Introduction

Oncology Letters

1792-1074 1792-1082

D.A. Spandidos

10.3892/ol.2025.15407

OL-31-2-15407

Articles

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

Alghamdi

Rana A.

1 2 Al-Zahrani

Maryam H.

1Department of Chemistry, Science and Arts College, King Abdulaziz University, Rabigh 21911, Saudi Arabia 2Regenerative Medicine Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 80216, Saudi Arabia 3Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia

Correspondence to: Dr Rana A. Alghamdi, Department of Chemistry, Science and Arts College, King Abdulaziz University, Rabigh Campus, Faculty Building 4, King Abdulaziz Road, Rabigh 21911, Saudi Arabia, E-mail: raalghamdi3@kau.edu.sa

022026

27112025

31 2

28072025 31102025

2025

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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.

CRISPR/Cas9 claudin-2 colorectal cancer tight junction protein gene expression knockout

Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, Saudi Arabia

G: 48-665-1443

The present study was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, Saudi Arabia (grant no. G: 48-665-1443).

Introduction

Colorectal cancer (CRC) is a major cause of cancer-associated mortality worldwide, In 2023, an estimated 1.97 million new cases of CRC and ~0.93 million deaths were reported globally (1). The burden of CRC is increasing in Eastern Asia, the Middle East, and parts of Southeast Asia, where rapid lifestyle transitions, Westernized diets, and limited early-screening coverage have contributed to rising incidence trends (2). In Asia, ~992,000 new CRC cases and ~498,000 related deaths were reported in 2020 (3). Proteomic and genomic studies have advanced cancer research by revealing molecular pathways involved in CRC particularly through dysregulated signaling cascades that drive tumor development and progression, such as the Wnt/β-catenin, PI3K/AKT signaling, MAPK/ERK pathway, and alterations in TP53-regulated stress response networks, all of which play key roles in cell proliferation, survival, and metastasis (4). Among key molecular players, claudin-2 (CLDN2), a component of tight junctions, has emerged as a contributor to malignancy by exhibiting aberrant expression (5).

The claudin family serves a key role in cell barriers, differentiation and proliferation. The expression patterns of claudin knockout (KO) vary throughout malignancies and organs (6). Thus, claudins have been proposed as targets for cancer treatment as well as diagnostic indicators. Previous research has demonstrated an increasing consensus on the potential value of CLDN2 as a biomarker for prognostic and therapeutic features in CRC (7–9). Notably, CLDN2 expression is elevated in inflammatory bowel disease and colorectal tumors compared with normal tissues (10).

CLDN2 has diverse biological functions beyond maintaining epithelial permeability. It contributes to paracellular water and ion transport, modulates epithelial proliferation and participates in signaling events associated with oncogenic transformation (8,11). Elevated CLDN2 levels have been associated with poor prognosis, advanced tumor stages and increased metastatic potential in several cancer types, including CRC (7,12). These findings highlight the importance of understanding the multifunctional role of CLDN2 in colorectal carcinogenesis. CLDN2 is the most distinct member of the claudin family and exhibits a unique expression pattern because its expression is limited to permeable epithelial tissues (11). In colon cancer, CLDN2 appears to function as an oncogene, enhancing cell proliferation and migration capacity through EGFR-mediated pathways (12). Notably, the increasing incidence of CRC has been connected to the presence of CLDN2 in cellular tight junctions.

Mechanistically, it has been demonstrated that CLDN2 inhibits the transcription of N-Myc downstream-regulated gene 1 (NDRG1), a well-established metastasis suppressor gene. This repression is mediated through the recruitment of zonula occludens-1 (ZO-1)-associated nucleic acid binding protein (ZONAB) to the NDRG1 promoter, where CLDN2 facilitates ZONAB binding and suppresses NDRG1 transcriptional activity (13).

Elevated CLDN2 expression has been observed in CRC compared with adjacent normal tissue and is associated with shorter cancer-specific survival and increased risk of recurrence in stage II/III CRC receiving adjuvant therapy (14). Although environmental and genetic variables can affect CRC, such as diet, obesity, and smoking, understanding the mechanisms by which CLDN2 functions during CRC development could help identify potential novel treatment options in the future. High CLDN2 expression patterns reported shorter survival outcomes, indicating a connection between high CLDN2 expression and CRC progression (13).

While genomic analyses have identified driver mutations in CRC, understanding the mechanisms by which specific proteins such as CLDN2 integrate into oncogenic signaling networks remains challenging (15–17). CRISPR/Cas9-based gene editing offers a key tool to dissect functional roles by enabling precise gene KO models (18). Furthermore, combining CRISPR approaches with gene expression profiling allows for further investigation into CRC biology and the identification of potential novel therapeutic targets in the future (19). Due to the limited mechanistic understanding of CLDN2 in CRC, the present study aimed to investigate the effects of CLDN2 deletion on gene expression patterns associated with migration, invasion and metastasis. A CRISPR/Cas9-mediated KO approach was used in HCT116 cells to assess both phenotypic changes and transcriptional alterations, with a focus on pathways implicated in CRC progression.

Materials and methods <sec> <title>Cell culture

The human CRC cell line HCT116 (cat. no. CCL-247; American Type Culture Collection) was obtained from Synthego. The HCT116 cell line is a widely used model of CRC repair deficiency due to a mutation in the mutL homolog 1 gene. Cells were authenticated by short tandem repeat profiling, and were confirmed to be mycoplasma-free prior to use.

Wild-type (Wt) and CLDN2-KO HCT116 cells were cultured in McCoy's 5A medium (cat. no. 16600082; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (cat. no. 10270106; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (cat. no. 15140-122; Thermo Fisher Scientific, Inc.). The cells were maintained at 37°C in a humidified incubator with 5% CO₂.

Generation of CRISPR/Cas9-mediated CLDN2-KO cells

CRISPR/Cas9 mediated KO of human CLDN2 (gene ID, 9075) in HCT116 cells was produced by Synthego. Guide RNA design and off-target prediction were performed using the Synthego CRISPR Design Tool (v1.2; Synthego). Ribonucleoprotein complexes consisting of Cas9 protein and a synthetic, chemically modified single-guide RNA (sgRNA) targeting exon 2 of human CLDN2 (sgRNA sequence, 5′-GGUGCUAUAGAUGUCACACU-3′) were electroporated into HCT116 cells. The sgRNA cut site was chrX:106,928,419. Following electroporation (20), single-cell clones were generated by fluorescence-activated cell sorting into 96- or 384-well plates and expanded (21). The assessment of editing efficiency was performed 48 h after electroporation. The process involved extracting genomic DNA (gDNA) from a subset of cells, followed by PCR amplification and sequencing using the Sanger sequencing method. Sequencing was performed in the forward direction using a 20-nt primer, generating 700–900 nt reads. Sanger sequencing services were provided by Synthego. PCR primers were as follows: Forward, 5′-CAGCCTGAAGACAAGGGAGC-3′; and reverse, 5′-TGTCTTTGGCTCGGGATTCC-3′. The sequencing primer used was as follows: 5′-CAGCCTGAAGACAAGGGAGC-3′. The chromatograms obtained were analyzed using the Synthego Inference of CRISPR edits (ICE; version 2.0 (Synthego) (22).

For insertion-deletion (indel), the indel examination was performed using the Illumina sequencing platform (Illumina, Inc.), according to the manufacturer's protocol (23). A total of 20 ng gDNA was utilized as a template for amplifying the region surrounding the sgRNA target site, using the specified primers. To generate monoclonal cell populations, cell pools with altered CLDN2 gene (CLDN2-KO) were distributed at a density of 1 cell/well using a single cell printer onto either 96- or 384-well plates (24). Every 3 days, all wells were recorded to verify the growth of a clone originating from a single cell. The PCR-Sanger-ICE genotyping technique was used to screen and identify clonal populations.

Wound healing assay

The migratory capacity of Wt and CLDN2-KO cells was assessed using a wound healing assay (25). Briefly, 3×10⁵ cells/well were seeded into 6-well plates and cultured until they reached 90% confluence. After which, a sterile pipette tip was used to scratch across the center of each well, creating a defined wound area. Detached cells were removed by washing with 500 µl PBS and fresh culture medium was added. Subsequently, cell migration into the scratched area was monitored and captured at 0 and 24 h using a Nikon E 600 phase-contrast microscope. Images of the wound area were analyzed using ImageJ software (version 2.9.0/1.53t; National Institutes of Health) (26). Wound closure percentage was calculated as [(A_t=0-At_=∆t)/A_t=0] ×100, where A_t=0 is the scratch area at 0 h and At_=∆t is the scratch area at 24 h.

RNA extraction and reverse transcription-quantitative PCR (RT-qPCR)

RNA was isolated in accordance with the manufacturer's protocol using an RNA extraction kit (cat. no. R1200; Beijing Solarbio Science & Technology Co., Ltd.). Subsequently, cDNA was synthesized using random nonamer primers and the First-Strand Synthesis System (MilliporeSigma; Merck KGaA). EvaGreen fluorescence-based RT-qPCR was performed using reagents purchased from Applied Biological Materials, Inc. RT-qPCR reactions were performed following standard protocols as previously described (27). The RT-qPCR primers used in the present study are listed in Table I. The gene expression levels in the samples were investigated using RT-qPCR and the cDNA synthesis and PCR procedures were optimized to ensure high-quality results.

Relative and normalized fold expression values calculation

The gene expression patterns of several target genes were examined in samples from both Wt and CLDN2-KO cells using RT-qPCR. Relative expression levels were calculated using the comparative Cq method (ΔΔCq), normalized to GAPDH as the reference gene (28). For every target gene, the Cq values that were derived from the amplification curves were used to compute ΔCq, copy number, ΔΔCq and fold change. ΔCq was calculated using the following formula: ΔCq=Cq_{(target gene; same sample)}-Cq_{(control gene; same sample)}. Copy number was calculated using the following formula: Copy number=100 × 2^−ΔCq. ΔΔCq was calculated using the following formula: ΔΔCq=ΔCq_{(same gene; target sample)}-ΔCq_{(same gene; control sample)}. Fold change was calculated using the following formula: Fold change=2^−ΔΔCq.

The housekeeping gene GAPDH was used as an internal control for normalization. The expression levels of GAPDH were relatively stable across all samples. Data are represented as the mean of three independent experiments.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software (version 8.0; Dotmatics). Data are presented as the mean ± SEM, with at least three independent biological replicates. Normality was assessed using the Shapiro-Wilk test. Statistical significance was assessed using Welch's unpaired t-test for ΔCq values Comparisons between two groups (Wt vs. CLDN2-KO) were made using an unpaired two-tailed Student's t-test if data were normally distributed or a Mann-Whitney U test for non-parametric comparisons. P<0.05 was considered to indicate a statistically significant difference.

Results <sec> <title>High efficiency of CRISPR/Cas9 editing of the CLDN2 gene

The Synthego ICE CRISPR Analysis tool was employed to assess the effectiveness of CRISPR CLDN2 gene KO (Fig. 1). The ICE value of 91% indicated a high degree of editing efficiency, suggesting that a notable proportion of the cells within the edited population harbored the intended CLDN2 gene KO. Particularly, 91% of cells contained the indel of the CLDN2 gene after CRISPR/Cas9 editing.

Fig. 2 presents the Sanger sequencing discordance plot, comparing sequencing traces from Wt and CLDN2-KO cells around the CRISPR cut site. Prior to the cut site, the Wt and CLDN2-KO traces overlapped. At the target site, a sharp increase in sequence discordance was observed in CLDN2-KO cells, evidenced by the divergence between Wt (green) and CLDN2-KO (orange) signal lines. This pattern is characteristic of successful genome editing. Furthermore, Fig. 3 displays the Sanger sequence alignment surrounding the sgRNA target site, with the sgRNA sequence underlined in black and the protospacer adjacent motif underlined in red. A vertical line marks the cut site, where sequence differences between Wt and CLDN2-KO cells become evident. These results confirmed that the CLDN2 gene was effectively knocked out in HCT116 cells with high efficiency. Consistent with the sequencing results, qPCR analysis demonstrated a significant reduction in CLDN2 expression in CLDN2-KO cells relative to Wt cells (Fig. S1).

CLDN2-KO impairs HCT116 cell migration

At 24 h, Wt cells exhibited near-complete wound closure (~96%), while CLDN2-KO cells achieved only ~41% closure. Quantification of wound healing (Fig. 4B) demonstrated a significant reduction in migratory capacity in CLDN2-KO cells compared with Wt controls (P=0.0027; unpaired t-test; n=3 independent experiments). These findings indicated that CLDN2 is key for the efficient migration of CRC cells.

Gene expression profiles in Wt and CLDN2-KO samples

To investigate the transcriptional effects of CLDN2 deletion, the expression levels of multiple genes implicated in invasion and metastasis was analyzed using RT-qPCR. Fig. 5 displays the copy numbers of target genes in Wt and CLDN2-KO cells. Analysis of copy number variations for all genes consistently demonstrated a significant decrease in copy number in CLDN2-KO samples compared with the Wt cells (all P<0.05). The observed patterns indicated that CLDN2-KO has a notable impact on its interacting partners, causing their downregulation.

Fig. 6 depicts the fold-change values of several genes in both Wt and CLDN2-KO samples. Of all the genes that were evaluated, IL-6 was the least downregulated in CLDN2-KO samples, with a fold-change of 0.718 and maintains a relatively stable expression pattern. By contrast, AF-6, which encodes Afadin, exhibited the most marked downregulation, with a fold-change of 0.008. These results suggested that CLDN2 loss disrupts a network of pro-metastatic gene expression programs that may contribute to impaired cellular migration and reduced metastatic potential.

Discussion

Claudins are key components of tight junctions, serving key roles in the maintenance of epithelial integrity and regulating paracellular permeability (8). Previous studies have suggested that claudins are involved in the development of malignancies. However, the specific process has not yet been fully elucidated (9,29,30).

In the present study, the role of CLDN2 in CRC progression was investigated. Using CRISPR/Cas9-mediated CLDN2-KO in HCT116 cells, it was demonstrated that the loss of CLDN2 leads to significant downregulation of genes associated with invasion, metastasis and cell motility. The findings of the present study aligned with previous studies suggesting that CLDN2 promotes tumorigenicity and metastasis in CRC (8–11,26,31). Particularly, it was observed that CLDN2-KO resulted in significantly reduced expression levels of ZONAB and NDRG1, two factors which regulate epithelial proliferation and metastasis suppression, respectively.

ZONAB is a Y-box transcription factor that interacts with the tight junction protein ZO-1. When retained at tight junctions, its transcriptional activity is restricted, whereas nuclear translocation of ZONAB promotes the expression levels of genes involved in proliferation and epithelial-mesenchymal transition (32,33). By contrast, NDRG1 functions as a metastasis suppressor. It inhibits invasion and migration, modulates epithelial differentiation and negatively regulates oncogenic pathways such as PI3K/AKT and Wnt/β-catenin (34,35). Downregulation of NDRG1 is associated with poor prognosis in CRC (13). Thus, CLDN2-driven suppression of NDRG1 through ZONAB activity provides a mechanistic association between tight junction dysregulation and enhanced tumor progression. These results support the hypothesis that CLDN2 may modulate the CLDN2/ZO-1/ZONAB signaling axis, thereby influencing transcriptional programs key for cancer cell migration and invasion (36). Previous studies suggested that the abundance of CLDN2 increases with cell confluence and the maturation of tight junctions and that ZO-1 (and ZO-2) help stabilize CLDN2 by reducing its turnover. Knockdown of ZO-1 leads to notable loss of CLDN2 abundance and promoter activity (37). In the present study, the downregulation of ZO-1 in CLDN2-KO cells indicated that downstream signaling is disrupted in the absence of CLDN2.

In addition to tight junction-related proteins, CLDN2-KO affected the expression levels of other metastasis-associated genes. Downregulation of CLDN14 and CLDN23, tight junction components previously associated with CRC progression (38,39), was observed. Furthermore, reduced expression level of AF-6 (encoding Afadin), a scaffolding protein involved in cell-cell adhesion and migration, was also notable. Previous research has demonstrated that CLDN2 physically interacts with Afadin via its PDZ-binding motif and this complex contributes to metastatic behavior in breast cancer models. In addition, loss of Afadin impairs colony formation and metastasis (40,41). This provides a precedent to consider a CLDN2-Afadin axis in CRC.

Furthermore, the data in the present study revealed a decreased expression level of CDK4, a cyclin-dependent kinase key for cell cycle progression. Since ZONAB (regulated by CLDN2) can associate with and influence CDK4 nuclear activity, reduced CDK4 expression may reflect the disruption of CLDN2/ZO-1/ZONAB signaling (32,33,42). Collectively, these transcriptional changes highlight the broad regulatory influence of CLDN2 on multiple pathways driving CRC metastasis.

Notably, several key oncogenic and tumor suppressor genes were also modulated by CLDN2 deletion. The proto-oncogene c-Myc, a master regulator of proliferation and metabolism, was significantly downregulated. Reduced c-Myc expression may contribute to the suppression of migratory and invasive phenotypes in CLDN2-deficient cells, consistent with its role in CRC aggressiveness (43).

An inverse association between p53 and CLDN2 has been reported in mouse colon tissue during dextran sulfate sodium-induced colitis (44), where reduced p53 expression was accompanied by increased CLDN2 levels, suggesting that loss of p53-mediated regulation may contribute to CLDN2 upregulation in inflamed and neoplastic intestinal epithelium (45). Furthermore, a previous study suggested a negative regulatory complex involving p53 and hepatocyte nuclear factor 4 α that may influence CLDN2 expression (44). However, in the present study, downregulation of p53 in the KO condition, indicated by a fold-change of 0.02, suggests that CLDN2 may have a regulatory role in its own expression. The reduced copy number further supports the notion of downregulation of p53 in the CLDN2-KO cells.

A previous study provided evidence for the qualitative and quantitative expression levels of Bcl-6 involvement in human CRC progression, and demonstrated that Bcl-6 appears to be involved in tumor development, from the earliest stage of carcinogenesis (46). In the present study, in the KO condition, Bcl-6 exhibited a downregulation, indicating a possible regulatory function for CLDN2 in its expression. Although direct evidence of a CLDN2-Bcl-6 axis is limited, to the best of our knowledge, perturbations in tight junction integrity and nuclear signaling have been associated with transcriptional repressor modulation in cancer (47,48), suggesting that CLDN2 loss may indirectly influence Bcl-6 expression.

Furthermore, the tumor suppressor gene adenomatous polyposis coli (APC), a key component of the Wnt/β-catenin signaling pathway, was markedly reduced. APC dysfunction is a hallmark of CRC initiation and progression (49,50). Its downregulation following CLDN2-KO highlights the complex interplay between tight junction integrity and oncogenic pathways. Loss of APC may also reflect feedback from junctional disruption on Wnt signaling regulation, further implicating CLDN2 in the modulation of oncogenic cascades. Activation of the Wnt/β-catenin signaling pathway has been documented in a notable proportion of gastric cancer cases. For example, nuclear β-catenin accumulation, a hallmark of Wnt pathway activation, has been reported in ~1/3 of gastric adenocarcinomas (51). Recent studies estimated that 30–50% of gastric tumor specimens exhibit hyperactivation of this pathway (52,53). In addition, activation of Wnt/β-catenin signaling has been reported to increase the expression levels of MMP7 at both the mRNA and protein levels in triple-negative breast cancer, providing a mechanistic association between Wnt signaling and invasive phenotypes (54,55). These findings further support the role of Wnt pathway activation in promoting cancer progression across multiple tumor types.

Furthermore, the vitamin D receptor (VDR), which regulates proliferation, differentiation and epithelial barrier function, exhibited decreased expression. Multiple studies have indicated that VDR can directly bind and regulate CLDN2 transcription in intestinal tissues, and that VDR dysregulation has been implicated in colorectal tumorigenesis (56,57). This supports a bidirectional regulatory association between VDR and CLDN2 expression, and suggests that CLDN2 loss may contribute to impaired VDR signaling in CRC.

Notably, elevated CLDN2 expression has been associated with poor prognosis in CRC, liver cancer and other gastrointestinal malignancies (7,12,58). The findings of the present study supported this clinical association by demonstrating that Cldn2 loss impairs migratory potential and suppresses pro-metastatic gene expression. Thus, targeting CLDN2 may represent a promising therapeutic strategy to inhibit CRC metastasis in the future, although careful evaluation of potential side effects on normal epithelial function is warranted in future studies.

It is key to acknowledge the limitations of the present study. While the present study results demonstrated a strong association between CLDN2 loss and altered gene expression, mechanistic experiments, such as rescue assays or pathway-specific inhibition, were not performed to directly establish causality. The findings were also based on in vitro analyses in a single CRC cell line; validation using in vivo models or additional cell lines in future studies would strengthen these conclusions. Another limitation is that CLDN2 protein loss was not validated by western blotting, although the CRISPR editing efficiency was demonstrated by qPCR, ICE analysis and Sanger sequencing. In addition, cell migration was assessed only by the wound healing assay. While this method reflects overall migratory capacity, it does not capture chemotactic or invasive behavior. Incorporating approaches such as Transwell migration and invasion assays would provide complementary evidence and these are planned for future research. Lastly, further studies are warranted to dissect the downstream mechanisms by which CLDN2 modulates signaling pathways. Rescue experiments restoring CLDN2 expression, pathway-specific analyses (such as PI3K/AKT, Wnt/β-catenin and yes-associated protein/transcriptional co-activator with PDZ-binding motif) and investigation of the interaction partners of CLDN2, such as ZONAB and Afadin, will be key to elucidate its precise role in CRC progression in the future.

In conclusion, the present study demonstrated that CLDN2 serves a key role in regulating metastasis-associated gene networks in CRC. Disruption of CLDN2 in HCT116 cells significantly impaired migration and was accompanied by the downregulation of key invasion- and metastasis-related genes, including ZONAB, NDRG1, AF-6, CLDN14, CLDN23, CDK4, Bcl−6 and APC. These transcriptional changes highlight the broad regulatory influence of CLDN2 on pathways governing cell adhesion, proliferation and Wnt/β-catenin signaling. While additional mechanistic studies, such as rescue assays and pathway-specific analyses, are warranted to further dissect these interactions, the present study findings provide notable genetic and phenotypic evidence that CLDN2 contributes to CRC progression. By directly associating CLDN2 deletion with the suppression of specific pro-metastatic genes, the present study suggests that targeting CLDN2 may represent a promising therapeutic strategy to potentially inhibit CRC metastasis in the future.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

RA conceived and designed the study. MA designed the study. RA and MA performed experiments, analyzed and interpreted the data and wrote the manuscript. RA and MA confirm the authenticity of all the raw data. Both authors have read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Use of artificial intelligence tools

During the preparation of this work, artificial intelligence tools were used to improve the readability and language of the manuscript or to generate images, and subsequently, the authors revised and edited the content produced by the artificial intelligence tools as necessary, taking full responsibility for the ultimate content of the present manuscript.

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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 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 sco...

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 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...

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 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 seq...

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 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 o...

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 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 t...

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.

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 t...

Table I.

Primers for RT-qPCR used in the present study.

Gene symbol	Oligo sequence (5′-3′)
AF-6 (AFDN)	F: AGTCGGTTGTGAAAGGAGGTGC
	R: TCCTGAGAGAGTCCAACCAGAC
APC	F: AGGCTGCATGAGAGCACTTGTG
	R: CACACTTCCAACTTCTCGCAACG
Bax	F: TCAGGATGCGTCCACCAAGAAG
	R: TGTGTCCACGGCGGCAATCATC
Bcl-2	F: ATCGCCCTGTGGATGACTGAGT
	R: GCCAGGAGAAATCAAACAGAGGC
Bcl-6	F: CATGCAGAGATGTGCCTCCACA
	R: TCAGAGAAGCGGCAGTCACACT
Caspase 3	F: GGAAGCGAATCAATGGACTCTGG
	R: GCATCGACATCTGTACCAGACC
CDK4	F: CCATCAGCACAGTTCGTGAGGT
	R: TCAGTTCGGGATGTGGCACAGA
CLDN2	F: GTGACAGCAGTTGGCTTCTCCA
	R: GGAGATTGCACTGGATGTCACC
CLDN14	F: CCAAGACCACCTTTGCCATCCT
	R: AGTTCTGCACCACGTCGTTGGT
CLDN23	F: CCTGGTGCGACGAGCGTTGTC
	R: GTCGCTGTAGTACTTGATGGCG
IL-6	F: AGACAGCCACTCACCTCTTCAG
	R: TTCTGCCAGTGCCTCTTTGCTG
MMP7	F: TCGGAGGAGATGCTCACTTCGA
	R: GGATCAGAGGAATGTCCCATACC
NDRG1	F: ATCACCCAGCACTTTGCCGTCT
	R: GACTCCAGGAAGCATTTCAGCC
p53	F: CCTCAGCATCTTATCCGAGTGG
	R: TGGATGGTGGTACAGTCAGAGC
PTMS	F: AGAAACTGCCGAGGATGGAGAG
	R: TGCCGTTTGGGATCCGCTTCAT
TCN1	F: CAGTGTGATGGAGAAAGCCCAG
	R: CCACTCAGAAGTTCCCAGTAGG
VDR	F: CGCATCATTGCCATACTGCTGG
	R: CCACCATCATTCACACGAACTGG
ZO-1 (TJP1)	F: GTCCAGAATCTCGGAAAAGTGCC
	R: CTTTCAGCGCACCATACCAACC
ZONAB (YBX3)	F: TGGTCCAAACCAGCCGTCTGTT
	R: GTTCTCAGTTGGTGCTTCACCTG
GAPDH (RG)	F: GTCTCCTCTGACTTCAACAGCG
	R: ACCACCCTGTTGCTGTAGCCAA

RG, reference gene; RT-qPCR, reverse transcription-quantitative PCR; F, forward; R, reverse; CLDN2, claudin-2; ZO-1, zonula occludens-1; VDR, vitamin D receptor; ZONAB, ZO-1-associated nucleic acid binding protein; YBX3, Y-box binding protein 3; NDRG1, N-Myc downstream-regulated gene 1; APC, adenomatous polyposis coli; AF-6/AFDN, Afadin; TJP1, tight junction protein 1; PTMS, parathymosin; TCN-1, transcobalamin 1.