Department of Biochemistry, Faculty of Science, Payame Noor University, Tehran, Iran.
Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran.
Mol Biol Rep. 2019 Oct;46(5):5371-5388. doi: 10.1007/s11033-019-04992-4. Epub 2019 Aug 5.
Drug resistance is a complex phenomenon during leishmaniasis chemotherapy. In this study, the genes and pathways involved in miltefosine (MIL)-resistant Leishmania were identified using microarray data and in silico approaches. GSE30685 and GSE45496 were obtained from GEO database and analyzed with GEO2R tool to identify genes involved in MIL-resistant Leishmania. 177 differentially expressed genes (DEGs) were selected from these GSEs, which about half of them were uncharacterized/hypothetical proteins. The interactions between DEGs were investigated using STRING database and protein-protein interaction (PPI) networks. Five hub nodes were found in the PPI network. The gene ontology (GO) analysis of the resulting network revealed that DNA replication (GO:0006260) and ATP hydrolysis coupled proton transport (GO:0015991) were the most enriched GO term. Iranian MIL-resistant Leishmania major (L. major) parasites were generated by exposure of wild-type isolates to the increasing concentrations of MIL over a period of 5 months. Proof of mRNA expression levels of the obtained hub genes was assessed in Iranian wild-type and acquired resistant L. major parasites by real-time PCR. A significant higher expression level of LDBPK_150170 (encoding protein phosphatase 2C, PP2C), was only observed in Iranian L. major parasites resistance to MIL. Moreover, the RT-PCR results showed that the expression of metacyclic marker (small hydrophilic endoplasmic reticulum-associated protein, SHERP) and MIL-resistant marker (Leishmania MIL-transporter, LMT) was significantly increased and decreased, respectively, in Iranian MIL-resistant L. major parasites. Taken together, these data suggested that PP2C as well as SHERP and LMT genes may be prospective targets for the treatment of MIL-resistant Leishmania.
耐药性是利什曼病化疗中的一种复杂现象。在这项研究中，使用微阵列数据和计算机方法鉴定了米替福新（MIL）耐药利什曼原虫涉及的基因和途径。从 GEO 数据库中获取 GSE30685 和 GSE45496，并使用 GEO2R 工具进行分析，以鉴定与 MIL 耐药利什曼原虫相关的基因。从这些 GSE 中选择了 177 个差异表达基因（DEGs），其中约一半为未鉴定/假设蛋白。使用 STRING 数据库和蛋白质-蛋白质相互作用（PPI）网络研究 DEGs 之间的相互作用。在 PPI 网络中发现了五个枢纽节点。对所得网络的基因本体（GO）分析表明，DNA 复制（GO：0006260）和 ATP 水解偶联质子转运（GO：0015991）是最丰富的 GO 术语。通过在 5 个月的时间内将野生型分离物暴露于越来越高的 MIL 浓度下，生成了伊朗 MIL 耐药利什曼原虫（L. major）寄生虫。通过实时 PCR 评估从伊朗野生型和获得的耐药 L. major 寄生虫中获得的枢纽基因的 mRNA 表达水平的证明。仅在伊朗 L. major 寄生虫对 MIL 耐药时，观察到 LDBPK_150170（编码蛋白磷酸酶 2C，PP2C）的表达水平显着升高。此外，RT-PCR 结果表明，伊朗 MIL 耐药 L. major 寄生虫中梅塔环标记（小亲水内质网相关蛋白，SHERP）和 MIL 耐药标记（利什曼 MIL 转运蛋白，LMT）的表达分别显着增加和减少。总之，这些数据表明 PP2C 以及 SHERP 和 LMT 基因可能是治疗 MIL 耐药利什曼的潜在靶标。
