Thyroid carcinoma (TC) is the most common endocrine tumor, accounting for 3.1% of worldwide cancer incidence[1]. In 2022, about 43,800 new cases in the United States were diagnosed with TC[2]. In Egypt, TC is the fifth most frequent cancer in females accounting for 3.6% of all malignancies in women[3]. It is classified as differentiated papillary (PTC), follicular (FTC), and undifferentiated or anaplastic (ATC)[4]. The incidence of DTC has been growing noticeably over the past 20 years worldwide, and is anticipated to be the fourth most common malignancy by 2030[5]. TC is initiated by genetic alterations and epigenetic changes in oncogenes or tumor suppressor genes[6]. These genetic alterations particularly engage a small set of genes whose protein products are typically members of the mitogen-activated protein kinase (MAPK) and PI3K/PTEN/AKT/mTOR signaling pathways[7].
The alpha Klotho (α-Klotho, KL) was identified in 1997 as an antiaging gene[8, 9]. Two other related genes, beta Klotho (β-Klotho) and gamma Klotho (γ-Klotho), have been identified as Klotho family members[10]. The α-Klotho gene spans 50 kb and is located on chromosome 13q12[8]. It encodes a type 1 single-pass transmembrane glycoprotein containing two large extracellular domains, KL1 and KL2, with a short single transmembrane and a short intracellular domain in its C-terminus. The γ-Klotho gene is located on chromosome 15q22.31 with 14 exons. It encodes a smaller single-pass transmembrane glycoprotein, with a small intracellular domain and a family 1 glycosidase-like extracellular KL1 domain[8, 10, 11].
The Klotho proteins are cofactors of fibroblast growth factors (FGF) 19, FGF21, and FGF23, which control specific metabolic activities of different tissues[11, 12]. The α-Klotho gene has been associated with several human malignancies including hepatocellular, renal cell, colorectal, pancreatic, breast, and lung cancers[13, 14]. The α-Klotho functions as a tumor suppressor gene mainly by regulating oxidative stress, insulin/IGF-1, FGF23, p53/p21 and Wnt signaling pathways[13, 15]. On the other hand, there are limited researches about the correlation between the γ-Klotho gene and cancer[11, 16].
The association between the α-Klotho gene and TC remains unclear. Up till now, there has been no study that investigated the role of the γ-Klotho gene in TC. The aim of this study was to assess the α-Klotho and γ-Klotho gene expressions in patients with DTC, which may help better understanding of the pathogenesis of this tumor.
The present case control study was carried out in Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt. This study included 40 DTC patients (31 PTC and 9 FTC patients) in addition to 40 age- and sex-matched subjects diagnosed as goiter and enrolled as a control group. All subjects were selected from Mansoura Oncology Center, Mansoura University, Mansoura, Egypt between December 2019 and March 2021. Informed consents were obtained from all participants in the study. The study protocol was approved by Institutional Research Board (IRB) of Mansoura Faculty of Medicine (IRB code MDP.19.09.25).
The included patients were diagnosed with primary DTC by preoperative palpation, fine-needle aspiration cytology (FNAC), and US. The diagnosis was confirmed by intraoperative rapid pathological and postoperative pathological examination. For all patients, full medical history was taken and careful clinical examination, including size and consistency of thyroid gland, assessment of lymph node, and body mass index (BMI) was done.
Patients with other primary malignancies or who previously underwent hemi-thyroidectomy, anti-TC chemotherapy, radiotherapy, or radioactive iodine treatment before surgery were excluded from this study. All laboratory investigations, including complete blood count (CBC), serum total triiodothyronine (T3), total thyroxine (T4), and thyroid stimulating hormone (TSH) were done in Mansoura Oncology Center in a time frame of less than 3 months preoperatively. The thyroid gland was evaluated by US characteristics, including margins, calcifications, and echogenicity.
Thyroid tissue specimens were obtained at the time of surgery from patients and controls and dissected immediately after surgery. The tumor tissues were collected from the center of the suspected viable cancer tissue[17]. The other part of the tissue samples was transported to the Pathology Department for confirmation of histopathological diagnosis and collection of other clinicopathological data. According to the American Joint Committee on Cancer (AJCC), 2010 recommendations, the TNM staging was accomplished (32 cases were stage I, three cases were stage II, one case was stage III, and four cases were stage IVA cancers).
To preserve RNA integrity, the fresh tissue samples were completely submerged as quickly as possible in an appropriate volume of RNAlater RNA Stabilization Reagent, approximately 10 μl of the reagent per 1 mg of tissue (Qiagen, Germany, Cat. No 76104). The samples were transported on ice and stored at 4°C for at least 24 hours, then stored at −80°C for subsequent total RNA extraction.
Referring to the modified Chomczyński and Sacchi's method[18], total RNA was extracted from thyroid tissue samples, using QIAzol Lysis Reagent kit (Qiagen, Germany, Cat No 79306) according to the kit's protocol. The integrity of RNA was evaluated by loading RNA samples on agarose gel electrophoresis. The RNA concentration and purity of the samples were assessed using the NanoDrop 2000c Spectrophotometer (Thermo Scientific, USA). The purity of RNA was assessed in each sample via two optical density (OD) ratios (A260/A280 and A260/A230). The RNA samples with 1.8 to 2.0 A260/A280 ratios were used. The isolated RNA was then stored at −80°C for subsequent reverse transcription.
Complementary DNA (cDNA) was synthesized using COSMO cDNA synthesis kit (Willowfort, COSMO cDNA synthesis kit, Birmingham Research and Development Park, Birmingham, WF-10205002), in accordance with manufacturer's instructions. The cDNA was synthesized using the thermal cycler (Applied Biosystem, Waltham, Massachusetts, USA) with the following program of 5 min at 25°C, 15 min at 45°C, and 5 min at 85°C. The synthesized cDNA samples were stored at −20°C.
The RT-qPCR assays were carried out on the 7500 Real Time PCR System, Applied Biosystem, USA, with HERAPLUS SYBR® Green qPCR Master Mix (2X), Birmingham Research and Development Park, Birmingham, WF10308001, and gene-specific real-time qPCR primers. It was performed according to the method described by Freeman et al.[19]. Each 20 μL reaction mix contained 10 μL of HERAPLUS SYBR® Green qPCR Master Mix (2X), 2 μL of the synthesized cDNA, 1 μL of forward primer, 1 μL of reverse primer, and the remaining 6 μL was RNase free water.
The primers for the α-Klotho and γ-Klotho genes were chosen from NCBI databases [
The real-time PCR reactions were performed with the following thermal cycling program of 2 min at 95°C, followed by 40 cycles of denaturation at 95°C for 10 s and annealing/extension at 60°C for 30 s. The analysis of melting curve of all the reactions was performed for evaluation of the specificity of the products.
Relative quantification (RQ) of mRNA expression was estimated using the comparative threshold method (ΔΔCt)[21]. The data were presented as RQ of the target mRNA, normalized as regards the mRNA of the reference gene GAPDH and in respect to the control samples. The fold change was calculated using the equation RQ = 2−ΔΔCt.
The collected data were introduced to a PC using Statistical Package for Social Science (SPSS) Version 25. Qualitative data were expressed as count and percent. Quantitative data were initially tested by Kolmogorov–Smirnov and Shapiro–Wilk's, then expressed as mean ± standard deviation (SD) for parametric numerical data or median and interquartile range (IQR) for nonparametric numerical data. Qualitative data were compared via Chi-square test (or Fisher's exact test), whereas quantitative data were compared via independent samples t-test or nonparametric Mann–Whitney U test. Receiver operating characteristic (ROC) curve analysis was used to determine the discrimination accuracy of the diagnostic test to distinguish between DTC and goiter[22]. Comparisons of area under ROC curve (AUC) were performed. Logistic and ordinal regression analyses were used for prediction of risk factors, using generalized linear models. Odds ratio (OR) and 95% confidence interval (95% CI) were calculated. The results were considered statistically significant if
Routine demographic and laboratory data of the patients and controls are presented in Table 1. DTC patients had a statistically significantly lower BMI than the controls (
Demographic and laboratory parameters of the studied groups.
41.08 ± 11.3 | 41.08 ± 11.3 | *1.000 | |
|
|||
|
9 (22.5%) | 9 (22.5%) | **1.000 |
|
31 (77.5%) | 31 (77.5%) | |
30.30 ± 6.2 | 33.80 ± 5.4 | ||
1.27 ± 0.4 | 1.23 ± 0.3 | *0.456 | |
7.24 (6.50–8.25) | 7.85 (6.30–8.85) | ***0.528 | |
1.10 (0.66–1.58) | 0.82 (1.03–2.45) |
p-value by **Chi-square test (data are presented as count and percent);
The α-Klotho gene expression was statistically significantly lower in DTC patients (median: 0.34; IQR: 0.22–0.54) compared to controls (median: 0.96; IQR: 0.83–1.19;
ROC curve analysis showed that the α-Klotho mRNA levels can discriminate between malignant and benign thyroid tissues by providing an AUC of 0.954 value (95% CI = 0.910–0.999;
With a specificity of 97.5% and a sensitivity of 87.5%, the α-Klotho cutoff value of ≤ 0.68 showed a significant diagnostic accuracy of DTC. However, the γ-Klotho cutoff value of ≤ 0.93 did not show significant diagnostic accuracy of the tumor with a specificity of only 55.0% and a sensitivity of only 50.0%.
DTC patients were classified into high and low α-Klotho gene expression groups, according to the cutoff value of the α-Klotho mRNA level (≤0.68). Low expression of the α-Klotho gene was identified in 35 of the 40 samples of the tumor tissues. The low α-Klotho expression group had larger tumor size (
Association between the demographics and the clinicopathological parameters and the two groups of the α-Klotho gene expression.
4 (80%) | 21 (60%) | **0.388 | ||
≥ |
1 (20%) | 14 (40%) | ||
1 (20%) | 8 (22.9%) | **0.886 | ||
4 (80%) | 27 (77.1%) | |||
30.3±5.4 | 30.3±6.4 | *0.988 | ||
0.94±0.4 | 2.91±0.9 | *<0.001 | ||
3 (60.0%) | 17 (49.0%) | **0.633 | ||
2 (40.0%) | 18 (51.0%) | |||
3 (60.0%) | 22 (62.9%) | **0.902 | ||
2 (40.0%) | 13 (37.1%) | |||
4 (80.0%) | 20 (57.1%) | **0.631 | ||
1 (20.0%) | 15 (42.9%) | |||
4 (80.0%) | 30 (85.7%) | **0.738 | ||
1 (20.0%) | 5 (14.3%) | |||
5 (100.0%) | 27 (77.1%) | **0.232 | ||
0 (0.0%) | 8 (22.9%) | |||
5 (100.0%) | 23 (65.7%) | **0.298 | ||
0 (0.0%) | 12 (34.3%) | |||
5 (100.0%) | 17 (48.6%) | **0.031 | ||
0 (0.0%) | 18 (51.4%) | |||
4 (80.0%) | 27 (77.1%) | **0.525 | ||
1 (20.0%) | 8 (22.9%) | |||
3 (60.0%) | 16 (45.7%) | **0.550 | ||
2 (40.0%) | 19 (54.3%) | |||
0 (0.0%) | 15 (42.9%) | **0.137 | ||
5 (100.0%) | 20 (57.1%) | |||
5 (100%) | 27 (77.1%) | **0.699 | ||
0 (0%) | 3 (8.6%) | |||
0 (0%) | 1 (2.9%) | |||
0 (0%) | 4 (11.4%) |
There was a statistically significant inverse correlation between the α-Klotho gene expression and the age of the studied groups (
Correlations of the α-Klotho gene expression with γ-Klotho gene expression and with different parameters.
γ |
−0.065 | 0.565 |
−0. 233 | ||
−0.210 | 0.194 | |
0.084 | 0.606 | |
0.101 | 0.534 | |
−0.258 | 0.108 | |
−0.346 | ||
−0.898 |
There were no statistically significant correlations between the γ-Klotho gene expression and the studied parameters (
Correlations of the γ-Klotho gene expression with different parameters.
γ |
||
---|---|---|
−0.028 | 0.866 | |
−0.254 | 0.114 | |
0.186 | 0.250 | |
0.244 | 0.130 | |
−0.156 | 0.338 | |
−0.104 | 0.525 | |
−0.089 | 0.586 |
Logistic regression analysis revealed that low α-Klotho mRNA expression was demonstrated to be significant predictor for the likelihood of DTC on top of goiter (
Logistic regression analysis to predict the likelihood of DTC on top of goiter.
0.998 | 0.99–1.006 | 0.626 | |
1.023 | 0.83–1.26 | 0.831 | |
1.004 | 0.97–1.039 | 0.836 | |
1.063 | 0.95–1.19 | 0.285 | |
α |
0.506 | 0.343–0.746 | |
γ |
1.059 | 0.893–0.1.256 | 0.508 |
Multivariate ordinal logistic regression was performed to ascertain the effects of the female gender, high-serum total T4, high-serum TSH and low α-Klotho mRNA expression on the likelihood of higher stage of DTC. Patients with higher α-Klotho mRNA expression had a lower odds (0.671) to exhibit higher stage of DTC (
Predictors of higher stage of DTC.
2.2395(0.747–7.675) | 0.142 | |
1.220(0.956–1.56) | 0.110 | |
0.812(0.587–1.125) | 0.211 | |
α |
0.671(0.246–0.835) |
Since Wolf et al.[23] had discovered that the α-Klotho had an inhibitory effect on breast cancer, the role of this gene in the pathogenesis, development, and prognosis of cancer has received more and more attention. It has been shown that the α-Klotho functions as a tumor suppressor gene in most malignancies, such as lung, gastric, pancreatic cancers, and melanoma[24,25,26]. However, some controversial results have also been reported, such as the effects of the gene on the angiogenesis and antiapoptosis that may play a part in the progression of the ovarian cancer[27].
In the present study, we evaluated the expression levels of the α-Klotho and γ-Klotho genes in DTC against goiter in the Egyptian population. We reported that the α-Klotho gene expression was statistically significantly decreased in DTC patients compared to controls (
Goiter can progress to TC in approximately 4–14% of cases[31]. Thus, advances in molecular biology are essential to provide new insights into diagnosis of goiter and prediction of malignant transformation[32]. The ROC curve analysis in our study indicated that the mRNA levels of α-Klotho gene with cutoff value of ≤0.68 had the potential to discriminate between benign and malignant thyroid tissue specimens with a specificity of 97.5% and a sensitivity of 87.5%. Our research showed that the α-Klotho can be used as a novel molecular marker to discriminate between DTC and goiter tissues.
Our study revealed that DTC patients with lower α-Klotho mRNA levels had larger tumor size (
Also, we reported the inverse correlation between the α-Klotho gene expression and the stage of DTC (
Zhou
There are few reports about the relationship between the γ-Klotho gene and cancers. For example, its effect on bladder cancer invasion and progression, colon cancer cell proliferation and its association with poor triple-negative breast cancer prognosis[11, 16].
In our study, we did not detect significant difference between the DTC patients and controls regarding the γ-Klotho gene expression (
In our study, there were no statistically significant correlations between the γ-Klotho gene expression and different parameters, as age, BMI, serum T3, T4, TSH, tumor stage, and size (
Trošt and his colleagues[16] had reported higher stage and worse progression in patients with triple-negative breast cancer, who expressed higher γ-Klotho expression. Also, Su et al.[38] had identified the aggressive behavior of the γ-Klotho, as its high expression predicted poor prognosis for glioma patients.
These different findings in our study and the others may be due to our small sample size, different tumor type, ethnicity, or presence of other factors.
To the best of our knowledge, the current study is the first one as regards the relationship between the γ-Klotho gene and DTC. The direct association between the γ-Klotho as a cofactor in FGF signaling pathway could not be illustrated in this research. Absence of significant association between the γ-Klotho gene expression and DTC in our study may be due to small sample size of our studied groups and population differences.
The logistic analysis of the current study revealed that low expression of α-Klotho mRNA, with a cutoff value ≤0.68 and quantified via qRT-PCR, was a significant predictor for the likelihood of DTC on top of goiter (
In conclusion, our results declared the tumor suppressor role of the α-Klotho gene in DTC and its importance as a promising novel biomarker for DTC. This also may provide opportunities for development of new Klotho-based therapies. To the best of our knowledge, there has been no study investigating the role of the γ-Klotho gene in DTC before. In our study, there was no significant difference between DTC and goiter as regards the γ-Klotho gene expression.