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Evaluation of the role of miRNAs related to cysplatin-induced nephrotoxicity in vitro

Grant number: 21/08205-7
Support type:Scholarships abroad - Research Internship - Master's degree
Effective date (Start): January 02, 2022
Effective date (End): May 01, 2022
Field of knowledge:Health Sciences - Pharmacy
Principal researcher:Patricia Moriel
Grantee:Nadine de Godoy Torso
Supervisor abroad: Luis Salazar Navarrete
Home Institution: Faculdade de Ciências Médicas (FCM). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
Research place: Universidad de La Frontera (UFRO), Chile  
Associated to the scholarship:19/20010-7 - MicroRNAs as possible cypplin-induced nephrotoxicity predictors in head and neck cancer patients, BP.MS

Abstract

Cisplatin is a highly effective antineoplastic agent, however, its use is limited due to its adverse reactions, especially nephrotoxicity. Therefore, there is a need to identify nephrotoxicity biomarker molecules, as classical pathological markers have low sensitivity and specificity. Among the biomarkers proposed in recent years, one of the most promising is miRNA. In a recent study carried out in our group, the sequencing technique was used with samples of miRNAs from plasma and urine extracted from 6 participants treated with cisplatin who showed nephrotoxicity, and from 6 participants who did not show nephrotoxicity after treatment (which constituted the group of cases and controls, respectively). Five miRNAs that had a fold regulation (FR)> 10 or <-10 were selected to evaluate their in vitro behavior. Furthermore, evaluating the current literature, a sixth miRNA was also selected. We believe that these miRNAs would be possible biomarkers of cisplatin-induced nephrotoxicity. Therefore, the objective of this study will be to evaluate, in vitro, the role of the six miRNAs (hsa-miR-6729-5p, hsa-miR-1238-5p, hsa-miR-4706, hsa-miR-6805-5p, hsa-miR-4322, hsa-miR-21-5p) possibly related to cisplatin-induced nephrotoxicity. The six miRNAs will be evaluated on TargetScan in combination with Ingenuity Pathway Analysis bioinformatics analysis to select for genes potentially related to nephrotoxicity. The interaction between the miRNA and the 3'UTR region of the selected target will be assessed by the luciferase assay, and the 3'UTR region will be cloned into the pmirGLO vector (Promega, Madison, Wi, USA), according to the instructions manufacturer. The hRluc-neo plasmid containing the Renilla luciferase coding region will be used as a reporter control for normalization. In cellularsHEK293, mimics miRNAs and the luciferase reporter vector will be cotransfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Luciferase activity will be tested using the Dual-Glo® Luciferase Assay System Kit (Promega, Madison, Wi, USA), according to the manufacturer's instructions. Results will be expressed as relative luciferase activity. Continuous variables will first be tested to assess the distribution of the value and then compared between groups using the t-test or the Mann-Whitney test. Paired data (prospective data) of more than 2 points will be analyzed using Friedman's test. P <0.05 will be considered statistically significant. (AU)

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