Gene Expression Analysis of Specific Genes Related to Genomic Stability in Colon Cancer Cell Lines-A Resource for Cancer Research

Appropriate cell models are the foundation of cancer research as all the cell lines are not the same in their ability to mimic primary tumor tissue. Molecular characteristics of various cell lines can help in selecting the right cell model for biomedical studies. This study describes the gene expression profile of nine genes, include the six-protein shelterin complex, tankyrases1and 2, and CTNNB1 in three different models of human colon cancer cell lines. Also, the relative telomere length in each cell line was evaluated. All genes' expression levels were analyzed using a real-time polymerase chain reaction. Nearly all test genes were significantly highly expressed in HT-29 as compared to HCT-116 and SW-620. SW-620 record a significantly increased expression of the CTNNB1 gene (P-value < 0.01) while most genes were poorly expressed in HCT-116. A positive correlation between CTNNB1 and hTERT expression was found in HCT-116 and SW-620 but not in HT-29. Telomere was found to be of the same length in all cell. Gene expression data presented in our results can assist in selecting appropriate cell lines in colon cancer research. Unique molecular properties of various cancer cell lines should be evaluated before selecting a cell line mmodelfor research purposes. Original Research Article Alamro et al.; JPRI, 34(2A): 10-19, 2022; Article no.JPRI.81664 11


INTRODUCTION
Colorectal cancer (CRC) is one of the most occurring types of cancer worldwide. According to GLOBOCAN 2018, CRC is the third most commonly diagnosed cancer with nearly 1.8 million new cases, and the second leading cause of cancer death, accounting for 9.2% of all cancer deaths [1].CRC is a heterogeneous disease with diverse molecular pathways typically due to loss of genomic stability either by genetic or epigenetic alterations. Telomeres play a significant role in maintaining genomic stability and their dysfunction can modulate many cell signals like the Wnt/β-catenin pathway leading to cancer [2].
The involvement of deregulated Wnt/β-catenin cascade with colorectal cancer has been well documented [3]. One of the most studied and critical Wnt pathways is canonical Wnt signaling, which functions by regulating the amount of the transcriptional co-activator β-catenin that controls the central developmental gene expression program [4]. Telomere dysfunction can occur due to impaired telomere length or by disruption of the shelterin complex. Many human diseases especially cancer are linked with mutations in shelterin. Shelterin complex is composed of six key individual proteins, the telomere repeatbinding factor 1 and 2 (TRF1 and TRF2), protection of telomeres 1 (POT1), repressor/activator protein 1 (RAP1), TRF1 and TRF2 interacting protein 1 (TIN2), and TIN2interacting protein 1 (TPP1) [5,6]. Telomere is also regulated by tankyrase which is involved in many cellular and molecular processes related to tumor suppressors.
Human cell lines obtained from malignant tumors are being used as a valuable in vitro model in cancer research for decades [7]. Cell line-based studies are being promoted all over the world to understand the genetic and epigenetic mechanisms involved in many kinds of malignancy [8]. Although cell lines are inexpensive and easy to handle in the laboratory, appropriate selection of proper cell line for a particular study need to be evaluated for best results [9]. Cell lines have their origin from tumor tissue samples of a patient [10,11]. Sorting the cell for a particular study is a very crucial step as cells in the experiment should truly represent the type of cancer they are supposed to represent.
Usually, cell lines represent gene expression patterns the same as their cellular origin [11]. Cell lines have their unique genetic characteristics like HCT116 has a mutation in codon 13 of the KRAS protooncogene and is mainly used for biomedical studies involving colon cancer proliferation and corresponding inhibitors [12,13]. These cells are most suitable for therapeutic research and drug screenings [14]. HT-29 has a mutation in the p53 gene [15]. These cells are mostly used to study the structural and molecular events involved in cell differentiation and are best fit for studies focusing on the anticancer effect of different food compounds [16,17]. SW-620 is used to study metastasis processes [18]. Physiological properties like cell proliferation rate, drug sensitivities, and metabolism depend on the gene expression patterns of particular cell lines [19]. HCT-116, HT-29, and SW-620 cell lines are the most commonly used cell lines in colorectal cancer studied but literature reporting the gene expression patterns of some specific genes related to genomic stability in these cells is not available. Therefore, different colorectal cancer cell line models were used, HCT-116, HT-29 and SW-620 to evaluate the telomere length and the expression of the telomere-associated proteins; telomerase, TRF1, TRF2, POT1, TPP1, TIN2, TNKL1, and TNKS2 and how they correlate with the transcriptional factor β-catenin expression level.

Cell Culture
Three human colon cancer cell lines HCT-116, HT-29, and SW-620 were used in the present study and were kindly provided by Stem Cell Unit, Department of Anatomy at King Khalid University Hospital, Riyadh, KSA. All cell lines were cultured in DMEM medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA) at 37˚C in a humidified atmosphere containing 5% CO2.

Nucleic Acid Isolation
The DNA and total RNA were isolated from each cell line using RNeasy Mini Kit and QIAamp DNA Mini Kit, respectively, according to the manufacturer's instructions (Qiagen). The isolated nucleic acids were quantified spectrophotometrically (NanoDrop 8000, Thermo Fisher Scientific).

Measurement of Relative Telomere Lengths
Telomere length was determined by a previously described qPCR protocol (42) .In brief, two PCR master mixes were prepared, one for telomere reaction with telomere (TEL) primer pair, and the other for single-copy gene reaction with human beta-globin gene (HBG) primer pair. The Primer sequences and annealing temperature are listed in Table 1. Each reaction contained 1X SYBR® Green Master-mix (Applied Biosystems, UK), 20 ng of DNA sample, Tel-1 (300 nM), Tel-2 (900 nM), HBG-1, and HBG-2 (400 nM). All reactions were performed by Light Cycler 480 II system (Roche Applied Science) using the following PCR cycling condition: initial denaturation at 95°C for 15 minutes followed by 45 cycles of (denaturation at 95°C for 15 sec, indicated annealing temperature for 2 min, and extension at 72°C for 30 sec).

Statistical Analysis
All statistical analyses were performed using GraphPad Prism® software (version 8.2.0 GraphPad Inc, USA). The relative gene expression was calculated using the copy number method. Nevertheless, the relative telomere length was calculated using 2 -ΔΔCt method (where ΔCt= Ct single copy gene-Ct telomere). Unpaired Student's t-test was used to estimate the difference in the gene expression and telomere length between colon cancer cell lines Pearson's correlation and Linear regression were used to test the association between the different variables. All data are presented as the mean ± standard error of the mean, and the Pvalue of less than 0.05 was considered statistically significant.

Assessment of β-catenin and Telomere Related Proteins Expression
The gene expression profile of telomere related proteins (hTERT, TERF1, TERF2, POT1, TIN2, TPP1, TNKS1, and TNKS2) and CTNNB1 of all cancer cell lines are presented in Fig. 1. SW-620 expressed a significantly higher level of β-catenin as compared to the other two cell lines. Comparing SW-620 and HT-29m there was a significant difference in the expression in all studied genes except hTERT and its negative regulator POT1. HT-29 showed a significantly high expression of TRF1, TRF2, TPP1, and TNKS2 as compared to SW-620 and HCT-116, where HCT-116 showed the least expression of almost all test genes. The expression level of hTERT in all cell lines was found to be the same (Fig. 1). Interestingly, POT1 which functions in telomere length regulation through controlling telomerase access to the overhang did not show any significant difference in its expression between the selected colon cancer cell lines, as the case in hTERT. One notable finding is that the expression pattern of TERF1, TERF2, TPP1, and TNKS2 was almost identical in all studied cell lines. Furthermore, the level of the previously mentioned genes was significantly low in HCT-116 and high in HT-29, the native and mutated TP53 models. Person correlation between all the genes of three cell lines is summarized and presented in Figure 3. CTNNB1 was positively correlated with all studied genes of all cell lines except POT1 in SW-620 and TERF1 and POT1 in HCT-116 it was found to be negatively correlated with its expression.
A negative correlation between hTERT and POT1 was found in all three cell lines.
In SW-620 and HCT-116, POT1 was negatively correlated with almost all the genes. Further, genes that were mainly involved in telomere structure regulation (TERF1, TERF2, TIN2 TPP1 and TNKS1) were found to be correlated in HT-29. Fig. 2 summarizes the correlation findings.

Relative Telomere Length (RTL) Measurement and Correlation with Gene Expression
Telomere was found to be of the same length in all cell lines as shown in Fig. 3. The correlation analysis was performed with Pearson's correlation test to demonstrate the association between the gene expression of the telomere related proteins and CTNNB1 with telomere length (Fig. 4). Interestingly, HT-29 and SW-620 show a counter result. While the telomere length in HT-29 shows a positive correlation with the expression of all studied genes, the telomere length of SW-620 on other hand was negatively correlated with the expression of all genes. In the case of HCT-116, no notable correlation or pattern was found between telomere length and the expression of all genes. Overall, the analysis showed that among the studied genes, no significant correlation was determined between telomere length and their expression.

DISCUSSION
Studies have found that telomere dysfunction is associated with chromosomal instability, a hallmark of cancer and causative of the acquisition of genetic lesions that are involved in the stepwise progression of cells into malignancy. Indeed, different factors and pathways may play a role in the regulation of telomere-related proteins and thus telomere biology. Previous studies have been documenting that Wnt/ β-catenin pathway is involved [20]. The present work aims to estimate the relative telomere length and the expression level of the telomere related proteins (hTERT, TERF1, TERF2, POT1, TIN2, TPP1, TNKS1 and TNKS2) using q-PCR in most commonly used colon cancer cell lines and how they correlate with the transcriptional factor β-catenin expression level. The mRNA level of β-catenin and TNKS1 was significantly higher in SW-620 as compared to the other two cells. SW620 is metastatic and poorly differentiated as compared to HT-29 and HCT116 which suggests that βcatenin may play an important role in metastasis. Lai et al explored the molecular marker for metastasis in human cancer tissue. He reported an elevated level of β-catenin expression in a poorly differentiated tissue. Similar results are reported in human hepatoma cell line HA22T which has high proliferation activity [21]. Many human cancers are reported with mutations and abnormal expression of β-catenin which is correlated with tumor invasion and metastasis [22]. Remarkably, it has been found that SW-620 owns a mutated APC [23,24] and wildtype CTNNB1status [24].
Previous studies have reported that both TNKS1 and TNKS2 reducing -catenin degradation by destabilizing the levels of Axin [25]; a key effector in the Wnt pathway and has been identified as a tumor suppressor and therefore hyperactivation Wnt/ -catenin signaling [26,27]. This finding is compatible with a previous study which found that the Wnt/βcatenin pathway is activated during CRC progression and accompanied by enhanced cell invasive behavior [28]. This makes the Wnt/βcatenin pathway become a potential therapeutic target for inhibiting CRC metastasis. Therefore, due to the role of tankyrase in Wnt/β-catenin signaling, Tankyrase 1 inhibitors may become a promising therapeutic target for CRC through suppressing Wnt signaling and tumor growth.
hTERT is one of the main catalytic subunits of the telomerase enzyme and is expressed in almost 90 % of human cancers. The expression level of hTERT in all cell lines was found to be the same in the presented data. Our results are well supported by Kawanishi-Tabata et.al (2002) results where he proved that the expression level of hTERT was not correlated with cancer progression in colon carcinoma [29]. Furthermore, copy number gain of the hTERT gene is highly correlated with hTERT expression found in neuroblastomas and lung cancer, but no such correlation has been found in colorectal carcinomas [30].
On examining the expression level of the six protein components of the shelterin complex we found that HT-29 showed significantly high expression of TRF1, TRF2, and TPP1 as compared to SW-620 and HCT-116, where HCT-116 showed the least expression of almost all components of shelterin complex. Many reports have shown overexpression of TRF1 and TRF2 in many kinds of cancer tissues [31]. HT-29 is a mutant for the p53 gene. Shelterins protect the chromosomes and prevent their ends but any kind of genotoxic stress or mutation can induce tumor [32]. The p53 protein is known for its tumor suppressor properties and plays a major role in the maintenance of genome integrity [33]. Mutation in p53 in HT-29 can be related to the high expression of these shelterin proteins as p53 protein-enhanced telomere stability even in presence of DNA damage. Some studies have also reported the inhibition of TRF2 in the induction of apoptosis in a p53 dependent manner. Impressively, there was a significant correlation between the expression of TERF1 and TERF2, TPP1 and TNKS1. Likewise, TERF2 expression was significantly correlated with the three previously mentioned genes. However, TPP1 has correlated with TERF1 and TERF2 expression but not tankyrases. These findings suggest that these telomeric proteins may play a role in cancer by enhancing the genomic instability through controlling DNA damage response (DDR) and manipulating the telomere structure but not length since we found that all the three colon cancer cell lines were almost having the same length. The suggestion is consistent with published data that found a significant correlation between TERF1 and TERF2 expression and demonstrates an inverse relation between TERF1 and TERF2 level with DDR activation [34].
Tankyrase proteins having poly(ADP-ribose) polymerase activity are the main components of the human telomeric complex among which Tankyrase-1 and 2 are most important. Change of expression level of these two proteins is identified in many cancers tissues. The expression level of the Tankyrase-1 and Tankyrase 2 were found to be significantly high in HT-29 followed by SW-620 whereas HCT-116 showed the least expression. The expression level of the Tankyrase protein depends on the tumor stage [35]. Cell lines used in the study were different in their ability to differentiate as HT-29 has an intermediate capacity to differentiate while SW-620 and HCT-116 are highly aggressive cell lines with the least differentiation. Our results agree with Abdulaziz, et al (35), who found high expression of TNKS1 in the early stages than late stages of CRC development. Also, Gelmini et al. (2006) [36], reported an inverse relation of TNKS with colon cancer stages.
As shown in Fig 2.C, CTNNB1 was significantly positively correlated with hTERT, while the last one was significantly correlated with TERF2. This finding is compatible with a study that found that the hTERT is a target of the Wnt/β-catenin pathway, and induction of β-catenin expression leads to increased hTERT expression while silencing endogenous β-catenin expression by βcatenin gene-specific shRNA effectively decreased hTERT expression [37]. Altered expression of β-catenin is found to be correlated with poor prognosis, aggressive phenotype and metastasis [38][39][40]. as clearly shown in our results of SW-620 cell lines where we found a strong correlation of CTNNB1 and with other genes as compared to the other two cells. Our results showed a strong correlation between βcatenin and hTERT concerning the different capacities of differentiation among the cell lines. The expression status of β-catenin significantly correlates with the de-differentiation process and prognosis in many human cancers tissues [41]. Gene Primer sequence (5′ -3′) Ta (°C)  Reference  TEL-1  TEL-2   GGT TTT TGA GGG TGA GGG TGA GGG TGA GGG TGA  GGG T  TCC CGA CTA TCC CTA TCC CTA TCC CTA TCC CTA  TCC CTA   55  [52] HBG-1 HGB-2 GCT TCT GAC ACA ACT GTG TTC ACT AGC CAC CAA CTT CAT CCA CGT TCA CC 60 [52] *For: forward, Rev: reverse, Ta: annealing temperature.

CONCLUSION
The selection of appropriate cell lines for molecular-based cancer studies is a very critical step to interpret and understand cancer biology. The gene expression patterns of some specific genes of HCT-116, HT-29, and SW-620 cell is reported to help researchers to choose the most appropriate cell line models for cancer studies.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.