Serum glycine dehydrogenase is associated with increased risk of lung cancer and promotes malignant transformation by regulating DNA methyltransferases expression
- Authors:
- Published online on: June 22, 2018 https://doi.org/10.3892/mmr.2018.9214
- Pages: 2293-2299
Abstract
Introduction
Lung cancer is the leading cause of cancer-associated mortality worldwide (1). Identifying novel risk factors is essential in order to prevent the disease. Abnormal overexpression of the metabolic enzyme glycine dehydrogenase (GLDC) has been associated with lung cancer and other types of tumors (2–4). GLDC is critical for the formation of cancer initiating cells in non-small cell lung cancer (NSCLC) (2). In animal models, its overexpression can induce malignant transformation of lung normal cells and promote the formation of tumors (2). However, there is currently no perspective study on the associations between GLDC and lung cancer.
DNA methylation is a covalent chemical modification, resulting in the addition of a methyl (CH3) group at the carbon 5 position of the cytosine ring, which is a hallmark of human diseases such as lung cancer (5–7). Methylation of DNA at position 5 of the cytosine ring is catalyzed by DNA methyltransferases (DNMTs) and is the predominant epigenetic modification in mammals (8). The mammalian DNMT family includes 4 active members: DNMT1, DNMT3A, DNMT3B and DNMT3L (9,10). DNMT1 is the most abundant DNMT and is involved in the maintenance of methylation (8,11,12). DNMT3 functions as a de novo methyltransferase and consists of 2 associated proteins encoded by the distinct genes, DNMT3A and DNMT3B (11). The expression levels of these DNMTs are reportedly elevated in cancers of the colon, prostate, breast, liver and in leukemia (13–16). Aberrant methylation of the tumor suppressive gene (TSG) is an early event in the development of lung cancer (17,18). MicroRNAs (miRNA/miRs) are a group of small non-coding RNAs (~22 nucleotides) that regulate gene expression (19–21). The expressions of miR-29a, −29b and −29c were downregulated in NSCLCs (7). Expression of the miR-29 family is inversely associated with DNMTs expression in lung cancer tissues and the miR-29 family directly targets DNMTs; the miR-29 family can revert aberrant methylation in lung cancer by targeting DNMTs (7). In addition, the enforced expression of the miR-29 family in lung cancer cell lines restored normal patterns of DNA and promoted the re-expression of TSGs silenced by methylation (7). However, the regulatory mechanism associated with miR-29 family expression has not been fully elucidated. The aim of the study was to assess the association between serum GLDC and lung cancer risk and study the mechanism underlying the effects of GLDC in lung cancer.
Materials and methods
Study cohort and serum samples
A nested case-control study was conducted in the well-characterized Chinese Cohort (22). The project included 300 invasive lung cancer cases, each of which were matched with 2 controls (n=600). The participants were recruited in Shandong Cancer Hospital and Shanghai cancer institute between 1998 and 2013, when they received physical examination and the physical characteristics are presented in Table I. Each participant donated more than one blood sample at the recruitment. For each case-subject match set, 2 control subjects closest to the case (based on matching criteria, age at time of sampling) with an available blood sample were chosen among the appropriate risk sets consisting of all cohort members alive and free of cancer at the time of diagnosis of the index case. Serum aliquots of 500 µl were stored at −180°C for measurements of GLDC. The matching criterion was age at the time blood was drawn. The Shandong (China) Ethical Board of the Shandong Academy of Occupational Health and Occupational Medicine (Shandong, China) approved the present study and written informed consent was obtained from each individual recruited.
Cell culture
Normal human bronchial epithelial (NHBE) cells were obtained from the America Type Culture Collection (ATCC; Manassas, VA, USA) were grown in RPMI-1640 medium (Sigma, Shanghai, China) containing 10% fetal bovine serum (FBS; Shanghai ExCell Biology, China) and 100 mg/ml penicillin and streptomycin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified atmosphere with 5% CO2 (23).
Pre-miR-29a/-29b/29c/Control miR, GLDC expressing plasmids/empty vectors and transfection experiments
Pre-miR-29a/-29b/29c/Control miR were purchased from Ambion, (Thermo Fisher Scientific, Inc.). GLDC expressing plasmids and empty vectors (mock) were purchased from Tiangen (Beijing, China). For transfection experiments, the cells were cultured in serum-free medium without antibiotics at 60% confluence for 24 h, and then transfected with transfection reagent (Lipofectamine 2000; Thermo Fisher Scientific, Inc.) according to manufacturer's instructions. After incubation for 6 h, the medium was removed and replaced with normal culture medium for 48 h.
Enzyme-linked immunosorbent assay (ELISA)
ELISA analysis was employed to detect the levels of serum GLDC protein in study cohort and was performed as described previously (24). The intra-batch and inter-batch coefficients of variation for GLDC protein were 4.54 and 8.73%, respectively. Multivariate unconditional logistic regression was performed to calculate the odds ratios (OR) and corresponding 95% confidence intervals (95% CI) for lung cancer occurrence, calculating ORs over the quartile levels and on a continuous log2 scale of circulating GLDC. The final multivariate models shown included 3 factors (years of smoking, asbestos exposure and sex) that affect the exposure and disease relation ≥10%. Ptrends was calculated by using the median values of GLDC quartiles.
Western blot analysis
Western blot analysis was performed as described previously (25). For membrane incubation the following primary antibodies were used: Rabbit anti-GLDC (cat. no. ab232989; 1:500), anti-DNMT1 (cat. no. ab87654; 1:500) anti-DNMT3A (cat. no. EPR18455; 1:500), anti-DNMT3B (cat. no. ab2851; 1:500) and anti-β-actin (cat. no. ab5694; 1:500; all Abcam, Cambridge, MA, USA) antibodies for overnight incubation at 4°C. Membranes were also incubated with IRDye™-800 conjugated anti-secondary antibodies (cat. no: ab6721; 1:10,000; Abcam) for 30 min at room temperature. For analysis, β-actin was a loading control. The specific proteins were visualized using the Odyssey™ Infrared Imaging System (Gene Company, Ltd., Hong Kong, China).
Colony formation assay
The colony formation assay was performed as described previously (26).
MTT assay
The effect on cell proliferation was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and was performed as described previously (27). Briefly, 1×104 cells were seeded onto 96-well plates and were incubated for 24 h in a 37°C, 5% CO2 cell culture incubator. MTT reagent (50 µl; 5 mg/ml) was added to each well and cells were incubated for a further 4 h. Then the formazan precipitate was dissolved in 150 µl dimethylsulfoxide and the absorbance rate was measured in a microplate reader at a wave length of 570 nm, with the reference wavelength set at 630 nm. Absorbance was directly proportional to the number of surviving cells. The viability of the control group.
Microarray analysis of miRNA
Microarray Analysis of miRNA was performed as described previously (28).
Northern blotting analysis
Northern blot analysis of miRNAs was performed as described previously (29,30). The probe sequences were as follows: miR-29a, 5′-TAACCGATTTCAGATGGTGCTA-3′; miR-29b, 5′-AACACTGATTTCAAATGGTGCTA-3′; miR-29c, 5′-TAACCGATTTCAAATGGTGCTA-3′; U6 small nuclear RNA, 5′-CCATGCTAATCTTCTCTGTATCGTTCCAA-3′.
Statistical analysis
The results of the colony formation and MTT assays were statistically analyzed, using a Student's t-test. Data were expressed as the mean ± standard error. Spearman correlation was performed for all other results in order to analyze the association between serum GLDC protein concentration and other variables (age, BMI and years of smoking). Multivariate logistic regression analysis was applied to assess the association between serum GLDC and lung cancer risk. All P-values presented are 2-sided and P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were conducted using SAS software, version 9.3 (SAS Institute, Inc., Cary, NC, USA).
Results
Higher serum GLDC is associated with increased risk in lung cancer
Baseline characteristics comparing 300 lung cancer cases and 600 matched controls are outlined in Table I; the matched variable was age. The median age was 60.1 years for lung cancer cases and 59.2 years for matched controls. The majority of the lung cancer cases and matched controls were men (82.7% for cases and 80.2% for controls). The cases had a lower average level of education, as well as a higher proportion of smokers (number of cigarettes per day or years of smoking) and greater occupational exposure to asbestos associated with lung cancer risk. However, no differences in body mass index (BMI) and physical activity were observed between the 2 groups. In addition, those with lung cancer had a higher energy intake rate as well as a lower consumption of fruit/carotenoids. In those with lung cancer, ~33% of cases were adenocarcinomas and 22% of the cases were squamous cell carcinomas (Table II). All other histological types of lung cancer accounted for <20% (Table II).
Serum GLDC was positively associated with the overall risk of lung cancer (Table III; OR=1.48; 95% CI, 1.01–2.04). The risk was elevated in the highest quartile (OR=1.59; 95% CI, 1.15–2.54) when compared with the lowest quartile (OR=0.99; 95% CI, 0.76–1.23). Tables IV and V present the associations between age, BMI, years of smoking and GLDC in the lung cancer cases and the matched controls. Serum GLDC levels were positively correlated with years of smoking (Spearman's ρ=0.81; Table IV); however, the association was attenuated in the sera of matched controls (Spearman's ρ=0.48; Table V).
Table III.Adjusted odds ratios for lung cancer by quartile levels and on a continuous log2 scale of circulating prolactin (n=300). |
Table IV.Spearman's correlation coefficients between age, body mass index, years of smoking and serum glycine dehydrogenase in patients with lung cancer (n=300). |
Table V.Spearman's correlation coefficients between age, body mass, years of smoking and serum glycine dehydrogenase in matched controls (n=600). |
GLDC promotes tumorigenesis in normal human bronchial epithelial (NHBE) cells
To investigate whether GLDC promotes proliferation in NHBE cells, western blotting was performed to determine whether GLDC expressing plasmids can upregulate GLDC protein expression in NHBE cells. The results of western blotting showed that GLDC protein was upregulated by GLDC expressing plasmids in cells (Fig. 1A). To identify the role of GLDC in regulating proliferation, an MTT assay was performed. Overexpressing GLDC significantly promoted proliferation in NHBE cells (Fig. 1B). A colony formation assay was employed to detect whether GLDC protein affected the colony formation rate of the cells. The results demonstrated that GLDC promoted colony formation in NHBE cells (Fig. 1C).
GLDC inhibits miR-29a/b/c expression in NHBE cells
To examine the role of GLDC in the regulation of miRNA expression, microarray analysis was performed in NHBE cells. A total of 19 evidently altered miRNAs were identified; 9 miRNAs were downregulated and 10 miRNAs were upregulated with >5 fold changes in cells transfected with GLDC expressing plasmids compared with control cells (Table VI). Northern blot analysis was conducted to confirm whether overexpressing GLDC affected miR-29 a/b/c expression in NHBE cells. The results revealed that overexpressing GLDC markedly downregulated their expression in cells (Fig. 2).
Table VI.microRNA expression and glycine dehydrogenase regulation in human bronchial epithelial cells. |
GLDC promotes DNMTs protein expression and miR-29a/b/c inhibits their expression in NHBE cells
In order to detect whether GLDC can affect DNMT1, DNMT3A and DNMT3B protein expression, western blot analysis was performed in NHBE cells transfected with GLDC expressing plasmids and empty vectors. The results of western blotting showed that DNMT1, DNMT3A and DNMT3B protein expression were upregulated by GLDC (Fig. 3A).
In addition, the results of western blotting were used to detect whether miR-29a/b/c can regulate DNMT1, DNMT3A and DNMT3B protein expression in NHBE cells. The results showed that miR-29a inhibited DNMT1, DNMT3A and DNMT3B protein expression in NHBE cells (Fig. 3B). However, miR-29b/c only downregulated DNMT3A and DNMT3B protein expression in NHBE cells (Fig. 3C and D).
GLDC is negatively correlated with miR-29a/b/c expression in the sera of participants
Having demonstrated that GLDC inhibited miR-29a/b/c expression in NHBE cells, the present study then used Spearman's correlation to analyze whether GLDC protein is negatively correlated with serum miR-29a/b/c expression. The results demonstrated that GLDC protein was negatively correlated with serum miR-29a/b/c expression in the serum of participants (Table VII).
Table VII.Spearman's correlation coefficients between serum glycine dehydrogenase and microRNA-29 family in all participants (n=891a). |
Discussion
Experimental evidence has revealed that GLDC may be an oncogene in lung cancer (2); however, up to now, there has not been a perspective study that determined whether it can promote the initiation of lung cancer. In this prospective study, an increased lung cancer risk was associated with higher serum GLDC concentrations. The results were in line with previous experimental evidence that demonstrated that exposure to GLDC can transform normal breast cells and primary NHBE cells to malignancy-like status (2). In order to reduce lung cancer mortality, prevention is one of the most effective strategies. Smoking is an important risk factor for lung cancer (31). Elucidating how carcinogens are produced by smoking and gaining a better understanding of this process will improve the scientific basis for the assessment of mechanisms associated with lung cancer development.
The present study showed that years of smoking were positively associated with the serum concentration of GLDC, which can increase lung cancer risk. The results implied that smoking may promote the initiation of lung cancer by upregulating serum GLDC concentration. Thus, developing an antagonist for serum GLDC may be helpful to prevent lung cancer in smokers.
In line with previous perspective and lab results (2), the present study revealed that GLDC promoted malignant transformation in NHBE cells. Increased proliferation and colony formation abilities are hallmarks of cancer (32). The results of the present study demonstrated that GLDC can promote proliferation and colony formation abilities in NHBE cells.
Aberrant methylation of TSG is an early event in the development of lung cancer (17,18). DNMTs control changes in methylation and 3 catalytically active DNMTs (DNMT1, DNMT3A and DNMT3B) have been identified (33). Recently, it has been reported that miRNA-29a/b/c can revert aberrant methylation in lung cancer by regulating DNMT3A and DNMT3B (7). In the present study, GLDC expression in serum, induced by smoking, was an upstream regulator of the miR-29 family. In addition, GLDC promoted DNMTs protein expression. Consistent with a previous report (7), the results of the present study showed that the miR-29 family may inhibit DNMTs protein expression in NHBE cells. Thus, the smoking/GLDC/miR-29 family/DNMTs signaling pathway may serve an important role in the early malignant transformation of normal lung cells.
Competing interests
The authors declare that they have no competing interests.
Glossary
Abbreviations
Abbreviations:
TSG |
tumor suppressive gene |
GLDC |
glycine dehydrogenase |
NSCLC |
non-small cell lung cancer |
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