Effect of miR-34b/c rs4938723 T > C on pediatric glioma susceptibility

Gliomas, the most common type of primary intracranial tumor, arise from the brain's glial cells and constitute 30% of all brain and central nervous system tumors. MiR-34b/c plays an important role in colorectal cancer and other cancers, but its role in glioma is unknown. In this study, we attempted to assess the effect of rs4938723 T > C in miR-34b/c on the susceptibility of glioma Chinese children. Odds ratios and 95% confidence intervals (CIs) were calculated to evaluate the association between the selected polymorphism and glioma susceptibility. Our results showed that there is no significant association between this polymorphism and the risk of glioma in Chinese children. However, stratified analysis showed that the rs4938723 TC/CC genotype significantly reduced the risk of glioma in participants under 60 months of age (adjusted OR = 0.56, 95% CI = 0.35 – 0.88, p = .013). Overall, our study indicates that miR-34b/c rs4938723 T > C polymorphism may have a weak influence on glioma susceptibility. Nevertheless, these findings need to be validated in well-designed studies with larger sample sizes and different populations.

METTL3 can activate Notch pathway and facilitate glioma occurrence through regulating its direct targets NOTCH3, DLL3, and HES1, and Notch pathway genes may serve as the potential treatment targets for glioma. 11 MicroRNA (miRNA) is a class of small non-protein-encoded single-stranded RNA with about 22 nucleotides. 12 miRNA participates in post-transcriptional regulation through various mechanisms, including promoting mRNA degradation, inhibiting translation, or leading to mRNA cleavage. [12][13][14] In the past decade, numerous miRNAs have been identified. miRNAs are thought to be involved in many biological processes that play a key role in carcinogenesis, including cell differentiation, proliferation, and apoptosis. Mutations in the physiological processes of miRNA may lead to the occurrence and development of tumors. A potentially functional polymorphism rs4938723 was found in the promoter region of miR-34b/c. The rs4938723 C/T polymorphism in the pri-miR-34b/c promoter region was predicted to affect the binding of transcription factor GATA-X with its target genes, thus affecting the expression level of target genes related to tumor differentiation and tumorigenesis. [15][16][17][18] In our previous study conducted recently, we firstly found that rs4938723 T > C polymorphism was associated with a significantly decreased neuroblastoma risk [37]. Here, we conducted a hospitalbased case-control study aiming to verify the association between miR-34b/c rs4938723 T > C and glioma risk in Chinese children.

| Study population
In the current case-control study, 314 patients with glioma and 380 cancer-free controls were included, and the demographic characteristics of all participants are shown in Table S1. All participants or their guardians provide written informed consent prior to the study.
The detailed information of selection criteria of study subjects was reported in our previous paper. 19

| SNP selection and genotyping
The rs4938723 T > C, located in the promoter region of pri-miR-34b/c, was selected for the study because of its well-known role in altering cancer susceptibility. Genotyping of SNP was performed with a TaqMan SNP Genotyping Assay (Applied Biosystems, USA), with details in a previous study. 20 We first used a TIANamp Blood DNA Kit (TianGen Biotech Co. Ltd., Beijing, China) to extract genomic DNA from participants' blood. Then the genomic DNA was added into a 384-well plate. The reactions were set under the following conditions: holding stage at 95 C for 10 min, followed by 40 cycles each of denaturation at 95 C for 15 s, annealing and extension at 60 C for 60 s. Negative controls (with water) and duplicate test samples (10% of all the samples) were included in each 384-well plate.
The 100% concordant of genotypes in replicates were achieved.

| Statistical analysis
The goodness-of-fit χ 2 test was used to detect whether the selected SNP in the control group deviated from Hardy-Weinberg equilibrium (HWE). The demographic distribution and allele frequency of all cases were compared with the control group by the two-sided chi-square test. The relationship between miR-34b/c polymorphism and glioma susceptibility was evaluated by logistic regression analysis using odds ratios (ORs) and 95% confidence intervals (CIs). In addition, adjusted OR and the corresponding 95% CIs (adjusted for age and gender) were calculated by unconditional multifactor logistic regression analysis. Finally, stratified analysis was performed according to age, gender, tumor origin site, and clinical stage. All statistical analyses were processed in SAS software (version 9.4, SAS Institute, NC, USA). When the p value <.05, the results were considered statistically significant.

| Associations between miR34b/c polymorphisms and glioma risk among Chinese children
The study included 314 glioma patients and 380 controls, as shown in

| Stratification analysis
We further demonstrate whether the association between rs4938723 T > C genotype and glioma risk varies by age, gender, tumor site, and clinical stage (  reported that miR-34b/c rs4938723 C allele was associated with reduced risk of acute lymphoblastic leukemia in an Iranian population of 110 children with acute lymphoblastic leukemia and 120 healthy children. 28 Polymorphisms can have different genetic effects on susceptibility to cancer, depending on cancer type, ethnicity, and region. [27][28][29] In a study involving 393 cases and 812 controls, miR-34b/c rs4938723 T > C was shown to be protective against neuroblastoma. 30 In 2019, Zhuo et al. 31  cancer, 32 and esophageal cancer. 33 The protective effect of miR-34b/c rs4938723 T > C may be determined by genetic, environmental factors, and gene-environment interaction. 34,35 In children younger than 60 months, glioma susceptibility is reduced, which is conducive to the prevention and treatment of glioma in children.
The low-sample size may be the reason why other observations were not positive.
As a preliminary study, this work has several limitations. First, we did not obtain any positive results in the logistic regression analysis comparing cases and controls, which may be due to insufficient However, due to the above limitations, it is necessary to conduct multi-ethnic population studies and functional analysis of rs4938723 polymorphism. Identification of glioma susceptibility genes will provide a better understanding of the underlying mechanism of glioma and lay the foundation for the development of new genetic markers and monitoring procedures.

| CONCLUSIONS
In conclusion, we provide the possibility of miR-34b/c rs4938723

FUNDING INFORMATION
This study was supported by grant from the National Natural Science Foundation of China (No: 81802346).