Construction and Optimization of shRNA/siRNA Lentivral Vectors for microRNA-486-5p Using miR-30/451 and miRNA Sponges
1. Department of Food Science and Engineering, Moutai Institute, Renhuai 564507, China
2. Department of Biology and Chemistry, College of Sciences, National University of Defense Technology, Changsha 410073, China
| Abstract: | By analyzing the hairpin structure of miR-486, it was found that the mature sequences of miR-486-5p and miR-486-3p are complementary. For this reason, it is probably the greatest challenge to express miR-486-5p using a lentiviral expression vector in C2C12 cells. Thus, we constructed shRNA/siRNA lentiviral expression vectors for microRNA-486-5p based on miR-30/451 and miRNA sponges. Oligonucleotide sequences (miR-486-5p TuD (tough decoy RNAs), miR-486-5p sponge, miR-486-5p-shRNA30 and miR-486-5p-shRNA451) were digested and cloned into the PLV-U6-MCS vector to generate U6-driven expression cassettes for mmu-miR-486-5p, and these vectors, including anti-miR-486-5p TuD, anti-miR-486-5p sponge, miR-486-5p-shRNA30 and miR-486-5p-shRNA451, were assessed by sequencing. The lentivirus vector was packaged in 293T cells according to the manufacturer’s instructions. Subsequently, C2C12 cells were infected using a lentivirus for the expression of miR-486-5p, and this lentivirus of PLV-U6-MCS served as a control. qRT-PCR analysis showed that anti-miR-486-5p TuD and the anti-miR-486-5p sponge decreased the expression level of miR-486-5p in C2C12 cells by 0.5-fold and 0.7-fold, respectively. Additionally, miR-486-5p-shRNA30 and miR-486-5p-shRNA451 upregulated the expression level of miR-486-5p in C2C12 cells by 15-fold and 3-fold, respectively. However, the expression level of miR-486-3p was increased up to 1000-fold in C2C12 cells containing the miR-486-5p-shRNA30 lentivirus, while other lentiviruses only led to 3-fold increases compared with the miR-486–5p-shRNA30 lentivirus in C2C12 cells. Additionally, western blot and gray density analysis demonstrated that the expression levels of AKT, and pAKT, ERK1/2 and pERK1/2, and Snail were upregulated (approximately 1.20-fold to 1.33-fold and 1.05-fold to 1.20-fold, respectively) in C2C12 cells infected with the anti-miR-486-5p TuD lentivirus and anti-miR-486-5p sponge lentivirus and downregulated (approximately 0.63–0.96 times and 0.72–0.92 times, respectively) in C2C12 cells infected with the miR-486-5p-shRNA30 lentivirus and miR-486-5p-shRNA451 lentivirus. In summary, anti-miR-486-5p TuD, which dramatically reduced the expression level of miR-486-5p and upregulated endogenous protein levels in C2C12 cells, was superior to the anti-miR-486-5p sponge in terms of interference expression of miR-486-5p. miR-486-5p-shRNA451, which did not lead to substantial expression of miR-486-3p and remarkably downregulated endogenous protein levels in C2C12 cells, was better than miR-486-5p-shRNA30 for overexpression of miR-486-5p. These results suggested that anti-miR-486-5p TuD and miR-486-5p-shRNA451 provide a simple way to build long-term effective overexpression or interference of miR-486-5p, to study miRNA function in C2C12 and mice, and to create a potential diagnostic method based on materials for diseases caused by miRNA deregulation. |
| Keywords: | miR-486; miR-486-5p; siRNA and shRNA Vector; C2C12; Lentivirus Vector; Tough Decoy (TuD) RNA; miR-486 spOnge; miR30-based and miR451-based shRNA Vector |
| DOI: | 10.57237/j.life.2025.01.002 |
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收稿日期: 2024-08-21
接受日期: 2024-11-29
在线出版日期: 2025-07-04
基金
| 1. | Guizhou Provincial Basic Research Program (Natural Science), (No. QiankeHeJiChu-ZK[2024]-YiBan-659) |
| 2. | The Youth Science and Technology Talent Growth Project of Guizhou Province Education Department (No. QianJiaoKe[2024]262) |
| 3. | Zunyi Technology and Big Data Bureau, Moutai Institute Joint Science and Technology Research and Development Project, grant number: (ZSKHHZ[2021]No.332) |
| 4. | The Natural Science Foundation of Hunan Province, grant number: (CX20190003) |
| 5. | The Research Foundation for Scientific Scholars of Moutai Institute, grant number: (mygccrc [2023]011) |
参考文献
| [1] | KIM V N, HAN J, SIOMI M C. Biogenesis of small RNAs in animals [J]. Nat Rev Mol Cell Biol, 2009, 10(2): 126-139. |
| [2] | KROL J, LOEDIGE I, FILIPOWICZ W. The widespread regulation of microRNA biogenesis, function and decay [J]. Nat Rev Genet, 2010, 11(9): 597-610. |
| [3] | PRITCHARD C C, CHENG H H, TEWARI M. MicroRNA profiling: approaches and considerations [J]. Nat Rev Genet, 2012, 13(5): 358-369. |
| [4] | DEVEALE B, SWINDLEHURST-CHAN J, BLELLOCH R. The roles of microRNAs in mouse development [J]. Nat Rev Genet, 2021, 22(5): 307-323. |
| [5] | DRAGOMIR M P, KNUTSEN E, CALIN G A. SnapShot: Unconventional miRNA Functions [J]. Cell, 2018, 174(4): 1038-1038 e1031. |
| [6] | KOZOMARA A, BIRGAOANU M, GRIFFITHS-JONES S. miRBase: from microRNA sequences to function [J]. Nucleic acids research, 2019, 47(D1): D155-D162. |
| [7] | ELKHOULY A M, YOUNESS R A, GAD M Z. MicroRNA-486-5p and microRNA-486-3p: Multifaceted pleiotropic mediators in oncological and non-oncological conditions [J]. Non-coding RNA research, 2020, 5(1): 11-21. |
| [8] | ALEXANDER M S, CASAR J C, MOTOHASHI N, et al. Regulation of DMD pathology by an ankyrin-encoded miRNA [J]. Skeletal muscle, 2011, 1(27): 1-17. |
| [9] | SMALL E M, O'ROURKE J R, MORESI V, et al. Regulation of PI3-kinase/Akt signaling by muscle-enriched microRNA-486 [J]. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(9): 4218-4223. |
| [10] | SHENOY A, BLELLOCH R H. Regulation of microRNA function in somatic stem cell proliferation and differentiation [J]. Nat Rev Mol Cell Biol, 2014, 15(9): 565-576. |
| [11] | ALEXANDER M S, CASAR J C, MOTOHASHI N, et al. MicroRNA-486-dependent modulation of DOCK3/PTEN/AKT signaling pathways improves muscular dystrophy-associated symptoms [J]. J Clin Invest, 2014, 124(6): 2651-2667. |
| [12] | SUN Y H, LIU Z F, YANG B B, et al. MicroRNA-486 inhibits cell proliferation, invasion and migration via down-regulating the TENM1 expressions and affecting ERK and Akt signaling pathways and epithelial-to-mesenchymal transition in papillary thyroid carcinoma [J]. Eur Rev Med Pharmacol Sci, 2019, 23(19): 8429-8439. |
| [13] | WANG A, ZHU J, LI J, et al. Downregulation of KIAA1199 by miR-486-5p suppresses tumorigenesis in lung cancer [J]. Cancer medicine, 2020, 9(15): 5570-5586. |
| [14] | ZHANG X, ZHANG T, YANG K, et al. miR-486-5p suppresses prostate cancer metastasis by targeting Snail and regulating epithelial-mesenchymal transition [J]. OncoTargets and therapy, 2016, 2016(9): 6909-6914. |
| [15] | CHANG K, MARRAN K, VALENTINE A, et al. Creating an miR30-based shRNA vector [J]. Cold Spring Harb Protoc, 2013, 2013(7): 631-635. |
| [16] | DOW L E, PREMSRIRUT P K, ZUBER J, et al. A pipeline for the generation of shRNA transgenic mice [J]. Nat Protoc, 2012, 7(2): 374-393. |
| [17] | CHANG K, ELLEDGE S J, HANNON G J. Lessons from Nature: microRNA-based shRNA libraries [J]. Nat Methods, 2006, 3(9): 707-714. |
| [18] | CHEUNG T H, QUACH N L, CHARVILLE G W, et al. Maintenance of muscle stem-cell quiescence by microRNA-489 [J]. Nature, 2012, 482(7386): 524-528. |
| [19] | TREIBER T, TREIBER N, MEISTER G. Regulation of microRNA biogenesis and its crosstalk with other cellular pathways [J]. Nat Rev Mol Cell Biol, 2019, 20(1): 5-20. |
| [20] | MIYOSHI K, MIYOSHI T, SIOMI H. Many ways to generate microRNA-like small RNAs: non-canonical pathways for microRNA production [J]. Mol Genet Genomics, 2010, 284(2): 95-103. |
| [21] | BARTEL D P. Metazoan MicroRNAs [J]. Cell, 2018, 173(1): 20-51. |
| [22] | CIFUENTES D, XUE H, TAYLOR D W, et al. A novel miRNA processing pathway independent of Dicer requires Argonaute2 catalytic activity [J]. Science, 2010, 328(5986): 1694-1698. |
| [23] | YANG J S, MAURIN T, ROBINE N, et al. Conserved vertebrate mir-451 provides a platform for Dicer-independent, Ago2-mediated microRNA biogenesis [J]. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(34): 15163-15168. |
| [24] | CHELOUFI S, DOS SANTOS C O, CHONG M M, et al. A dicer-independent miRNA biogenesis pathway that requires Ago catalysis [J]. Nature, 2010, 465(7298): 584-589. |
| [25] | BOFILL-DE ROS X, GU S. Guidelines for the optimal design of miRNA-based shRNAs [J]. Methods, 2016, 103(157-166. |
| [26] | HERRERA-CARRILLO E, BERKHOUT B. Dicer-independent processing of small RNA duplexes: mechanistic insights and applications [J]. Nucleic acids research, 2017, 45(18): 10369-10379. |
| [27] | HERRERA-CARRILLO E, HARWIG A, BERKHOUT B. Silencing of HIV-1 by AgoshRNA molecules [J]. Gene Ther, 2017, 24(8): 453-461. |
| [28] | HARWIG A, KRUIZE Z, YANG Z, et al. Analysis of AgoshRNA maturation and loading into Ago2 [J]. PloS one, 2017, 12(8): 1-15. |
| [29] | SHANG R, ZHANG F, XU B, et al. Ribozyme-enhanced single-stranded Ago2-processed interfering RNA triggers efficient gene silencing with fewer off-target effects [J]. Nature communications, 2015, 6(2014): 8430-8443. |
| [30] | HARWIG A, HERRERA-CARRILLO E, JONGEJAN A, et al. Deep Sequence Analysis of AgoshRNA Processing Reveals 3' A Addition and Trimming [J]. Molecular therapy Nucleic acids, 2015, 4(2015): e247-e255. |
| [31] | LIU Y P, SCHOPMAN N C, BERKHOUT B. Dicer-independent processing of short hairpin RNAs [J]. Nucleic acids research, 2013, 41(6): 3723-3733. |
| [32] | SONG L, LIN C, GONG H, et al. miR-486 sustains NF-kappaB activity by disrupting multiple NF-kappaB-negative feedback loops [J]. Cell research, 2013, 23(2): 274-289. |
| [33] | MOCKENHAUPT S, GROSSE S, RUPP D, et al. Alleviation of off-target effects from vector-encoded shRNAs via codelivered RNA decoys [J]. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(30): E4007-4016. |
| [34] | HARAGUCHI T, OZAKI Y, IBA H. Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells [J]. Nucleic acids research, 2009, 37(6): e43-e56. |
| [35] | CAO H, YU W, LI X, et al. A new plasmid-based microRNA inhibitor system that inhibits microRNA families in transgenic mice and cells: a potential new therapeutic reagent [J]. Gene Ther, 2016, 23(6): 527-542. |
| [36] | ZAHM A M, MENARD-KATCHER C, BENITEZ A J, et al. Pediatric eosinophilic esophagitis is associated with changes in esophageal microRNAs [J]. Am J Physiol Gastrointest Liver Physiol, 2014, 307(8): G803-G812. |
| [37] | XIE J, AMERES S L, FRIEDLINE R, et al. Long-term, efficient inhibition of microRNA function in mice using rAAV vectors [J]. Nature methods, 2012, 9(4): 403-409. |
| [38] | GOU D, ZHANG H, BAVISKAR P S, et al. Primer extension-based method for the generation of a siRNA/miRNA expression vector [J]. Physiol Genomics, 2007, 31(3): 554-562. |
| [39] | LIU Y P, KARG M, HERRERA-CARRILLO E, et al. Towards Antiviral shRNAs Based on the AgoshRNA Design [J]. PloS one, 2015, 10(6): e0128618. |
| [40] | GENTNER B, SCHIRA G, GIUSTACCHINI A, et al. Stable knockdown of microRNA in vivo by lentiviral vectors [J]. Nat Methods, 2009, 6(1): 63-66. |
| [41] | TISCORNIA G, SINGER O, VERMA I M. Design and cloning of lentiviral vectors expressing small interfering RNAs [J]. Nat Protoc, 2006, 1(1): 234-240. |
| [42] | QIU H, ZHONG J, LUO L, et al. A PCR-Based Method to Construct Lentiviral Vector Expressing Double Tough Decoy for miRNA Inhibition [J]. PloS one, 2015, 10(12): e0143864. |
| [43] | MATVEEVA O V, NAZIPOVA N N, OGURTSOV A Y, et al. Optimized models for design of efficient miR30-based shRNAs [J]. Frontiers in genetics, 2012, 3(163): 1-12. |
| [44] | ELEGHEERT J, BEHIELS E, BISHOP B, et al. Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins [J]. Nat Protoc, 2018, 13(12): 2991-3017. |
| [45] | SINGER O, TISCORNIA G, IKAWA M, et al. Rapid generation of knockdown transgenic mice by silencing lentiviral vectors [J]. Nat Protoc, 2006, 1(1): 286-292. |
| [46] | LESCH H P, LAITINEN A, PEIXOTO C, et al. Production and purification of lentiviral vectors generated in 293T suspension cells with baculoviral vectors [J]. Gene Ther, 2011, 18(6): 531-538. |
| [47] | TISCORNIA G, SINGER O, VERMA I M. Production and purification of lentiviral vectors [J]. Nat Protoc, 2006, 1(1): 241-245. |
| [48] | LALIT P A, RODRIGUEZ A M, DOWNS K M, et al. Generation of multipotent induced cardiac progenitor cells from mouse fibroblasts and potency testing in ex vivo mouse embryos [J]. Nat Protoc, 2017, 12(5): 1029-1054. |
| [49] | SUN N, LEE A, WU J C. Long term non-invasive imaging of embryonic stem cells using reporter genes [J]. Nat Protoc, 2009, 4(8): 1192-1201. |
| [50] | RICHNER M, VICTOR M B, LIU Y, et al. MicroRNA-based conversion of human fibroblasts into striatal medium spiny neurons [J]. Nat Protoc, 2015, 10(10): 1543-1555. |
| [51] | KRAMER M F. Stem-loop RT-qPCR for miRNAs [J]. Curr Protoc Mol Biol, 2011, Chapter 15(10): 151000-151015. |
| [52] | MOSER J J, CHAN E K, FRITZLER M J. Optimization of immunoprecipitation-western blot analysis in detecting GW182-associated components of GW/P bodies [J]. Nat Protoc, 2009, 4(5): 674-685. |
| [53] | GIRARDI F, TALEB A, EBRAHIMI M, et al. TGFbeta signaling curbs cell fusion and muscle regeneration [J]. Nature communications, 2021, 12(1): 750-766. |
| [54] | STEINKRAUS B R, TOEGEL M, FULGA T A. Tiny giants of gene regulation: experimental strategies for microRNA functional studies [J]. Wiley Interdiscip Rev Dev Biol, 2016, 5(3): 311-362. |
| [55] | HENSHALL D C, HAMER H M, PASTERKAMP R J, et al. MicroRNAs in epilepsy: pathophysiology and clinical utility [J]. Lancet Neurol, 2016, 15(13): 1368-1376. |
| [56] | LU T X, ROTHENBERG M E. MicroRNA [J]. J Allergy Clin Immunol, 2018, 141(4): 1202-1207. |
| [57] | ZHAO J, WANG H, ZHOU J, et al. miR-130a-3p, a Preclinical Therapeutic Target for Crohn's Disease [J]. J Crohns Colitis, 2021, 15(4): 647-664. |
| [58] | LIN S, GREGORY R I. MicroRNA biogenesis pathways in cancer [J]. Nature reviews Cancer, 2015, 15(6): 321-333. |
| [59] | MICHLEWSKI G, CACERES J F. Post-transcriptional control of miRNA biogenesis [J]. Rna, 2019, 25(1): 1-16. |
| [60] | ZHANG S, LIU Y, YU B. New insights into pri-miRNA processing and accumulation in plants [J]. Wiley Interdiscip Rev RNA, 2015, 6(5): 533-545. |
