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      Droplet digital PCR-based circulating microRNA detection serve as a promising diagnostic method for gastric cancer

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          Abstract

          Background

          Novel non-invasive biomarkers for gastric cancer (GC) are needed, because the present diagnostic methods for GC are either invasive or insensitive and non-specific in clinic. The presence of stable circulating microRNAs (miRNAs) in plasma suggested a promising role as GC biomarkers.

          Methods

          Based on the quantitative droplet digital PCR (ddPCR), four miRNAs (miR-21, miR-93, miR-106a and miR-106b) related to the presence of GC were identified in plasma from a training cohort of 147 participants and a validation cohort of 28 participants.

          Results

          All circulating miRNA levels were significantly higher in the plasma of GC patients compared to healthy controls ( P < 0.05). Through a combination of four miRNAs by logistic regression model, receiver operating characteristic (ROC) analyses yielded the highest AUC value of 0.887 in discriminating GC patients from healthy volunteers. Furthermore, miR-21, miR-93 and miR-106b levels were significantly related to an advanced TNM stage in GC patients. ROC analyses of the combined miRNA panel also showed the highest AUC value of 0.809 in discriminating GC patients with TNM stage I and II from stage III and IV. Through combining four miRNAs and clinical parameters, a classical random forest model was established in the training stage. In the validation cohort, it correctly discriminated 23 out of 28 samples in the blinded phase (false rate, 17.8%).

          Conclusions

          Using the ddPCR technique, circulating miR-21, miR-93, miR-106a and miR-106b could be used as diagnostic plasma biomarkers in gastric cancer patients.

          Electronic supplementary material

          The online version of this article (10.1186/s12885-018-4601-5) contains supplementary material, which is available to authorized users.

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          Most cited references27

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          Gastric cancer: prevention, screening and early diagnosis.

          Gastric cancer continues to be an important healthcare problem from a global perspective. Most of the cases in the Western world are diagnosed at late stages when the treatment is largely ineffective. Helicobacter pylori (H. pylori) infection is a well-established carcinogen for gastric cancer. While lifestyle factors are important, the efficacy of interventions in their modification, as in the use of antioxidant supplements, is unconvincing. No organized screening programs can be found outside Asia (Japan and South Korea). Although several screening approaches have been proposed, including indirect atrophy detection by measuring pepsinogen in the circulation, none of them have so far been implemented, and more study data is required to justify any implementation. Mass eradication of H. pylori in high-risk areas tends to be cost-effective, but its adverse effects and resistance remain a concern. Searches for new screening biomarkers, including microRNA and cancer-autoantibody panels, as well as detection of volatile organic compounds in the breath, are in progress. Endoscopy with a proper biopsy follow-up remains the standard for early detection of cancer and related premalignant lesions. At the same time, new advanced high-resolution endoscopic technologies are showing promising results with respect to diagnosing mucosal lesions visually and targeting each biopsy. New histological risk stratifications (classifications), including OLGA and OLGIM, have recently been developed. This review addresses the current means for gastric cancer primary and secondary prevention, the available and emerging methods for screening, and new developments in endoscopic detection of early lesions of the stomach.
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            microRNA-21 promotes tumor proliferation and invasion in gastric cancer by targeting PTEN

            Gastric cancer is one of the most common carcinomas in China. microRNAs, a type of non-coding RNA, are important specific regulators and are involved in numerous bioprocesses of an organism. microRNA-21 (miR-21) has been identified as the most suitable choice for further investigation because it is overexpressed in nearly all solid tumors; furthermore, it has been demonstrated that miR-21 is involved in the genesis and progression of human cancer. It has been reported that PTEN, an important tumour suppressor, is regulated by multiple miRNAs. Thus, in this study we focused on the expression and significance of miR-21 in gastric cancer tissues, and the role of miR-21 in the biological behaviour and the expression of PTEN in gastric cancer cells. Real-time PCR was used to detect miR-21 expression in gastric cancer tissues, the adjacent normal tissues, and the gastric cell lines. The gastric cancer cell line BGC-823 was transfected with pre-miR-21/miR-21 inhibitor to overexpress/downregulate miR-21. The influence of miR-21 on the biological behaviour of gastric cancer cells was evaluated using the CCK-8 kit, FCMs, the scratch healing assay and the transwell test. Western blotting and the Luciferase Reporter Assay were used to evaluate the change of PTEN expression after lowered expression of miR-21 in gastric cancer cell lines. Real-time PCR analysis indicated that miR-21 exhibited higher expression in gastric cancer tissues compared to the adjacent non-tumor tissues. miR-21 expression was significantly associated with the degree of differentiation of the tumour tissues (P=0.004), as well as local invasion and lymph node metastasis (P<0.01). After transfection, pre-miR21 BGC-823 cells grew faster than the negative and control groups (P<0.01). The reduction in miR-21 expression demonstrated a remarkable effect on the biological behaviour of gastric cancer cells (P<0.05); the pre-miR-21-transfected cells healed more quickly compared to the control cells in the scratch healing assay, whereas the transwell test indicated that cell migration in vitro was notably inhibited with the downregulation of miR-21 (P<0.05). The western blot results and Luciferase Reporter Assay demonstrated that PTEN expression was remarkably increased after miR-21 inhibition (P<0.05). microRNA-21 expression was upregulated in gastric carcinoma tissues and was significantly associated with the degree of differentiation of tumour tissues, local invasion and lymph node metastasis. Overexpression of miR-21 promoted BGC-823 cell growth, invasion and cell migration in vitro, whereas downregulation of miR-21 exhibited a stronger inhibitory effect on the biological behaviour of gastric cancer cells; additionally, miR-21 inhibition may upregulate the PTEN expression level, which indicates that PTEN may be a target gene for gastric cancer initiation and development.
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              Serum microRNAs as powerful cancer biomarkers.

              MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression at the posttranscriptional level by either degrading or blocking translation of messenger RNA targets. Recent expression profiling studies have revealed that miRNAs play important regulatory roles in a variety of cellular functions as well as in every cancer type studied so far. Lately, the discovery of miRNAs in the serum of cancer patients opened up the exciting prospective of using miRNAs as powerful and non-invasive cancer biomarkers. In this article, we review the current literature on serum miRNAs in different cancer types and the approaches used to detect and quantify these molecules. We then discuss the potential of miRNA biomarkers to improve disease diagnosis by distinguishing healthy from malignant tissues, identifying the tissue of origin in poorly differentiated tumors or tumors of unknown origin and distinguishing between different subtypes of the same tumor. We will also compare the advantages and disadvantages of potential serum miRNA biomarker molecules for cancer classification, estimation of prognosis and prediction of therapeutic efficacy. Finally, we will establish a set of criteria that these new molecules and clinical studies that use them must fulfill before they can be used as reliable tools in diagnostic and prognostic settings. Copyright © 2010 Elsevier B.V. All rights reserved.
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                Author and article information

                Contributors
                +86-28-87393805 , gzhao@uestc.edu.cn
                490397636@qq.com
                comelina123@163.com
                1358271315@qq.com
                191197067@qq.com
                87239657@qq.com
                gqjia2009@163.com
                258380648@qq.com
                +86-28-87393315 , 37771782@qq.com
                Journal
                BMC Cancer
                BMC Cancer
                BMC Cancer
                BioMed Central (London )
                1471-2407
                22 June 2018
                22 June 2018
                2018
                : 18
                : 676
                Affiliations
                [1 ]ISNI 0000 0004 1808 0950, GRID grid.410646.1, Department of Gastrointestinal Surgery, , Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, ; Chengdu, 610072 China
                [2 ]ISNI 0000 0004 0369 4060, GRID grid.54549.39, School of Medicine, , University of Electronic Science and Technology of China, ; Chengdu, 610054 China
                [3 ]ISNI 0000000119573309, GRID grid.9227.e, Institute of Chengdu Biology, and Sichuan Translational Medicine Hospital, , Chinese Academy of Sciences, ; Chengdu, 610041 China
                [4 ]Department of General Surgery, Qionglai Medical Center Hospital, Chengdu, Sichuan Province, China, Chengdu, 611530 China
                [5 ]ISNI 0000 0004 1808 0950, GRID grid.410646.1, Department of General Surgery, , Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, ; Chengdu, 610072 China
                Author information
                http://orcid.org/0000-0001-5899-9230
                Article
                4601
                10.1186/s12885-018-4601-5
                6013872
                29929476
                28c5f84c-064a-4118-97a6-766c9e8a88df
                © The Author(s). 2018

                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
                : 2 April 2018
                : 15 June 2018
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 81172832
                Award ID: 81771723
                Funded by: Sichuan Youth Science and Technology Foundation
                Award ID: 2013JQ0020
                Funded by: Special Program for Sichuan Youth Science and Technology Innovation
                Award ID: 2014TD0010
                Funded by: Health and Family Planning Commission of Sichuan Province
                Award ID: 110190
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2018

                Oncology & Radiotherapy
                gastric cancer,liquid biopsy,ddpcr,mir-21,mir-93,mir-106a,mir-106b
                Oncology & Radiotherapy
                gastric cancer, liquid biopsy, ddpcr, mir-21, mir-93, mir-106a, mir-106b

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