let­7f­1 targeted AKT2 in PC3 cells and upregulation of AKT2 could attenuate the effects induced by miR­let­7f­1. The results of the current study indicate that miR­let­7f­1 is involved in the anticancer effects of lycopene and serves an important role in the inhibition of prostate cancer progression through the downregulation of AKT2.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis , Carotenoids/pharmacology , Cell Proliferation , MicroRNAs/genetics , Cell Line, Tumor , Down-Regulation , Drug Screening Assays, Antitumor , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lycopene , Male , MicroRNAs/metabolism , Prostatic Neoplasms , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Transcriptional Activation
4.
Biomed Res Int ; 2014: 475379, 2014.
Article in English | MEDLINE | ID: mdl-25133163

ABSTRACT

The formation and death of macrophages and foam cells are one of the major factors that cause coronary heart disease (CHD). In our study, based on the Edinburgh Human Metabolic Network (EHMN) metabolic network, we built an enzyme network which was constructed by enzymes (nodes) and reactions (edges) called the Edinburgh Human Enzyme Network (EHEN). By integrating the subcellular location information for the reactions and refining the protein-reaction relationships based on the location information, we proposed a computational approach to select modules related to programmed cell death. The identified module was in the EHEN-mitochondria (EHEN-M) and was confirmed to be related to programmed cell death, CHD pathogenesis, and lipid metabolism in the literature. We expected this method could analyze CHD better and more comprehensively from the point of programmed cell death in subnetworks.


Subject(s)
Apoptosis , Coronary Disease/metabolism , Coronary Disease/pathology , Metabolic Networks and Pathways , Coronary Disease/enzymology , Genes, Reporter , Humans , Molecular Sequence Annotation , Scotland
5.
Prostate ; 67(15): 1621-9, 2007 Nov 01.
Article in English | MEDLINE | ID: mdl-17823924

ABSTRACT

BACKGROUND: Prostate cancer frequently metastasizes to bone. Androgen suppression treatment is initially highly effective, but eventually results in resistant cancer cells. This study evaluates the effects of androgen suppression on the bone and bone marrow (BM). In particular we questioned whether the androgen therapy could adversely facilitate prostate cancer progression through an increase growth factor secretion by the bone microenvironment. METHODS: Global gene expression is analyzed on mPEDB DNA microarrays. Insulin-like growth factor binding protein-5 (IGFBP5) is detected by immunohistochemistry in mouse tissues and its regulation measured by qPCR and Western blotting in human BM stromal cells. Effects of extracellular matrix-associated IGFBP5 on human prostate epithelial cells are tested in an MTS cell-growth assay. RESULTS: Castration increases expression of 159 genes (including 4 secreted cytokines) and suppresses expression of 84 genes. IGFBP5 is most consistently increased and the increase in expression is reversed by testosterone administration. IGFBP5 protein is detected in vivo in osteoblasts, BM stromal cells, and endothelial cells. Primary human stromal cell cultures secrete IGFBP5. In vitro, treatment of immortalized human marrow stromal cells with charcoal-stripped serum increases IGFBP5 mRNA expression, which is reversed by androgen supplementation. IGFBP5 is incorporated into the extracellular matrix. Further, IGFBP5 immobilized on extracellular matrices of stromal cells enhances the growth of immortalized prostate epithelial cells. CONCLUSIONS: Androgen suppressive therapy increases IGFBP5 in the BM microenvironment and thereby may facilitate the progression of prostate cancer.


Subject(s)
Androgens/pharmacology , Bone Marrow/drug effects , Carrier Proteins/genetics , Gene Expression/drug effects , Testosterone/pharmacology , Androgens/blood , Animals , Bone Marrow/metabolism , Bone Marrow/pathology , Bone and Bones/drug effects , Bone and Bones/metabolism , Bone and Bones/pathology , Carrier Proteins/metabolism , Cell Line , Cell Proliferation/drug effects , Epithelial Cells/drug effects , Epithelial Cells/pathology , Gene Expression Profiling , Humans , Male , Mice , Mice, Inbred C57BL , Oligonucleotide Array Sequence Analysis , Orchiectomy , Prostate/drug effects , Prostate/pathology , RNA, Messenger/metabolism , Seminal Vesicles/drug effects , Seminal Vesicles/pathology , Testosterone/blood
6.
J Urol ; 171(6 Pt 1): 2477-81, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15126879

ABSTRACT

PURPOSE: We investigated whether antisense (AS) oligodeoxynucleotide (ODN) targeting the clusterin gene enhances the cytotoxic effect of gemcitabine in human bladder cancer KoTCC-1 cells in vitro and in vivo, and evaluated the usefulness of the combined administration of AS clusterin ODN and gemcitabine using an intraperitoneal tumor cell injection model. MATERIALS AND METHODS: The cytotoxic effect of combined treatment with AS clusterin ODN and gemcitabine on in vitro KoTCC-1 growth was examined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The in vivo growth inhibitory effects of combined AS clusterin ODN and gemcitabine therapy on subcutaneous KoTCC-1 tumor was also examined. The intraperitoneal tumor cell injection model, which mimics intravesical administration therapy against bladder cancer, was used to evaluate the efficacy of combined AS clusterin ODN and gemcitabine therapy. RESULTS: AS clusterin ODN treatment of KoTCC-1 cells significantly enhanced gemcitabine chemosensitivity in a dose dependent manner, decreasing gemcitabine IC50 by approximately 90%. In vivo systemic administration of AS clusterin ODN and gemcitabine significantly decreased subcutaneous KoTCC-1 tumor volume compared with scramble control ODN plus gemcitabine. Furthermore, combined administration of AS clusterin ODN plus gemcitabine resulted in significantly delayed formation of hemorrhagic ascites compared with scramble control ODN plus gemcitabine in an intraperitoneal tumor cell injection model. CONCLUSIONS: These findings suggest that AS clusterin ODN may be useful for enhancing the cytotoxicity of gemcitabine in patients with bladder cancer, particularly as a novel therapeutic strategy for intravesical instillation therapy.


Subject(s)
Antimetabolites, Antineoplastic/therapeutic use , Deoxycytidine/analogs & derivatives , Deoxycytidine/therapeutic use , Glycoproteins/genetics , Molecular Chaperones/genetics , Neoplasm Proteins/genetics , Oligodeoxyribonucleotides, Antisense/therapeutic use , Urinary Bladder Neoplasms/therapy , Administration, Intravesical , Animals , Cell Line, Tumor , Clusterin , Combined Modality Therapy , Female , Humans , Mice , Mice, Inbred BALB C , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/genetics , Gemcitabine
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