|Year : 2019 | Volume
| Issue : 4 | Page : 899-903
Methylation of RUNX3 and RASSF1A and the risk of Malignancy in small solitary pulmonary nodules
Jinglan Zhao, Xiangyu Cui, Xiuying Huang, Ruizhen Lu, Peng Chen, Zhixue Zhang
Department of Thoracic Surgery, The Affiliated Central Hospital of Qingdao University, Qingdao, Shandong, China
|Date of Web Publication||14-Aug-2019|
The Affiliated Central Hospital of Qingdao University, Si Liu Nan Road 127#, Qingdao, Shandong Province 266033
Source of Support: None, Conflict of Interest: None
Background: This study aimed to evaluate the methylation of RUNX3 and RASSF1A gene promoter regions as a marker to distinguish between benign and malignant of small solitary pulmonary nodule (SPN) ≤10 mm in size.
Materials and Methods: A total of 147 patients with pathologically confirmed SPNs were enrolled. DNA samples were extracted from biopsy tissues or serum. Methylation of RUNX3 and RASSF1A gene promoter regions was detected by the methylation-specific polymerase chain reaction. The expression of RUNX3 and RASSF1A in SPN tissues was detected by western blot.
Results: Of the 147 patients, 89 had benign SPNs and 58 had malignant SPNs. The rate of serum RUNX3 and RASSF1A gene methylation in malignant SPNs was significantly higher than that in benign SPNs (65.5% vs. 12.3%, and 67.2% vs. 10.1%, respectively; P < 0.05). The expression of RUNX3 and RASSF1A in malignant SPN tissues was lower than that in benign SPN tissues. The hypermethylation status of RUNX3 or RASSF1A genes was not significantly associated with age, gender, and smoking.
Conclusions: The methylation level of the RUNX3 and RASSF1A gene promoter regions is a promising marker for assessing SPNs.
Keywords: DNA methylation, RASSF1A gene, RUNX3 gene, small solitary pulmonary nodule
|How to cite this article:|
Zhao J, Cui X, Huang X, Lu R, Chen P, Zhang Z. Methylation of RUNX3 and RASSF1A and the risk of Malignancy in small solitary pulmonary nodules. J Can Res Ther 2019;15:899-903
|How to cite this URL:|
Zhao J, Cui X, Huang X, Lu R, Chen P, Zhang Z. Methylation of RUNX3 and RASSF1A and the risk of Malignancy in small solitary pulmonary nodules. J Can Res Ther [serial online] 2019 [cited 2020 Oct 30];15:899-903. Available from: https://www.cancerjournal.net/text.asp?2019/15/4/899/264304
| > Introduction|| |
Solitary pulmonary nodule (SPN) is defined as a round or ovoid lung lesion <3 cm in diameter, without concomitant pneumonia and atelectasis of involved lung segments and lobes., Recently, many much smaller SPNs have been identified on computed tomography (CT) of the chest, often as incidental findings. The SPN <1 cm in diameter is designated as small SPN. Diagnoses of benign and malignant small SPNs have become a concern and a challenge.,
Some SPNs are indicated pathologically in the early stages of lung cancers. Although some SPNs are malignant, in most age groups, the majority are benign. These commonly include granulomas, hamartomas, or intrapulmonary lymph nodes and less commonly include benign bronchial adenomas. Moreover, a host of other lesions, ranging from rheumatoid nodules to sarcoid lesions, can appear as SPNs. Therefore, preoperative characterization of SPNs is extremely important.
DNA methylation is a key regulator of gene transcription and genomic stability. Alterations in DNA methylation patterns are frequently detected in human tumors, including lung cancer., Some published studies suggest a relationship between the methylation status of several genes and cancer diagnoses. The hypermethylation of specific genes might be expected to serve as a biomarker for these diagnoses.
The RUNX gene family consists of three members: RUNX1/AML1, RUNX2, and RUNX3. All three RUNX family members have pivotal roles in normal developmental processes and carcinogenesis. RUNX3 is located on human chromosome 1p36, a region that has long been suggested to be a tumor suppressor locus in various tumors. Unlike many tumor suppressors, such as p53, which are inactivated mainly by deletion and mutation, RUNX3 is unique, in that it is inactivated primarily by epigenetic silencing. Inactivation of RUNX3 due to DNA hypermethylation was recently reported in various tumors, including lung cancer, gastric cancer, breast cancer, pancreatic cancer, colon cancer, and hepatocellular carcinoma.,,,,,
RASSF1A is an important tumor suppressor gene that is involved in the regulation of intracellular biological events, such as cell growth, regulation of cell cycle, differentiation, and apoptosis, to inhibit the occurrence and development of tumors. The hypermethylation of RASSF1A gene promoter region can down-regulate this gene. Consequently, RASSF1A loses its tumor suppressor function, which can induce lung cancer.
In the present study, we first examined the relationship between the methylation status of RUNX3 and RASSF1A and malignancy in small SPNs. Using bisulfite sequencing, as well as nested methylation-specific polymerase chain reaction (nested-MSP), we further analyzed the different methylation status of RUNX3 and RASSF1A in small SPNs.
| > Materials and Methods|| |
A total of 147 cases of SPNs collected and confirmed by clinical pathology or biopsy included 89 benign lesions and 58 malignant lesions. Each pulmonary nodule was <10 mm in size. These 147 cases comprised 102 men and 45 women aged 26–80 years (median: 63.7 years). Written informed consent was obtained from all participants, and all procedures were approved by the hospital's ethics board.
The venous blood samples and pulmonary nodule tissues were collected from the patients with SPNs detected by CT at the Center Medical Group of Qingdao. These tissues were collected after reconfirmation by a senior pathologist from the Center Medical Group of Qingdao.
Methylation-specific polymerase chain reaction
Genomic DNA was extracted using SepaGene (Sankojun-Yaku Co., Tokyo). DNA was extracted from the blood using the Blood Genomic DNA Extraction Kit (centrifugal column type) from Beijing Tiangen Biotech, according to the operating instructions. The methylation status of the examined genes was determined by MSP as described previously.
The primers were designed, according to the literature, to identify the methylation-specific sequence (M) and unmethylation-specific sequence (U). The RUNX3-M sense primer (5'-GCGTCGTATAGTTAATCGG-3') and RUNX3-M antisense primer (5-'CTCCTCCGCGAAATAAC-3') produced a 123 bp amplification product. The RUNX3-U sense primer (5'-GTGTTGTATAGTTAATTGG-3') and RUNX3-U antisense primer (5'-CTCCTCCACAAAATAAC-3') produced a 123 bp amplification product. The RASSF1A-M sense primer (5'-GGGTTTTGCGAGAGCGCG-3') and RASSF1A-M antisense primer (5'-CCCGATTAAACC CGTACTTCG-3') produced a 202 bp amplification product. The RASSF1A-U sense primer (5'-GGTTTTGTGAGAGTGTGTTTAG-3') and RASSF1A-U antisense primer (5'-CCCAATTAAACC CATACTTCAC-3') produced a 202 bp amplification product.
DNA samples were treated with bisulfite to convert all unmethylated cytosine to uracil while leaving methylated cytosines unaffected. Briefly, 2 μg of genomic DNA was denatured by the treatment with NaOH and modified by sodium bisulfite. DNA samples were then purified using a Wizard DNA purification resin (Promega), treated with NaOH, precipitated with ethanol, and resuspended in 30 μl.
Modified DNA was amplified in a total volume of 20 μl using GeneAmp PCR Gold Buffer (PE Applied Biosystems) containing 1.0 mM MgCl2, 20 μM of each primer, 0.2 mM dNTPs, and 1 unit of Taq polymerase (AmpliTag Gold DNA Polymerase, PE Applied Biosystems). After activation of the Tag polymerase at 95°C for 8 min, polymerase chain reaction (PCR) was performed in a GeneAmp 2400 thermal cycler (PE Applied Biosystems) for 40 cycles, with each cycle consisting of denaturation at 95°C for 30 s, annealing at 55°C–60°C for 30 s, and extension at 72°C for 30 s, followed by a final 7 min extension at 72°C. A negative control (water without DNA) and a positive control were included for each amplification. The PCR products were loaded onto a nondenaturing 6% polyacrylamide gel, stained with ethidium bromide, and visualized under ultraviolet illumination.
Protein extraction and western blotting
Total protein from all tissue samples was extracted using the standard method. The protein concentration was routinely analyzed by 12% SDS-PAGE. Protein was transferred to a nitrocellulose membrane, which was blocked with 5% nonfat dry milk and incubated with rabbit anti-N-Myc downstream regulated 1 (NDRG1) primary antibody (Zymed, San Francisco, CA, USA) overnight at 4°C. The secondary horseradish peroxidase-conjugated antirabbit antibody was visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). Equal protein loading was confirmed through the inclusion of β-actin.
Promoter methylation of NDRG1 was analyzed using Chi-square test using SPSS 13.0 software (SPSS Inc, Chicago, IL). The correlation of the clinicopathological data using unconditional logistic regression was used to estimate odds ratios and 95% confidence intervals. A P < 0.05 was considered statistically significant.
| > Results|| |
[Table 1] lists the baseline characteristics of the 147 patients. The mean age of patients was 63.7 years (range 26–80).
Methylation status of RUNX3 and RASSF1A in SPN tissues
Among the 58 malignant SPNs, 43 cases featured hypermethylation of the RUNX3 gene, representing a methylation rate of 74.1%. Of the 89 benign SPNs, 15 cases featured hypermethylation of the RUNX3 gene promoter region, representing a methylation rate of 16.9%. The rate of RUNX3 gene methylation in malignant SPNs was significantly higher than that in benign SPNs (P < 0.05) [Figure 1]a. RASSF1A hypermethylation in malignant SPNs was detected in 41 of 58 cases (70.7%), which was more frequent than that in benign SPNs (13 of 89, 14.6%) [Figure 1]b.
|Figure 1: Methylation-specific polymerase chain reaction analysis for the methylation of RUNX3 (a) and RASSF1A (b) in solitary pulmonary nodule tissues. Genomic DNA from b solitary pulmonary nodule tissues treated with sodium bisulfite was amplified using methylated (M) and unmethylated primers (U). P was used as a positive control for methylated reaction, and N was used as a positive control for unmethylated reaction. Ma denotes malignant solitary pulmonary nodule and Be denotes benign solitary pulmonary nodule|
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RUNX3 and RASSF1A expression in solitary pulmonary nodule tissues
Expression of RUNX3 and RASSF1A in SPN tissues by western blot revealed a significant decline of RUNX3 and RASSF1A in malignant SPNs [Figure 2].
|Figure 2: Expression of RUNX3 and RASSF1A in solitary pulmonary nodule tissues detected by western blot. Expression of the β-actin gene was used as internal control. The levels of RUNX3 and RASSF1A in malignant solitary pulmonary nodules decreased compared with that in benign solitary pulmonary nodules. Lane 1 displays benign solitary pulmonary nodule and lane 2 displays malignant solitary pulmonary nodule|
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Relationship between RUNX3 and RASSF1A methylation and age, gender, and smoking
[Table 2] summarizes the relationships between RUNX3 methylation status and age, gender, and smoking. RUNX3 hypermethylation was not significantly associated with patient age, gender, and smoking status.
|Table 2: Relationship between the methylation status of RUNX3 and age, gender, and smoking|
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[Table 3] shows the relationship between RASSF1A methylation status and age, gender, and smoking. RASSF1A hypermethylation was not significantly associated with patient age, gender, and smoking status.
|Table 3: Relationship between the methylation status of RASSF1A and age, gender, and smoking|
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Methylation status of RUNX3 and RASSF1A in serum
The expression of RUNX3 in malignant SPNs was significantly lower than that in benign SPNs (P < 0.05) [Figure 3]. Among the 58 malignant SPNs, 38 cases featured hypermethylation of the RUNX3 gene, representing a methylation rate of 65.5%. In the 89 benign SPNs, 11 cases featured RUNX3 gene promoter region hypermethylation, representing a methylation rate of 12.3%.
|Figure 3: Methylation-specific polymerase chain reaction analysis for the methylation of RUNX3 (a) and RASSF1A (b) in serum. Genomic DNA from b solitary pulmonary nodule tissues treated with sodium bisulfite was amplified using methylated (M) and unmethylated primers (U). P was used as a positive control for methylated reaction, and N was used as a positive control for unmethylated reaction. Ma denotes malignant solitary pulmonary nodule and Be denotes benign solitary pulmonary nodule|
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The expression of RASSF1A was also decreased in malignant SPNs compared with that in benign SPNs (P < 0.05). RASSF1A hypermethylation in malignant SPNs was detected in 39 of [Table 4] shows 58 cases (67.2%), which was more frequent than that in benign SPNs (nine of 89 or 10.1%).
|Table 4: Comparison of methylation rate of RUNX3 and RASSF1A between tissue and serum|
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The hypermethylation of the two genes in the serum was consistent with the results of tissue hypermethylation. However, the detection rate of serum hypermethylation was slightly lower. There was no statistical significance between the two methylation rates (P > 0.05).
| > Discussion|| |
The diagnosis of patients with small SPNs remains poor because of the small size and the limitations of imaging examination. Many markers have been suggested that may be predictive of disease progression and survival. Some of them, such as p53, have been proposed as independent markers, while others do not appear to have a role as a diagnostic indicator in a clinical setting. Diagnosis of benign and malignant SPNs has become both a concern and a challenge. Some SPNs have been implicated pathologically in the early stages of lung cancers. Although there have been reports of the prognostic value of methylation patterns in several tumors, limited data are available on the diagnostic value of gene methylation in human SPNs. Therefore, it is necessary to find a preoperative method in the characterization of SPNs.
We evaluated the hypermethylation status of two tumor suppressor genes associated with lung cancer, RUNX3 and RASSF1A, to distinguish benign from possibly malignant SPNs, without a biopsy when possible. RUNX3 is a human Runt-related transcription factor (RUNX). Little or no expression of RUNX3 due to CpG island hypermethylation was observed in carcinomas of the liver, breast, lung, colon, and prostate., RUNX3 is more frequently methylated in lung cancers and RUNX3 methylation occurred at an early stage. As well, RUNX3 methylation status has been associated with decreased survival. RASSF1A is one of the Ras families of genes, which are associated with the signal transduction from G protein-coupled receptors and activation of the Ras signal transduction pathway in human tumors. In the present study, the Ras effectors/tumor suppressors RASSF1 and NORE1 were found to be inactivated by promoter hypermethylation in a variety of human tumors. RASSF1A plays a role in cancer progression in its early stage. The methylation status of RASSF1A has been reported in lung cancers.,,,
In this study, we demonstrated the methylation level of RUNX3 and RASSF1A genes in patients with SPNs. The methylation status of RUNX3 and RASSF1A in tissue or serum of patients with malignant SPNs was much higher than that of patients with benign SPNs. The difference was statistically significant. Our results are promising because the study population was primarily patients with SPNs. In the clinical setting, new relevant biomarkers must provide cost-effective and noninvasive monitoring in patients at low risk and identify high-risk refractory tumors before they progress. The present success using blood specimens from patients with SPN is encouraging since it is easy to acquire such samples, patient discomfort is eased, and the examination of blood may detect other diseases, such as hypertension and coronary heart disease.
| > Conclusions|| |
We analyzed gene promoter methylation status using MSP. We found that RUNX3 and RASSF1A were hypermethylated in 65.5% and 67.2% of the malignant SPN samples, respectively. Methylation of the RUNX3 and RASSF1A gene promoters was more frequent in malignant SPNs. The methylation level of RUNX3 and RASSF1A gene promoter region could be promising markers for assessing SPNs. However, because we only examined a small number of cases, further studies will be necessary to both clarify and confirm its usefulness.
Financial support and sponsorship
This work was completed with the support of the people's livelihood project of Qingdao Municipal Science and Technology Bureau (No. 13-1-3-45nsh).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]