A novel approach to identify the mechanism of miR-145-5p toxicity to podocytes based on the essential genes targeting analysis


Posted: 2021-11-03 19:00:00
Mol Ther Nucleic Acids . 2021 Sep 20;26:749-759. doi: 10.1016/j.omtn.2021.09.005. eCollection 2021 Dec 3. Sipan Zhang 1 , Junnan Wu 1 , Xiaodong Zhu 1 , Hui Song 1 , Lu Ren 1 , Qiaoli Tang 1 , Xiaodong Xu 1 , Chunbei Liu 1 , Jiong Zhang 1 , Weixin Hu 1 , Zhihong Liu 1 , Shaolin Shi 1 Affiliations Expand Affiliation 1 National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu 21002, China. Item in Clipboard Sipan Zhang et al. Mol Ther Nucleic Acids. 2021. Show details Display options Display options Format Mol Ther Nucleic Acids . 2021 Sep 20;26:749-759. doi: 10.1016/j.omtn.2021.09.005. eCollection 2021 Dec 3. Authors Sipan Zhang 1 , Junnan Wu 1 , Xiaodong Zhu 1 , Hui Song 1 , Lu Ren 1 , Qiaoli Tang 1 , Xiaodong Xu 1 , Chunbei Liu 1 , Jiong Zhang 1 , Weixin Hu 1 , Zhihong Liu 1 , Shaolin Shi 1 Affiliation 1 National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu 21002, China. Item in Clipboard CiteDisplay options Display options Format Abstract MicroRNAs (miRNAs) are emerging as effective therapeutic agents. When testing whether miR-145-5p could alleviate kidney injury, we unexpectedly found that extracellular vesicles loaded with miR-145-5p induced proteinuria and podocyte foot process effacement in normal control mice. To explore the mechanism of miR-145-5p's toxicity to podocytes, we hypothesized that miR-145-5p could enter podocytes and inhibit genes essential for podocytes. We demonstrated that systemically administered miRNA can enter podocytes. Next, we predicted 611 podocyte essential genes based on single-cell RNA sequencing (RNA-seq) and found that 32 of them are predicted to be targeted by miR-145-5p. Functional annotation of the 32 podocyte essential genes revealed small GTPase-mediated signal transduction as the top pathway. We experimentally validated that miR-145-5p targeted Arhgap24 and Srgap1, the essential regulators of the Rho family of small GTPases, increased the activity of Rac1 and Cdc42, and reduced RhoA activity, accompanied by cellular injury, in podocytes. These results explain how miR-145-5p has deleterious effect on podocytes. Most importantly, our study provides a novel approach to investigate how a miRNA affects a given cell type, allowing not only identification of the molecular mechanism underlying an observed side effect of a miRNA drug but also prediction of miRNA drug toxicity on various cell types. Keywords: Arhgap24; essential genes; miR-145-5p; podocyte; single-cell RNA-seq; small GTPases; toxicity. © 2021 The Author(s). Conflict of interest statement The authors declare no competing interests. Figures Graphical abstract Graphical abstract Graphical abstract Figure 1 miR-145-5p induced podocyte injury in… Figure 1 miR-145-5p induced podocyte injury in mice (A) Time course of the development of… Figure 1 miR-145-5p induced podocyte injury in mice (A) Time course of the development of proteinuria urine albumin/creatinine ratio (ACR) in the mice treated with miR-145-5p EVs in the presence (miR-145-5p EVs +inhibitor) and absence (miR-145-5p EVs) of miR-145-5p inhibitor. miR-145-5p EV sample and inhibitor were administered on days 1, 2, 3, 4, 5, and 6. (B and C) Electronic microscopy (EM) examination revealed apparent foot process effacement of the podocytes in mice treated with miR-145-5p EVs, which was reversed by miR-145-5p inhibitor. ∗p 0.05. Data are represented as mean ± SD. Figure 2 Tissue distribution of injected EVs… Figure 2 Tissue distribution of injected EVs and the miRNA in a mouse (A) Fluorescence… Figure 2 Tissue distribution of injected EVs and the miRNA in a mouse (A) Fluorescence imaging of the mouse injected with DiR-labeled EVs through tail vein. The image was taken 30 min after the injection, showing predominant fluorescence signal in liver as well as other tissues. (B) Fluorescence imaging of individual organs from the same mouse, showing that EVs were accumulated in liver, kidney, and spleen but not muscles. (C) Confocal imaging of Cy5-miR-145-5p in an isolated glomerulus 48 h after the Cy5-miR-145-5p-loaded EVs were injected via tail vein to a Npsh2-cre/eGFP transgenic mouse whose podocytes express eGFP. Arrows: Cy5-miR-145-5p-loaded EVs accumulated in podocytes. Scale bar: 20 μm. (D) In situ hybridization of artificial miR# in kidney after the miR#-containing EVs were injected via tail vein to a mouse. It was clearly shown that miR# was delivered to all glomerular cells, including podocytes that are localized at periphery of each glomerular tuft. Arrows: miR#-loaded EVs accumulated in podocytes. G, glomerulus; T, tubule. Scale bar: 20 μm. Figure 3 Toxicity of miR-145-5p on podocytes… Figure 3 Toxicity of miR-145-5p on podocytes in culture (A) Fluorescent EVs were incubated with… Figure 3 Toxicity of miR-145-5p on podocytes in culture (A) Fluorescent EVs were incubated with podocytes in culture, and the fluorescence was observed in the cells after 12 h. Scale bar: 100 μm. (B) Phalloidin staining of cultured podocytes treated with miR-145-5p EVs, showing a reduction of F-actin stress fibers in the cells. Scale bar: 50 μm. (C) Quantifications of F-actin stress fibers in cultured podocytes after transfection of miR-145-5p mimic and scramble control, which shows that miR-145-5p mimic induced a reduction of F-actin stress fibers. The results represented data from three independent experiments. ∗p 0.05, statistically significant. Data are represented as mean ± SD. Figure 4 Schematic diagram of the approach… Figure 4 Schematic diagram of the approach for prediction of the toxicity of a miRNA… Figure 4 Schematic diagram of the approach for prediction of the toxicity of a miRNA based on its targeting of the essential genes of a cell type Figure 5 GO analysis of the 32… Figure 5 GO analysis of the 32 podocyte essential genes of miR-145-5p potential target Figure 5 GO analysis of the 32 podocyte essential genes of miR-145-5p potential target Figure 6 miR-145-5p targeted Arhgap24 and increased… Figure 6 miR-145-5p targeted Arhgap24 and increased activity of Rac1 and Cdc42 in podocytes (A)… Figure 6 miR-145-5p targeted Arhgap24 and increased activity of Rac1 and Cdc42 in podocytes (A) Luciferase reporter assay: the reporter constructs contained Arhgap24 3′ UTRs with or without mutations in the miR-145-5p binding site downstream of Luc open reading frame (ORF) were prepared and were co-transfected with miR-145-5p mimic into cultured podocytes. (B) Immunoblotting of the proteins of Arhgap24 in the cultured podocytes after transfection with miR-145-5p mimic and scramble control, showing that the Arhgap24 protein was significantly downregulated by miR-145-5p. (C) Immunofluorescence staining of cultured podocytes showed downregulation of Arhgap24 protein by the miR-145-5p in EVs, and the effect of miR-145-5p EVs was abolished by the inhibitor. Scale bar: 100 μm. (D) Immunohistochemical staining of the Arhgap24 proteins in the glomeruli of mice treated as indicated. Scale bar: 40 μm. ∗p 0.05, statistically significant. (E) miR-145-5p upregulated Rac1 and Cdc42 activities but reduced RhoA activity as shown by immunoblotting of the total and active form (PD, pulldown) of the proteins in cultured podocytes. ∗p 0.05, statistically significant. Data are represented as mean ± SD. Figure 7 miR-145-5p significantly reduced spreading and… Figure 7 miR-145-5p significantly reduced spreading and adhesion of the podocytes and induced injury in… Figure 7 miR-145-5p significantly reduced spreading and adhesion of the podocytes and induced injury in cultured podocytes (A) RTCA of cultured podocytes transfected with miR-145-5p mimic. (B) RTCA of cultured podocytes transfected with scramble control. (C) Immunofluorescence staining of vinculin, a marker and component of focal adhesion, showing a great reduction of the protein in the cultured podocytes transfected with miR-145-5p. Scale bar: 50 μm. (D) Annexin V analysis of podocytes treated with miR-145-5p indicated increased apoptosis of the cells compared with scramble control. (E) Immunoblotting of synaptopodin and CD2AP showing their downregulation in cultured podocytes treated with miR-145-5p. Data are represented as mean ± SD. The results represented data from three independent experiments. ∗p 0.05, statistically significant. Figure 8 The top 50 miRNAs detected… Figure 8 The top 50 miRNAs detected in mouse podocytes by RNA sequencing Figure 8 The top 50 miRNAs detected in mouse podocytes by RNA sequencing All figures (9) References Lu J., Getz G., Miska E.A., Alvarez-Saavedra E., Lamb J., Peck D., Sweet-Cordero A., Ebert B.L., Mak R.H., Ferrando A.A. MicroRNA expression profiles classify human cancers. Nature. 2005;435:834–838. - PubMed Johnson S.M., Grosshans H., Shingara J., Byrom M., Jarvis R., Cheng A., Labourier E., Reinert K.L., Brown D., Slack F.J. RAS is regulated by the let-7 microRNA family. Cell. 2005;120:635–647. - PubMed Tazawa H., Tsuchiya N., Izumiya M., Nakagama H. Tumor-suppressive miR-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells. Proc. Natl. Acad. Sci. USA. 2007;104:15472–15477. - PMC - PubMed Xu N., Papagiannakopoulos T., Pan G., Thomson J.A., Kosik K.S. MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells. Cell. 2009;137:647–658. - PubMed Chang T.C., Wentzel E.A., Kent O.A., Ramachandran K., Mullendore M., Lee K.H., Feldmann G., Yamakuchi M., Ferlito M., Lowenstein C.J. Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis. Mol. Cell. 2007;26:745–752. - PMC - PubMed Show all 47 references LinkOut - more resources Research MaterialsMiscellaneous

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バイオクイックニュース日本語版:エクソソーム特集

バイオクイックニュース日本語版
8月 01, 2019 バイオアソシエイツ

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