Skip to content
2000
image of Novel Perspective of Regulating p53/Bcl2/Caspase-3 via In vitro Targeted AFP Gene Knocks Out in HepG2 Cells Using CRISPR/Cas9 Editing Tool

Abstract

Introduction

Hepatocellular carcinoma (HCC) is a major health burden worldwide, with a persistent need for molecular target drugs. Alpha-fetoprotein (AFP) is a major concern during HCC, as it has an incompletely solved action. CRISPR/Cas9 is a gene editing tool that aids in cancer treatment research; thus, this study evaluated the effect of knockout of AFP on HCC using CRISPR/Cas9 technique.

Methods

Two sgRNAs targeting specific sites in AFP exon 2 were separately cloned to the mammalian expression vector pSpCas9 (BB)-2a-GFP (PX458). HepG2 cells were transfected with CRISPR constructs I and II, and a pool of the two constructs (M) for 6 -, 24- and 39 hours using liopfectamine3000. AFP editing was evaluated regarding genomic DNA sequence, RNA, and protein expression levels. In addition, the effect of AFP knocking out on HepG2 viability, and apoptotic genes mRNA and protein expression levels were evaluated using crystal violet assay, real-time PCR, and western blot analysis respectively.

Results

The results revealed efficient delivery of the AFP/CRISPR constructs to HepG2 cells. Insertion and deletion mutations introduced to the AFP genomic sequence were analyzed using TIDE software analysis and the Expasy translation tool. The viability of the HepG2 cells was reduced 39 hours post-transfection with significant modulation in the expression of the apoptotic markers p53, BAX, Bcl2, and caspase-3.

Conclusion

This study succeeded in developing AFP/CRISPR constructs that could disrupt the AFP genomic sequence, reduce its expression, and restore the activity of cell-specific apoptotic factors, demonstrating the potential inhibitory effect of AFP downregulation on HCC progression.

© 2025 Bentham Science Publishers
Loading

Article metrics loading...

/content/journals/cgt/10.2174/0115665232366303250529164610
2025-06-12
2025-09-13

  • From This Site

    /content/journals/cgt/10.2174/0115665232366303250529164610
    dcterms_title,dcterms_subject,pub_keyword
    -contentType:Contributor -contentType:Concept -contentType:Institution
    10
    5
Loading full text...

Full text loading...

References

  1. Llovet J.M. Kelley R.K. Villanueva A. Hepatocellular carcinoma. Nat. Rev. Dis. Primers 2021 7 1 6 10.1038/s41572‑020‑00240‑3 33479224
    [Google Scholar]
  2. Cho Y. Kim B.H. Park J.W. Overview of Asian clinical practice guidelines for the management of hepatocellular carcinoma: An Asian perspective comparison. Clin. Mol. Hepatol. 2023 29 2 252 262 10.3350/cmh.2023.0099 36907570
    [Google Scholar]
  3. Li H. Yang Y. Hong W. Huang M. Wu M. Zhao X. Applications of genome editing technology in the targeted therapy of human diseases: Mechanisms, advances and prospects. Signal Transduct. Target. Ther. 2020 5 1 1 10.1038/s41392‑019‑0089‑y 32296011
    [Google Scholar]
  4. Wei J. Li Y. CRISPR-based gene editing technology and its application in microbial engineering. Engineering Microbiology 2023 3 4 100101 10.1016/j.engmic.2023.100101 39628916
    [Google Scholar]
  5. Li Q. Qin Z. Wang Q. Xu T. Yang Y. He Z. Applications of genome editing technology in animal disease modeling and gene therapy. Comput. Struct. Biotechnol. J. 2019 17 689 698 10.1016/j.csbj.2019.05.006 31303973
    [Google Scholar]
  6. Naeimi Kararoudi M. Hejazi S.S. Elmas E. Clustered regularly interspaced short palindromic repeats/Cas9 gene editing technique in xenotransplantation. Front. Immunol. 2018 9 9 1711 10.3389/fimmu.2018.01711 30233563
    [Google Scholar]
  7. Hille F. Richter H. Wong S.P. Bratovič M. Ressel S. Charpentier E. The biology of CRISPR-Cas: Backward and forward. Cell 2018 172 6 1239 1259 10.1016/j.cell.2017.11.032 29522745
    [Google Scholar]
  8. Liu G. Lin Q. Jin S. Gao C. The CRISPR-Cas toolbox and gene editing technologies. Mol. Cell 2022 82 2 333 347 10.1016/j.molcel.2021.12.002 34968414
    [Google Scholar]
  9. Allen D.B. Rosenberg M. Hendel A. Using synthetically engineered guide RNAs to enhance CRISPR genome editing systems in mammalian cells. Front Genome Ed 2020 2 617910 10.3389/fgeed.2020.617910
    [Google Scholar]
  10. Xue C. Greene E.C. DNA repair pathway choices in CRISPR-Cas9 mediated genome editing. Trends Genet. 2021 37 7 639 656 10.1016/j.tig.2021.02.008 33896583
    [Google Scholar]
  11. Lalonde S. Stone O.A. Lessard S. Frameshift indels introduced by genome editing can lead to in-frame exon skipping. PLoS One 2017 12 6 e0178700 10.1371/journal.pone.0178700 28570605
    [Google Scholar]
  12. Martinez-Lage M. Puig-Serra P. Menendez P. Torres-Ruiz R. Rodriguez-Perales S. CRISPR/Cas9 for cancer therapy: Hopes and challenges. Biomedicines 2018 6 4 105 10.3390/biomedicines6040105 30424477
    [Google Scholar]
  13. Li T. Yang Y. Qi H. CRISPR/Cas9 therapeutics: Progress and prospects. Signal Transduct. Target. Ther. 2023 8 1 36 10.1038/s41392‑023‑01309‑7 36646687
    [Google Scholar]
  14. Sharony R. Zadik I. Parvari R. Congenital deficiency of alpha feto-protein. Eur. J. Hum. Genet. 2004 12 10 871 874 10.1038/sj.ejhg.5201246 15280901
    [Google Scholar]
  15. Pardee A.D. Shi J. Butterfield L.H. Tumor-derived α-fetoprotein impairs the differentiation and T cell stimulatory activity of human dendritic cells. J. Immunol. 2014 193 11 5723 5732 10.4049/jimmunol.1400725 25355916
    [Google Scholar]
  16. Gillespie J.R. Uversky V.N. Structure and function of α-fetoprotein: A biophysical overview. Biochim. Biophys. Acta Protein Struct. Mol. Enzymol. 2000 1480 1-2 41 56 10.1016/S0167‑4838(00)00104‑7 11004554
    [Google Scholar]
  17. Lin B. Wang Q. Liu K. Dong X. Zhu M. Li M. Alpha-fetoprotein binding mucin and scavenger receptors: An available bio-target for treating cancer. Front. Oncol. 2021 11 625936 10.3389/fonc.2021.625936 33718192
    [Google Scholar]
  18. Li P. Wang S.S. Liu H. Elevated serum alpha fetoprotein levels promote pathological progression of hepatocellular carcinoma. World J. Gastroenterol. 2011 17 41 4563 4571 10.3748/wjg.v17.i41.4563 22147961
    [Google Scholar]
  19. Hu X. Chen R. Wei Q. Xu X. The landscape of alpha fetoprotein in hepatocellular carcinoma: Where are we? Int. J. Biol. Sci. 2022 18 2 536 551 10.7150/ijbs.64537 35002508
    [Google Scholar]
  20. Hanif H. Ali M.J. Susheela A.T. Update on the applications and limitations of alpha-fetoprotein for hepatocellular carcinoma. World J. Gastroenterol. 2022 28 2 216 229 10.3748/wjg.v28.i2.216 35110946
    [Google Scholar]
  21. Chen T. Dai X. Dai J. AFP promotes HCC progression by suppressing the HuR-mediated Fas/FADD apoptotic pathway. Cell Death Dis. 2020 11 10 822 10.1038/s41419‑020‑03030‑7 33009373
    [Google Scholar]
  22. Cazzetta V. Franzese S. Carenza C. Della Bella S. Mikulak J. Mavilio D. Natural killer–dendritic cell interactions in liver cancer: Implications for immunotherapy. Cancers 2021 13 9 2184 10.3390/cancers13092184 34062821
    [Google Scholar]
  23. Głowska-Ciemny J. Szymański M. Kuszerska A. Malewski Z. von Kaisenberg C. Kocyłowski R. The role of alpha-fetoprotein (AFP) in contemporary oncology: The path from a diagnostic biomarker to an anticancer drug. Int. J. Mol. Sci. 2023 24 3 2539 10.3390/ijms24032539 36768863
    [Google Scholar]
  24. Ran F.A. Hsu P.D. Wright J. Agarwala V. Scott D.A. Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 2013 8 11 2281 2308 10.1038/nprot.2013.143 24157548
    [Google Scholar]
  25. Doench J.G. Fusi N. Sullender M. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 2016 34 2 184 191 10.1038/nbt.3437 26780180
    [Google Scholar]
  26. Sander J.D. Joung J.K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 2014 32 4 347 355 10.1038/nbt.2842 24584096
    [Google Scholar]
  27. Sambrook J. Russell D.W. Molecular Cloning. 3rd ed Cold Spring Harbor, NY, USA Cold Spring Harbor Laboratory Press 2001
    [Google Scholar]
  28. Akin Y. Hacer I. Ebru A. Real-TimePCR for gene expression analysis. In:Polymerase Chain Reaction. IntechOpen 2012 229 254 10.5772/37356
    [Google Scholar]
  29. Ros J. Salva F. Dopazo C. Liver transplantation in metastatic colorectal cancer: Are we ready for it? Br. J. Cancer 2023 128 10 1797 1806 10.1038/s41416‑023‑02213‑1 36879000
    [Google Scholar]
  30. Xu L. Chen L. Zhang W. Neoadjuvant treatment strategies for hepatocellular carcinoma. World J. Gastrointest. Surg. 2021 13 12 1550 1566 10.4240/wjgs.v13.i12.1550 35070063
    [Google Scholar]
  31. Motomura K. Kuwano A. Tanaka K. Koga Y. Masumoto A. Yada M. Potential predictive biomarkers of systemic drug therapy for hepatocellular carcinoma: Anticipated usefulness in clinical practice. Cancers 2023 15 17 4345 10.3390/cancers15174345 37686621
    [Google Scholar]
  32. Kudo M. Recent advances in systemic therapy for hepatocellular carcinoma in an aging society: 2020 Update. Liver Cancer 2020 9 6 640 662 10.1159/000511001 33442538
    [Google Scholar]
  33. Chan Y.T. Zhang C. Wu J. Biomarkers for diagnosis and therapeutic options in hepatocellular carcinoma. Mol. Cancer 2024 23 1 189 10.1186/s12943‑024‑02101‑z 39242496
    [Google Scholar]
  34. Bialecki E.S. Di Bisceglie A.M. Diagnosis of hepatocellular carcinoma. HPB 2005 7 1 26 34 10.1080/13651820410024049 18333158
    [Google Scholar]
  35. Lin B. Dong X. Wang Q. Li W. Zhu M. Li M. AFP-inhibiting fragments for drug delivery: The promise and challenges of targeting therapeutics to cancers. Front. Cell Dev. Biol. 2021 9 635476 10.3389/fcell.2021.635476 33898423
    [Google Scholar]
  36. Parra N.S. Ross H.M. Khan A. Advancements in the Diagnosis of hepatocellular carcinoma. Int J Transl Med 2023 3 1 51 65 10.3390/ijtm3010005
    [Google Scholar]
  37. Mengstie M.A. Wondimu B.Z. Mechanism and applications of CRISPR/Cas-9-mediated genome editing. Biologics 2021 15 353 361 10.2147/BTT.S326422 34456559
    [Google Scholar]
  38. McCarty N.S. Graham A.E. Studená L. Ledesma-Amaro R. Multiplexed CRISPR technologies for gene editing and transcriptional regulation. Nat. Commun. 2020 11 1 1281 10.1038/s41467‑020‑15053‑x 32152313
    [Google Scholar]
  39. Xu Y. Li Z. CRISPR-Cas systems: Overview, innovations and applications in human disease research and gene therapy. Comput. Struct. Biotechnol. J. 2020 18 2401 2415 10.1016/j.csbj.2020.08.031 33005303
    [Google Scholar]
  40. Homann S. Hofmann C. Gorin A.M. A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells. PLoS One 2017 12 9 e0182941 10.1371/journal.pone.0182941 28863132
    [Google Scholar]
  41. Shalem O. Sanjana N.E. Hartenian E. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 2014 343 6166 84 87 10.1126/science.1247005 24336571
    [Google Scholar]
  42. Brinkman E.K. Chen T. Amendola M. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 2014 42 22 e168 10.1093/nar/gku936 25300484
    [Google Scholar]
  43. Fajrial A.K. He Q.Q. Wirusanti N.I. Slansky J.E. Ding X. A review of emerging physical transfection methods for CRISPR/Cas9-mediated gene editing. Theranostics 2020 10 12 5532 5549 10.7150/thno.43465 32373229
    [Google Scholar]
  44. Fierro J. DiPasquale J. Perez J. Dual-sgRNA CRISPR/Cas9 knockout of PD-L1 in human U87 glioblastoma tumor cells inhibits proliferation, invasion, and tumor-associated macrophage polarization. Sci. Rep. 2022 12 1 2417 10.1038/s41598‑022‑06430‑1 35165339
    [Google Scholar]
  45. Liang X. Potter J. Kumar S. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J. Biotechnol. 2015 208 44 53 10.1016/j.jbiotec.2015.04.024 26003884
    [Google Scholar]
  46. Feoktistova M Geserick P Leverkus M Crystal violet assay for determining viability of cultured cells. Cold Spring Harb Protoc 2016 2016 4 pdb-rot087379 10.1101/pdb.prot087379 27037069
    [Google Scholar]
  47. Śliwka L. Wiktorska K. Suchocki P. The comparison of MTT and CVS assays for the assessment of anticancer agent interactions. PLoS One 2016 11 5 e0155772 10.1371/journal.pone.0155772 27196402
    [Google Scholar]
  48. Lu Y. Lin B. Li M. The role of alpha-fetoprotein in the tumor microenvironment of hepatocellular carcinoma. Front. Oncol. 2024 14 1363695 10.3389/fonc.2024.1363695 38660138
    [Google Scholar]
  49. Shamas-Din A. Kale J. Leber B. Andrews D.W. Mechanisms of action of Bcl-2 family proteins. Cold Spring Harb. Perspect. Biol. 2013 5 4 a008714 10.1101/cshperspect.a008714 23545417
    [Google Scholar]
  50. Vishnoi K. Kumar S. Ke R. Rana A. Rana B. Dysregulation of immune checkpoint proteins in hepatocellular carcinoma: Impact on metabolic reprogramming. Curr. Opin. Pharmacol. 2022 64 102232 10.1016/j.coph.2022.102232 35526340
    [Google Scholar]
  51. Dirican E. Özcan H. Karabulut Uzunçakmak S. Takım U. Evaluation expression of the caspase-3 and caspase-9 apoptotic genes in schizophrenia patients. Clin. Psychopharmacol. Neurosci. 2023 21 1 171 178 10.9758/cpn.2023.21.1.171 36700323
    [Google Scholar]
  52. Li M. Li H. Li C. Alpha fetoprotein is a novel protein‐binding partner for caspase‐3 and blocks the apoptotic signaling pathway in human hepatoma cells. Int. J. Cancer 2009 124 12 2845 2854 10.1002/ijc.24272 19267404
    [Google Scholar]
/content/journals/cgt/10.2174/0115665232366303250529164610
Loading
Novel Perspective of Regulating p53/Bcl2/Caspase-3 via In vitro Targeted AFP Gene Knocks Out in HepG2 Cells Using CRISPR/Cas9 Editing Tool
Bentham Science Publishers ; https://doi.org/10.2174/0115665232366303250529164610
/content/journals/cgt/10.2174/0115665232366303250529164610
/content/journals/cgt/10.2174/0115665232366303250529164610
Loading

Data & Media loading...

Supplements

Supplementary material is available on the publisher’s website along with the published article.

file format download Download

  • Article Type:     Research Article
Keywords: Bcl2 ; BAX ; caspase-3 ; AFP ; p53 ; hepatocellular carcinoma ; CRISPR/Cas9

Most Cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error
Please enter a valid_number test