Volume 31, Issue 1 (3-2024)
RJMS 2024, 31(1): 1-9 |
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Research code: مصوب دانشگاه آزاد
Ethics code: IR.TUMS.EMRI.REC.1398.008
Clinical trials code: کارآزمایی نمی باشد
Pediatric Growth and Development Research Center, Institute of Endocrinology and Metabolism, Iran University of Medical Sciences, Tehran, Iran , nekouian.r@iums.ac.ir
Abstract: (405 Views)
Background & Aims: Papillary thyroid carcinoma (PTC) is the most prevalent thyroid gland tumor arising from thyroid follicular cells. MicroRNAs (miRs) are endogenous single‑stranded non‑coding RNAs attaching to the different sites of the target mRNAs, leading to regulation of gene expression. Hence, miRs play a role in biological functions. Diagnostic role of miRs in PTC has been previously studied. However, the results of such molecular studies should be reinvestigated among different ancestries worldwide. Despite the fact, other than diagnosis, it is vital to have capability to discriminate aggressive cases from the non-aggressive ones to prevent over-diagnosis and over-treatment. Hence, the current investigation was performed to study the association of let-7f, miR-146b-5p, miR-34b, miR-16 and miR-877-5p expression in blood serum and tumor tissue with aggressiveness of PTC in an Iranian population.
Methods: This was a case control study. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was used to compare blood and tumor tissue expression of the miRs between aggressive and non-aggressive PTC samples. Eighteen patients with aggressive PTC were considered as the case group and 18 patients with non-aggressive PTC as the control group. Samples were taken from three hospitals as Toos, Shariyati and Firoozgar (Tehran, Iran). A written informed consent was obtained from each participant. Fresh tissue specimens of aggressive and non-aggressive PTC tissues were obtained from tumors and instantly kept at -80 °C until assessment. Two independent pathologists assessed the samples. PTC was confirmed by light microscope using hematoxylin and eosin staining. Total RNA was extracted with columnar extraction method using total RNA purification kit (Norgen, Canada) according to the manufacturer's instructions. Sample quality control was using cel-mir-39 Spike-In Kit (Norgen, Canada) which offers a quantified synthetic RNA (cel-miR-39) for spike-in. Cel-mir-39 was used for normalization in RT-qPCR assay as the reference to state ∆Ct. cDNA was synthesized with poly A polymerase technique using microScript microRNA cDNA Synthesis kit (Norgen, Canada). Afterwards, the cDNA was used for real-time reverse transcriptase polymerase chain reaction (RT-PCR) using SYBR Green mastermix (Norgen, Canada). Fold changes (FC) of each miR were computed via the formula 2-∆∆CT in Excel 2013 calibrating with the mean of expression in non-aggressive PTC patients. Significance of individual FCs was assessed with one sample t test (FC = 1 was the null hypothesis). Besides, FC between blood serum and tumor tissue was performed through independent t test at the significance level of 0.05.
Results: MiR-16 had a meaningful surge in blood (FC = 2.85; P = 0.024), while miR-34 showed a meaningful down-regulation in blood (FC = 0.19; P < 0.001) and tumor tissue (FC = 0.19; P < 0.001). In addition, miR-146 had a major up-regulation in blood (FC = 48.10; P < 0.001) and tumor tissue (FC = 60.61; P < 0.001), miR-877 a momentous down-regulation in blood (FC = 0.22; P < 0.001), and let-7 a meaningful down-regulation in blood (FC = 0.09; P < 0.001) and tumor tissue (FC = 0.13; P < 0.001). Independent t test was used to compare FC of each miR (blood expression versus tumor tissue expression). MiR-16 exhibited a meaningful up-regulation in aggressive PTC samples (2.85 vs 0.92; P = 0.020). However, no major difference was reported between blood and tumor tissue FCs for other miRs.
Conclusion: MiRs can help to differentiate invasive PTC from non-invasive PTC. Concisely, miR-16 and miR-877 had an upregulation in blood, miR-34 and let-7 a down regulation in blood and tumor tissue, miR-146 an upregulation in blood and tumor tissue, and miR-146 a significant up regulation in blood and tumor tissue. Expression alteration of miR-16 was meaningfully more prevailing in blood compared with tumor tissue in favor of upregulation. Among the studied miRs, miR-146 was the most frequently occurring result in this study and previous studies. Predictive role of these biomarkers should be assessed in further well-designed cohort investigations, then the results can be utilized as personalized medicine in the management of PTC.
Methods: This was a case control study. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was used to compare blood and tumor tissue expression of the miRs between aggressive and non-aggressive PTC samples. Eighteen patients with aggressive PTC were considered as the case group and 18 patients with non-aggressive PTC as the control group. Samples were taken from three hospitals as Toos, Shariyati and Firoozgar (Tehran, Iran). A written informed consent was obtained from each participant. Fresh tissue specimens of aggressive and non-aggressive PTC tissues were obtained from tumors and instantly kept at -80 °C until assessment. Two independent pathologists assessed the samples. PTC was confirmed by light microscope using hematoxylin and eosin staining. Total RNA was extracted with columnar extraction method using total RNA purification kit (Norgen, Canada) according to the manufacturer's instructions. Sample quality control was using cel-mir-39 Spike-In Kit (Norgen, Canada) which offers a quantified synthetic RNA (cel-miR-39) for spike-in. Cel-mir-39 was used for normalization in RT-qPCR assay as the reference to state ∆Ct. cDNA was synthesized with poly A polymerase technique using microScript microRNA cDNA Synthesis kit (Norgen, Canada). Afterwards, the cDNA was used for real-time reverse transcriptase polymerase chain reaction (RT-PCR) using SYBR Green mastermix (Norgen, Canada). Fold changes (FC) of each miR were computed via the formula 2-∆∆CT in Excel 2013 calibrating with the mean of expression in non-aggressive PTC patients. Significance of individual FCs was assessed with one sample t test (FC = 1 was the null hypothesis). Besides, FC between blood serum and tumor tissue was performed through independent t test at the significance level of 0.05.
Results: MiR-16 had a meaningful surge in blood (FC = 2.85; P = 0.024), while miR-34 showed a meaningful down-regulation in blood (FC = 0.19; P < 0.001) and tumor tissue (FC = 0.19; P < 0.001). In addition, miR-146 had a major up-regulation in blood (FC = 48.10; P < 0.001) and tumor tissue (FC = 60.61; P < 0.001), miR-877 a momentous down-regulation in blood (FC = 0.22; P < 0.001), and let-7 a meaningful down-regulation in blood (FC = 0.09; P < 0.001) and tumor tissue (FC = 0.13; P < 0.001). Independent t test was used to compare FC of each miR (blood expression versus tumor tissue expression). MiR-16 exhibited a meaningful up-regulation in aggressive PTC samples (2.85 vs 0.92; P = 0.020). However, no major difference was reported between blood and tumor tissue FCs for other miRs.
Conclusion: MiRs can help to differentiate invasive PTC from non-invasive PTC. Concisely, miR-16 and miR-877 had an upregulation in blood, miR-34 and let-7 a down regulation in blood and tumor tissue, miR-146 an upregulation in blood and tumor tissue, and miR-146 a significant up regulation in blood and tumor tissue. Expression alteration of miR-16 was meaningfully more prevailing in blood compared with tumor tissue in favor of upregulation. Among the studied miRs, miR-146 was the most frequently occurring result in this study and previous studies. Predictive role of these biomarkers should be assessed in further well-designed cohort investigations, then the results can be utilized as personalized medicine in the management of PTC.
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