Open Access

Polymorphisms of ‑800G/A and +915G/C in TGF‑β1 gene and lung cancer susceptibility

  • Authors:
    • Qing‑Guo Di
    • Bao‑Hua Sun
    • Ming‑Ming Jiang
    • Jun‑Feng Du
    • Zhi‑Tao Mai
    • Xin  Zhang
    • Li‑Rong Zhou
    • Yu‑Min Chi
    • Jing Lv
  • View Affiliations

  • Published online on: May 16, 2017     https://doi.org/10.3892/ol.2017.6173
  • Pages: 733-736
  • Copyright: © Di et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

We studied the relationship between the polymorphisms of ‑800G/A and +915G/C in transforming growth factor‑β1 (TGF‑β1) gene and lung cancer susceptibility. The sequence‑specific primer polymerase chain reaction (PCR‑SSP) technique was used to test 156 non‑small cell lung cancer (NSCLC) patients that were selected as the observation group and 156 patients with pneumonia and tuberculosis that were selected as the control group (age and gender 1:1 proximal matching principle) and the polymorphisms of the first exon ‑800G/A and +915G/C TGF‑β1 genes. The expression of TGF‑β1 levels in peripheral blood was detected using ELISA. The proportion of ‑800G/A gene AA subtype and A allelic gene in the observation group was significantly higher than that in the control group, while the proportion of +915G/C gene CC subtype and C allelic gene was also significantly higher than that in the control group (P<0.05). The cancer risk [odds ratio (OR)] of patients with A allelic gene in ‑800G/A gene was 4.8 (95% CI=2.563‑6.537, P<0.05), while the cancer risk (OR) of patients with C allelic gene in +915G/C gene was 4.7 (95% CI=2.317‑5.864, P<0.05). The serum TGF‑β1 expression levels of ‑800G/A gene AA subtype in the observation group was significantly higher than the GG type, GA type and the control group, while the TGF‑β1 level of +915G/C gene CC subtype was significantly higher than the GG type, GC type and the control group (P<0.05). Therefore, the polymorphisms of ‑800G/A and +915G/C in TGF‑β1 gene are closely related to the lung cancer susceptibility.

Introduction

Lung cancer is ranked as the highest malignant tumor that threatens human health in the world. The morbidity and mortality associated with lung cancer increases year by year with an increase in environmental pollution as well as the age of the population (1). The early detection of lung cancer is hindered due to a lack of biochemical markers with typical clinical features, and high sensitivity and specificity. In addition, the imaging examination often lags behind the occurrence and development of tumor, all of which are important factors that can lead to a poor survival prognosis (2). The transforming growth factor-β1 (TGF-β1), as a type of multifunctional cytokine which plays important role in inducing local angiogenesis, extracellular matrix secretion, immune evasion, cell heterogeneity adhesion, cell proliferation, invasion, metastasis and other aspects (3). The TGF-β1 gene, which is located on chromosome 19 of the long arm, contains seven exons and six introns. Among them, the first exon has a higher gene mutation rate and single-nucleotide polymorphism (SNP), which was confirmed to be closely related to the occurrence of a variety of malignant tumors, such as esophageal, breast, prostate, liver cancer and others (4,5). Based on this, our study analyzed the relationship between polymorphisms of −800G/A and +915G/C in the TGF-β1 gene and lung cancer susceptibility in order to provide a reference for the early diagnosis of lung cancer.

Subjects and methods

Subject information

A total of 156 patients that were admitted to Cangzhou Central Hospital and diagnosed with non-small cell lung cancer (NSCLC) from January 2013 to January 2016, were selected as part of the observation group, and 156 patients with pneumonia and tuberculosis during the same period were selected as the control group. Surgery, radiotherapy, chemotherapy and other treatments were not carried out. Tissue samples were obtained, and the tissue sections were created using the conventional method and stored at −80°C. The ratio of age and gender of the patients in both groups was 1:1 according to the proximal matching principle. In the observation group, there were 82 males and 74 females, aged 42–77 years, with an average age of 62.3±14.5 years. Based on the tumor pathologic type, there were 95 cases with squamous carcinoma and 61 cases with adenocarcinoma. Based on the clinical TNM stage, there were 15 cases in stage I, 46 cases in stage II, 62 cases in stage III and 33 cases in stage IV, with a maximum diameter of 0.5–3.6 cm, and an average of 2.4±1.2 cm. In the control group, there were 80 males and 76 females, aged 40–80 years, with an average age of 63.5±15.7 years. The study was approved by the Ethics Committee at Cangzhou Central Hospital and written informed consent rights were obtained from the patients or their families.

Research methods

The sequence-specific primer polymerase chain reaction (PCR-SSP) technique was used to test the polymorphisms of the first exon −800G/A and +915G/C in the TGF-β1 gene. The expression levels of TGF-β1 in peripheral blood were detected using ELISA.

PCR-SSP technique

Tissue DNA was extracted using the kit purchased from Sigma (St. Louis, MO, USA). The main steps were conducted as follows: 20 mg tissue were taken, and after being ground, 500 µl of tissue lysate were added. The solution was soaked in the water at 50°C for 1 h. Then, proteinase K was added to reach a final concentration of 100 µg/ml with soaking in 50°C water for 3 h. Then, the extraction was respectively performed using an equal volume of saturated phenol, phenol-chloroform (volume ratio as 1:1) and chloroform-isoamyl alcohol each time. Sodium ethylate (1/10 volume) and ice ethanol (2-fold volume) were added to precipitate the DNA. The appropriate amount of TE solution was added to dissolve the precipitation. Subsequently, DNA concentration and purity were detected using an ultraviolet spectrophotometer (Applied Biosystems Life Technologies, Foster City, CA, USA). The primers were designed by referring to the sequence on PubMed, and the primer design was synthesized by Shanghai Bioengineering Co., Ltd. (Shanghai, China), which is shown in Table I. A total of 25 µl of the PCR amplification system included 10X buffer 2.5 µl + dNTPs (2.5 mmol/l) 2.0 µl + upstream and downstream primers (20 pmol for each) + cDNA 200 ng + Taq polymerase 1.25U and distilled water. The reaction conditions were as follows: 94°C for 5 min, 94°C for 40 sec, annealing for 1 min (temperature is shown in Table I), 72°C for 1 min, 35 cycles in total, and 72°C for 5 min. The enzyme digestion system included −800G/A and +915G/C gene expansion products (10 µl for each). The restriction enzyme (as shown in Table I; was used, after which the enzymatic digestion was performed in water at 37°C for 3 h. The product, which was detected by 2% agarose gel electrophoresis and ethidium bromide staining method, was identified under ultraviolet light.

Table I.

Primer sequences of polymorphisms of −800G/A and +915G/C in TGF-β1 gene.

Table I.

Primer sequences of polymorphisms of −800G/A and +915G/C in TGF-β1 gene.

SequenceReaction temperature (°C)Enzyme digestionLength (bp)
−800G/AF (G): 5′-ACAGTTGGCACGGGCTTTCG-3′57HpyCh4 IVG: 182 and 206
F (A): 5′-CAGACTCTAGAGACTGTCAG-3′ A: 388
      R: 5′-GTCACCAGAGAAAGAGGAC-3′
+915G/CF (G): 5′-GTGCTGACGCCTGGCCG-3′58Bsu36 IG: 233
F (C): 5′-GTGCTGACGCCTGGCCC-3′ C: 233
      R: 5′-GGCTCCGGTTCTGCACTC-3′

[i] TGF-β1, transforming growth factor-β1.

ELISA method

Peripheral blood (5 ml) was collected and performed by centrifugation at 3,000 × g for 20 min. The upper layer of the serum was taken and stored at −20°C. The kits were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and the microplate reader was purchased from the Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The steps were carried out strictly in accordance with the specifications.

Statistical analysis

The normal data were expressed as mean ± standard deviation, t-test was used for comparison, while the measurement data were expressed by rate. The Chi-square test was used for comparison. The exposure risk was tested using a single factor logistic model and expressed by the odds ratio (OR) value. P<0.05 indicated that the difference was statistically significant. The SPSS 20.0 software (IBM, Armonk, NY, USA) was applied for statistical analysis.

Results

Analysis of the polymorphisms of −800G/A and +915G/C gene

The proportion of −800G/A gene AA subtype and A allelic gene in the observation group was significantly higher than that in the control group, while the proportion of +915G/C gene CC subtype and C allelic gene was also significantly higher than that in the control group; differences were statistically significant (P<0.05). The cancer risk (OR) of patients with A allelic gene in −800G/A gene was 4.8 (117×96/39×60) (95% CI=2.563–6.537, P<0.05) while the cancer risk (OR) of the patients with C allelic gene in +915G/C gene was 4.7 (102×111/54×45), (95% CI=2.317–5.864, P<0.05) (Table II).

Table II.

Analysis on the polymorphisms of −800G/A and +915G/C gene [n (%)].

Table II.

Analysis on the polymorphisms of −800G/A and +915G/C gene [n (%)].

−800G/A+915G/C


GroupsCasesGGGAAAGAGGGCCCGC
Observation group156281110639 (25.0)117 (75.0)312379  54 (34.6)102 (65.4)
Control group1567026  3496 (61.5)  60 (38.5)961530111 (71.2)  45 (28.8)
χ2 60.471 42.423 56.794 41.793
P-value <0.001 <0.001 <0.001 <0.001
Comparison of serum TGF-β1 expression levels

The TGF-β1 expression levels of −800G/A gene AA subtype serum in the observation group were significantly higher than GG type, GA type and the control group, while the TGF-β1 levels of +915G/C gene CC subtype were significantly higher than GG type, GC type and the control group, and the differences were statistically significant (P<0.05) (Fig. 1).

Discussion

SNP is characterized in the third generation of genetic markers found currently. A previous study has shown that there are 10 TGF-β1 gene SNPs at least, including −988 (C>A), +72 (insC), −509 (C>T), −800 (G>A), codon 327 (Thr>Pro), codon 10 (Leu>Pro), codon 47 (Gly>Glu), codon 263 (Thr>Ile), codon 25 (Arg>Pro) and 713-8delC (6). The detection methods of SNPs include restriction fragment length polymorphism (RFLP), single-stranded conformation polymorphism (SSCP) and allele-specific oligonucleotide (ASO) probe.

A study has demonstrated that the number of TGF-β1 SNP sites and the pattern of manifestation are specific to both the population and disease. The TGF-β1 promoter −800G/A and −509C/T polymorphisms were related to the occurrence of esophageal cancer (7), the codon 10 site was correlated with the occurrence of breast cancer (8), and −509 (C>T) and codon 10 (Leu>Pro) polymorphisms were associated with the occurrence of bladder cancer (9). This study indicates that the proportion of −800G/A gene AA subtype and A allelic gene in the observation group were significantly higher than that in the control group, while the proportion of +915G/C gene CC subtype and C allelic gene were significantly higher than that in the control group; differences were statistically significant. The cancer risk (OR) of patients with A allelic gene in −800G/A gene was 4.8, while the cancer risk (OR) of patients with C allelic gene in +915G/C gene was 4.7, which suggests that −800G/A (A>G) and +915G/C (C>G) are closely related to the lung cancer susceptibility. The serum TGF-β1 expression levels of −800G/A gene AA subtype in the observation group were significantly higher than GG type, GA type and the control group, while TGF-β1 levels of +915G/C gene CC subtype were significantly higher than GG type, GC type and the control group; differences were statistically significant. High levels of TGF-β1 play an important role in promoting the occurrence and development of lung cancer (10). Through the reconstruction of the eukaryotic expression recombinant of TGF-β1 promoter codon 10 in vitro and the transfection of HeLa cell line, it was found that the mutation of single SNP site could lead to abnormally high expression of circulating TGF-β1 levels, the pathogenesis of which is closely related to the processing, modification, transport and release of proteins after translation rather than the transcriptional activity of the gene. In addition, the different expression of amino acids caused by an adjustment of polymorphism of relative sites would form different primary peptide chain structures, which would eventually decide the biological behavior of the translated protein (11).

The study on the mechanism of polymorphic sites and tumorigenesis has considered that activator protein 1 (AP1) and hypoxia-inducible factor-1 (HIF-1) are important factors in the regulation of the expression of TGF-β1 (12). When −800G/A (A>G) exist, the binding activity of AP1 with this site is increased, which would promote the transcription and translation of the TGF-β1 gene (12). For HIF-1, the presence of either A or G is not relevant and can be combined, however, for G, the competitive binding with AP1 would be formed (13). TGF-β1 gene polymorphism is also associated with self-autoimmune diseases, repair in trauma, inflammatory reaction, organ fibrosis and other diseases (14,15). After HBV infection, secondary cirrhosis may be related to the SNP of codon 10, and the possibility of codon 10 (Leu>Pro) developed to liver cirrhosis would be increased, while its severity may be associated with the SNP of −509 site, and the disease condition at −509 (C>T) may become more serious (16).

References

1 

Yung KW, Yung TT, Chung CY, Tong GT, Liu Y, Henderson J, Welbeck D and Oseni S: Principles of cancer staging. Asian Pac J Surg Oncol. 1:1–16. 2015.

2 

Ghoneum M, Felo N, Nwaogu OM, Fayanju IY, Jeffe JA and Margenthaler DB: Clinical trials in surgical oncology. Asian Pac J Surg Oncol. 1:73–82. 2015.

3 

Eberlein C, Rooney C, Ross SJ, Farren M, Weir HM and Barry ST: E-cadherin and EpCAM expression by NSCLC tumour cells associate with normal fibroblast activation through a pathway initiated by integrin αvβ6 and maintained through TGFβ signalling. Oncogene. 34:704–716. 2015. View Article : Google Scholar : PubMed/NCBI

4 

Wan PQ, Wu JZ, Huang LY, Wu JL, Wei YH and Ning QY: TGF-β1 polymorphisms and familial aggregation of liver cancer in Guangxi, China. Genet Mol Res. 14:8147–8160. 2015. View Article : Google Scholar : PubMed/NCBI

5 

Amani D, Khalilnezhad A, Ghaderi A, Niikawa N and Yoshiura K: Transforming growth factor beta1 (TGFβ1) polymorphisms and breast cancer risk. Tumour Biol. 35:4757–4764. 2014. View Article : Google Scholar : PubMed/NCBI

6 

Zhou TB, Zhao HL, Fang SL and Drummen GP: Association of transforming growth factor-β1 T869C, G915C, and C509T gene polymorphisms with rheumatoid arthritis risk. J Recept Signal Transduct Res. 34:469–475. 2014. View Article : Google Scholar : PubMed/NCBI

7 

Xiao Y, Yuan X, Qiu H and Li Q: Single-nucleotide polymorphisms of TGFβ1 and ATM associated with radiation-induced pneumonitis: A prospective cohort study of thoracic cancer patients in China. Int J Clin Exp Med. 8:16403–16413. 2015.PubMed/NCBI

8 

Parvizi S, Mohammadzadeh G, Karimi M, Noorbehbahani M and Jafary A: Effects of two common promoter polymorphisms of transforming growth factor-β1 on breast cancer risks in Ahvaz, West South of Iran. Iran J Cancer Prev. 9:e52662016.PubMed/NCBI

9 

Gautam KA, Pooja S, Sankhwar SN, Sankhwar PL, Goel A and Rajender S: c.29C>T polymorphism in the transforming growth factor-β1 (TGFB1) gene correlates with increased risk of urinary bladder cancer. Cytokine. 75:344–348. 2015. View Article : Google Scholar : PubMed/NCBI

10 

Shen ZT, Shen JS, Ji XQ, Li B and Zhu XX: TGF-β1 rs1982073 polymorphism contributes to radiation pneumonitis in lung cancer patients: A meta-analysis. J Cell Mol Med. 20:2405–2409. 2016. View Article : Google Scholar : PubMed/NCBI

11 

Zhou J, You W, Sun G, Li Y, Chen B, Ai J and Jiang H: The marine-derived oligosaccharide sulfate MS80, a novel transforming growth factor β1 inhibitor, reverses epithelial mesenchymal transition induced by transforming growth factor-β1 and suppresses tumor metastasis. J Pharmacol Exp Ther. 359:54–61. 2016. View Article : Google Scholar : PubMed/NCBI

12 

Shah R, Hurley CK and Posch PE: A molecular mechanism for the differential regulation of TGF-beta1 expression due to the common SNP −509C-T (c. −1347C>T). Hum Genet. 120:461–469. 2006. View Article : Google Scholar : PubMed/NCBI

13 

Wang HB, Song WG, Liu HQ, Fang F and Xiao Y: Role of TGFB1 polymorphism in the development of metastatic brain tumors in non-small cell lung cancer patients. Genet Mol Res. 14:3545–3550. 2015. View Article : Google Scholar : PubMed/NCBI

14 

Saad MN, Mabrouk MS, Eldeib AM and Shaker OG: Effect of MTHFR TGFβ1, and TNFB polymorphisms on osteoporosis in rheumatoid arthritis patients. Gene. 568:124–128. 2015. View Article : Google Scholar : PubMed/NCBI

15 

Doğru-Abbasoğlu S, Vural P, Baki M, Özderya A, Karadağ B and Uysal M: Arg25Pro (c.915G>C) polymorphism of transforming growth factor β1 gene increases the risk of developing Graves' disease. Int Immunopharmacol. 20:366–369. 2014. View Article : Google Scholar : PubMed/NCBI

16 

Wu XD, Zeng K, Gong CS, Chen J and Chen YQ: Transforming growth factor-β genetic polymorphisms on development of liver cirrhosis in a meta-analysis. Mol Biol Rep. 40:535–543. 2013. View Article : Google Scholar : PubMed/NCBI

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Spandidos Publications style
Di QG, Sun BH, Jiang MM, Du JF, Mai ZT, Zhang X, Zhou LR, Chi YM and Lv J: Polymorphisms of ‑800G/A and +915G/C in TGF‑β1 gene and lung cancer susceptibility. Oncol Lett 14: 733-736, 2017
APA
Di, Q., Sun, B., Jiang, M., Du, J., Mai, Z., Zhang, X. ... Lv, J. (2017). Polymorphisms of ‑800G/A and +915G/C in TGF‑β1 gene and lung cancer susceptibility. Oncology Letters, 14, 733-736. https://doi.org/10.3892/ol.2017.6173
MLA
Di, Q., Sun, B., Jiang, M., Du, J., Mai, Z., Zhang, X., Zhou, L., Chi, Y., Lv, J."Polymorphisms of ‑800G/A and +915G/C in TGF‑β1 gene and lung cancer susceptibility". Oncology Letters 14.1 (2017): 733-736.
Chicago
Di, Q., Sun, B., Jiang, M., Du, J., Mai, Z., Zhang, X., Zhou, L., Chi, Y., Lv, J."Polymorphisms of ‑800G/A and +915G/C in TGF‑β1 gene and lung cancer susceptibility". Oncology Letters 14, no. 1 (2017): 733-736. https://doi.org/10.3892/ol.2017.6173