NUDT21, as a tumor suppressor gene,which could inhibit the occurrence and development of various cancers, including small cell lung cancer, bladder cancer, and breast cancer. NUDT21 is a 3'-terminal processing factor of mRNA precursor (targeted mRNA precursors 3'contain multiple poly-A splicing sites), regulating mRNA expression through selective alternative polyadenylation [40]. Ultimately, the normal physiological process of NULDT21 was involved in regulating proliferation, differentiation and apoptosis. However, the overexpression of tumor suppressor genes in cancer patients has attracted more and more attention. But the mechanism of them has not yet been elucidated. Studies had found that it might be closely related to genetic alters and DNA methylation [41, 42]. However, the expression and gene regulation network of NUDT21 in patients with LUAD was rarely reported.
In our study, the expression level of NUDT21 and the correlation between differential expression of NUDT21 and pathological stage were first explored in LUAD patients. We found that NUDT21 was up-regulated expression in patients with LUAD compared with normal human. In addition, our results showed a significant correlation between the expression of NUDT21 and pathological stage. Furthermore, the survival of LUAD patients with the low expression level of NUDT21 was better prognostic value than LUAD patients with a high expression level of NUDT21. In brief, overexpression of NUDT21 may play an essential role in survival with LUAD patients. Contrary to other studies, NUDT21 was low expressed in varies cancer [43]. To further explore the mechanism of NUDT21 overexpression in LUAD patients, we found that genetic alteration of NUDT21 was high (12%). Moreover, the promoter methylation level of NUDT21 in LUAD was lower compared to normal human. Therefore, genetic alteration and methylation of NUDT21 may be the leading cause of NUDT21 overexpression. Moreover, NUDT21-neighboring genes (the 50 most frequently altered neighbor genes) were altered at frequencies > 17% in LUAD. We then explore the potential interactions and function of NUDT21 and its neighboring genes. We found that NUDT21 and its neighboring genes have complex and tight networks of connections. Their primary functions mainly include the regulatory region DNA binding, transcription regulatory region DNA binding, and regulatory region nucleic acid binding. The above evidence reveals that they might affect the processes of gene binding, transcription, and regulation involved in the occurrence and development of cancer.
Moreover, GO enrichment analysis revealed that these genes' functions are mainly connected with inflammatory response caused by the activation of inflammatory factors. As expected, the expression of NUDT21 was positively associated with the immune cell infiltration, including CD8 + T cells, macrophages, neutrophils and dendritic cell. Immunotherapy has been recognized as the fourth pillar of cancer therapy, and a large number of preclinical and clinical studies have been revealed the efficacy of immunotherapy for lung cancer [44]. Targeting and regulating the tumor immune microenvironment can divert the immune system to anticancer and increase the sensitivity to established chemotherapy. Chemokines and their receptors are crucial for inflammation and anti-tumor immunity, thus influencing angiogenesis, tumor occurrence, progression, metastasis, therapeutic efficacy, and patient outcomes [45–47]. Studies have shown that the CXC family of chemokines and their receptors are associated with tumour metastasis and therapy resistance [48, 49]. Besides, our results showed that The KEGG pathway of NUDT21 and its neighboring genes involved in the apelin signaling pathway, PI3K-Akt signaling pathway, and axon guidance. Apelin is an endogenous ligand of G protein coupled receptor APJ. It is widely expressed in many tissues, especially the lungs. More and more evidence revealed that the apelin signaling pathway is closely associated with the occurrence of respiratory diseases [50]. Studies have found that inhibiting apelin effectively remodeled the tumor microenvironment, reduced angiogenesis, and effectively inhibited tumor growth [51]. Importantly, apelin prevents resistance to antiangiogenic receptor tyrosine kinase (RTK) inhibitor therapy in lung cancer [51]. PI3K-Akt signaling pathways play a key role in the regulation of cell proliferation, differentiation and apoptosis. AKT is a crucial regulator of cell survival and apoptosis [52, 53]. AKT is activated by PDK1 through phospholipid binding and activation loop phosphorylation at Thr308 [52, 53]. AKT inhibits apoptosis and promotes survival of malignant cells by phosphorylating and inactivating multiple targets, including Bad, C-Raf and Caspase-9 [54]. In recent years, research has shown that the axon guidance cue molecule Slit2 could suppress lung cancer cell invasion and growth [55].
We also focus on their targets and regulators. We then explored the transcription factor targets, kinase targets and miRNA targets of NUDT21 and its neighboring genes, and found that DNMT1, HDAC1, and MYC were their key regulated factor in LUAD. DNMT1 is a major epigenetic enzyme, maintains the genomic stability and the epigenetic state of DNA by copying CpG methylation markers and generating heritable methylation patterns through cell metabolism [56, 57]. Approaches for inhibition of DNMT1 may become novel strategies for treating cancers [58]. In recent years, abnormal expression of HDAC1 has been found to have potential clinical prognostic value in various cancers. Studies have shown that down-regulation of HDAC1 can inhibit cell proliferation, migration, invasion, angiogenesis and induce cell apoptosis [59]. MYC is a transcription factor that is overexpressed in tumors and participates in preventing immune cells from attacking tumor cells by inducing the expression of PD-L1. MYC expression's role is an alternative marker for evaluation of treatment with non-small cell lung cancer [60]. Therefore, Targeting DNMT1, HDAC1, and MYC may be a promising approach to the treatment of LUAD. We also found that Kinase CDK1, Kinase ATM and Kinase PLK1 were the top three targets in the NUDT21 kinase-target network. Kinase CDK1, Kinase ATM and Kinase PLK1 are involved in DNA repair and progression through the cell cycle. Kinase ATM and RNF8/RNF168 ubiquitin-ligase modify chromatin extensively on the DNA damaged side. In mitosis, phosphorylation of 53BP1 by Kinase PLK1 and Kinase CDK1 impairs the ability of 53BP1 to bind to ubiquitination H2A and locate DNA damage sites properly [61]. The NUDT21 miRNA-target network was associated with MIR-302C, MIR-9, and MIR-330. They are involved in tumor proliferation, differentiation, and invasion. It had been reported that Mir-302C-5P act as a cancer suppressor in a variety of cancers [62]. Moreover, Mir-302c-3p is a tumor suppressor gene of malignant glioma and can be used as a new biomarker to predict glioma patients' clinical prognosis [63]. miR-9 includes three family members, including miR-9-1, miR-9-2 and miR-9-3. Researchers recently found that the promoter region of Mir-9-3 was hypermethylated in lung cancer, leading to the down-regulation of Mir-9-3, and poor prognosis of patients and miR-9 may be a potential lung cancer biomarker [64, 65]. MiR-330 acts as a tumor-suppressor microRNA (miRNA) in various cancers and is a promising candidate for miRNA replacement therapy in lung cancer patients [66]. In conclusion, the transcription factor targets, kinase targets and miRNA targets of NUDT21 and its neighboring genes may be potential therapeutic targets and biomarker for LUAD.
Pinellia ternata is a commonly used Chinese medicine for treating infection, inflammation, cough, and vomiting,which had been documented by the Chinese Pharmacopoeia (2015 edition) [21, 22]. However, more and more studies have found that Pinellia ternata has a therapeutic effect on various cancers [23, 24]. Our results found that 13 active compounds of Pinellia ternata were retrieved base on OB and DL from the TCMSP. Among them, baicalein was the best combination with NUDT21 (Score =-8.8 kcal/mol). Hydrogen bonding was their main interaction force (Six hydrogen bonds were found in our study). Baicalein was a bioactive component extracted from various traditional Chinese medicines, including Pinellia ternata and Scutellaria baicalensis Georgi [67, 68]. In recent years, anti-tumor, anti-inflammatory, and antimicrobial activities of baicalein were found [69]. It had been reported that baicalein could reverse cisplatin resistance of human lung adenocarcinoma A549 cells via PI3K/Akt/NF-κB pathway[70]. Although our study has not been further verified the anti-LUAD of baicalein by cells or animal experiments, our results preliminarily indicate that it may affect NUDT21 against LUAD.
In conclusion, by showing the overexpression and gene regulation network of NUDT21 in LUAD, we expect to provide a new perspective on drug selection for clinical workers from the standpoint of immunotherapy for LUAD. Furthermore, identifying new therapeutic targets and prognostic biomarkers to more accurately predicts the survival in patients with LUAD. We also provide potential therapeutic drugs from traditional Chinese medicine in the treatment of LUAD.