Transforming growth factor-β1 gene promoter -509C/T polymorphism in association with expression affects colorectal cancer development and depends on gender

Abstract

It is widely known that sporadic colorectal cancer (CRC) is age-related diseases with higher incidence rate among men. Transforming growth factor-β1 (TGF-β1) is a major immune regulatory cytokine with a great impact and dual role in gastrointestinal carcinogenesis. In this context, the aim of the study was to explore the role of circulating TGF-β1 and the -509C/T functional promoter polymorphism (rs1800469) within the TGF-β1 gene (TGFB1) in the susceptibility, progression, and prognosis of CRC among Bulgarian male and female patients. Patients with sporadic CRC and healthy controls were genotyped by polymerase-chain reaction-restriction fragment length polymorphism. Serum TGF-β1 levels before and after curative surgery were determined by ELISA. Total RNA was extracted from paired tumor, normal mucosa and distant metastasis samples and was used for quantitative detection of TGFB1 mRNA by TaqMan qPCR.We observed that TGF-β1 serum levels depend on the -509C/T genotype in combination with gender. TGF-β1 serum levels in CRC patients were decreased compared to controls, but statistical significance was reached only for men. In the stratified analysis by gender and genotype, a significant association was found for the CC genotype. Overall, our results indicate that the -509C allele increased the cancer risk, particularly for advanced stages (OR = 1.477; p = 0.029). The results from the relative mRNA quantification showed a significant upregulation of TGFB1 in distant metastases compared to primary tumor tissues and higher TGFB1 mRNA levels in men (RQ = 4.959; p = 0.022). In conclusion, we present data that diminished circulating TGF-β1 due to the CC genotype could be a possible risk factor for tumor susceptibility and progression. This association is more pronounced in males than in females. Colorectal cancer tissue expression of TGFB1 gene mRNA correlates with tumor progression and metastasis.

Fig 1. Gender-related correlation between the TGFB1-509 polymorphism and serum TGF-β1 levels in healthy controls.

(A) CC genotype. (B) CT genotype. (C) TT genotype. (D) the total group. The individual data from females are presented as empty circles, and those from males are presented as filled squares.

Fig 2. TGF-β1 serum levels in controls, aged under and over 50 in relation to TGFB1-509 polymorphism.

(A) Females over 50 (>50) compared to females under 50 (<50). (B) Males over 50 (>50) compared to males under 50 (<50). The results are presented as the mean value ±SE (box) with a ±0.95 confidential interval (whisker).The statistical significance of the U-test is shown as the p-value.

Fig 3. Serum levels of TGF-β1 in CRC patients.

(A) Subdivided according to the gender. (B) CRC stage. (C) operative status. (D) operative status in men and women with CRC. The results are presented as the mean value ±SE (box) and ±SD (whisker). The statistical significance of the U-test is shown. * p-value < 0.05, ** p-value < 0.01, and *** p-value < 0.001.

Fig 4. Serum levels of TGF-β1 in cases and controls over the age of 50 yrs.

(A) Male and female carriers of TGFB1 -509CC genotype. (B) Male and female carriers of TGFB1 -509CT genotype. (C) Male and female carriers of TGFB1 -509TT genotype. The results are presented as the mean value ±SE (box) and ±SD (whisker). * p-value < 0.05, ** p-value < 0.01.

Fig 5. TGFB1 mRNA relative expression in tumor tissues and distant metastases.

mRNA quantification was performed in tissue samples from CRC patients with 4 stage CRC, carriers of TGFB1 -509CC+CT genotypes in relation to the gender. The relative gene expression was determined by the 2^−ΔΔCt method in primary tumor tissue (Tu tis) and in distant metastases (Met) after normalization to the housekeeping reference genes (GAPDH and 18srRNA) and calibrated to the paired non-tumoral mucosa or to the primary tumor tissue (Met vs. Tu tis). Statistical significance is shown compared to the calibrator. * p<0.05.