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      Fluorescence activated cell sorting followed by small RNA sequencing reveals stable microRNA expression during cell cycle progression

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          Abstract

          Background

          Previously, drug-based synchronization procedures were used for characterizing the cell cycle dependent transcriptional program. However, these synchronization methods result in growth imbalance and alteration of the cell cycle machinery. DNA content-based fluorescence activated cell sorting (FACS) is able to sort the different cell cycle phases without perturbing the cell cycle. MiRNAs are key transcriptional regulators of the cell cycle, however, their expression dynamics during cell cycle has not been explored.

          Methods

          Following an optimized FACS, a complex initiative of high throughput platforms (microarray, Taqman Low Density Array, small RNA sequencing) were performed to study gene and miRNA expression profiles of cell cycle sorted human cells originating from different tissues. Validation of high throughput data was performed using quantitative real time PCR. Protein expression was detected by Western blot. Complex statistics and pathway analysis were also applied.

          Results

          Beyond confirming the previously described cell cycle transcriptional program, cell cycle dependently expressed genes showed a higher expression independently from the cell cycle phase and a lower amplitude of dynamic changes in cancer cells as compared to untransformed fibroblasts. Contrary to mRNA changes, miRNA expression was stable throughout the cell cycle.

          Conclusions

          Cell cycle sorting is a synchronization-free method for the proper analysis of cell cycle dynamics. Altered dynamic expression of universal cell cycle genes in cancer cells reflects the transformed cell cycle machinery. Stable miRNA expression during cell cycle progression may suggest that dynamical miRNA-dependent regulation may be of less importance in short term regulations during the cell cycle.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12864-016-2747-6) contains supplementary material, which is available to authorized users.

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          Most cited references37

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          Control of translation and mRNA degradation by miRNAs and siRNAs.

          The control of translation and mRNA degradation is an important part of the regulation of gene expression. It is now clear that small RNA molecules are common and effective modulators of gene expression in many eukaryotic cells. These small RNAs that control gene expression can be either endogenous or exogenous micro RNAs (miRNAs) and short interfering RNAs (siRNAs) and can affect mRNA degradation and translation, as well as chromatin structure, thereby having impacts on transcription rates. In this review, we discuss possible mechanisms by which miRNAs control translation and mRNA degradation. An emerging theme is that miRNAs, and siRNAs to some extent, target mRNAs to the general eukaryotic machinery for mRNA degradation and translation control.
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            Identification of genes periodically expressed in the human cell cycle and their expression in tumors.

            The genome-wide program of gene expression during the cell division cycle in a human cancer cell line (HeLa) was characterized using cDNA microarrays. Transcripts of >850 genes showed periodic variation during the cell cycle. Hierarchical clustering of the expression patterns revealed coexpressed groups of previously well-characterized genes involved in essential cell cycle processes such as DNA replication, chromosome segregation, and cell adhesion along with genes of uncharacterized function. Most of the genes whose expression had previously been reported to correlate with the proliferative state of tumors were found herein also to be periodically expressed during the HeLa cell cycle. However, some of the genes periodically expressed in the HeLa cell cycle do not have a consistent correlation with tumor proliferation. Cell cycle-regulated transcripts of genes involved in fundamental processes such as DNA replication and chromosome segregation seem to be more highly expressed in proliferative tumors simply because they contain more cycling cells. The data in this report provide a comprehensive catalog of cell cycle regulated genes that can serve as a starting point for functional discovery. The full dataset is available at http://genome-www.stanford.edu/Human-CellCycle/HeLa/.
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              Targeting the cell cycle: a new approach to cancer therapy.

              The cell cycle represents a series of tightly integrated events that allow the cell to grow and proliferate. Critical parts of the cell cycle machinery are the cyclin-dependent kinases (CDKs), which, when activated, provide a means for the cell to move from one phase of the cell cycle to the next. The CDKs are regulated positively by cyclins and regulated negatively by naturally occurring CDK inhibitors (CDKIs). Cancer represents a dysregulation of the cell cycle such that cells that overexpress cyclins or do not express the CDKIs continue to undergo unregulated cell growth. The cell cycle also serves to protect the cell from DNA damage. Thus, cell cycle arrest, in fact, represents a survival mechanism that provides the tumor cell the opportunity to repair its own damaged DNA. Thus, abrogation of cell cycle checkpoints, before DNA repair is complete, can activate the apoptotic cascade, leading to cell death. Now in clinical trials are a series of targeted agents that directly inhibit the CDKs, inhibit unrestricted cell growth, and induce growth arrest. Recent attention has also focused on these drugs as inhibitors of transcription. In addition, there are now agents that abrogate the cell cycle checkpoints at critical time points that make the tumor cell susceptible to apoptosis. An understanding of the cell cycle is critical to understanding how best to clinically develop these agents, both as single agents and in combination with chemotherapy.
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                Author and article information

                Contributors
                +36-1-4591500 , patocs.attila@med.semmelweis-univ.hu
                Journal
                BMC Genomics
                BMC Genomics
                BMC Genomics
                BioMed Central (London )
                1471-2164
                27 May 2016
                27 May 2016
                2016
                : 17
                : 412
                Affiliations
                [ ]2nd Department of Medicine, Semmelweis University, Szentkirályi utca 46, 1088 Budapest, Hungary
                [ ]“Lendület” Hereditary Endocrine Tumours Research Group, Hungarian Academy of Sciences, Semmelweis University, Szentkirályi utca 46, 1088 Budapest, Hungary
                [ ]Department of Immunology, Eötvös Loránd University, Pázmány Péter sétány 1/C, 1117 Budapest, Hungary
                [ ]1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, 1085 Budapest, Hungary
                [ ]Molecular Medicine Research Group, Hungarian Academy of Sciences – Semmelweis University, Szentkirályi utca 46, 1088 Budapest, Hungary
                [ ]Department of Laboratory Medicine, Semmelweis University, Nagyvárad tér 4, 1089 Budapest, Hungary
                Article
                2747
                10.1186/s12864-016-2747-6
                4884355
                27234232
                42069524-3851-4d11-99f6-c4bff5691e99
                © The Author(s). 2016

                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
                : 28 September 2015
                : 17 May 2016
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100003825, Magyar Tudományos Akadémia (HU);
                Award ID: Lendület grant
                Funded by: Technology Innovation Fund, National Developmental Agency
                Award ID: KTIA-AIK-2012-12-1-0010
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100003549, Országos Tudományos Kutatási Alapprogramok (HU);
                Award ID: PD100648
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2016

                Genetics
                fluorescence-activated cell sorting (facs),cell cycle,dynamic expression,dna staining,mirna

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