Skip to main content

Advertisement

SpringerLink
Log in

Engineering HEK293T cell line by lentivirus to produce miR34a-loaded exosomes

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

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

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

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

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

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Data Availability

All the data are reported as they were obtained. And the original data will be presented upon a reasonable request. The RNA folding (Fig. 1) was obtained from http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi website.

References

  1. Huppi K, Martin SE, Caplen NJ (2005) Defining and assaying RNAi in mammalian cells. Mol Cell 17(1):1–10

    Article  CAS  PubMed  Google Scholar 

  2. 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

    Article  CAS  PubMed  Google Scholar 

  3. 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

  4. 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

    Article  CAS  PubMed  Google Scholar 

  5. 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

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  6. O’Neill CP, Dwyer RM (2020) Nanoparticle-based delivery of tumor suppressor microRNA for cancer therapy. Cells 9(2):521

    Article  PubMed  PubMed Central  Google Scholar 

  7. 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

    Article  CAS  Google Scholar 

  8. Tian Z et al (2021) Insight into the prospects for RNAi therapy of Cancer. Front Pharmacol, 12(308)

  9. Suh JH et al (2021) Therapeutic application of exosomes in inflammatory diseases. Int J Mol Sci 22(3):1144

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Munagala R et al (2021) Exosome-mediated delivery of RNA and DNA for gene therapy. Cancer Lett 505:58–72

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Sinha D et al (2021) Trends in Research on Exosomes in Cancer Progression and Anticancer Therapy. Cancers 13(2):326

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Wahlgren J et al (2012) Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes. Nucleic Acids Res 40(17):e130–e130

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Lotvall J, Valadi H (2007) Cell to cell signalling via exosomes through esRNA. Cell Adhes Migr 1(3):156–158

    Article  Google Scholar 

  14. 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

  15. 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

    Article  ADS  PubMed  Google Scholar 

  16. Coughlan C et al (2020) Exosome isolation by Ultracentrifugation and Precipitation and techniques for downstream analyses. Curr Protoc Cell Biol 88(1):e110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 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

    Article  CAS  PubMed  Google Scholar 

  18. O’Brien J et al (2018) Overview of MicroRNA Biogenesis, Mechanisms of actions, and circulation. Front Endocrinol (Lausanne) 9:402

    Article  PubMed  Google Scholar 

  19. Fu Z et al (2021) MicroRNA as an important target for anticancer drug development. Front Pharmacol, : p. 2212

  20. Holjencin C, Jakymiw A (2022) MicroRNAs and their big therapeutic impacts: delivery strategies for Cancer intervention. Cells 11(15):2332

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Sharma P et al (2020) Nanomaterials for autophagy-related miRNA-34a delivery in cancer treatment. Front Pharmacol 11:1141

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 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

    Article  CAS  Google Scholar 

  23. 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

    Article  Google Scholar 

  24. Fu S et al (2020) Exosome engineering: current progress in cargo loading and targeted delivery. NanoImpact 20:100261

    Article  Google Scholar 

  25. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Misso G et al (2014) Mir-34: a new weapon against cancer? Mol therapy-nucleic acids 3:e195

    Article  Google Scholar 

  27. Hermeking H (2010) The miR-34 family in cancer and apoptosis. Cell Death & Differentiation 17(2):193–199

    Article  CAS  Google Scholar 

  28. Li M 34a: potent tumor suppressor, cancer stem cell inhibitor, and potential anticancer therapeutic, front. Cell Dev Biol, (9): p. 322

  29. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 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

    Article  CAS  PubMed  Google Scholar 

  32. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Amiri A et al (2022) Exosomes as bio-inspired nanocarriers for RNA delivery: Preparation and applications. J Translational Med 20(1):1–16

    Article  Google Scholar 

  34. 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

    Article  CAS  PubMed  Google Scholar 

  35. 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

    Article  Google Scholar 

  36. Munir J, Yoon JK, Ryu S (2020) Therapeutic miRNA-enriched extracellular vesicles: current approaches and future prospects. Cells 9(10):2271

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Zhang J et al (2015) Exosome and Exosomal MicroRNA: trafficking, sorting, and function. Proteom Bioinf 13(1):17–24Genomics

    CAS  Google Scholar 

  38. Bolukbasi MF et al (2012) miR-1289 and “Zipcode”-like sequence enrich mRNAs in microvesicles. Mol Therapy-Nucleic Acids 1:e10

    Article  Google Scholar 

  39. Koppers-Lalic D et al (2014) Nontemplated nucleotide additions distinguish the small RNA composition in cells from exosomes. Cell Rep 8:1649–1658

    Article  CAS  PubMed  Google Scholar 

  40. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Dufait I et al (2012) Retroviral and lentiviral vectors for the induction of immunological tolerance Scientifica, 2012

Download references

Acknowledgements

This study was supported financially by Grant No. #2837 from Mazandaran University of Medical Sciences, Sari, Iran.

Author information

Authors and Affiliations

  1. Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Mazandaran University of Medical Sciences, Sari, Iran

    Sahar Abdi Sarkami

  2. Pharmaceutical Sciences Research Center, Hemoglobinopathy Institute, Mazandaran University of Medical Sciences, Sari, Iran

    Sajjad Molavipordanjani

  3. Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Mazandaran University of Medical Sciences, FarahAbad Road, Sari, Iran

    Saeed Abediankenari, Javad Akhtari, Pooria Gill, Hossein Ghalehnoei & Shabanali Khodashenas Lemoni

Authors
  1. Sahar Abdi Sarkami
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sajjad Molavipordanjani
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Saeed Abediankenari
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Javad Akhtari
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Pooria Gill
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hossein Ghalehnoei
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shabanali Khodashenas Lemoni
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shabanali Khodashenas Lemoni.

Ethics declarations

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All animal experiments were approved by the Research and Ethical Committee of Mazandaran University of Medical Sciences, Sari, Iran.

Disclosure of potential conflicts of interest

The authors declare no competing interest.

Research involving human participants and/or animals

This research does not include human tissue or animals.

Informed consent

All the authors consent to publish this article’s findings.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sarkami, S.A., Molavipordanjani, S., Abediankenari, S. et al. Engineering HEK293T cell line by lentivirus to produce miR34a-loaded exosomes. Mol Biol Rep 50, 8827–8837 (2023). https://doi.org/10.1007/s11033-023-08754-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-023-08754-1

Keywords

Search

Navigation