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      Overexpression of miR-584-5p inhibits proliferation and induces apoptosis by targeting WW domain-containing E3 ubiquitin protein ligase 1 in gastric cancer

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          Abstract

          Background

          MicroRNAs are endogenously expressed, small non-coding RNAs that modulate gene expression by targeting specific mRNAs, resulting in translational repression or mRNA degradation. Although miR-584-5p has been reported to play a vital role in various malignancies, its role and the molecular mechanisms underlying the effects of miR-584-5p in gastric cancer (GC) remain to be clarified. In this study, we investigated the role of miR-584-5p in GC.

          Methods

          The expression of miR-584-5p and its specific target gene were determined in human GC specimens and cell lines by microRNA real-time polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR) and Western blot. The effects of miR-584-5p depletion or ectopic expression on GC proliferation were evaluated in vitro using CCK-8 proliferation assays, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, colony formation assays and cell-cycle assays and the in vivo effects were investigated using a mouse tumorigenicity model. Cell apoptosis was evaluated by in vitro flow cytometric analysis, cell viability assays and in vivo TUNEL assays. Luciferase reporter assays were employed to identify interactions between miR-584-5p and its specific target gene.

          Results

          A series of in vitro and in vivo gain- and loss-of-function assays revealed that miR-584-5p inhibited GC cell proliferation, while apoptosis was induced. Luciferase reporter assays and Western blot analysis revealed WWP1 to be a direct target of miR-584-5p. The effects of miR-584-5p-mimic were rescued by WWP1 overexpression. In contrast, the effects of the miR-584-5p-inhibitor were impaired by WWP1-shRNA. Furthermore, miR-584-5p expression levels correlated negatively with WWP1 protein expression in GC tissues and GC cell lines. A series of investigations indicated that miR-584-5p promoted senescence and activated the TGFβ signaling pathway by downregulation of WWP1.

          Conclusion

          Taken together, these results suggest that downregulation of miR-584-5p contributes to tumor progression by downregulation of WWP1, thus, highlighting the potential of miR-584-5p as a therapeutic target for human GC.

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          Most cited references24

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          microRNAs: Master regulators as potential therapeutics in cancer.

          It has been demonstrated that all the known processes involved in cancer, including apoptosis, proliferation, survival, and metastasis, are regulated by small regulatory noncoding RNAs consisting of approximately 19-25 nucleotides; these are named microRNAs (miRNAs). Both loss and gain of miRNA function contribute to cancer development through the upregulation and silencing, respectively, of different target genes. Experimental evidence indicates that the use of miRNA mimics or anti-microRNAs may represent a powerful therapeutic strategy to interfere with key molecular pathways involved in cancer. This review provides insights about how micro- RNAs act as oncogenes and tumor suppressor genes and how these findings, along with our increasing understanding of miRNA regulation, can be applied to optimize recent miRNA-based technologies and make them suitable for clinical applications.
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            miR-506 acts as a tumor suppressor by directly targeting the hedgehog pathway transcription factor Gli3 in human cervical cancer.

            Although significant advances have recently been made in the diagnosis and treatment of cervical carcinoma, the long-term survival rate for advanced cervical cancer remains low. Therefore, an urgent need exists to both uncover the molecular mechanisms and identify potential therapeutic targets for the treatment of cervical cancer. MicroRNAs (miRNAs) have important roles in cancer progression and could be used as either potential therapeutic agents or targets. miR-506 is a component of an X chromosome-linked miRNA cluster. The biological functions of miR-506 have not been well established. In this study, we found that miR-506 expression was downregulated in approximately 80% of the cervical cancer samples examined and inversely correlated with the expression of Ki-67, a marker of cell proliferation. Gain-of-function and loss-of-function studies in human cervical cancer, Caski and SiHa cells, demonstrated that miR-506 acts as a tumor suppressor by inhibiting cervical cancer growth in vitro and in vivo. Further studies showed that miR-506 induced cell cycle arrest at the G1/S transition, and enhanced apoptosis and chemosensitivity of cervical cancer cell. We subsequently identified Gli3, a hedgehog pathway transcription factor, as a direct target of miR-506 in cervical cancer. Furthermore, Gli3 silencing recapitulated the effects of miR-506, and reintroduction of Gli3 abrogated miR-506-induced cell growth arrest and apoptosis. Taken together, we conclude that miR-506 exerts its anti-proliferative function by directly targeting Gli3. This newly identified miR-506/Gli3 axis provides further insight into the pathogenesis of cervical cancer and indicates a potential novel therapeutic agent for the treatment of cervical cancer.
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              Conformational flexibility underlies ubiquitin ligation mediated by the WWP1 HECT domain E3 ligase.

              Ubiquitin ligases (E3) select proteins for ubiquitylation, a modification that directs altered subcellular trafficking and/or degradation of the target protein. HECT domain E3 ligases not only recognize, but also directly catalyze, ligation of ubiquitin to their protein substrates. The crystal structure of the HECT domain of the human ubiquitin ligase WWP1/AIP5 maintains a two-lobed structure like the HECT domain of the human ubiquitin ligase E6AP. While the individual N and C lobes of WWP1 possess very similar folds to those of E6AP, the organization of the two lobes relative to one another is different from E6AP due to a rotation about a polypeptide hinge linking the N and C lobes. Mutational analyses suggest that a range of conformations achieved by rotation about this hinge region is essential for catalytic activity.
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                Author and article information

                Contributors
                15720801585@163.com
                lizheng2281@163.com
                weisong5660@163.com
                wanglinjun8831@163.com
                zekuanxu8860@163.com
                zhanglei4832@163.com
                wangweizhi180@163.com
                libowen6901@163.com
                sunguangli0324@163.com
                xujianghao0982@163.com
                liqiang88321@163.com
                wanglu021332@163.com
                zekuanxu5188@163.com
                2921910298@qq.com
                diancaizhang@163.com
                haoxu5348@163.com
                xuzekuan@njmu.edu.cn
                Journal
                J Exp Clin Cancer Res
                J. Exp. Clin. Cancer Res
                Journal of Experimental & Clinical Cancer Research : CR
                BioMed Central (London )
                0392-9078
                1756-9966
                21 April 2017
                21 April 2017
                2017
                : 36
                : 59
                Affiliations
                [1 ]ISNI 0000 0004 1799 0784, GRID grid.412676.0, Department of General Surgery, , The First Affiliated Hospital of Nanjing Medical University, ; No.300, Guangzhou road, Nanjing, Jiangsu province China
                [2 ]ISNI 0000 0004 1936 9916, GRID grid.412807.8, Department of Surgery, , Vanderbilt University Medical Center, ; Nashville, Tennessee USA
                Article
                532
                10.1186/s13046-017-0532-2
                5401563
                28431583
                bb9d5aa0-ee1f-42ed-b347-fb12d6f9082a
                © The Author(s). 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 15 January 2017
                : 17 April 2017
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 81272712, 81572362
                Award Recipient :
                Funded by: the National Natural Science Foundation Project of International Cooperation
                Award ID: NSFC-NIH, 81361120398
                Award Recipient :
                Funded by: Project of Jiangsu Province
                Award ID: BRA2015474
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2017

                Oncology & Radiotherapy
                mir-584-5p,wwp1,proliferation,apoptosis,cellular senescence,tgfβ signaling pathway,gastric cancer

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