Introduction
Neuroblastoma, which appears during the development of the sympathetic nervous system, is a frequently occurring pediatric solid tumor.1 With a comparatively low morbidity and affecting approximately 10.2 per million children younger than 15 years of age, neuroblastoma accounts for approximately 10% of all childhood cancers.2,3 However, neuroblastoma, a frequently fatal form of malignancy, is the third most common cause of childhood cancer-related mortality.4 Both in terms of prognosis and clinical presentation, neuroblastoma exhibits significant heterogeneity.5 It is well recognized that spontaneous regression is routinely observed in localized neuroblastoma and is not limited to stage 4S.6 Neuroblastoma, on the other hand, can be classified into different subgroups with varying survival rates based on clinical and biological characteristics.3,7 The long-term survival of neuroblastoma with low risk is approximately 90%, whereas survival in high-risk neuroblastoma is less than 40%, even with aggressive and comprehensive therapy.8,9 Neuroblastoma not only can have a devastating impact on the family, but it also poses a severe threat to public health and is a global burden.10,11
Unfortunately, the exact etiology of neuroblastoma continues to be incompletely determined.12 Potential environmental exposure, including parental exposure to hydrocarbons, turpentine, lacquer thinner, wood dust, solder, sources of radiation, and electrical devices, have been reported to alter neuroblastoma susceptibility in descendants, although the reasons are not entirely clear.13,14 On the other hand, increasing verification regarding studies in molecular epidemiology demonstrates that genetic polymorphisms associated with genetic risk factors may have an essential impact on susceptibility to neuroblastoma.15,16 A multitude of susceptibility genes for neuroblastoma have been discovered based on previous genome-wide association studies, including CASC15, DUSP12, DDX4, IL31RA, HSD17B12, BARD1, LMO1, MLF1, CPZ, HACE1, LIN28B, and RSRC1.17–22 In addition to genome-wide association studies, candidate gene methods have also served as powerful tools to identify potential inherited genetic variants that are related to neuroblastoma. Indeed, several previous studies have revealed the susceptibility of genes such as NEFL, CDKN1B, TP53, BARD1, and ALKBH5 to neuroblastoma.15,23–26
MicroRNAs (miRNAs) are endogenous small noncoding RNAs with approximately 19–25 nucleotides.27 miRNA bind to the 3′-untranslated region (UTR) of targeted messenger RNAs (mRNAs) and regulate gene expression post-transcriptionally by either degrading or inhibiting the translation of corresponding mRNAs.28,29 With the discovery of miRNAs, evidence that they have significant and diverse roles in multiple biological processes, including cell proliferation, development, differentiation, and apoptosis, as well as their crucial implication in cancer development has accumulated.30–32 Single nucleotide polymorphisms (SNPs) within miRNAs influence the ways in which miRNAs function. They have an impact on primary transcription, the procedure for processing and maturation of precursor (pre)-miRNAs and primary (pri)-miRNAs, as well as the interactions between miRNAs and their targets, thereby modulating gene expression.33 Based on the type of cancer, the region, and the ethnicity, polymorphisms may have a variety of genetic impacts on cancer risk. In recent years, the correlation between miR-618 rs2682818 and cancer risk has been extensively studied in a variety of malignancies, including breast cancer, follicular lymphoma, colorectal cancer, and acute lymphocytic leukemia,34–37 but not in neuroblastoma. To evaluate the relevance of the rs2682818 polymorphism to the risk of neuroblastoma in Chinese children, we conducted research involving 402 neuroblastoma patients and 473 control individuals.
Methods
Study subjects
The individuals involved were selected from the Nanjing Medical University Children’s Hospital in Jiangsu Province, as previously mentioned in our investigations.38,39 All the included individuals were unrelated ethnic Chinese who came from Jiangsu Province, China. The study included 402 individuals with neuroblastoma who were diagnosed histopathologically and 473 cancer-free controls (Table S1). The institutional review board of the Children’s Hospital of Nanjing Medical University approved the study protocol. All participants signed a written informed consent form for our research with the signature of their parent or legal guardian.
SNP screening and genotyping
In a previous study, we provided a detailed description regarding the selection of potentially functioning polymorphisms.40 Briefly, to screen for any potentially functioning polymorphisms of miR-618, we selected the 3′ UTR, 5′ UTR, 5′-flanking region, exons, and intron of the miR-618 according to the criteria. The rs2682818 polymorphism in the miR-618 was available and ultimately chosen. As previously mentioned in our study, TaqMan real-time polymerase chain reaction was performed to accurately genotype miR-618 rs2682818 C>A using the 7900 Sequencing Detection System from Applied Biosystems (Foster City, CA, USA).40,41 To ensure accurate genetic typing of the results, we randomly selected 10% of the DNA samples for further confirmation, and the results were 100% concordant. A goodness-of-fit chi-square test was performed to test for deviations from Hardy-Weinberg equilibrium (HWE) for the genotype frequencies in control subjects. Differences in the distribution of selected demographic variables and genotypes between neuroblastoma cases and control individuals were assessed with two-sided chi-square tests. Adjusted odds ratios (AORs) and 95% confidence intervals (CIs) were calculated by multivariate logistic regression and were used to estimate the correlation of the rs2682818 C>A polymorphism with neuroblastoma risk. In addition, analysis was performed after stratification by age, sex, tumor origin, and clinical stage. SAS (version 9.4; SAS Institute, Cary, NC, USA) was used to perform the statistical analysis, and p-values <0.05 were considered statistically significant.
Results
Association of miR-618 rs2682818 C>A with neuroblastoma risk
Table 1 shows the genotype distribution of miR-618 rs2682818 C>A in all participants and the association with the risk of neuroblastoma. In the controls, the data were in accord with the HWE (p = 0.358). No significant correlation was detected between miR-618 rs2682818 C>A polymorphism and neuroblastoma.
Table 1miR-618 rs2682818 C>A polymorphism and neuroblastoma risk in children from Jiangsu Province
| Genotype | Cases, n = 400 | Controls, n = 473 | pa | Crude OR (95% CI) | p | Adjusted OR (95% CI)b | p b |
|---|
rs2682818 (HWE = 0.358)
|
|---|
| CC | 234 (58.50) | 252 (53.28) | | 1.00 | | 1.00 | |
| CA | 132 (33.00) | 181 (38.27) | | 0.79 (0.59–1.05) | 0.098 | 0.78 (0.59–1.05) | 0.097 |
| AA | 34 (8.50) | 40 (8.46) | | 0.92 (0.56–1.50) | 0.724 | 0.92 (0.56–1.50) | 0.729 |
| Additive | | | 0.239 | 0.88 (0.72–1.09) | 0.239 | 0.88 (0.72–1.09) | 0.239 |
| Dominant | 166 (41.50) | 221 (46.72) | 0.122 | 0.81 (0.62–1.06) | 0.122 | 0.81 (0.62–1.06) | 0.121 |
| CC/CA | 366 (91.50) | 433 (91.54) | | 1.00 | | 1.00 | |
| AA | 34 (8.50) | 40 (8.46) | 0.982 | 1.01 (0.62–1.62) | 0.982 | 1.01 (0.62–1.62) | 0.979 |
Stratification analyses
To further investigate the relationship between the rs2682818 C>A polymorphism and neuroblastoma susceptibility under different stratification conditions, we analyzed the results following stratification by age, sex, site of tumor origin, and clinical stage (Table 2). Compared with the CC genotype, the CA/AA genotypes had a lower risk of neuroblastoma developing in the adrenal gland (AOR = 0.57, 95% CI: 0.35–0.91, p = 0.018). No other significant correlations with the risk of neuroblastoma were found.
Table 2Stratification analysis for the association between miR-618 rs2682818 C>A polymorphism and neuroblastoma susceptibility
| Variable | rs2682818, cases/controls
| OR (95% CI) | p
| AOR (95% CI)a | pa |
|---|
| CC | CA/AA |
|---|
| Age, months |
| ≤18 | 83/70 | 54/69 | 0.66 (0.41–1.06) | 0.088 | 0.66 (0.41–1.06) | 0.088 |
| >18 | 151/182 | 112/152 | 0.89 (0.64–1.23) | 0.475 | 0.89 (0.64–1.23) | 0.479 |
| Sex |
| Female | 117/130 | 74/95 | 0.87 (0.58–1.28) | 0.472 | 0.87 (0.58–1.28) | 0.472 |
| Male | 117/122 | 92/126 | 0.76 (0.53–1.10) | 0.148 | 0.76 (0.53–1.10) | 0.145 |
| Site of origin |
| Adrenal gland | 62/252 | 31/221 | 0.57 (0.36–0.91) | 0.019 | 0.57 (0.35–0.91) | 0.018 |
| Retroperitoneal | 91/252 | 75/221 | 0.94 (0.66–1.34) | 0.732 | 0.94 (0.66–1.35) | 0.749 |
| Mediastinum | 68/252 | 51/221 | 0.86 (0.57–1.28) | 0.450 | 0.85 (0.57–1.28) | 0.442 |
| Other | 10/252 | 8/221 | 0.91 (0.35–2.35) | 0.849 | 0.92 (0.36–2.37) | 0.859 |
| Clinical stage |
| I+II+4s | 94/252 | 77/221 | 0.93 (0.66–1.33) | 0.703 | 0.96 (0.67–1.36) | 0.811 |
| III+IV | 93/252 | 70/221 | 0.86 (0.60–1.23) | 0.404 | 0.84 (0.59–1.21) | 0.348 |
Discussion
In this study, we investigated the association between miR-618 rs2682818 C>A polymorphism and neuroblastoma predisposition in 402 cases and 473 controls. The rs2682818 C>A polymorphism CA/AA genotypes were associated with a reduced risk of neuroblastoma in the adrenal gland compared with the CC genotype.
In a variety of cancers, miR-618 is an oncogene or a tumor-suppressing gene. The deregulation of miR-618, as described in previous studies, is directly correlated with tumorigenesis in breast cancer, esophageal Barrett’s cancer, bladder cancer, prostate cancer, hepatocellular carcinoma, and thyroid cancer.36,42–46 The rs2682818 polymorphism, which is found in the stem-loop sequence of premiR-618 may disrupt the structural development of the secondary stem-loop and consequently affect the further transformation of the precursor miR-618 into the mature form, which may have an impact on miR-618 expression.34
Fu et al.34 described a variant rs2682818 T allele that interfered with pri-miR-618 stem-loop generation as well as the progress of interactions of miR-618 with its targets, caused lower levels of mature miR-618 and increased the risk of follicular lymphoma. In addition, miR-618 rs2682818 was related to increased breast cancer susceptibility.36 In contrast, as reported by Chen et al.,37 rs2682818 polymorphism participated the development of colorectal cancer. They discovered that, compared with the CC genotype, the AA or AC/AA genotype of rs2682818 in the Chinese population was correlated with a decreased susceptibility to colorectal cancer. In addition, Shao et al.47 reported that the rs2682818 polymorphism influenced on the degree of expression of mature miR-618 in colorectal cancer cells, which in turn affected the capacity of miR-618 to target TIMP1 expression and inhibited the development of colorectal cancer. As mentioned above, these findings indicate that rs2682818 polymorphism may affect cancer susceptibility. Nevertheless, previous studies reported the association of miR-618 rs2682818 with neuroblastoma susceptibility. Our results indicate that the rs2682818 polymorphism decreases the susceptibility to neuroblastoma in tumors originating from the adrenal gland.
Although this is the first study to find an association of the miR-618 rs2682818 C>A polymorphism with susceptibility to neuroblastoma. Some limitations of these preliminary findings must be recognized. First, some important environmental factors, including parental exposure, dietary habits, and living conditions, were not taken into consideration. Second, the study subjects only included Chinese individuals from Jiangsu Province, and the conclusion may not be generalized to the Chinese and other ethnic populations. Finally, only the rs2682818 C>A polymorphism was investigated. Other SNPs that may be functional need to be evaluated in the future.
Conclusions
In summary, the miR-618 rs2682818 C>A polymorphism was significantly associated with decreased risk of neuroblastoma in tumors originating from the adrenal gland. Our findings indicate that miR-618 rs2682818 may be a neuroblastoma susceptibility gene. Large multiregional and multicenter studies with populations of various ethnicities are required to verify our results.
Supporting information
Supplementary material for this article is available at https://doi.org/10.14218/CSP.2023.00035 .
Table S1
Demographic characteristics of neuroblastoma patients and cancer-free controls from Jiangsu province.
(DOC)
Abbreviations
- AOR:
adjusted odds ratio
- CI:
confidence interval
- HWE:
Hardy-Weinberg equilibrium
- miRNA:
microRNA
- mRNA:
messenger RNA
- OR:
odds ratio
- pre:
precursor
- pri:
primary
- SNP:
single nucleotide polymorphism
- UTR:
untranslated region
Declarations
Ethical statement
This study was performed in accordance with the ethical principles of Declaration of Helsinki (as revised in 2013). The institutional review board of the Children’s Hospital of Nanjing Medical University approved the study protocol (202112141-1). All participants signed a written informed consent form for our research with the signature of their parent or legal guardian.
Data sharing statement
The corresponding author will make the datasets produced during the study and used to support the conclusions accessible on reasonable request.
Funding
This work was conducted with financial support from the Guangzhou Science and Technology Project (No: 202201020622), the Postdoctoral Science Foundation of China (No: 2021M691649), and the Postdoctoral Science Foundation of Jiangsu Province (No: 2021K524C).
Conflict of interest
One of the authors, Jing He has been an editorial board member of Cancer Screening and Prevention since April 2023. The authors have no other conflict of interest.
Authors’ contributions
Designed the study (HYW, JH), wrote the paper (LLX, CMD, JH), performed the experiments (JMC, XXZ), collected the clinical information and samples (CLZ, HYW), provided the data analysis and prepared the tables (CMD, JH). All authors approved this version to be published.