Doutorado MicroRNAa221 e MicroRNA29
Description
ABSTRACT Profile of microRNA-221 and microRNA-29a in individuals with carotid atherosclerotic disease treated with endarterectomy and carotid angioplasty. Introduction: It is known that microRNAs (miRNA) are involved in the processes of endothelial inflammation and atheroma plaque formation, and are also responsible for regulating its stability, with its expression being specific to each tissue and disease. The miRNA profile can be used as a marker for diagnosis and prognosis and influence the course of diseases, through its stimulation or suppression. Our objective was to study the relationship between the expression of miRNA-221 and 29a in the pre- and postoperative periods of patients with carotid disease, the surgical treatment used and the clinical evolution of this patient. Population and Methods: we selected 61 individuals with unilateral or bilateral carotid stenosis, above 70%, of which 43 underwent endarterectomy (Group 1) and 18 underwent angioplasty (Group 2). We collected blood to measure serum miRNAs preoperatively and 6 months after surgery. In the 1-month postoperative follow-up, we carried out a clinical evaluation and at 6 months, a Carotid Artery Doppler Ultrasound. Results: of the selected patients, the majority were male (62.3%); hypertensive (91.4%); diabetic (52.6%) and with a previous history of smoking (69.2%). Furthermore, 49% of patients were symptomatic and 23.4% reported coronary artery disease. MiRNA-29a showed a statistically significant drop in expression 6 months after surgical correction (95% CI, from 17.83 to 1.20; p<0.001). miRNA-221 behaved in a similar way (95% CI, from 150.99 to 8.67; p<0.001). Regarding the technique used, Group 1 (95% CI, from 1232.5 to 62.6; p < 0.001) showed a greater drop than Group 2 (95% CI, from 847.6 to 47.5; p < 0.001 ). In the ultrasound evaluation, the drop in the group with normal ultrasound at 6 months in miRNA-29a (95% CI, from 20.4 to 1.2; p = 0.001) and miRNA-221 (95% CI, from 153.9 to 9.5; p<0.001) was greater than the drops in the group with restenosis, both in miRNA-29a (95% CI, from 291.1 to 1.2; p=0.04) and in miRNA-221 (IC 95%, from 243.8 to 8.8; p=0.02). We did not observe any interference from the presence of symptoms preoperatively, coronary artery disease or smoking. Conclusion: the drop in the expression of these miRNAs in the postoperative period may suggest the use of these molecules as serum biomarkers for monitoring these patients.
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Laboratory Examinations Peripheral venous blood samples were obtained on the day of the surgery and 6 months after the procedure. Whole blood was stored in a tube containing ethylenediaminetetraacetic acid (EDTA) and frozen at 80 C. RNA Extraction Total RNA was extracted using the Trizol (Life Technologies, USA) according to the manufacturer’s instructions. Blood samples were collected in EDTA tubes (approximately 4 mL) and were transferred to a 50-mL falcon sterile tube free of RNase, and 20 mL of red cell lysis buffer was added. After 15 min on ice, the mixture was centrifuged again for 10 min at 3000 rpm. The supernatant was discarded, and the pellet was resuspended in 250 mL of phosphate- buffered saline and 750 mL of Trizol and transferred to an Eppendorf tube and processed according to the Trizol protocol. MicroRNA Complementary DNA (cDNA) Synthesis Reverse transcription was performed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For the miRNA cDNA synthesis, for each 5 ng of RNA, we added 0.75 mL of RT buffer, 0.075 mL of dNTP’s, 1.5 mL of specific primers (miRNA or endogenous control) and 0.5 mL of the MultiScribeTM enzyme, 0.094 mL of RNase out (1.9U), filling it up with diethylpyrocarbonate-treated water to amount a total of 7.5 ml. The samples were then placed in a thermal circulator for 30 min at 16°C, 30 min at 42°C, and 5 min at 85°C, cooled to 4°C, and stored in a -20°C freezer. Before using the synthesized cDNA in real-time polymerase chain reaction (PCR), it was diluted in a 1:4 diethylpyrocarbonate-treated water proportion. RQ-PCR of MicroRNAs Real-time PCR was used to confirm differential miRNA expression. Commercially available TaqMan Assay-on-demand (Applied Biosystems) was used for the quantitative expression analysis. The comparative CT method (DDCT) was used to calculate the relative quantification of expression. U6 housekeeping gene was used in this study. Amplification reactions by real-time quantitative polymerase chain reaction (RQ-PCR) were performed twice with Taqman Master Mix (Applied Biosystems, Waltham, Massachusetts, USA) reagent. The reaction had a final volume of 10 mL, using 5 mL of the specific reagent Taqman Master Mix, 0.5 mL of each specific probe, and 4.5 mL of diluted cDNA. The 7500 Fast Real Time PCR System (Applied Biosystems) detection equipment was used with the 7500 Sequence Detection System (Applied Biosystems) to obtain Ct values. The amplification standard conditions were 95°C for 10 min, followed by 40 cycles of 95C for 15 sec and 60C for 1 min. The data were then exported to Excel spreadsheets to calculate the 2-DDCT values. A threshold of 0.1 for the endogenous and studied miRNAs, and the value considered for the deviation between samples was up to 0.5.