Overexpression of Klotho gene using CRISPR/Cas9 induces apoptosis and inhibits cell motility in the human colorectal cancer cells

Despite advances in early detection and treatment, colorectal cancer remains one of the leading causes of cancer‐related deaths. The klotho (KL) gene plays a critical role in the development and progression of colorectal cancer. This study investigates the role of the KL gene in colorectal cancer by using the CRISPR/Cas9 system to overexpress and knock out (KO) the KL gene in human colorectal cancer cells (Caco‐2). The effects of the changes were assessed by gene expression analysis, flow cytometry, scratch wound closure assays, colony formation assays, and immunofluorescence staining. Our results showed that overexpression of the KL gene increased apoptosis and decreased cell motility in cancer cells, whereas knockout of the KL gene had the opposite role. The present study elucidates the mechanisms underlying this role and highlights the potential of the CRISPR/Cas9 system as a gene editing tool in cancer research. Our data suggest that activation of the KL gene may serve as a novel therapeutic strategy and biomarker for studies in colorectal cancer.

tumorigenesis mechanisms. [9,10]15][16][17][18][19][20][21][22] Currently, CRISPR-Cas9 is used in vitro and in animal models to understand single-gene diseases such as cystic fibrosis, hemophilia, and sickle cell anemia and to determine whether this approach is effective and safe for clinical use.In addition, this technology shows promise for the treatment and prevention of more complex human diseases such as cancer and heart disease. [23,24]The aim of this study was to overexpress the KL gene in apoptosis-resistant human colon cancer cells (Caco-2) with the CRISPR/Cas9 technique and to evaluate whether the activation and inactivation of the KL gene have an apoptotic effect in Caco-2 cells.

Transfection of CRISPR/Cas9 plasmid constructs for the activation and knockout of the KL gene
For the activation of the KL gene, a CRISPR activation plasmid kit (Cat no: sc-400970-ACT, Santa Cruz Biotechnology, Texas, USA) was used in Caco-2 cells.KL gene activation plasmid kit includes the SAM transcription activation system (dCas9-VP64-Blast, MS2-P65-HSF1-Hygro, sgRNA (MS2)-Puro) (Figure 1A).The protocol of the manufacturer's manual was applied.Transfection of constructs in Caco-2 cells was performed using Transfectamine 5000 transfection reagent (AAT Bioquest, California, USA), according to the manufacturer's recommendation.Cells were seeded at 2 × 10 5 cells per well in a six-well tissue culture plate for transfection.Cells were grown to 70%-80% confluence in fresh antibiotic-free growth media 24 h prior to transfection.For transfection, 2. For CRISPR gene KO, sgRNA sequences direct the Cas9 protein to induce a site-specific double-strand break (DSB) in the genomic DNA.
The transfection was performed according to the protocol in the manufacturer's manual.Transfectamine 5000 transfection reagent (AAT Bioquest) was used for KL gene transfection into Caco-2 cells.Cells were seeded at 2 × 10 5 cells per well in a 6-well tissue culture plate for transfection.Cells were grown to 70%-80% confluence in fresh antibiotic-free growth media 24 h prior to transfection.Furthermore, the standard growth medium was changed 24 h before transfection to a fresh antibiotic-free growth medium.For transfection, 2.5 μg of plasmid DNA and 10 μL of transfection reagent per well were used.

RNA extraction and qRT-PCR analysis
Total RNA from cells was isolated using the total RNA extraction kit (Blirt SA, Gdansk, Poland).The protocol was as per the manufacturer's recommendation.Then, total RNA was reverse transcribed into cDNA with the transcriptome RNA kit (Blirt SA).To obtain cDNA, 10 μL of 2xRT master mix, 2 μL of transcriptome enzyme mix, nuclease-free water, and mRNA were mixed.The mixture was incubated at 25  1).
The expression level was determined by quantitative real-time PCR

2.5
In-vitro scratch wound closure assay 2 × 10 5 Caco-2 cells were plated in a 6-well plate.Then, cells were grown to 70% confluency and scratched with a pipette tip to create a monolayer cell-free scratched area.Cells were gently washed with PBS to remove floating cells, followed by transfection of plasmid constructs for overexpression and knockdown of the KL gene.After 24 h, photographs were taken from three random positions.Cells transfected with knockout plasmids were used as control cells.Inverted microscope photographs (Carl Zeiss Axio Observer D1, Oberkochen, Germany) of the migration of cells were taken at the same locations every 24 h for 3 days. [25]The wound areas in the cells were calculated every 24 h using Zen 2 Pro software.The relative surface area of the wound area was calculated by considering the apertures in the photographs taken at the end of the first 24 h after the wound was created as 100%.

Colony formation assay
Colon cancer cells were seeded in 6-well plates at 1 × 10 5 cells per well.Plasmid transfection was performed to overexpress and knockout the KL gene in colon cancer cells.The medium was changed 24 h after transfection.The fresh culture medium was replaced every 3 days, and colonies were counted if they contained ≥50 cells.The number of colonies was counted on the sixth day after seeding, and they were stained using 1.25% crystal violet.The colony formation rate was calculated with the equation: colony formation rate = (number of colonies/number of seeded cells) × 100%.

Immunofluorescence staining
To determine the status of the apoptotic pathway (active caspase 3 and

Statistical analysis
For each experiment, three independent experiments were carried out at different times.Results were expressed as mean ± SE.SPSS 20.0 software was used for statistical analysis.A one-way ANOVA with Tukey's post hoc analysis and a paired t-test were used to analyze the data.Differences between experimental and control groups were considered statistically significant when p < 0.05.

Determination of the overexpression KL gene in colon cancer cells
To determine the effects of overexpression and knockout of the

Colony formation assay
After regulating the expression of the KL gene, colony formation analysis was performed to determine the functional effect of KL gene on colon cancer cells.As shown in Figure 3A, the number of colonies in KL-OE Caco-2 cells was significantly lower than in the control group.
Colony formation in KL-KO cells was similar to the control (Figure 3B).

Detection of KL gene induced apoptosis in Caco-2 cells by flow cytometry
To determine the effects of overexpression or knockout of KL on apoptosis in Caco-2 cells, the apoptosis rate was evaluated by Annexin V/PI flow cytometry analysis (Figure 4A,B).As a result of the analysis, it is seen as 2.1% for Caco-2 cells, 20.6% for KL-OE cells, and 4.8% for KL-KO.We found a significantly higher percentage of apoptosis in the KL-OE compared with the control cells (p < 0.01).At the same time, there was no significant difference KL-KO (p > 0.05).

Cell migration scratch wound healing
The effect of the regulation of KL gene expression on cell invasion capacity in Caco-2 cells was evaluated by an in vitro scratch wound

Apoptosis-related gene expression levels after the regulation of KL gene expression by CRISPR/Cas9
We investigated the gene expression of potential apoptotic proteins involved in KL-induced apoptosis.When KL gene was knocked out in cells, caspase-8 activity was significantly decreased (p < 0.001).When KL gene was overexpressed in the cell, caspase-3 activity increased (p < 0.05).BCL-2 gene expression showed up-regulation in the KL-KO cells in comparison with the untreated cells (Caco-2, control), but it was lower in comparison with the KL-OE cells.These results may show that the increase in the KL gene expression level leads to the triggering of the mitochondrial pathway of apoptosis in cells (Figure 6).

Immunofluorescence staining of active caspase-3 and 8
Increasing the expression of the KL gene in Caco-2 cells caused apoptosis via effector caspase activity.As a result of the regulation of KL gene expression in Caco-2 cells, in both the KL-OE and KL-KO cells groups, immunoreactivity for caspase-3 and 8 was observed (Figure 7).

DISCUSSION
Regulation of KL gene expression affects cell functions related to maturation, differentiation, proliferation, and apoptosis.In the present study, the KL gene was both activated and knocked out using the CRISPR/Cas9 technique in apoptosis-resistant human colon cancer cells.
The KL gene is mainly expressed in the kidney and brain, but has also been detected in a limited number of other tissues, including placenta, ovary, prostate, and colon.Notably, KL gene expression is lower in tumor tissue than in nontumor regions.Studies have shown that a 50% reduction in KL gene expression is observed in ovarian and lung cancers, while KL gene expression is 80% in breast, pancreatic, and cervical cancers. [11,14,15,20,25,26]Suggesting that the reduction or loss of KL gene expression in some cancers may cause malignancy. [5]e tumor suppressor property of KL was first shown in a study on breast cancer. [12]Later, the overexpression of the KL gene was determined in the thyroid, [27] liver, [7,16] lung, [28] kidney, [29] and colon cancers [10] and has been shown to reduce the proliferation of cancer cells and cause apoptosis.In our flow cytometry analyses, early and late apoptosis rates resulting from the overexpression of the KL gene in colon cancer cells were 3.8% and 20.6%, respectively.This result showed that the KL gene has a tumor-suppressive effect by leading colon cancer cells to apoptosis.Our results were similar to those of studies performed in lymphoma, lung, liver, and colon cancer cells. [10,28,30,31]ll motility is particularly important for cancer invasion and metastasis.In our study, we evaluated for the first time the effect of altering KL gene overexpression on cell invasion capacity in colon cancer cells using the in vitro scratch-closure assay, which is an accepted in vitro model to evaluate epithelial cell migration during invasion.Our results showed that overexpression of the KL gene in Caco-2 cells had a positive effect on inhibiting cancer cell motility (p < 0.001) at 72 h.When the KL gene was knocked out in the cells, it was observed that the wound area began to close, but this closure was less than in nontransfected Caco-2 cells (p < 0.001).Studies have shown that increased KL gene expression in cancer cells decreases colony formation. [15,31]In a study of HCT-116 and HT-29 colon cancer cells, it was shown that increased KL gene overexpression inhibited the proliferation of cancer cells and reduced their colony forming potential.In SW480 and HT-29 colon cancer cells, in which KL gene expression was increased by virusmediated transfection, colony formation was shown to be reduced by increasing KL gene expression. [10]Similarly, our study found that when KL expression was increased in Caco-2 cells, the number of colonies formed was lower than in the control group.
In a study conducted in lung cancer cells, it has been shown that increasing the expression of the KL gene leads to an increase in the expression of the BCL-2 gene and a decrease in the expression of BAX.
However, when the KL gene was silenced, the expression of the BCL-2 gene increased while the BAX gene decreased. [28]A study conducted on lymphoma cells has shown that increasing the KL gene decreases the expression of the antiapoptotic MCL-1 gene and activates the expression of the active CASPASE-3 gene. [30]In our study, the gene expression levels of CASPASE-3, CASPASE-8, BID, BAD, BCL-2, and BAX genes were expression of CASPASE-3 has been reported in lymphoma cells. [30]The BCL-2 gene expression has been found to be reduced in lung cancer cells. [28]creasing the expression of the KL gene in Caco-2 cells caused apoptosis via effector caspase activity.In our immunofluorescence analyses, CASPASE-3 and 8 showed negative immunoreactivity in colon cancer cells and positive immunoreactivity in KL-OE cells.In addition, less positivity was observed in the KL-KO cells compared to the KL-OE cell group.Over the past decade, studies investigating the antitumor effect of KL in colon cancer cells have shown that KL gene overexpression inhibits cancer cell proliferation.KL overexpression was shown to suppress proliferation and invasion in HT-29 colon cancer cells through inhibition of the PI3K/AKT pathway mediated by IGF1R. [5]Similarly, in one of our studies, we demonstrated for the first time that KL selectively induces apoptosis in human colorectal adenocarcinoma cells (HT-29) and induces programmed cell death in cancer cells (CCD 841 CoN) without affecting healthy colon cells. [32]In our subsequent studies, we found that overexpression of the KL gene in Caco-2 cells using CRISPR/Cas9 sensitizes the TRAIL death receptor DR4, leading to cell apoptosis and suppressing cell proliferation. [33]on KL overexpression, the unfolded protein response pathway mediates KL gene activities in cancer suppression. [34]

CONCLUSION
The CRISPR/Cas9 technique was used in this study to investigate the potential apoptotic effect of overexpression and knockout of the KL gene in human adenocarcinoma colon cancer cells (Caco-2).Our 5 μg of plasmid DNA and 10 μL of transfection reagent per well were used.For the cell selection used, puromycin dihydrochloride (Cat no: sc-108071, Santa Cruz Biotechnology), hygromycin B (Cat no: sc-29067, Santa Cruz Biotechnology), and blasticidin S HCl (Cat no: sc-495389, Santa Cruz Biotechnology) antibiotics were used.Nontransfected cells were killed with the help of antibiotics.Puromycin dihydrochloride, hygromycin B, and blasticidin F I G U R E 1 Klotho CRISPR/Cas9 plasmid constructs.(A) Klotho CRISPR activation plasmid, (B) klotho CRISPR/Cas9 knockout and HDR plasmid (https://www.scbt.com/whats-new/crispr-systems).S HCl antibiotics concentrations ranged from 2-10, 200-500, and 1-20 μg mL −1 , respectively.A selective antibiotic titration was used for every cell type.The medium was replaced with a fresh medium containing selective antibiotics every 3-4 days until resistant colonies were detected.CRISPR knockout (KO) and HDR plasmid kits were used to KO the KL gene (Cat no: sc-400970 and sc-400970-HDR, Santa Cruz Biotechnology) (Figure 1B) in Caco-2 cells.The CRISPR/Cas9 knockout plasmid consists of KL-specific 20 nt guide RNA sequences derived from the GeCKO (v2) library.KL CRISPR/Cas9 KL-KO plasmids and KL HDR plasmids were co-transfected and designed to repair the site-specific Cas9-induced DNA cleavage within the human KL gene.CRISPR/Cas9 KO plasmids consist of klotho-specific 20 nt guide RNA sequences.
After incubation, successful transfection of the CRISPR/Cas9 plasmid was confirmed by detecting the green fluorescent protein (GFP) via fluorescent microscopy.The co-transfection of the CRISPR/Cas9 knockout plasmid and the HDR plasmid was confirmed by fluorescent microscopy detection of the red fluorescent protein (RFP).Cells were selected for a minimum of 3-5 days.In vivo, fluorescence imaging using GFP and RFP was done to select the labeled gene visually.TA B L E 1 Sequence and characterization of primers used in this study.
using the SYBR green qRT-PCR kit (Blirt SA) in a StepOnePlus (Thermo Scientific, Carlsbad, USA) PCR system.This study PCR protocol used, following 3 min of initial denaturation at 95 • C for 5 s, the cycles of 40 reactions were held at 60 • C for 30 s, at 95 • C for 15 s, and at 60 • C for 1 min.Ct values (threshold cycle) were obtained after qRT-PCR, and raw data used the GAPDH reference gene as a control.qRT-PCR data based on the 2 −ΔΔCt method were analyzed.
Annexin V-PI staining was performed to determine apoptosis following regulation of the KL gene.Flow cytometry was used to detect apoptotic cells using the Cell MeterTM live-cell caspases and phosphatidylserine detection kit (Cat No. 22850, AAT Bioquest).Briefly, cells and trans-fected cells were suspended in the washing buffer.Then, Annexin V and the PI working solution were added, respectively.The cells were gently mixed and incubated for 15 min at room temperature in the dark.The stained cells were analyzed by flow cytometry (Accuri C6, BD Biosciences, USA).

caspase 8 )
in colon cancer cells (Caco-2), KL-OE cells, and KL-KO cells, the immunohistochemical staining method was applied.Caco-2 cells were seeded on coverslips, overexpression constructs were created, and the KL gene was knocked out.Furthermore, after the transfected cells reached sufficient confluency, the cells were washed with PBS three times and then fixed with methanol for 20 min.After fixation, the cells were washed three times again with PBS and incubated for 30 min in PBS containing 1.5% normal block serum.Next, primary antibodies (caspase-3:100, caspase-8:100, ABclonal, Massachusetts, USA) were added and incubated overnight at +4 • C.After incubation, it was washed three times with PBS.It was incubated at room temperature for 60 min with the appropriate fluorescent secondary antibodies (FITC and CF594, Santa Cruz Biotechnology) for immunofluorescence staining.Subsequently, the cells were sealed with a capping solution (AAT Bioquest) containing DAPI (4,6-diamidino-2-phenylindole) dye to stain the nuclei.After the cells reached sufficient confluence, they were examined and photographed under a fluorescence microscope (Leica DM2500, Wetzlar, Germany).Micrographs were taken from 10 randomly selected areas for morphometric analysis.Caspase 3 and caspase 8 positivity in cells was determined according to the total number of cells in 10 photographs taken from each group (cells were counted in Image J).

KL
gene in Caco-2 cells, the mRNA levels of KL were analyzed by qRT-PCR.When the transfected cells were compared to the nontransfected Caco-2 cells, the relative expression of KL gene after F I G U R E 2 qRT-PCR analysis of relative KL gene transcript expression.Untreated cells were used as the control group (n = 3, *p < 0.05, **p < 0.01).(Caco-2 = untreated cells, KL-OE cells = overexpression KL gene+Caco-2, KL-KO cells = knockout KL gene+Caco-2).transfection was 86.87 (p < 0.01) in KL-OE and 0.72 (p < 0.05) in KL-KO, respectively.Since KL-OE gene expression in KL-KO Caco-2 cells was thought to be able to maintain the existence of mRNAs produced from the KL gene in the cell genome before transfection, the decrease in expression in this group was accepted as transfection success.These results prove the successful transfection of KL activation vectors (KL-OE cells) and KL knockout vectors (KL-KO cells) (Figure 2).

F I G U R E 5
Wound healing assay to evaluate the migration rate of Caco-2 cells after KL-OE and KL-KO.The relative migration distance of cells to the denuded area was measured 24, 48, and 72 h after scratch formation.(A) Inverted microscope images of the wound area (scale bars: 200 μm).(B) The effect of changes in KL gene expression on wound surface area in Caco-2 cells.Caco-2 cells (untreated cells) were used as control (n = 3, ***p < 0.001).(Caco-2 = untreated cells, KL-OE cells = overexpression KL gene + Caco-2, KL-KO cells = knockout KL gene + Caco-2).
investigated to determine the apoptosis relation to the change in KL gene expression in colon cancer cells.The expression of CASPASE-3 (p < 0.05), CASPASE-8 (p < 0.001), BCL-2 (p < 0.05), BAX, and BAD genes was found to be increased in KL-OE cells compared to KL-KO, while the expression of the BID gene was decreased.These results suggest that KL induces apoptosis in Caco-2 cells by upregulating the expression of CASPASE-3 and CASPASE-8 genes.Similar to our results, increased