This study combined the modular genes selected by DEGs and WGCNA to explore the key difference genes between MYCN amplification and MYCN normal NB. First of all, it was confirmed that there is a big difference in gene expression between MYCN amplified and MYCN unamplified samples. There are 76 intersection genes between DEGs and WGCNA blue-green modules. Secondly, GO analysis confirmed that the intersection genes in BP and CP are mainly distributed in glial cell regeneration and myelination. Molecular function (MF) shows that the main intersection genes are concentrated in DNA binding transcription activation and RNA polymerase II specificity. The results of KEGG show intersection. Genes are mainly enriched in the interaction between PI3K-Akt signaling pathway and extracellular matrix receptors. Based on these results, we infer that MYCN amplified NB can regulate DNA binding transcription activity and RNA polymerase specificity, affect myelination and glial cell regeneration, and participate in PI3K-Akt and extracellular matrix receptor interactions, etc. Signal pathways regulate tumor cell proliferation and differentiation[13, 14], resulting in MYCN-expanded NB cells that have stronger proliferation, differentiation and metastasis capabilities than normal NB cells , which ultimately leads to poor prognosis for MYCN-expanded NB patients.
In this study, 76 differentially expressed hub genes were uploaded to String to draw a diagram of the interaction network, and the top 20 core genes in the network were screened through the CytoHubba plug-in in Cytoscape. Univariate COX regression analysis showed that 4 genes in 20 cores (HBA2, FOS, NR4A2 and EMP1) were closely related to the overall survival of patients with MYCN amplified NB (P value<0.05). In addition, these four key genes are closely related to the clinical stage of NB patients. The higher the clinical stage of the patient, the higher the expression of HBA2 gene, while the expression of FOS, NR4A2 and EMP1 decreases. Multivariate COX regression analysis confirmed that as the expression of HBA2 increased or the expression of NR4A2 decreased, the patient's survival time was shorter, indicating that HBA2 and NR4A2 not only expressed significant differences in MYCN amplified and MYCN normal NB patients, but also compared with MYCN amplified NB patients The survival time is closely related. In other words, HBA2 and NR4A2 can be used as potential prognostic markers for MYCN-amplified NB patients.
HBA2 is located on chromosome 16 with a base length of about 30 kb and mainly encodes human hemoglobin. Retrospective studies have confirmed that NB patients are easily misdiagnosed as leukemia due to abnormal blood routine results such as increased hemoglobin, which is mainly due to the fact that NB tumor cells invade the bone marrow, their morphology is similar to primitive and naive lymphocytes, and glycogen staining Positive. The results of this study indicate that the overexpression of HBA2 gene in NB cells may lead to active proliferation of bone marrow in patients with NB, and an increase in the proportion of erythroid. The higher the expression level, the more active the metabolism of NB tumor cells. The expression of HBA2 gene in MYCN amplified NB patients was significantly higher than in MYCN normal patients, and the higher the clinical stage, the higher the expression level. Therefore, in the process of treating patients with MYCN amplified NB, attention should be paid to changes in the patient's blood picture to prevent misdiagnosis of the disease and delay treatment.
Studies have confirmed that the protein encoded by the NR4A2 gene can act as a transcription factor to limit the function of CAR T cells in solid tumors[19–21]. In other solid tumors, the role of NR4A2 is contradictory. On the one hand, NR4A2 can be used for cancer treatment drug screening, for example, as a drug target for glioblastoma. On the other hand, the high expression of NR4A2 in gastric cancer cells confers chemoresistance and attenuates 5-fluorouracil-induced apoptosis. In this study, NR4A2 decreased with the increase of INSS stage, while the expression of NR4A2 decreased, the shorter the patient's survival time, indicating that the expression of NR4A2 in NB is different from other solid tumors. However, The form or pathway of NR4A2 in MYCN expansion of NB affects cell proliferation and differentiation. These mechanisms still need to be further studied.
This study constructed a prognostic risk score for patients with MYCN amplified NB based on HBA2 and NR4A2. According to the risk score, patients can be divided into high-risk and low-risk groups. The results of survival analysis show that the survival time of patients in the high-risk group is shorter than that in the low-risk group, and the area under the ROC curve AUC is 0.745 (P<0.05, 95%CI: 0.687-0.798), the sensitivity is 0.916, and the specificity is 0.877. There have been previous studies on the overall prognosis model of neuroblastoma patients[9, 26], and the use of bioinformatics to identify key MYCN-related genes[10, 27], but temporarily There is no risk score prediction for the MYCN amplified NB population. This study is aimed at MYCN amplified NB, using only two genes HBA2 and NR4A2 to construct a risk score with an AUC value greater than 0.7. In general, HBA2 and NR4A2 are potential prognostic marker genes for MYCN amplified NB patients.
The results of this study have certain limitations, that is, even if the expression profile data in the GEO database can be used to quickly mine the potential prognostic marker genes of MYCN amplified NB patients, the bioinformatics algorithm is different, so the final result will be different. The difference. This study has initially successfully verified the expression of HBA2 and NR4A2 on clinical tissue samples. Therefore, the next step will focus on the functional experiments of HBA2 and NR4A2 genes in MYCN to expand NB cells to obtain more biological processes., To provide more evidence for treating NB and predicting the prognosis of children with NB.