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      A novel mechanism of lncRNA and miRNA interaction: CCAT2 regulates miR-145 expression by suppressing its maturation process in colon cancer cells

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          Abstract

          Background

          Although both long and micro RNAs are emerging as important functional components in colorectal cancer (CRC) progression and metastasis, the mechanism of their interaction remains poorly understood. CCAT2 (Colon cancer-associated transcript-2), a long noncoding RNA (lncRNA), has been reported to be over-expressed in CRC and is found to promote tumor growth. miRNAs, a class of naturally occurring short RNAs negatively control the expression of target genes by cleaving mRNA or through translation repression. Recently, we reported that miR-145 and miR-21 cooperate to regulate colon cancer stem cell (CSC) proliferation and differentiation. Considering that CCAT2 is mainly located in the nucleus and miRNA maturation process begins in the nucleus, we hypothesize that CCAT2 selectively blocks miR-145 maturation process, resulting in decreased mature miR-145 affecting colon CSC proliferation and differentiation.

          Methods

          The levels of CCAT2 were manipulated by transfection of CCAT2 expression plasmid or knockdown by siRNA or by CRISPR/Cas9. Quantitative RT-PCR was performed to examine the expression of CCAT2 and pri-, pre- and mature miR-145/21. Fluorescence in situ hybridization (FISH) was used to visualize CCAT2 in the cells. In vitro processing of pri-miRNA-145 was performed using T7 RNA polymerase and recombinant human Dicer.

          Results

          We have observed that modulated expression of CCAT2 regulates the expression of miR-145 in colon cancer HCT-116 and HT-29 cells. Knockout of CCAT2 increases miR-145 and negatively regulates miR-21 in HCT-116 cells, impairs proliferation and differentiation. In contrast, stable up-regulation of CCAT2 decreases mature miR-145 and increases the expression of several CSC markers in colon cancer cells. We have also observed that CCAT2 is enriched in the nucleus and correlates with the expression of pre-miR-145 but not pre-miR-21 in HCT-116 cells. These results indicate CCAT2 selectively blocks miR-145 maturation by inhibiting pre-miR-145 export to cytoplasm. Further, we revealed that CCAT2 blocks cleavage of pre-miR-145 by Dicer in vitro.

          Conclusions

          Our results identify CCAT2 as a negative regulator of miRNA-145 biogenesis, and expose a novel mechanism of lncRNA-miRNA crosstalk.

          Electronic supplementary material

          The online version of this article (10.1186/s12943-017-0725-5) contains supplementary material, which is available to authorized users.

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          Most cited references27

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          Non-coding RNAs: the architects of eukaryotic complexity.

          J. Mattick (2001)
          Around 98% of all transcriptional output in humans is non-coding RNA. RNA-mediated gene regulation is widespread in higher eukaryotes and complex genetic phenomena like RNA interference, co-suppression, transgene silencing, imprinting, methylation, and possibly position-effect variegation and transvection, all involve intersecting pathways based on or connected to RNA signaling. I suggest that the central dogma is incomplete, and that intronic and other non-coding RNAs have evolved to comprise a second tier of gene expression in eukaryotes, which enables the integration and networking of complex suites of gene activity. Although proteins are the fundamental effectors of cellular function, the basis of eukaryotic complexity and phenotypic variation may lie primarily in a control architecture composed of a highly parallel system of trans-acting RNAs that relay state information required for the coordination and modulation of gene expression, via chromatin remodeling, RNA-DNA, RNA-RNA and RNA-protein interactions. This system has interesting and perhaps informative analogies with small world networks and dataflow computing.
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            A genome-wide association scan of tag SNPs identifies a susceptibility variant for colorectal cancer at 8q24.21.

            Much of the variation in inherited risk of colorectal cancer (CRC) is probably due to combinations of common low risk variants. We conducted a genome-wide association study of 550,000 tag SNPs in 930 familial colorectal tumor cases and 960 controls. The most strongly associated SNP (P = 1.72 x 10(-7), allelic test) was rs6983267 at 8q24.21. To validate this finding, we genotyped rs6983267 in three additional CRC case-control series (4,361 affected individuals and 3,752 controls; 1,901 affected individuals and 1,079 controls; 1,072 affected individuals and 415 controls) and replicated the association, providing P = 1.27 x 10(-14) (allelic test) overall, with odds ratios (ORs) of 1.27 (95% confidence interval (c.i.): 1.16-1.39) and 1.47 (95% c.i.: 1.34-1.62) for heterozygotes and rare homozygotes, respectively. Analyses based on 1,477 individuals with colorectal adenoma and 2,136 controls suggest that susceptibility to CRC is mediated through development of adenomas (OR = 1.21, 95% c.i.: 1.10-1.34; P = 6.89 x 10(-5)). These data show that common, low-penetrance susceptibility alleles predispose to colorectal neoplasia.
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              Re-evaluation of the roles of DROSHA, Export in 5, and DICER in microRNA biogenesis.

              Biogenesis of canonical microRNAs (miRNAs) involves multiple steps: nuclear processing of primary miRNA (pri-miRNA) by DROSHA, nuclear export of precursor miRNA (pre-miRNA) by Export in 5 (XPO5), and cytoplasmic processing of pre-miRNA by DICER. To gain a deeper understanding of the contribution of each of these maturation steps, we deleted DROSHA, XPO5, and DICER in the same human cell line, and analyzed their effects on miRNA biogenesis. Canonical miRNA production was completely abolished in DROSHA-deleted cells, whereas we detected a few DROSHA-independent miRNAs including three previously unidentified noncanonical miRNAs (miR-7706, miR-3615, and miR-1254). In contrast to DROSHA knockout, many canonical miRNAs were still detected without DICER albeit at markedly reduced levels. In the absence of DICER, pre-miRNAs are loaded directly onto AGO and trimmed at the 3' end, yielding miRNAs from the 5' strand (5p miRNAs). Interestingly, in XPO5 knockout cells, most miRNAs are affected only modestly, suggesting that XPO5 is necessary but not critical for miRNA maturation. Our study demonstrates an essential role of DROSHA and an important contribution of DICER in the canonical miRNA pathway, and reveals that the function of XPO5 can be complemented by alternative mechanisms. Thus, this study allows us to understand differential contributions of key biogenesis factors, and provides with valuable resources for miRNA research.
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                Author and article information

                Contributors
                aa5142@wayne.edu
                makkerp@karmanos.org
                lfarhana@med.wayne.edu
                a.majumdar@wayne.edu
                Journal
                Mol Cancer
                Mol. Cancer
                Molecular Cancer
                BioMed Central (London )
                1476-4598
                30 September 2017
                30 September 2017
                2017
                : 16
                : 155
                Affiliations
                [1 ]Department of Veterans Affairs Medical Center, 4646 John R, Detroit, MI 48201 USA
                [2 ]ISNI 0000 0001 1456 7807, GRID grid.254444.7, Karmanos Cancer Institute, ; Detroit, MI 48201 USA
                [3 ]ISNI 0000 0001 1456 7807, GRID grid.254444.7, Departments of Internal Medicine, , Wayne State University, ; Detroit, MI 48201 USA
                Article
                725
                10.1186/s12943-017-0725-5
                5622467
                28964256
                40405a79-2dd3-415f-9265-f18442cc50dc
                © 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
                : 23 May 2017
                : 20 September 2017
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000054, National Cancer Institute;
                Award ID: 1R21CA175916
                Funded by: FundRef http://dx.doi.org/10.13039/100000738, U.S. Department of Veterans Affairs;
                Award ID: I101BX001927
                Categories
                Research
                Custom metadata
                © The Author(s) 2017

                Oncology & Radiotherapy
                mirna biogenesis,lncrna-mirna crosstalk,cancer stem cells
                Oncology & Radiotherapy
                mirna biogenesis, lncrna-mirna crosstalk, cancer stem cells

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