Associations of CCAT2 gene polymorphisms with neuroblastoma susceptibility in children from Jiangsu province

Abstract

Background

The CCAT2 gene is associated with carcinogenesis, but its effect on neuroblastoma, the most common extracranial tumor in children, remains unclear.

Methods

We conducted a case–control study involving 402 children with neuroblastoma and 473 children without neuroblastoma. TaqMan genotyping of two CCAT2 polymorphisms (rs3843549 A > G and rs6983267 T > G) was conducted for all participants. Correlations were analyzed by calculating the odds ratio (OR) and 95% confidence interval (CI). Furthermore, we performed stratified analyses for both polymorphisms to evaluate their associations more comprehensively.

Results

We performed a statistical analysis employing three distinct genetic models to evaluate the rs3843549 A > G polymorphism and the rs6983267 T > G polymorphism. Moreover, we further investigated the potential protective polymorphisms (rs3843549 AG/GG and rs6983267 TG/GG) by stratified analysis. There was no significant association between CCAT2 gene polymorphisms and neuroblastoma susceptibility.

Conclusion

CCAT2 gene polymorphisms (rs3843549 A > G and rs6983267 T > G) were not associated with susceptibility to neuroblastoma. However, the accuracy of this conclusion may be limited by various confounding factors. Future analyses would benefit from a more comprehensive approach that accounts for additional variables.

Supplementary Information

The online version contains supplementary material available at 10.1186/s40001-025-03693-y.

Introduction

Neuroblastoma represents the most common solid extracranial tumor among children and typically arises from neural crest elements of the sympathetic nervous system [1]. The global incidence has risen to 0.28 per 100,000 individuals. Furthermore, the age group with the highest mortality rate comprises those under 1 year of age, with a neuroblastoma-related mortality rate of 0.41 per 100,000 [2]. Despite accounting for 10% of all pediatric cancers, this malignancy accounts for 15% of pediatric cancer deaths because of its marked clinical heterogeneity. Tumors commonly occur in the adrenal gland and exhibit diverse degrees of differentiation along normal differentiation trajectories, reflecting distinct clinical phenotypes and prognoses. Most cases are predominantly sporadic, and their clinical manifestations are often subtle and highly variable. The median age at diagnosis for neuroblastoma is approximately one and a half years, with most cases identified only through physical examination. These findings imply that undetected disease progression contributes to lower survival rates and distant metastasis in older children [3, 4]. Fortunately, significant advances in treatment methods have been made in recent decades. For instance, multimodal treatment for high-risk patients can more precisely target tumor cells and decrease the metastatic disease burden although minimizing systemic toxicity. Anti-GD2 antibodies, MIBG, ALK inhibitors, and differentiation therapy have significantly improved neuroblastoma survival rates from less than 20% to more than 50% [5]. Other approaches, such as CAR-T-cell therapy and mesenchymal stem cell-based TRAIL delivery, have also shown promise in preclinical and clinical studies [6, 7].

Single-nucleotide polymorphisms (SNPs) represent the most prevalent form of DNA variation in the genome and serve as crucial genetic markers for investigating complex human diseases. For example, the variant rs78378222 is associated with neuroblastoma because of its disruption of the correct termination and polyadenylation of TP53 transcripts [8]. Genome-wide association studies (GWASs) have become indispensable for analyzing neuroblastoma susceptibility, revealing significant common and rare genetic variants, including CASC15, BARD1, CHEK2, LMO1, LIN28B, AXIN2, BRCA1, TP53, SMARCA4, and CDK1NB [9]. Our recent study revealed four MYCN gene polymorphisms (rs57961569 G > A, rs9653226 T > C, rs13034994 A > G, and rs60226897 G > A) and TRMT10C rs4618204 C > T, which are significantly associated with neuroblastoma susceptibility [10, 11]. However, these discoveries have not fully elucidated the pathogenesis of neuroblastoma from a genetic perspective.

Long noncoding RNAs (lncRNAs) are involved in the cell cycle, differentiation, and metabolism through various molecular mechanisms. The overexpression or aberrant activation of these molecules may contribute to carcinogenesis [12, 13]. Several lncRNAs (CASC7, CASC15, LncNB1, MEG3, etc.) are related to neuroblastoma [14, 15]. However, few studies have investigated whether colon cancer-associated transcript 2 (CCAT2) is also involved in neuroblastoma. In the past decade, studies have demonstrated that the overexpression of CCAT2 plays an oncogenic role in many types of cancer [16, 17]. Evaluating the association between the CCAT2 polymorphism and neuroblastoma susceptibility is essential for a deeper understanding of its biological mechanisms in this disease.

Elevated expression levels of CCAT2 have been observed in neuroblastoma, where it exerts biological effects on cell growth, including antiapoptotic effects, and is correlated with poor prognosis [18]. Moreover, the expression of miR-424, which can play dual roles (promoting/inhibiting) in carcinogenesis under various conditions, is reduced in neuroblastoma. By acting as a molecular sponge for this miRNA, CCAT2 potentially downregulates miR-424 expression in neuroblastoma [19]. As a transcript, CCAT2 has demonstrated significant potential as both a biomarker and a prognostic indicator in other cancer types. Unfortunately, the precise molecular mechanism through which CCAT2 influences susceptibility to neuroblastoma remains unconfirmed. Studies of CCAT2 SNPs have focused primarily on rs6983267. Studies have demonstrated that rs6983267 can modulate the functionality of the partner RNA-binding protein/complex. This variant binds the cleavage Factor I complex in an allele-specific way, influencing cancer metabolism [20]. The secondary structure and enrichment level of CCAT2 are affected by the G/T allele of the rs6983267 polymorphism [18]. Moreover, it is associated with pT3-T4 thyroid cancer, myeloid malignancies, lung cancer, and uterine cervical cancer [21–24]. Studies have shown that it is involved in downregulating the chemosensitivity to 5'-FU [25]. In contrast, research on rs3843549 as a potential carcinogenic factor remains relatively scarce, with no established associations with susceptibility to recurrent miscarriage [26]. Interestingly, a study revealed that carrying at least one polymorphic allele of rs3843549 prevents the development of hepatocellular carcinoma in specific subgroups, including individuals under 65 years of age, males, and regular drinkers [27]. These two SNPs may influence neuroblastoma susceptibility by potentially altering the function or expression of CCAT2. To date, no studies have investigated the relationship between CCAT2 gene SNPs and neuroblastoma susceptibility. Therefore, we conducted a case–control study within a Chinese population cohort from Jiangsu province to explore the connection of rs3843549 and rs6983267 with neuroblastoma susceptibility.

Methods

Study subjects

The current study included 402 cases and 473 cancer-free controls from the Children’s Hospital of Nanjing Medical University in Jiangsu province (Table S1). All the participants met the same selection criteria as those in the previous study [28, 29]. The case cohort consisted of patients with neuroblastoma confirmed through biopsy or histopathological analysis and age-matched patients without neuroblastoma randomly selected from among contemporaneous physical examination participants. We subsequently compared demographic characteristics between the case and control groups on the basis of variables such as age and sex. We obtained written informed consent from all the participants or their legal guardians during recruitment, and the study protocols were approved by the Institutional Review Boards of Children’s Hospital of Nanjing Medical University (Approval Number: 202412006-1).

Polymorphism selection and genotyping

Two genetic variants of the CCAT2 gene (rs3843549 A > G and rs6983267 T > G) were chosen from previous literature [20, 26]. These patients met the conditions of having a minor allele frequency greater than 5% and low linkage disequilibrium. The genomic DNA of all the research subjects was extracted using the TIANamp DNA Kit (TianGen Biotech Co. Ltd., Beijing, China), after which the two selected SNPs were genotyped via the TaqMan PCR method [30–32]. To ensure the quality control and precision of the genotyping analysis, we retested a randomly selected subset (10% of samples), and the results were 100% consistent with those of the previous genotyping results.

Statistical analysis

The goodness-of-fit χ² test was used to evaluate the conformity of the genotype frequency distributions in the control group to the Hardy–Weinberg equilibrium (HWE). To analyze demographic characteristics and genotype frequency distributions between the case group and the control group, we used the two-sided χ² test for detection. Moreover, we calculated the study data via multivariate logistic regression to estimate the odds ratios (ORs) and 95% confidence intervals (CIs) to investigate the associations between the selected SNPs and neuroblastoma susceptibility. We subsequently conducted stratified analyses of risk genotypes by age, sex, tumor location, and clinical stage. All the statistical analyses used two-sided tests with a significance threshold of P < 0.05. The results were considered statistically significant when the P value fell below this critical value.

Results

Associations of CCAT2 polymorphisms with neuroblastoma risk

A total of 402 case patients and 473 controls were included in this study and we successfully genotyped all 402 case patients and 472 controls (genetic typing failed for one individual). We explored the potential associations between CCAT2 polymorphisms and neuroblastoma susceptibility (Table 1). The genotype frequencies of two selected SNPs (rs3843549 A > G and rs6983267 T > G) were in accordance with the HWE (rs3843549: P_HWE = 0.267; rs6983267: P_HWE = 0.751). Moreover, we analyzed the two selected SNPs using three genetic models: additive, dominant and recessive. For the rs3843549 A > G polymorphism the models are defined as follows; additive: per additional G allele (vs. A allele), dominant: (AG + GG) vs. AA, and recessive: GG vs. (AA + AG). For the rs6983267 T > G polymorphism the models are defined as follows; additive: per additional G allele (vs. T allele), dominant: (TG + GG) vs. TT, and recessive: GG vs. (TT + TG). A comparative analysis adjusting for age and sex revealed no significant correlation between CCAT2 genetic polymorphisms and neuroblastoma according to the dominant models (AG/GG vs. AA: adjusted OR = 0.97, 95% CI 0.72–1.30, P = 0.831 for the rs3843549 A > G polymorphism; TG/GG vs. TT: adjusted OR = 0.94, 95% CI 0.71–1.24, P = 0.641 for the rs6983267 T > G polymorphism) or other comparisons. On the basis of the statistical data, the rs3843549 AG/GG and rs6983267 TG/GG genotypes (OR value < 1.00) may confer protective effects. Unfortunately, we observed no correlation between the CCAT2 SNPs and a reduced risk of neuroblastoma. Nevertheless, we further performed a combined analysis of the potential protective polymorphisms (rs3843549 AG/GG and rs6983267 TG/GG), but no significant associations were detected (adjusted OR = 0.94, 95% CI 0.71–1.24, P = 0.641).

Table 1.

Associations of CCAT2 gene polymorphisms with neuroblastoma susceptibility in children from Jiangsu province

Genotype	Cases (N = 402)	Controls (N = 472)	Pa	Crude OR (95% CI)	P	Adjusted OR (95% CI)b	Pb
rs3843549 A > G (HWE = 0.267)
AA	288 (71.64)	335 (70.97)		1.00		1.00
AG	109 (27.11)	129 (27.33)		0.98 (0.73–1.33)	0.910	0.98 (0.73–1.33)	0.913
GG	5 (1.24)	8 (1.69)		0.73 (0.24–2.25)	0.580	0.73 (0.24–2.25)	0.580
Additive			0.737	0.96 (0.73–1.25)	0.737	0.96 (0.73–1.26)	0.739
Dominant	114 (28.36)	137 (29.03)	0.828	0.97 (0.72–1.30)	0.828	0.97 (0.72–1.30)	0.831
AA/AG	397 (98.76)	464 (98.31)		1.00		1.00
GG	5 (1.24)	8 (1.69)	0.583	0.73 (0.24–2.25)	0.584	0.73 (0.24–2.25)	0.585
rs6983267 T > G (HWE = 0.751)
TT	138 (34.33)	155 (32.84)		1.00		1.00
TG	193 (48.01)	228 (48.31)		0.95 (0.71–1.28)	0.740	0.95 (0.71–1.28)	0.739
GG	71 (17.66)	89 (18.86)		0.90 (0.61–1.32)	0.579	0.90 (0.61–1.32)	0.579
Additive			0.574	0.95 (0.78–1.15)	0.574	0.95 (0.78–1.15)	0.574
Dominant	264 (65.67)	317 (67.16)	0.642	0.94 (0.71–1.24)	0.642	0.94 (0.71–1.24)	0.641
TT/TG	331 (82.34)	383 (81.14)		1.00		1.00
GG	71 (17.66)	89 (18.86)	0.649	0.92 (0.65–1.30)	0.650	0.92 (0.65–1.30)	0.651
Protective genotypesc
0	138 (34.33)	155 (32.84)		1.00		1.00
1–2	264 (65.67)	317 (67.16)	0.642	0.94 (0.71–1.24)	0.642	0.94 (0.71–1.24)	0.641

OR odds ratio, CI confidence interval, HWE Hardy–Weinberg equilibrium

^aχ² test for genotype distributions between neuroblastoma cases and cancer-free controls

^bAdjusted for age and gender

^cProtective genotypes were carriers with rs3843549 AG/GG and rs6983267 TG/GG genotypes

Stratification analysis

To evaluate the impact of these two SNPs on neuroblastoma in different subgroups, we conducted a stratified analysis on the basis of age, sex, tumor origin site, and INSS stage (Table 2). Unfortunately, no significant associations were detected between the rs3843549 A > G and rs6983267 T > G polymorphisms and the risk of neuroblastoma.

Table 2.

Stratification analysis for the association between CCAT2 gene polymorphisms and neuroblastoma susceptibility in children from Jiangsu province

Variables	rs3843549 (cases/controls)		AOR (95% CI)a	Pa	rs6983267 (cases/controls)		AOR (95% CI)a	Pa	Combined genotypes (cases/controls)		AOR (95% CI)a	Pa
	AA	AG/GG			TT	TG/GG			0	1–2
Age, month
≤ 18	105/98	34/41	0.77 (0.45–1.32)	0.348	57/46	82/93	0.71 (0.44–1.16)	0.174	57/46	82/93	0.71 (0.44–1.16)	0.174
> 18	183/237	80/96	1.08 (0.76–1.54)	0.675	81/109	182/224	1.09 (0.77–1.55)	0.615	81/109	182/224	1.09 (0.77–1.55)	0.615
Gender
Females	125/147	66/78	1.00 (0.66–1.49)	0.980	64/71	127/154	0.92 (0.61–1.38)	0.672	64/71	127/154	0.92 (0.61–1.38)	0.672
Males	163/188	48/59	0.94 (0.61–1.45)	0.767	74/84	137/163	0.95 (0.65–1.40)	0.802	74/84	137/163	0.95 (0.65–1.40)	0.802
Sites of origin
Adrenal gland	73/335	20/137	0.67 (0.39–1.14)	0.139	38/155	55/317	0.71 (0.45–1.12)	0.135	38/155	55/317	0.71 (0.45–1.12)	0.135
Retroperitoneal	110/335	57/137	1.27 (0.87–1.85)	0.222	49/155	118/317	1.18 (0.80–1.73)	0.405	49/155	118/317	1.18 (0.80–1.73)	0.405
Mediastinum	88/335	32/137	0.89 (0.57–1.40)	0.622	44/155	76/317	0.85 (0.56–1.28)	0.430	44/155	76/317	0.85 (0.56–1.28)	0.430
Others	14/335	4/137	0.69 (0.22–2.14)	0.514	5/155	13/317	1.27 (0.45–3.64)	0.652	5/155	13/317	1.27 (0.45–3.64)	0.652
Clinical stages
I + II + 4 s	125/335	48/137	0.91 (0.61–1.34)	0.623	64/155	109/317	0.83 (0.57–1.19)	0.302	64/155	109/317	0.83 (0.57–1.19)	0.302
III + IV	118/335	45/137	0.95 (0.64–1.42)	0.801	51/155	112/317	1.07 (0.73–1.57)	0.726	51/155	112/317	1.07 (0.73–1.57)	0.726

AOR adjusted odds ratio, CI confidence interval

^aAdjusted for age and gender, omitting the correspondence factor

Discussion

CCAT2 has already been extensively studied as a lncRNA with established regulatory functions across many types of cancer. It is located on chromosome 8q24.21 and contributes to tumorigenesis by modulating key signaling pathways or other complex mechanisms that govern cell proliferation, migration, apoptosis, and other biological behaviors [17]. Furthermore, CCAT2 inhibits the maturation process of miR-145, which consequently facilitates the proliferation and differentiation of colon cancer stem cells [33]. It also participates as a key regulator of tumor suppression or growth processes by competitively binding other miRNAs, such as miR-4496, miR-493, miR-424, miR-216b, miR-23b, miR-34a, miR-145, miR-200b, and miR-143 [18]. Moreover, it contributes to tumorigenesis in lung, breast, and hepatocellular carcinomas, among other malignancies [16, 17]. Above all, the abnormal expression of CCAT2 frequently correlates with both the onset and progression of cancer. In neuroblastoma, CCAT2 may not be related only to miR-424 regulation. Research has revealed a concurrent decrease in p53 protein expression and an increase in Bcl-2 protein expression within this tumor type, suggesting a profound change in the apoptotic regulatory pathway [18]. Moreover, Chen et al. successfully demonstrated this approach by utilizing si-CCAT2, which confirmed that CCAT2 may negatively regulate p53 and positively regulate Bcl-2 to participate in the regulation of neuroblastoma [34]. Altered expression of p53 and Bcl-2, which are core regulators of apoptosis, directly influences tumor cell survival. These findings suggest that CCAT2 likely influences neuroblastoma pathogenesis by disrupting the critical p53/Bcl-2 apoptotic axis. Whether CCAT2 is involved in regulating the malignant behaviors of neuroblastoma cells remains unknown. To investigate this connection, we designed and conducted a hospital-based case–control study among Chinese children from Jiangsu province. This study represents the first research into the potential association between CCAT2 polymorphisms (rs3843549 A > G and rs6983267 T > G) and neuroblastoma. Chen et al. demonstrated that increased CCAT2 expression inhibits the proliferation of neuroblastoma cells although inducing their apoptosis [34]. Interestingly, this observation appears to contradict both the well-established carcinogenic role of CCAT2 in various other cancer types and the initial premise of this gene study. However, our findings revealed no significant correlation between the rs3843549 A > G or rs6983267 T > G variant and the risk of neuroblastoma. The possible reasons for this outcome are as follows: First, these CCAT2 SNPs may not contribute to expression or biological function in the neural crest lineage. Second, the genetic germline variation examined does not always align precisely with the functional outcomes of somatic overexpression, since context-specific signaling pathways also influence their expression levels.

Several shortcomings may have influenced the results. First, the relatively small sample size and the regional restriction of participants to Jiangsu province may limit the generalizability of the findings. It does not represent the susceptibility of other regions or ethnic groups. Second, clinical variability persisted in our study cohort because of inherent differences in physician decision-making and patient heterogeneity. Dietary habits, socioeconomic status, and other environmental factors were not included in our logistic regression model. Moreover, the insufficient effect size, the presence of population stratification, and differences in linkage disequilibrium may have contributed to these negative results. Third, the CCAT2 gene may harbor additional unidentified SNPs that contribute to the occurrence of neuroblastoma. Moreover, the sample size for the homozygous genotype (rs3843549 GG: 5 cases, 8 controls) was relatively small, resulting in limited statistical power. This also means that determining rs3843549 GG as a protective genotype solely on the basis of an OR value < 1.00 may be unreliable Notably, our experimental results reveal an observable trend (e.g., adrenal gland tumors, OR = 0.67 ~ 0.71; ≤ 18 months subgroup, OR = 0.71 ~ 0.77) within some subgroups, despite the absence of statistical significance (Table 2). This is a significant point that warrants our attention in future experiments. Unfortunately, no association was found in the other subgroups or when the results were combined with the results of the genotype analysis. The limitations or shortcomings in our research methodology may limit the identification of associations within these subgroups.

Collectively, our results indicate that the CCAT2 genes rs3843549 A > G and rs6983267 T > G are not significantly associated with neuroblastoma susceptibility. Given that CCAT2 has demonstrated strong oncogenic potential in other cancers, we will explore its association with neuroblastoma more thoroughly by expanding both the sample size and geographical coverage, and by conducting a more systematic study on genetic variations to dispel confusion.

Conclusion

In summary, this study confirmed for the first time that two SNPs (rs3843549 A > G and rs6983267 T > G) of the CCAT2 gene do not significantly correlate with neuroblastoma susceptibility. To confirm these findings more accurately, future research should incorporate more considerations.

Abbreviations

SNP

Single-nucleotide polymorphism

GWAS

Genome-wide association study

lncRNA

Long noncoding RNA

CCAT2

Colon cancer-associated transcript 2

HWE

Hardy–Weinberg equilibrium

OR

Odds ratio

CI

Confidence interval

Author contributions

All the authors included in this manuscript contributed significantly to this work. PY and JH: conceptualized and designed the research study; WZ, JC, XZ and JH: funding acquisition; CZ: collected the samples and clinical data; JC and XZ: DNA extraction and TaqMan genotyping; WZ and JH: analyzed the data and prepared all the tables; SC, ZW, PY and JH: wrote the paper. All the authors read and approved the final article.

Funding

This work was supported by the National Natural Science Foundation of China (No: 32300473) and the Guangzhou Science and Technology Project (Nos: 2025A04J4537, 2025A04J4696, 202201020622) and the Guangdong Basic and Applied Basic Research Foundation (No: 2023A1515220053).

Data availability

All the data are available upon request from the corresponding authors (Jing He or Peng Yi).

Declarations

Ethics approval and consent to participate

The research protocol was in accordance with the guidelines of the Declaration of Helsinki; all participants’ guardians signed an informed consent form during recruitment, and the study protocol was approved by the institutional review board of the Children’s Hospital of Nanjing Medical University (Approval No: 202412006-1).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.