Decreased HD-MIR2911 absorption in human subjects with the SIDT1 polymorphism fails to inhibit SARS-CoV-2 replication

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CAS Article Google Scholar Download references This work was supported by grants from the Chinese Science and Technology Major Project of China (2015ZX09102023-003), National Key Research and Development Plan (2018YFA0507100), National Basic Research Program of China (973 Program) (2014CB542300 and 2012CB517603), National Natural Science Foundation of China (81250044, 81602697, and 31741075), the Natural Science Foundation of Jiangsu Province (BE2016737), and the Fundamental Research Funds for the Central Universities (020814380130, 020814380133, 020814380137, and 020814380146). These authors contributed equally: Zhen Zhou, Yu Zhou, Xia-Ming Jiang, Yanbo Wang Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), NJU Institute of AI Biomedicine and Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu 210023, China Zhen Zhou, Yu Zhou, Yanbo Wang, Xi Chen, Chen-Yu Zhang & Liang Li State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, Hubei 430071, China Xia-Ming Jiang, Gengfu Xiao & Lei-Ke Zhang Department of Critical Care Medicine and Nanjing infectious Disease Center, The Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210003, China Yongxiang Yi You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar L.L., L.Z, Y.Y., and C.Z. conceived and designed the experiments; Z.Z., Z.Y., X.J., Y.W. participated in multiple experiments; Z.Z., G.X., C.Z., L.L., X.C. analyzed the data. Z.Z., C.Z., L.L. wrote the manuscript. C.Z., Y.Y., L.Z., and L.L. provided the final approval of the manuscript. Correspondence to Chen-Yu Zhang or Yongxiang Yi or Lei-Ke Zhang or Liang Li. The authors declare that they have no conflict of interest. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and Permissions Zhou, Z., Zhou, Y., Jiang, X. et al. Decreased HD-MIR2911 absorption in human subjects with the SIDT1 polymorphism fails to inhibit SARS-CoV-2 replication. Cell Discov 6, 63 (2020). https://doi.org/10.1038/s41421-020-00206-5 Download citation Received: 01 June 2020 Accepted: 14 August 2020 Published: 11 September 2020 DOI: https://doi.org/10.1038/s41421-020-00206-5


Low pH treatment
The pH of the culture medium was adjusted with hydrochloric acid to pH 3.5, and an unmodified culture medium (approximately pH 7.4) was used as a control. Synthetic miRNAs were directly added to the medium with different pH values (40 pmol/ml). Then miRNA and culture medium mixture was incubated with cultured cells for 30 min. At last, the cells were washed three times and incubated with FBS-free medium containing RNase A at 37 ℃ for 1 h to remove extracellularly attached miRNAs.

Human blood Sample Collection
To detect the baseline level of exogenous miRNA in serum, we obtained 135 random adult volunteers seeking routine checkups at Jinling Hospital, Nanjing, China, according to protocols approved by the ethics committee of Jinling Hospital. All blood donors contribute one whole blood sample and one serum sample, respectively.
To test the impact of SIDT1 polymorphism on miRNA uptake, 10 volunteers were divided into two groups, the control group (SIDT1 wt , n=5) and the SIDT1 polymorphism group (SIDT1 poly , n=5). Each donor was fed 200 ml honeysuckle decoction. Serum samples were collected before and 0.5, 1, 3, 6 hours after honeysuckle decoction treatment, respectively.
To assess the function of exosomal MIR2911 in serum, 8 volunteers were divided into two groups, the control group (SIDT1 wt , n=5) and the SIDT1 polymorphism group (SIDT1 poly , n=3). Each donor was fed 200 ml honeysuckle decoction. Serum samples were collected before and 2 hours after honeysuckle decoction treatment.

Genotyping
To analyze the SNP rs2271496 of sidt1, genomic DNA was extracted from 100 μl whole blood sample of each volunteer and purified with the DNeasy Blood & Tissue kit (TIAGEN), according to the manufacturer's instruction. For genotyping, we designed a pair of primers with the sequence TACAGTCCTTGGAAGAGCAGAG/TGTGCATGTGCACGTTTTGCAT. After 35 cycles of amplification by multiplex PCR, the PCR product was purified by ExoI (Fermentas) and FastAP (Fermentas) to remove excess primers and dNTPs. 2μL of purified PCR product was mixed with 1μL Snapshot Mix (ABI), 0.1μL extension primer (CCCTGCCATGCAGGTGACAGCC) and 2.9μL milliQ water. The following extension reaction was employed: 30 cycles (96 °C for 10 sec, 52°C for 5 sec, 60°C for 30 sec). After the extension product was added to the loading buffer, it was denatured at 95 degrees Celsius for 3 minutes and ice cold immediately, then sequenced by a 3730XL sequencer (ABI).

Exosome isolation
For exosome collection from culture cells, HEK293T cells were transfected with miRNA by using Lipofectomine 2000 (invitrogen). After 36 hours, exosomes were isolated from the cell culture medium using a Total Exosome Isolation Reagent (Invitrogen) according to the manufacturer's instructions.
For human serum exosome collection, exosomes were isolated from human serum samples using a Serum Exosome Isolation Kit (Vazyme) according to the manufacturer's instructions.
Exosome pellets were collected and re-suspended in PBS.

RNA isolation and quantitative RT-PCR assays
Small RNAs (<100 nt) from honeysuckle decoction were extracted using the Universal Plant MicroRNA Kit (Biotech) according to manufacturer's instructions. Total RNAs from human serum, human exosomes, cell exosomes were extracted using TRIZOL reagent according to the manufacturer's instructions.
To detect MIR2911 level, quantitative RT-PCR was performed using TaqMan miRNA probes (Applied Biosystems) using an LC96 PCR machine according (Roche) to the manufacturer's instructions. A series of synthetic MIR2911 oligonucleotides at known concentrations was reverse transcribed and amplified to build standard curve. The absolute amount of MIR2911 was then calculated in reference to the standard curve.

miRNA target prediction
SARS-CoV-2 genome sequence was acquired from the NCBI database (Refseq ID: MN908947). RNAhybrid 1 was used to search potential MIR2911 binding sites on SARS-COV-2 S gene.

Evaluation of inhibitory effect on S protein of synthetic MIR2911 and exosomes
To test the inhibitory effect on S protein of synthetic MIR2911, HEK293 cells were transfected with GFP-S protein plasmid (Genscript) and synthetic MIR2911 or NC by using Lipofectamine 2000. At 24 h post treatment, fluorescent and cytometric analysis were employed to test the expression level of GFP-S protein.
To test the inhibitory effect on S protein of exosomes, HEK293T cells were cultured overnight in 12-well cell culture petridish and then incubated with exosomes isolated from 6 ml cell medium or 250 μl serum from different volunteers for 8 hours. The cells were then transfected with GFP-S protein plasmid by using Lipofectamine 2000. At 24 h post treatment, fluorescent and cytometric analysis were employed to test the expression level of GFP-S protein.