Circulating Micro RNA (106, 21) as Biomarker for Helicobacter pylori associated gastric cancer Ibrahim A. Altamemi, Rana Masheel Salim, Osamah Tahir Muslim Annals of Tropical Medicine and Public Health, 2020 Background: gastric cancer (GC) residue one. of the major health burdens accounting for of all cancers and is the third leading cause of cancer-related deaths worldwide. Chronic inflammation due to Helicobacter pylori infection plays the role in triggering carcinogenesis. Gastric cancer managements must be quickly and the time of gastric cancer diagnosis is necessary for treatment, unfortunately, the existing circulating biomarker for gastric cancer diagnosis, prognosis display low sensitivity and specificity, the gastric cancer diagnosis is based on only on the invasive procedures such as upper digestive endoscopy and diagnosis for the majority of patients is made at an advanced stage when only limited treatment options can be offered, therefore other the recent studies are suggests using miRNAs as gastric cancer biomarker. Objective: The study aims to evaluate miRNA2l, miRNA-106 as a non invasive diagnostic biomarker for Helicobacter pylori associated gastritis and gastric cancer. Patients and methods: A case control study have been conducted and based on 3 group, The first group was included 20 patients with Helicobacter pylori infection associated gastric cancer, who were observation in Oncology Center at Al-Diwaniyah City, Second group was include 20 patients with Helicobacter pylori infection associated Gastritis who visited Endoscopy Department of Gastroenterology and Hepatology of Al-Diwanyiah Teaching hospital. Third group was include 40 healthy volunteers. Venipuncture used to collect samples of blood from these groups. The collection of three milliliters blood was payload out in plain non EDTA tube in order to clot. Further, centrifuges was utilized to separate the serum and then stored at -20οC which was further used to identify free miRNA-106 and miRNA-21 qPCR. Results: Current study reveals that miR-21 expression .was. significantly highest in patient with gastric .cancer , and then patients with gastritis and then control group (P < 0.001), 6.28 (4.78) fold change versus 2.45 (1.79) fold change versus 1 (---) fold change In addition, the miR-106 expression was significantly highest in patients with gastric cancer and then patients with gastritis and control group (P < 0.001), 7.03 (4.81)fold change versus 3.02 (1.82)fold change versus 1 (---) fold change, respectively, the diagnostic value of both miR21 and miR-106 was evaluate using receiver operator characteristic ROC curve analysis the cutoff value of miR21 was > 4.32 fold change with an area under the curve (AUC) of 0.973, an accuracy of 97.3 %, a sensitivity of 100 % and a specificity of 95 %, Moreover, the cutoff value of miR-106 was > 4.84 fold change with an area under the curve (AUC) of 0.981, an accuracy of 98.1 %, a sensitivity of 100 % and a specificity of 96 %. Conclusion: We can obtain diagnostic biomarkers with high specificity and sensitivity, by using two miRNA miRNA-21 which show high sensitivity, and miRNA-106, which show high specificity, Perhaps this could complement each other in testing whilst increasing sensitivity when individually related to miRNA. Keyword: Circulating Micro RNA, Biomarker, Helicobacter pylori, gastric cancer How to cite this article: Altamemi IA, Salim RM, Muslim OT (2020): Circulating Micro RNA (106,21) as biomarkers for Helicobacter pylori associated gastric cancer, Ann Trop Med & Public Health; 23(S14): .231438 http://doi.org/10.36295/ASRO.2020 . DOI: SP231438 Introduction The vast majority of gastric cancer worldwide is attributable to H. pylori, chronic inflammation due to H. pylori .infection play a crucial role in causing carcinogeness according to a multi-stage cycle first described by Correa, H. pylori-driven inflammation triggers the development of gastric cancer leading to typical stages of mucosal alterations such as chronic gastritis, glandular atrophy, intestinal metaplasia, dysplasia before reaching the final stage of invasive gastric cancer [1]. Despite recent advances in diagnostic techniques and preoperative management, these methods, however, are costly, invasive, and can put too much risk on the patient. Improvement in GC's prompt diagnosis and care has a critical effect on the patients' optimum management and long-term survival [2], thus the establishment .of novel strong specific biomarkers with sufficient sensitivity is an ideal strategy for improving the early detection of GC and the cure rates for patients. Many of these biomarkers are not specific for the early stages; however, some of possible non-invasive diagnostic biomarkers Altamemi et al (2020): Biomarker of H Pylori associated gastric cancer Oct 2020 Vol. 23 Issue 14 http://doi.org/10.36295/ASRO.2020.231438 Annals of Tropical Medicine & Public Health for early-stage gastric cancer discovered circulating molecules genetic and epigenetic alterations [3]. Thus miRNAs present inside the bloodstream are called circulatory miRNAs. Therefore, these miRNAs were considered important biomarkers for early detection of cancer [4-5]. There are numerous reports that specify various miRNAs involved in GC tumerogenesis such as miR-106a [6] miR-106a can be followed in salid tumor, stool, and plasma/serum sample of patients with gastrointestinal tumor [7]. The new studies show that miR-106a levels are significantly correlated with the stage of tumor node metastasis (TNM), tumor size and differentiation, lymphatic and distant metastasis, and invasion [8]. The miR-21 is over expressed in various cancers and has been causally associated to cellular proliferation, apoptosis, and migration and it had been reported that miR-21 induces invasion, intravasation, and metastasis [9]. Materials and Methods Patient group and sample collection This case control study comprised of 3 groups, 1st consisted of20 patients with H. pylori-associated Gastric Cancer who were observation in Oncology Hospital in Diwaniya in the period from March 2018to February 2019. Under the supervision of specialists, Lesions observed by endocytoscopy underwent endoscopic biopsy or endoscopic resection. Second group was 20 patients with H. pylori-associated Gastritis who visited Endoscopy Department of Gastroenterology and Hepatology of Al-diwanyia Teaching hospital. Individuals with gastritis were recruited consecutively from health checkup examinees that had undergone gastro copy and serologic analysis as part of a screening program for gastritis under the supervision of internal medicine specialists. While, 3rd group was include 40 healthy volunteers. Venipuncture used to collect samples from groups; 3 milliliter blood was collected in plain tubes without EDTA. Blood was permitted to clot in order to separate the serum through five minutes centrifugation at 13000rpm. The separated serum was then gathered in Eppendorf tubes and stored at -20 for further usage in miRNA-106 & miRNA-21 qPCR. Total RNA extraction The TRIzol® reagent kit (Bioneer, Korea) was used to extract the total RNA as per the instruction of the company, further, Nanodrop spectrophotometer (THERMO USA) was used to check the extracted genomic DNA. Thus, purity of DNA & concentration of DNA was estimated by reading the absorbance at 260/280nm. STEM-LOOP RT-qPCR The expression analysis of 106 miRNA & 21miRNA was quantified using stem loop RT-qPCR which was further normalized with GAPDH housekeeping genes in normal samples, blood patients & serum using the technique of Real-time PCR. This method was done according to the described method of [10].
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