Cervical cancer is one of the common gynecological malignancies, with an incidence among female malignant tumors second only to breast cancer. About 500,000 new cervical cancer cases occur worldwide each year, and more than 85% of cervical cancer cases occur in developing countries such as China (Yu et al., 2012). Reliable and specific biomarkers for the early diagnosis and prognosis prediction of cervical cancer are still lacking. Therefore, novel biomarkers and therapeutic targets are an urgent need.
NEIL2 is a member of the endonuclease family of genes encoding DNA repair enzymes, mainly involved in the BER pathway in mammalian cells(Canugovi et al., 2012). NEIL function often involves protecting cells from the accumulation of oxidative DNA damage in the genome to maintain genomic stability. Loss of NEIL2 may lead to decreased DNA stability in tumor cells, and cells exhibit a significant increase in DNA damage after the deletion of the NEIL2 gene, which in turn affects tumor development and prognosis(Sayed et al., 2020)..
In this study, we used “cervical cancer” as the keyword to search TCGA and GEO databases and screened cervical cancer and normal tissue samples in three datasets: TCGA-CC, GSE7410, and GSE63514.286. cervical cancer tissue samples and three normal tissue samples were retained in the TCGA, of which 271 had survival information; 40 cervical cancer samples and three normal tissue samples were retained in the GSE7410 dataset; and 76 cervical cancer tissue samples and 24 normal tissue samples were retained in the GSE63514 dataset. There were 402 cervical cancer samples and 30 normal tissue samples in the three datasets combined. Thereafter, integration and in-depth analysis of these datasets using R software and bioinformatics was performed for NEIL2 gene expression and diagnostic analysis to identify key modules and key genes associated with NEIL2.
Research has found that compared with normal tissues, NEIL2 expression was significantly decreased in colorectal cancer, gastric cancer, lung cancer, and other tissues(Broderick et al., 2006; Shinmura et al., 2004; Vartanian et al., 2006). Shinmura et al. used the TCGA database to study the relationship between NEIL2 and 13 common cancers, including bladder urothelial carcinoma, breast invasive carcinoma, and colon adenocarcinoma, and concluded that the reduced expression level of NEIL2 was a risk factor for the progression of various cancers(Shinmura et al., 2016). In this study, we discovered that NEIL2 showed low expression in cervical cancer. We therefore speculate that NEIL2 may play a tumor suppressor role in cervical cancer. There was a strong correlation between the NEIL2 expression level and the cervical cancer diagnosis. We also found that NEIL2 expression was associated with patient age and pathological type, with low expression in patients aged below 50 and squamous carcinoma patients. As both are influencing factors of NEIL2 expression, when assessing NEIL2 expression for cervical cancer diagnosis, age and pathological type should be considered.
Researchers used tools such as GEPIA2 to find that low expression of NEIL2 was associated with poor prognosis in HER2-positive patients. This implies that low expression of NEIL2 may affect tumor treatment response and survival(Shinmura et al., 2016).This is in line with our findings. Our study also showed that OS, PFI, and DFS were all significantly better in the NEIL2 high-expression group than in the low-expression group (P < 0.05). Multivariate Cox regression analysis found that only pathological factors N and T and high NEIL2 expression had significant differences (P < 0.05), indicating that in addition to pathological factors N and T, high expression of NEIL2 is also an independent risk factor affecting the prognosis of cervical cancer patients. Based on the identification of these three independent prognostic factors, we constructed a 1-year, 3-year, and 5-year survival nomogram model independent of the factor. Preliminary evaluation of the nomogram model showed high prediction accuracy for cervical cancer prognosis evaluation. The fact that longer time increased the accuracy further illustrates the importance of NEIL2 expression for cervical cancer prognosis prediction. We analyzed the expression and prognosis of NEIL2 in common cancers among women, such as breast invasive cancer, cervical cancer, ovarian serous cystadenocarcinoma, uterine endometrial cancer, and uterine carcinoma sarcoma, and found that the differential expression of NEIL2 may be an independent risk factor for the prognosis of these common cancers. This indicates that the differential expression of NEIL2 is not only associated with the prognosis of cervical cancer but also with that of other cancers among women.
As for the mechanism by which NEIL2 expression changes lead to cancer initiation and progression, multiple studies have suggested that low expression levels of NEIL2 may cause somatic DNA mutations and copy number variation, thus leading to genomic instability, activation of oncogenes, and downregulation of tumor suppressor genes. In cervical cancer, when the mRNA and protein expression of NEIL2 is significantly reduced, the level of damaged genome repair will decrease, leading to genomic instability and the initiation of cervical cancer[8]. SNP mapping to the NEIL2 promoter region may lead to an increased risk of breast cancer in BRCA2 mutation carriers by increasing the level of genomic instability, and genetic variation in the SNP 5' untranslated region onNEIL2 is associated with breast cancer risk, prognosis, and response to treatment. These variants may affect the expression level and function of NEIL2 and thus, the tumor prognosis(Benitez-Buelga et al., 2017; Cumova et al., 2021).
With the help of GO and KEGG enrichment analysis, we can functionally classify genes and explore the relevant signaling pathways involved to better study the mechanisms associated with NEIL2. The most differential genes were associated with the occurrence and development of cancer. Among them, key module genes in TCGA were enriched in mucin-type o-glycan biosynthesis, secretion and action of growth hormone, SLC transporter disorders, and phospholipid metabolism, while key module genes of GSE7410 were enriched in ECM receptor interaction pathways and ECM proteoglycan and MET-activated PTK2 signaling. Antigenic and structural changes in mucin-type carbohydrate chains (O-glycans) are associated with rectal cancer(Yang et al., 1994). Studies have shown that elevated growth hormone levels may be associated with the development of a variety of cancers. Excess growth hormone can promote cell proliferation and division, inhibit apoptosis, and stimulate angiogenesis, all of which are beneficial to tumor formation. On the other hand, growth hormone can also indirectly promote tumor progression by regulating tumor-promoting growth factors, such as insulin-like growth factors. Overall, the dysregulation of growth hormone activity may be an important factor leading to the development of some cancers(Boguszewski and Boguszewski, 2019). Tumor cell metabolism is demanding and needs large amounts of phospholipids and other substances to be synthesized. SLC transporters play important roles in regulating nutrient uptake in tumor cells(Jang et al., 2013). Therefore, SLC transporters are potential tumor therapeutic targets, and the disorders of SLC transporters and phospholipid metabolism have important associations with the development of many cancers(Meng et al., 2018). PTK2 is also related to the development of tumors(Gomez et al., 2016).According to the GO and KEGG results, these functions and pathways may be involved in the carcinogenesis of cervical cancer, thus requiring further investigation.
We screened for related genes that may also have important relationships with cancer through the online jvenny website. Among the genes most associated with NEIL2 are ENTPD2 and PLA2G6. There are eight members of the ENTPD family, but only ENTPD1 has been extensively reported in hepatocellular carcinoma, lung adenocarcinoma, prostate cancer, and other cancers(Stagg and Smyth, 2010). However, ENTPD2 is significantly overexpressed in hepatocellular carcinoma, and hypoxia is a cause of the accumulation of myeloid-derived suppressor cells (MDSCs), which upregulates ENTPD2 through HIF1D, thus maintaining undifferentiated MDSCs. Thus, blocking ENTPD2 in cancer cells significantly inhibited hepatocellular carcinoma growth in vivo(Chiu et al., 2017). ENTPD2 is rarely reported with cervical cancer, so we can use it as the next prognostic marker and treatment method when studying the immunosuppressive tumor microenvironment of cervical cancer. However, PLA2G6, like the famous c-myc, as a target of c-Myc, may have a positive correlation with the expression of c-Myc. By inducing the dysfunction of arachidonic acid metabolism, it activates inflammatory responses through the modulation of inflammatory mediators, leading to impaired liver detoxification and malignant transformation into cancer(Li et al., 2017). Moreover, other evidence that PLA2G6 may be related to the growth and prognosis of esophageal cancer has been collected(Du QC et al., 2022). Since there are few reports on ENTPD2 and PLA2G6 in cervical cancer, these two genes can be studied further.