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Validation of specific miRNAs as effectors of Inflammation and angiogenesis in the diabetic retina in type 2 Diabetes Mellitus, by means of an epidemiologic, observational, analytical and multicenter case-control study
Spain (IFMSA-SPAIN)-University of Valencia, Valencia
Ophthalmic Research Unit Santiago Grisolia /FISABIO
Pedro Sanz Solana
Mª Dolores Pinazo Durán
Required: English Accepted: Spanish, Italian
Type of Research Project
- Basic science
What is the background of the project?
Diabetic retinopathy (DR) is the first global cause of blindness in people in its working age. Noncoding ribonucleic acids (RNAs) are known as microRNAs (miRNAs). They act by regulating gene expression at the transcriptional/post-transcriptional level. Its aberrant expression has been linked to various diseases. Our objective is the validation of previous results in which we identified some new microRNAs in tears, involved in the pathogenic mechanisms of the DR in type 2 diabetics (DM2) with/without DR and healthy subjects. After RNA extraction, libraries construction, next generation sequencing and bioinformatics analyses we identified 64 miRNAs, among them three involved in inflammation and angiogenesis with strong statistical association with DR.
What is the aim of the project?
We propose to continue the validation phases of these results by means of an epidemiologic, observational, analytical and multicenter case-control study in 225 patients with DM2 (with/without RD) and 225 control subjects.
What techniques and methods are used?
Once the samples from the patients with DM2 (with/without RD) and the samples from the control subjects have arrived at the lab we will proceed for the analysis of them. The student we will first learn how to analyze the expression of three specific microRNAs: (hsa-miR-15b-5p, hsa-miR-155-5p, hsa-miR-195-3p) by Quantiative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) We will also quantify by qRT-PCR the expression of their target genes: - TGF-beta3 Receptor (TGFBR3) , Bcl-2-like protein 2 (BCL2L2), Suppressor of cytokine signaling 6 (SOCS69) in the case of the hsa-miR-15b-5p micro-RNA - Interleukin-17 receptor B precursor (IL17RB), Interleukin-36 gamma precursor (IL36G), Interleukin-6 Receptor (IL6R), Fibroblast Growth Factor 2 (FGF2) in case of hsa-miR 155-5p microRNA - Fibroblast Growth Factor 9 (FGF9), Fibroblast Growth Factor 14 (FGF14), Vascular Endotherlial Growth Factor C (VEGFC) in case of hsa-miR-195-3p microRNA. Then, we will quantify the expression of the proteins encoded by these genes by Enzyme-linked Immunosorbent assay (ELISA). Finally, we will analyse the results and variations by making the predetermined calculations and we will deevelope the statistic study by using the Statistical Package for the Social Sciences (SPSS) program.
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
What are the tasks expected to be accomplished by the student?
To analyze the expression of several proteins encoded by genes target of miRNAS selcted, by Enzyme-linked Immunosorbent assay (ELISA) technique. The student will have the opportunity to work with the samples, manipulating them in the sense of store them use them in the provided kit. For the ELISA kit procedure, the sudent will follow the manufacture instructions: Basically, defrost sampls, incubate in 96 well dish precoated at ½ dilution. Primary and secondary antibody incubation and absorbance measure. Then, the student will make the specific calculations by accurate ecuations and, finally, the student will develope the statistic study by using the Statistical Package for the Social Sciences (SPPS) program.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Yes. The tutor of the project will provide during the first week research papers related to the basis of the project that will help the student to understand the project itself.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a scientific report - The student will have the opportunity to present the results together with the supervisor at a conference
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Hardworking, sociable, eager to learn and enthusiastic about science and specifically the eyes and vision. Previous experience with: laboratory tasks
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Pre-Medical students from the American-British system - Students in biomedical fields
- Genetic systems for a new approach to risk of progression of diabetic retinopathy. Pinazo-Durán MD; Zanón-Moreno V; Lleó-Perez A; García-Medina JJ; Galbis-Estrada C; Roig-Revert MJ; Marco-Ramírez C; López-Gálvez M; Dolz-Marco R; Duarte L; Campos Borges C; Salgado-Borges J; Gallego-Pinazo R. Arch Soc Esp Oftalmol. 2016 May;91(5):209-16. doi: 10.1016/j.oftal.2016.01.016. Epub 2016 Mar 3. English; Spanish. PMID: 26948458
- MicroRNAs as potential biomarkers of eye diseases. Pinazo-Durán MD; Galbis-Estrada C; Zanón-Moreno V; García-Medina JJ; Bendala-Tufanisco E; Vinuesa-Silva I; Benítez del Castillo FJ. Arch Soc Esp Oftalmol. 2015 Dec;90(12):604-5. doi: 10.1016/j.oftal.2015.07.016. Epub 2015 Nov 18. English; Spanish. No abstract available. PMID: 26596419
- Present and future of ribonucleic acid interference Pinazo-Durán MD; Zanón-Moreno V; Vinuesa Silva I; García Medina JJ; Benítez del Castillo J. Arch Soc Esp Oftalmol. 2015 Jan;90(1):1-3. doi: 10.1016/j.oftal.2014.11.015. Spanish. No abstract available. PMID: 25660458
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