Engineering HEK293T cell line by lentivirus to produce miR34a-loaded exosomes.
Exosome
HEK293T
Lentiviral vector
miR34a
Journal
Molecular biology reports
ISSN: 1573-4978
Titre abrégé: Mol Biol Rep
Pays: Netherlands
ID NLM: 0403234
Informations de publication
Date de publication:
Nov 2023
Nov 2023
Historique:
received:
25
04
2023
accepted:
10
08
2023
medline:
10
11
2023
pubmed:
4
9
2023
entrez:
2
9
2023
Statut:
ppublish
Résumé
RNA (ribonucleic acid) antisense is developing as a possible treatment option. As an RNA, miR-34a is involved in P53 function and cancer cell apoptosis. Although the therapeutic applications of miRNAs have several limitations, such as structural instability and susceptibility to nucleases. To resolve these issues, this study aims to apply exosomes as a delivery vehicle for miR-34a. This study aims to create a cell factory to generate miR34a-enriched exosomes. The produced nanoparticles act as a delivery system and improve the structural stability of miR34a. First exosome specific sequences were inserted into miR34a. The resulting miR34a oligonucleotide was transduced HEK293T cells genome with a lentiviral system. In the structure of miR34a oligonucleotide, six nucleotides were substituted to increase its packaging rate into exosomes. To maintain the secondary structure, stability, and expression of the miRNA gene, changes to the miR34a oligonucleotide were made using PCR (polymerase chain reaction) Extension. The forward-34a (5-TGGGGAGAGGCAGGACAGG-3) and Reverse-34a primers (5-TCCGAAGTCCTGGCGTCTCC-3) were used for amplification of the miR34a gene from DNA. The results confirmed that the changes in miR34a oligonucleotide do not affect its secondary structure. The energy level of the manipulated miR34a oligonucleotide was kept the same compared to the original one. Moreover, the loading of miR34a to the exosomes was increased. Our findings revealed that normal HEK293T did not express miR34a. However, lentiviral transduced miR34a oligonucleotide induced the loading of miR34a into the exosome. Moreover, replacing six nucleic acids in the 3' end of miR34a increased the loading of miR34a to exosome.
Sections du résumé
BACKGROUND
BACKGROUND
RNA (ribonucleic acid) antisense is developing as a possible treatment option. As an RNA, miR-34a is involved in P53 function and cancer cell apoptosis. Although the therapeutic applications of miRNAs have several limitations, such as structural instability and susceptibility to nucleases. To resolve these issues, this study aims to apply exosomes as a delivery vehicle for miR-34a.
AIMS
OBJECTIVE
This study aims to create a cell factory to generate miR34a-enriched exosomes. The produced nanoparticles act as a delivery system and improve the structural stability of miR34a.
METHODS
METHODS
First exosome specific sequences were inserted into miR34a. The resulting miR34a oligonucleotide was transduced HEK293T cells genome with a lentiviral system. In the structure of miR34a oligonucleotide, six nucleotides were substituted to increase its packaging rate into exosomes. To maintain the secondary structure, stability, and expression of the miRNA gene, changes to the miR34a oligonucleotide were made using PCR (polymerase chain reaction) Extension. The forward-34a (5-TGGGGAGAGGCAGGACAGG-3) and Reverse-34a primers (5-TCCGAAGTCCTGGCGTCTCC-3) were used for amplification of the miR34a gene from DNA.
RESULTS
RESULTS
The results confirmed that the changes in miR34a oligonucleotide do not affect its secondary structure. The energy level of the manipulated miR34a oligonucleotide was kept the same compared to the original one. Moreover, the loading of miR34a to the exosomes was increased.
CONCLUSION
CONCLUSIONS
Our findings revealed that normal HEK293T did not express miR34a. However, lentiviral transduced miR34a oligonucleotide induced the loading of miR34a into the exosome. Moreover, replacing six nucleic acids in the 3' end of miR34a increased the loading of miR34a to exosome.
Identifiants
pubmed: 37658928
doi: 10.1007/s11033-023-08754-1
pii: 10.1007/s11033-023-08754-1
doi:
Substances chimiques
MicroRNAs
0
Oligonucleotides
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
8827-8837Subventions
Organisme : Mazandaran University of Medical Sciences
ID : 2837
Informations de copyright
© 2023. The Author(s), under exclusive licence to Springer Nature B.V.
Références
Huppi K, Martin SE, Caplen NJ (2005) Defining and assaying RNAi in mammalian cells. Mol Cell 17(1):1–10
doi: 10.1016/j.molcel.2004.12.017
pubmed: 15629712
Chen F, Hu SJ (2012) Effect of microRNA-34a in cell cycle, differentiation, and apoptosis: a review. J Biochem Mol Toxicol 26(2):79–86
doi: 10.1002/jbt.20412
pubmed: 22162084
Yamakuchi M, Ferlito M, Lowenstein CJ (2008) miR-34a repression of SIRT1 regulates apoptosis Proceedings of the National Academy of Sciences, 105(36): p. 13421–13426
Akao Y et al (2011) Dysregulation of microRNA-34a expression causes drug-resistance to 5-FU in human colon cancer DLD-1 cells. Cancer Lett 300(2):197–204
doi: 10.1016/j.canlet.2010.10.006
pubmed: 21067862
Fan YN et al (2014) Mir-34a mimics are potential therapeutic agents for p53-mutated and chemo-resistant brain tumour cells. PLoS ONE 9(9):e108514
doi: 10.1371/journal.pone.0108514
pubmed: 25250818
pmcid: 4177398
O’Neill CP, Dwyer RM (2020) Nanoparticle-based delivery of tumor suppressor microRNA for cancer therapy. Cells 9(2):521
doi: 10.3390/cells9020521
pubmed: 32102476
pmcid: 7072816
Reshke R et al (2020) Reduction of the therapeutic dose of silencing RNA by packaging it in extracellular vesicles via a pre-microRNA backbone. Nat biomedical Eng 4(1):52–68
doi: 10.1038/s41551-019-0502-4
Tian Z et al (2021) Insight into the prospects for RNAi therapy of Cancer. Front Pharmacol, 12(308)
Suh JH et al (2021) Therapeutic application of exosomes in inflammatory diseases. Int J Mol Sci 22(3):1144
doi: 10.3390/ijms22031144
pubmed: 33498928
pmcid: 7865921
Munagala R et al (2021) Exosome-mediated delivery of RNA and DNA for gene therapy. Cancer Lett 505:58–72
doi: 10.1016/j.canlet.2021.02.011
pubmed: 33610731
pmcid: 8005491
Sinha D et al (2021) Trends in Research on Exosomes in Cancer Progression and Anticancer Therapy. Cancers 13(2):326
doi: 10.3390/cancers13020326
pubmed: 33477340
pmcid: 7829710
Wahlgren J et al (2012) Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes. Nucleic Acids Res 40(17):e130–e130
doi: 10.1093/nar/gks463
pubmed: 22618874
pmcid: 3458529
Lotvall J, Valadi H (2007) Cell to cell signalling via exosomes through esRNA. Cell Adhes Migr 1(3):156–158
doi: 10.4161/cam.1.3.5114
Lässer C, Eldh M, Lötvall J (2013) The role of exosomal shuttle RNA (esRNA) in cell-to-cell communication. Emerg Concepts Tumor Exosome–Mediated Cell-Cell Communication, : p. 33–45
Villarroya-Beltri C et al (2013) Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs. Nat Commun 4(1):2980
doi: 10.1038/ncomms3980
pubmed: 24356509
Coughlan C et al (2020) Exosome isolation by Ultracentrifugation and Precipitation and techniques for downstream analyses. Curr Protoc Cell Biol 88(1):e110
doi: 10.1002/cpcb.110
pubmed: 32633898
pmcid: 8088761
Limoni SK et al (2019) Engineered Exosomes for targeted transfer of siRNA to HER2 positive breast Cancer cells. Appl Biochem Biotechnol 187(1):352–364
doi: 10.1007/s12010-018-2813-4
pubmed: 29951961
O’Brien J et al (2018) Overview of MicroRNA Biogenesis, Mechanisms of actions, and circulation. Front Endocrinol (Lausanne) 9:402
doi: 10.3389/fendo.2018.00402
pubmed: 30123182
Fu Z et al (2021) MicroRNA as an important target for anticancer drug development. Front Pharmacol, : p. 2212
Holjencin C, Jakymiw A (2022) MicroRNAs and their big therapeutic impacts: delivery strategies for Cancer intervention. Cells 11(15):2332
doi: 10.3390/cells11152332
pubmed: 35954176
pmcid: 9367537
Sharma P et al (2020) Nanomaterials for autophagy-related miRNA-34a delivery in cancer treatment. Front Pharmacol 11:1141
doi: 10.3389/fphar.2020.01141
pubmed: 32792960
pmcid: 7393066
Li F et al (2018) miR-221 suppression through nanoparticle-based miRNA delivery system for hepatocellular carcinoma therapy and its diagnosis as a potential biomarker. Int J Nanomed 13:2295
doi: 10.2147/IJN.S157805
Chaudhary V, Jangra S, Yadav NR (2018) Nanotechnology based approaches for detection and delivery of microRNA in healthcare and crop protection. J Nanobiotechnol 16(1):1–18
doi: 10.1186/s12951-018-0368-8
Fu S et al (2020) Exosome engineering: current progress in cargo loading and targeted delivery. NanoImpact 20:100261
doi: 10.1016/j.impact.2020.100261
Kalfert D et al (2020) Multifunctional roles of miR-34a in cancer: a review with the emphasis on head and neck squamous cell carcinoma and thyroid cancer with clinical implications. Diagnostics 10(8):563
doi: 10.3390/diagnostics10080563
pubmed: 32764498
pmcid: 7459507
Misso G et al (2014) Mir-34: a new weapon against cancer? Mol therapy-nucleic acids 3:e195
doi: 10.1038/mtna.2014.47
Hermeking H (2010) The miR-34 family in cancer and apoptosis. Cell Death & Differentiation 17(2):193–199
doi: 10.1038/cdd.2009.56
Li M 34a: potent tumor suppressor, cancer stem cell inhibitor, and potential anticancer therapeutic, front. Cell Dev Biol, (9): p. 322
Hong DS et al (2020) Phase 1 study of MRX34, a liposomal miR-34a mimic, in patients with advanced solid tumours. Br J Cancer 122(11):1630–1637
doi: 10.1038/s41416-020-0802-1
pubmed: 32238921
pmcid: 7251107
Song B-W, Oh S, Chang W (2022) Multiplexed targeting of microRNA in stem cell-derived extracellular vesicles for regenerative medicine. BMB Rep 55(2):65
doi: 10.5483/BMBRep.2022.55.2.182
pubmed: 35000674
pmcid: 8891620
Alvarez-Erviti L et al (2011) Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol 29(4):341–345
doi: 10.1038/nbt.1807
pubmed: 21423189
Wang J-H et al (2018) Anti-HER2 scfv-directed extracellular vesicle-mediated mRNA-based gene delivery inhibits growth of HER2-positive human breast tumor xenografts by prodrug activation. Mol Cancer Ther 17(5):1133–1142
doi: 10.1158/1535-7163.MCT-17-0827
pubmed: 29483213
pmcid: 5932266
Amiri A et al (2022) Exosomes as bio-inspired nanocarriers for RNA delivery: Preparation and applications. J Translational Med 20(1):1–16
doi: 10.1186/s12967-022-03325-7
Lamichhane TN et al (2016) Oncogene knockdown via active loading of small RNAs into extracellular vesicles by sonication. Cell Mol Bioeng 9(3):315–324
doi: 10.1007/s12195-016-0457-4
pubmed: 27800035
Raghav A, Jeong G-B (2021) A systematic review on the modifications of extracellular vesicles: a revolutionized tool of nano-biotechnology. J Nanobiotechnol 19(1):1–19
doi: 10.1186/s12951-021-01219-2
Munir J, Yoon JK, Ryu S (2020) Therapeutic miRNA-enriched extracellular vesicles: current approaches and future prospects. Cells 9(10):2271
doi: 10.3390/cells9102271
pubmed: 33050562
pmcid: 7601381
Zhang J et al (2015) Exosome and Exosomal MicroRNA: trafficking, sorting, and function. Proteom Bioinf 13(1):17–24Genomics
Bolukbasi MF et al (2012) miR-1289 and “Zipcode”-like sequence enrich mRNAs in microvesicles. Mol Therapy-Nucleic Acids 1:e10
doi: 10.1038/mtna.2011.2
Koppers-Lalic D et al (2014) Nontemplated nucleotide additions distinguish the small RNA composition in cells from exosomes. Cell Rep 8:1649–1658
doi: 10.1016/j.celrep.2014.08.027
pubmed: 25242326
Chen L et al (2020) Exosomes derived from GDNF-modified human adipose mesenchymal stem cells ameliorate peritubular capillary loss in tubulointerstitial fibrosis by activating the SIRT1/eNOS signaling pathway. Theranostics 10(20):9425
doi: 10.7150/thno.43315
pubmed: 32802201
pmcid: 7415791
Dufait I et al (2012) Retroviral and lentiviral vectors for the induction of immunological tolerance Scientifica, 2012