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CRISPR/Cas9‑mediated claudin‑2 knockout in HCT116 cells reveals its key role in colorectal cancer progression

  • Authors:
    • Rana A. Alghamdi
    • Maryam H. Al‑Zahrani
  • View Affiliations / Copyright

    Affiliations: Department of Chemistry, Science and Arts College, King Abdulaziz University, Rabigh 21911, Saudi Arabia, Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia
    Copyright: © Alghamdi et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 54
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    Published online on: November 27, 2025
       https://doi.org/10.3892/ol.2025.15407
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Abstract

Colorectal cancer (CRC) progression involves complex mechanisms of invasion and metastasis. Claudin‑2 (CLDN2), a tight junction protein, has emerged as a key regulator paracellular permeability and its dysregulation is implicated in chronic inflammatory diseases and cancer. The present study aimed to determine the mechanisms by which CLDN2 deletion affects genes associated with motility and invasion of colon cancer cells. CRISPR/Cas9 was used to knock out CLDN2 in HCT116 cells. Subsequently, gene expression was analyzed using reverse transcription‑quantitative PCR and migratory capacity was assessed using wound healing assays. CLDN2 deletion led to the downregulation of genes associated with motility and metastasis, including zonula occludens‑1‑associated nucleic acid binding protein, N‑Myc downstream‑regulated gene 1, CLDN14, CLDN23, Bcl‑2, p53 and Bcl‑6, suggesting that CLDN2 supports pro‑migratory gene networks. These findings demonstrated that CLDN2 regulates metastatic gene expression in CRC. Although further mechanistic studies are warranted, the present study provided notable genetic and phenotypic evidence of the role of CLDN2 in promoting cancer cell migration and invasion, offering a potential foundation for future studies into its signaling interactions and therapeutic potential.
View Figures

Figure 1

ICE analysis of CRISPR/Cas9-mediated
CLDN2-KO efficiency in HCT116 cells. ICE software analysis
confirmed successful editing of the CLDN2 locus with an
efficiency of ~91%. The editing score reflects the proportion of
indels detected in the cell population. ICE, Inference of CRISPR
Edits; CLDN2, claudin-2; Wt, wild-type; KO, knockout; indel,
insertion-deletion.

Figure 2

Sanger discordance plot displays the
alignment/base between the Wt and CLDN2-KO samples and
inside the inference window (the area around the cute site)
indicates the average proportion of signal that differs from the
reference sequence obtained from the Wt trace. In the plot, the
proximity between the green line and orange line is seen before to
the cut site. However, a typical CRISPR edit leads to a notable
increase in sequence discordance at the cut site, causing the green
and orange lines to stay far apart afterwards. Wt, wild-type;
CLDN2-KO, claudin-2 knockout.

Figure 3

Sanger sequence view displays the
knockout and Wt sequences in the immediate area of the guide
sequence. This displays the sequence base calls obtained from Wt)
and CLDN2-KO). The guide sequence is represented by the
horizontal black underlined section. The PAM location is indicated
with a horizontal red underline. The vertical line, indicated by
the black dots, depicts the precise location of the incision.
Performing a cut and attempting to repair it often leads to the
presence of mixed sequencing bases. PAM, protospacer adjacent
motif; Wt, wild-type; CLDN2-KO, claudin-2 knockout.

Figure 4

Wound healing assay assessing cell
migration in Wt and CLDN2-KO HCT116 cells. (A)
Representative images of wound closure at 0 and 24 h post-scratch
(scale bar, 100 µm). (B) Quantification of wound closure
percentage. Wt cells achieved ~96% closure, while CLDN2-KO
cells demonstrated 41% closure after 24 h (P=0.0027; unpaired
two-tailed t-test; n=3). Data are presented as the mean ± SEM.
**P<0.01. Wt, wild-type; CLDN2-KO, claudin-2 knock
out.

Figure 5

Gene expression analysis of invasion-
and metastasis-related genes in CLDN2-KO vs. Wt HCT116
cells. Reverse transcription-quantitative PCR exhibited significant
downregulation of multiple target genes, including ZONAB, NDRG1,
CLDN14, CLDN23, Bcl-2, p53 and Bcl-6. Gene expression
levels were normalized to GAPDH. Data are represented as mean ± SEM
(n=3). Statistical comparisons were made using unpaired two-tailed
t-tests. *P<0.05. ns, not significant; Wt, wild-type;
CLDN2-KO, claudin-2 knock out; ZO-1, zonula occludens-1;
VDR, vitamin D receptor; ZONAB, ZO-1-associated
nucleic acid binding protein; NDRG1, N-Myc downstream-regulated
gene 1; APC, adenomatous polyposis coli; AF-6/AFDN, Afadin;
TJP1, tight junction protein 1; YBX3, Y-box binding protein 3;
PTMS, parathymosin; TCN-1, transcobalamin 1.

Figure 6

Relative expression levels of
metastasis-associated genes in Wt and CLDN2-KO HCT116 cells.
Gene expression was quantified using reverse
transcription-quantitative PCR. Each bar represents the mean ± SEM
of three independent experiments. Values were calculated using the
2−ΔΔCq method and normalized to GAPDH. Expression in Wt
cells was set to 1.0 and knock out values were expressed relative
to this baseline. Each bar corresponds to a specific gene and the
height of the bars indicates the magnitude of the fold-change
observed in the CLDN2-KO samples compared with the Wt
samples. The highest bars on the figure represent the gene with the
lowest degree of expression variation. IL-6 exhibited the
lowest degree of downregulation (fold-change, 0.718), whereas AF-6
demonstrated the most pronounced reduction (fold-change, 0.008).
Wt, wild type; ZO-1, zonula occludens-1; VDR, vitamin
D receptor; ZONAB, ZO-1-associated nucleic acid binding
protein; NDRG1, N-Myc downstream-regulated gene 1; APC, adenomatous
polyposis coli; AF-6, Afadin; PTMS, parathymosin; TCN-1,
transcobalamin 1; CLDN2-KO, claudin-2 knock out; AU,
arbitrary Units.
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Copy and paste a formatted citation
Spandidos Publications style
Alghamdi RA and Al‑Zahrani MH: CRISPR/Cas9‑mediated claudin‑2 knockout in HCT116 cells reveals its key role in colorectal cancer progression. Oncol Lett 31: 54, 2026.
APA
Alghamdi, R.A., & Al‑Zahrani, M.H. (2026). CRISPR/Cas9‑mediated claudin‑2 knockout in HCT116 cells reveals its key role in colorectal cancer progression. Oncology Letters, 31, 54. https://doi.org/10.3892/ol.2025.15407
MLA
Alghamdi, R. A., Al‑Zahrani, M. H."CRISPR/Cas9‑mediated claudin‑2 knockout in HCT116 cells reveals its key role in colorectal cancer progression". Oncology Letters 31.2 (2026): 54.
Chicago
Alghamdi, R. A., Al‑Zahrani, M. H."CRISPR/Cas9‑mediated claudin‑2 knockout in HCT116 cells reveals its key role in colorectal cancer progression". Oncology Letters 31, no. 2 (2026): 54. https://doi.org/10.3892/ol.2025.15407
Copy and paste a formatted citation
x
Spandidos Publications style
Alghamdi RA and Al‑Zahrani MH: CRISPR/Cas9‑mediated claudin‑2 knockout in HCT116 cells reveals its key role in colorectal cancer progression. Oncol Lett 31: 54, 2026.
APA
Alghamdi, R.A., & Al‑Zahrani, M.H. (2026). CRISPR/Cas9‑mediated claudin‑2 knockout in HCT116 cells reveals its key role in colorectal cancer progression. Oncology Letters, 31, 54. https://doi.org/10.3892/ol.2025.15407
MLA
Alghamdi, R. A., Al‑Zahrani, M. H."CRISPR/Cas9‑mediated claudin‑2 knockout in HCT116 cells reveals its key role in colorectal cancer progression". Oncology Letters 31.2 (2026): 54.
Chicago
Alghamdi, R. A., Al‑Zahrani, M. H."CRISPR/Cas9‑mediated claudin‑2 knockout in HCT116 cells reveals its key role in colorectal cancer progression". Oncology Letters 31, no. 2 (2026): 54. https://doi.org/10.3892/ol.2025.15407
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