Hojati Zohreh, Ganjalikhani-Hakemi Mazdak, Ameri Mahnaz, Alimohammadi-Jelodar Seyedeh Fatemeh, Dehbashi Moein, Mohammad Ganji Maryam, Homayouni Vida, Khanahmad Hossein
Division of Genetics, Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, 81746-73441 Iran.
Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
Indian J Clin Biochem. 2020 Jul;35(3):359-366. doi: 10.1007/s12291-019-00834-z. Epub 2019 Jun 4.
Acute myelogenous leukemia (AML) is a complex blood malignancy leading to immature leukemic stem cells (LSCs) proliferation. T cell immunoglobulin mucin-3 (TIM-3) is known as a biomarker of AML LSCs. Several microRNAs (miRNAs) can affect gene expression in AML. In this study, the silencing effect of miR-133a-5p on TIM-3 expression in AML cell lineage (HL-60) was investigated. It's been hypothesized that miR-133a-5p may suppress the TIM-3 expression in AML cell line. Initially, miRNA-TIM-3 prediction, enrichment, and network analysis were done. Then, miR-133a-5p mimic was transfected into HL-60 cells. The TIM-3 protein and gene expression were measured by flow cytometry analysis and real-time PCR, respectively. MTT assay was also carried out. Based on the Bioinformatics predictions, miR-133a-5p was able to silence TIM-3 expression. Also, significant pathways pertained to miR-133a-5p were obtained using enrichment analysis. According to this, miR-133a-5p was mainly engaged in the MAPK signaling pathway and Nicotine addiction pathway using the KEGG database. The TIM-3 protein expression of the transfected cells was measured as 17.15 ± 8.87% ( = 0.001). A 52.48% significant gene silencing in mRNA level was obtained in comparison to the negative control. Despite of down regulation of TIM-3, HL-60 cell viability has not been significantly changed. It has been finally confirmed that miR-133a-5p could strongly suppress TIM-3 expression in AML cell line. Presumably, down regulation of TIM-3 could affect MAPK and Nicotine addiction signaling pathways.
急性髓系白血病（AML）是一种复杂的血液恶性肿瘤，可导致不成熟的白血病干细胞（LSC）增殖。T细胞免疫球蛋白粘蛋白-3（TIM-3）是AML-LSC的生物标志物。几种微小RNA（miRNA）可影响AML中的基因表达。在本研究中，研究了miR-133a-5p对AML细胞系（HL-60）中TIM-3表达的沉默作用。据推测，miR-133a-5p可能抑制AML细胞系中的TIM-3表达。首先，进行了miRNA-TIM-3预测、富集和网络分析。然后，将miR-133a-5p模拟物转染到HL-60细胞中。分别通过流式细胞术分析和实时PCR测量TIM-3蛋白和基因表达。还进行了MTT试验。基于生物信息学预测，miR-133a-5p能够沉默TIM-3表达。此外，使用富集分析获得了与miR-133a-5p相关的重要信号通路。据此，使用KEGG数据库，miR-133a-5p主要参与丝裂原活化蛋白激酶（MAPK）信号通路和尼古丁成瘾通路。转染细胞的TIM-3蛋白表达测定为17.15±8.87%（P=0.001）。与阴性对照相比，在mRNA水平上获得了52.48%的显著基因沉默。尽管TIM-3下调，但HL-60细胞活力未发生显著变化。最终证实，miR-133a-5p可强烈抑制AML细胞系中的TIM-3表达。据推测，TIM-3的下调可能影响MAPK和尼古丁成瘾信号通路。
