Introduction
Lung cancer (LC) is the most common malignant tumor
worldwide and represents the leading cause of cancer-related
mortality, with ~1.8 million new cases of LC diagnosed globally,
resulting in ~1.6 million deaths annually (1). More than 85% of LC cases are
classified as non-small cell LC (NSCLC), with lung adenocarcinoma
(LUAD) becoming the most prevalent pathologic subtype (2). Late diagnosis remains a major cause
of high mortality for LC. Early detection, particularly at stage I,
significantly improves patient outcomes, resulting in 81-85% 5-year
survival rates, while only 15-19% at stage IV (3,4).
As the prognosis of LUAD remains generally poor, it
is becoming increasingly important to investigate the molecular
mechanisms of tumor initiation and progression. In this context,
DNA methylation biomarkers are widely used for early-stage cancer
diagnosis and disease recurrence monitoring. Methylation within CpG
islands of DNA gene promoters can induce transcriptional silencing
of tumor suppressor genes, thereby playing a critical role in
oncogenesis (5). On the basis of
these findings, two commercially available assays utilizing plasma
cell-free DNA methylation levels have been introduced into clinical
practice to detect colorectal cancer and LC: ‘Epi proColon’, which
assesses SEPT9 methylation for colorectal cancer, and ‘Epi
proLung’, which evaluates PTGER4 and SHOX2 methylation for
LC (6,7).
SHOX2 and RASSF1A methylation were
preliminarily used for LC diagnosis. A study was conducted to
compare SHOX2 methylation in lung tumors vs. normal tissues,
and found that 96% of matched pairs had elevated methylation levels
in tumor tissues (8). Similarly,
the RASSF1A gene promoter hypermethylation was found in 63%
of NSCLC cell lines, but it remained hypomethylated in normal
epithelial cells (9).
Therefore, the present study aimed to investigate
the diagnostic value of SHOX2 and RASSF1A methylation
in early-stage LUAD. Using methylation-specific PCR (MSP), the
methylation status of SHOX2 and RASSF1A in tumor
tissues and the corresponding non-tumor lung tissues from
early-stage LUAD cases were evaluated. Additionally, the diagnostic
value of SHOX2 and RASSF1A methylation was analyzed
and their relationship with clinicopathological characteristics was
examined.
Materials and methods
Patients
The present study was approved by the Ethics
Committee of Foshan First People's Hospital (approval no. 145;
date, 2025; Foshan, China). In total, 25 participants (6 males and
19 females) were included in the present study. The clinical data
of these 25 patients, suffering from early-stage LUAD and
hospitalized in Foshan First People's Hospital from November 2022
to March 2023, were retrospectively analyzed. The corresponding
clinicopathological characteristics are shown in Table I. Informed consent was provided by
all patients prior to undergoing surgery. Postoperatively, the
tumor tissues and paired non-tumor lung tissues were collected and
stored at -80˚C. The diagnosis of LUAD was confirmed by two
experienced pathologists.
 | Table ICorrelation between methylation
status and diverse parameters. |
Table I
Correlation between methylation
status and diverse parameters.
| Clinicopathological
characteristic | Total (n=25) | Methylation level
of SHOX2, n (%) | Methylation level
of RASSF1A, n (%) |
|---|
| Sex | | | |
|
Male | 6 | 3 (50.0) | 1 (16.7) |
|
Female | 19 | 16 (84.2) | 7 (36.8) |
|
P-value | | 0.125 | 0.624 |
| Age, years | | | |
|
<60 | 11 | 9 (81.8) | 3 (27.3) |
|
≥60 | 14 | 10 (71.4) | 5 (35.7) |
|
P-value | | 0.661 | >0.999 |
| TNM stage | | | |
|
I | 22 | 16 (72.7) | 6 (27.3) |
|
II | 3 | 3 (100.0) | 2 (66.7) |
|
P-value | | 0.554 | 0.231 |
| Lymph node
metastasis | | | |
|
Positive | 2 | 2 (100.0) | 1 (50.0) |
|
Negative | 22 | 16 (72.7) | 6 (27.3) |
|
P-value | | >0.999 | 0.507 |
| Differentiation
degree | | | |
|
Moderately
/poorly | 11 | 9 (81.8) | 4 (36.4) |
|
Well | 6 | 5 (83.3) | 2 (33.3) |
|
P-value | | >0.999 | >0.999 |
DNA extraction
According to the manufacturer's instructions, total
genomic DNA was isolated from tissue samples using the TIANamp
Genomic DNA kit (Tiangen Biotech Co., Ltd.). The DNA quality and
integrity were determined by measuring the A260/280 ratios, with a
ratio of >1.8 indicating high purity, and total DNA
concentrations were calculated by NanoPhotometer N60 Touch (Implen,
https://implen.cn/).
Genomic DNA modifications by bisulfite
treatment
The genomic DNA, ranging from 200-500 ng per
reaction, was modified with sodium bisulfite by using the EpiJET
Bisulfite Conversion Kit (Thermo Fisher Scientific, Inc.). The
complete conversion of unmethylated cytosine to uracil was carried
out at 98˚C for 10 min and 60˚C for 150 min. Then the converted DNA
was purified and stored at -80˚C.
MSP
MSP was performed to assess the methylation status
of CpG islands in the promoter regions of RASSF1A and
SHOX2 using 2 sets of sequence-specific primers, one
targeting the methylated DNA sequence, and the other one targeting
the unmethylated DNA sequence. PCR-grade water was included as a
negative control. A total volume of 20 µl for each reaction,
including 1 µl of converted DNA, 10 µl of 2X Rapid Taq Master Mix
(Vazyme Biotech Co., Ltd.), 0.5 µl of each primer (10 µM), and 8 µl
of PCR-grade water. The PCR program was set for all the methylated
and unmethylated primers, consisting of initial denaturation at
95˚C for 5 min, 35 cycles of amplification at 95˚C for 30 sec, 60˚C
for 50 sec, and 72˚C for 45 sec, and final extension of 72˚C for 10
min. Subsequently, 8 µl of each PCR product was electrophoresed on
a 3% agarose gel and visualized with SYBR Safe (cat. no. S33102;
Invitrogen; Thermo Fisher Scientific, Inc.) staining method
(Fig. S1), and the images of the
bands were captured using Azure 300 (Azure Biosystems, Inc.).
SHOX2 and RASSF1A were considered methylated when the
MSP amplification products were detected in reactions with
methylation-specific primers. The primer sequences were as follows:
RASSF1A methylation-specific (10) sense, 5'-GGGTTTTGCGAGAGCGCG-3 and
antisense, 5'-GCTAACAAACGCGAACCG-3'; RASSF1A
unmethylation-specific sense, 5'-GGTTTTGTGAGAGTGTGTTTAG-3' and
antisense, 5'-CACTAACAAACACAAACCAAA-3'; SHOX2
methylation-specific (11) sense,
5'-CGTACGAGTATAGGCGTTTACG-3' and antisense,
5'-AAAACGATTACTTTCGCCCG-3'; SHOX2 unmethylation-specific
sense, 5'-TGTATGAGTATAGGTGTTTATG-3' and antisense,
5'-AAAACAATTACTTTCACCCA-3'.
Statistical analysis
The experiments were conducted with three
independent replicates. Categorical data are presented as number
(percentage) [n (%)]. All the statistical analyses were performed
using Fisher's exact test. All the data were analyzed by SPSS
Statistics software version 13.0 (IBM Corp.). P<0.05 was
considered to indicate a statistically significant difference.
Results
Methylation status of SHOX2 and
RASSF1A
The methylation status of SHOX2 and
RASSF1A in lung tumor tissues and paired non-tumor lung
tissues from 25 patients diagnosed with early-stage LUAD were
examined. Among them, 19 cases were found to have SHOX2
methylation in tumor tissues, whereas only 3 paired non-tumor lung
tissue samples were tested positive (P<0.001). Furthermore,
RASSF1A methylation was detected in 8 tumor tissue samples,
compared with only 1 positive paired non-tumor tissue sample
(P=0.023). Additionally, combined methylation of SHOX2 and
RASSF1A was observed in 20 tumor tissue samples, compared
with only 4 paired non-tumor lung tissue samples were tested
positive (P<0.001). Fisher's exact test showed that there was a
statistically significant difference in methylation status between
lung tumor tissues and paired non-tumor lung tissues (Table II). Moreover, in tumor tissues,
SHOX2 exhibited complete methylation in 9 cases and partial
methylation in 10 cases, while RASSF1A showed complete
methylation in 1 case and partial methylation in 7 cases. In paired
non-tumor lung tissues, SHOX2 showed complete methylation in
1 case and partial methylation in 2 cases, whereas RASSF1A
exhibited partial methylation in 1 case, and no sample showed
complete methylation (Table
SI).
| Table IIMethylation of SHOX2 and
RASSF1A in early-stage lung adenocarcinoma. |
Table II
Methylation of SHOX2 and
RASSF1A in early-stage lung adenocarcinoma.
| Group | Total | Methylation level
of SHOX2, n (%) | Methylation level
of RASSF1A, n (%) | Methylation level
of SHOX2 + RASSF1A, n (%) |
|---|
| Lung cancer | 25 | 19(76) | 8(32) | 20(80) |
| Normal | 25 | 3(12) | 1(4) | 4(16) |
| P-value | | <0.001 | 0.023 | <0.001 |
Diagnostic performance of SHOX2 and
RASSF1A methylation
The diagnostic performance of SHOX2 and
RASSF1A methylation in early-stage LUAD was further
analyzed. The sensitivity and specificity of SHOX2
methylation were 76.0 and 88.0%, respectively. However, the
sensitivity of RASSF1A methylation was only 32.0%, with a
specificity of 96.0%. Finally, the specificity and sensitivity of
SHOX2 and RASSF1A combined methylation were also
analyzed. The result revealed that the specificity and sensitivity
were 84.0 and 80.0%, respectively (Table III).
| Table IIIDiagnostic value of SHOX2 and
RASSF1A methylation. |
Table III
Diagnostic value of SHOX2 and
RASSF1A methylation.
| | Sensitivity
(%) | Specificity
(%) | PPV (%) | NPV (%) | Accuracy (%) |
|---|
| SHOX2
methylation | 76.0 | 88.0 | 86.4 | 78.6 | 82.0 |
| RASSF1A
methylation | 32.0 | 96.0 | 88.9 | 58.5 | 64.0 |
| SHOX2 + RASSF1A
methylation | 80.0 | 84.0 | 83.3 | 80.8 | 82.0 |
Correlation of methylation status with
clinicopathological parameters
The potential associations between SHOX2
methylation, RASSF1A methylation, and the
clinicopathological parameters in LUAD cases were also evaluated by
using Fisher's test. No statistically significant associations were
observed between SHOX2 methylation, RASSF1A
methylation and the clinicopathological parameters (Table I).
Discussion
DNA methylation, as the most prevalent epigenetic
mechanism that regulates gene expression, plays a critical role in
tumor initiation and development (12,13).
DNA methylation is emerging as a potential biomarker to detect LC
(14,15). For instance, the methylation of
SHOX2 and RASSF1A were identified as two potential
biomarkers for LC diagnosis (16,17).
Nevertheless, the value of SHOX2 and RASSF1A
methylation in screening and diagnosis of early-stage LUAD cases
require further investigation.
In the present study, MSP was used to detect the
methylation status of the SHOX2 and RASSF1A genes in
tumor tissues and paired non-tumor lung tissues from patients
diagnosed with early-stage LUAD. However, it is noteworthy that the
MSP method has inherent limitations. MSP requires two separate
primer reactions, can only detect pre-designed CpG sites, and
suffers from low throughput and efficiency. Moreover, it provides
qualitative rather than quantitative results. A primary limitation
of the present study is its reliance on a single methodological
approach, MSP, for methylation detection, which inherently
constrains the interpretation and scope of the conclusions. Further
research employing other techniques is essential for confirmation.
Additionally, gene expression levels of SHOX2 and RASSF1A were not
evaluated, and only six male patients were included, which
represent a limitation of the present study and also highlight a
critical direction for future research and refinement. The results
showed that methylation levels of SHOX2 and RASSF1A
were significantly elevated in tumor tissues. These findings were
in line with previous studies (18,19),
suggesting that the aberrant methylation of SHOX2 and
RASSF1A may play an important role in the initiation and
development of LUAD.
SHOX2, a transcriptional regulator whose
dysregulation was driven by promoter hypermethylation, has been
implicated in LC (12).
Functionally, SHOX2 was a modulator for key cellular
processes such as cell proliferation, apoptosis and
epithelial-mesenchymal transition (20). With upregulated SHOX2
expression in LC, it enhanced LC cell viability, migration and
invasion in vitro, and promoted LC tumorigenesis and
pulmonary nodule metastasis in vivo (16). Notably, although SHOX2
methylation was significantly elevated in LC, which appeared
contradictory given that DNA methylation was typically associated
with gene silencing and considered to downregulate SHOX2
expression. It was reported that transcriptional activity was
closely associated with DNA methylation patterns in different
genomic regions: The hypermethylation in promoter and enhancer
regions generally restrained gene expression, while the
hypermethylation of gene body was positively related to the active
gene transcription (21,22). As a matter of fact, the elevated
methylation level of SHOX2 (gene body) in LC cells suggested
that the hypermethylation of SHOX2 (gene body) may promote
the mRNA expression of SHOX2 in this context (16).
As a well-known tumor suppressor gene that regulates
the cell cycle and apoptosis (23), the methylation of RASSF1A
gene promoter region caused RASSF1A expression deficiency,
which led to the activation of Yes-associated protein (YAP) and
contributed to the development of malignant characteristics,
including invasiveness, apoptosis resistance, and ultimate
transformation into cancer cells (24).
In addition, the diagnostic performance of
SHOX2 and RASSF1A methylation was evaluated. The
sensitivity and specificity of SHOX2 methylation were 76.0
and 88.0%, respectively. By contrast, RASSF1A methylation
exhibited a lower sensitivity of 32.0%, but a higher specificity of
96.0%. Moreover, the sensitivity and specificity of SHOX2
and RASSF1A combined methylation were 80.0 and 84.0%,
respectively.
In consistent with the present findings, the
positive detection rate of RASSF1A in histological specimens
of LUAD was 39% (25).
SHOX2 methylation in formalin-fixed paraffin-embedded (FFPE)
tissues could distinguish between benign and malignant lung
diseases with 60% sensitivity and 90.4% specificity (26). Similarly, Shi et al
(19) confirmed the diagnostic
performance of SHOX2 methylation in FFPE samples of LUAD,
reporting a sensitivity of 64.3% and a specificity of 92.1%. A
comprehensive meta-analysis comprising 2,296 participants
(including 1,129 LC cases) demonstrated the diagnostic accuracy of
SHOX2 methylation by showing a sensitivity of 70% at a
specificity of 96% in various samples, such as lymph nodes,
bronchial aspirates, pleural effusion, plasma and tumor tissues
from LC (27).
Furthermore, a diagnostic sensitivity of 69.6-81.0%
and a specificity of 90-97.4% for the SHOX2 and
RASSF1A combined methylation in bronchoalveolar lavage fluid
of LC were achieved (28,29). In FFPE samples, 66.2% sensitivity
and 94.1% specificity for LUAD were shown for the SHOX2 and
RASSF1A combined methylation (18). The combination of SHOX2 and
RASSF1A methylation in 251 FFPE LC specimens yielded a
diagnostic sensitivity of 89.8% and a specificity of 90.4%
(19).
Although no statistically significant association
was found between SHOX2 methylation, RASSF1A methylation and
the clinicopathological parameters, it has been reported that there
are associations between them. RASSF1A methylation was found
to have a higher frequency in male patients (30). In addition, SHOX2 gene
hypermethylation showed a correlation with smoking history, and
RASSF1A and SHOX2 methylation were confirmed to serve
as promising biomarkers for predicting malignant lung diseases and
might potentially help with the distinguishment of aggressive
phenotypes in early-stage LUAD (31).
The positive correlation between the invasiveness of
LUAD and SHOX2 and RASSF1A combined methylation level
was confirmed, with greater invasiveness leads to higher
methylation positivity rates (18). A positive association between the
combined methylation levels of SHOX2 and RASSF1A and
the expression of Ki-67 in early-stage LUAD was demonstrated by
further research (32). As
inferred, patients who tested positive for SHOX2 and
RASSF1A methylation may experience more rapid tumor
progression (18). Additional
analysis showed that the SHOX2 methylation level was
positively correlated with the stage of LC (19). Moreover, a high correlation was
found between high frequency of RASSF1A promoter methylation
and cancer pathogenesis as well as more aggressive clinical
phenotype (33,34). It was also indicated in the
research that lower sensitivity in stage I tumors was exhibited for
SHOX2 methylation, and a markedly lower sensitivity of LUAD
than that of small cell LC and squamous cell carcinoma (19).
The statistical power of the present study was
limited by the relatively small cohort size; and future validation
in a larger population is warranted. Further investigations were
warranted to validate the clinical value of RASSF1A and SHOX2
methylation assays to improve the diagnosis and treatment of LC. In
conclusion, the detection of RASSF1A and SHOX2
methylation might become a complementary approach to improve
conventional pathological diagnosis of LUAD and facilitate earlier
disease diagnosis and treatment of LUAD.
Supplementary Material
Representative DNA gel image of
polymerase chain reaction products of SHOX2 and RASSF1A
methylation. SHOX2, short stature homeobox 2; RASSF1A, Ras
association domain family 1 isoform A; M, methylated products; U,
unmethylated products.
Methylation status of SHOX2 and
RASSF1A.
Acknowledgements
Not applicable.
Funding
Funding: The present study was supported by the Guangdong
Medical Science and Technology Research Fund (grant no. B2024087)
and the Foshan Medical Research Fund (grant no. 20240356, 20230315
and 20240071).
Availability of data and materials
The data generated in the present study may be
requested from the corresponding author.
Authors' contributions
YX and SC conceived and designed the study. YC wrote
the manuscript. YC, XZ and PL performed the experiments and data
analysis. ZC and ZL assisted with sample processing and data
analysis. All authors read and approved the final version of the
manuscript. YC and YX confirm the authenticity of all the raw
data.
Ethics approval and consent to
participate
Informed consent was obtained from all participants
before sample collection. The present study was approved by the
Ethics Committee of Foshan First People's Hospital (approval no.
145; date, 2025; Foshan, China).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
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