Singh Alok Kumar, Pandey Rajeev Kumar, Shaha Chandrima, Madhubala Rentala
a School of Life Sciences, Jawaharlal Nehru University , New Delhi , India.
b Cell Death and Differentiation Research Laboratory, National Institute of Immunology , New Delhi , India.
Autophagy. 2016 Oct 2;12(10):1817-1831. doi: 10.1080/15548627.2016.1203500. Epub 2016 Jul 26.
Leishmania is an obligate intracellular parasite that replicates inside phagolysosomes or parasitophorous vacuoles (PV) of the monocyte/macrophage lineage. It reprograms macrophages and produces a metabolic state conducive to successful infection and multiplication. MicroRNAs (miRNAs), a class of small 22 to 24 nucleotide noncoding regulatory RNAs alter the gene expression and consequently proteome output by targeting mRNAs, may play a regulatory role in modulating host cell functions. In the present study, we demonstrate the novel regulatory role of host microRNA, MIR30A-3p in modulation of host cell macroautophagy/autophagy after infection with L. donovani. Our in vitro studies showed that MIR30A-3p expression was significantly enhanced after L. donovani infection in a time-dependent manner. Transient transfection with a MIR30A-3p inhibitor followed by L. donovani infection promoted the autophagic response and decreased the intracellular parasite burden in both THP-1 cells and human monocyte-derived macrophages (HsMDM). BECN1/Beclin 1, the mammalian ortholog of yeast Vps30/Atg6, is a key autophagy-promoting protein that plays a key role in the regulation of cell death and survival. We report BECN1-dependent modulation of host cell autophagy in response to L. donovani infection. Pretreatment of L. donovani-infected macrophages with the MIR30A-3p mimic decreased and with antagomir increased the expression of BECN1 protein. We demonstrate that BECN1 is a potential target of MIR30A-3p and this miRNA negatively regulates BECN1 expression. Our present study reveals for the first time a novel role of MIR30A-3p in regulating autophagy-mediated L. donovani elimination by targeting BECN1. The present study has significant impact for the treatment of visceral leishmaniasis.
利什曼原虫是一种专性细胞内寄生虫，在单核细胞/巨噬细胞系的吞噬溶酶体或寄生泡（PV）内复制。它对巨噬细胞进行重编程，并产生有利于成功感染和增殖的代谢状态。微小RNA（miRNA）是一类22至24个核苷酸的小非编码调节RNA，通过靶向mRNA改变基因表达，进而影响蛋白质组输出，可能在调节宿主细胞功能中发挥调节作用。在本研究中，我们证明了宿主微小RNA MIR30A-3p在杜氏利什曼原虫感染后对宿主细胞巨自噬/自噬的新型调节作用。我们的体外研究表明，杜氏利什曼原虫感染后，MIR30A-3p的表达呈时间依赖性显著增强。用MIR30A-3p抑制剂进行瞬时转染，随后感染杜氏利什曼原虫，可促进自噬反应，并降低THP-1细胞和人单核细胞衍生巨噬细胞（HsMDM）中的细胞内寄生虫负荷。BECN1/Beclin 1是酵母Vps30/Atg6的哺乳动物同源物，是一种关键的自噬促进蛋白，在细胞死亡和存活的调节中起关键作用。我们报告了杜氏利什曼原虫感染后宿主细胞自噬的BECN1依赖性调节。用MIR30A-3p模拟物预处理杜氏利什曼原虫感染的巨噬细胞会降低BECN1蛋白的表达，而用拮抗剂处理则会增加其表达。我们证明BECN1是MIR30A-3p的潜在靶点，这种miRNA负向调节BECN1的表达。我们目前的研究首次揭示了MIR30A-3p通过靶向BECN1在调节自噬介导的杜氏利什曼原虫清除中的新作用。本研究对内脏利什曼病的治疗具有重要意义。
