Although there has been great progress in surgical and medical therapy for lung cancer, the latest global cancer statistics (2018) reported that lung cancer is still the most frequently diagnosed cancer, and its mortality ranks first globally . As LUAD is the most common histological subtype of lung cancer, this low 5-year survival rate is far from satisfactory. LUAD is often diagnosed at an advanced or metastatic stage, which makes early detection and treatment impossible. However, cancer mortality can be vastly reduced if cases are detected or treated early . Therefore, it is urgent and necessary to seek innovative biomarkers and precise molecular mechanisms for the early diagnosis, treatment, and prognosis of LUAD.
In recent years, due to the applications of RNA-Seq data and microarray-based expression profiling data, the ceRNA hypothesis proposes a new RNA interaction mechanism, which further contribute to the better understanding of the tumourigenesis and prognosis of LUAD at the molecular level . In our study, DEGs, DELs, and DEMs were first identified by using the raw sequencing data of LUAD cases and normal controls from the TCGA database. Then, the DEL-DEM-DEG ceRNA network was constructed on the basis of DEL-DEM interactions and DEM-DEG interactions. This initial ceRNA network consisted of 340 DELs, 29 DEMs, and 218 DEGs. Subsequently, GO and KEGG pathway analyses of those 218 DEGs were conducted by DAVID. The GO analysis of DEGs indicated that the DEGs were significantly enriched in “nucleoplasm”, “transcription factor complex”, “protein binding”, and “metal ion binding”, which further suggested that LUAD might be considered a metabolism-related disease. The KEGG pathway results showed that the DEGs involved in the ceRNA network were associated with microRNAs in cancer, pathways in cancer, cell cycle, HTLV-1 infection, and the PI3K-Akt signalling pathway. These results also provide significant clues to explore the molecular mechanisms of tumourigenesis and prognosis in LUAD patients. Indeed, many studies have illustrated that the cell cycle, HTLV-1 infection, and PI3K-Akt signalling pathway are highly related to various types of cancer, especially lung cancer [30–33]. For example, Weimiao Li et al. proposed that overexpression of cell cycle-related proteins in tumours is always related to tumour proliferation behaviours and poor prognosis in non-small-cell lung cancer (NSCLC) . Furthermore, Hiromitsu M et al. in 1990 validated that human T-cell leukaemia virus type 1 (HTLV-1) is associated with small cell lung cancer , while no studies have explored the relationship between HTLV-1 infection and LUAD. In addition, the PI3K-Akt signalling pathway regulates the normal physiological activities of cells. Some evidence has also validated that aberrant activation of the PI3K-Akt signalling pathway always leads to tumourigenesis and metastasis in many types of cancer, such as LUAD , gastric carcinoma , and bladder carcinoma . For LUAD, the PI3K-Akt pathway may affect cell apoptosis and proliferation . Additionally, the PPI network in this study consisted of 218 nodes and 455 edges. This provides useful information on the PPIs involved in our ceRNA network. All of the above evidence suggests that our ceRNA network might play essential roles in exploring the mechanisms of LUAD.
After conducting these functional analyses and PPI construction, K-M survival analysis of all differentially expressed genes involved in the ceRNA network screened 24 DELs, 4 DEMs, and 29 DEGs, all of which were significantly correlated with progression in LUAD patients (P < 0.05). Then, a new ceRNA network was constructed based on the significant genes in the survival analysis. Moreover, for the residual 29 DEGs, we used cBioPorta to explore the alteration information and the drug-gene interactions. In this study, we found that multiple alterations, deep deletions, and amplifications occurred frequently for those 29 DEGs in LUAD patients. It is well known that the tumourigenesis and prognosis of LUAD is mostly the consequence of multiple and cooperative genomic alterations . For example, the famous tumour suppressor TP53 is often deactivated and deleted in the majority of LUAD patients [37, 38]. In this study, the large tumour suppressor 2 (LATS2) gene suffered from deep deletion in LUAD patients (Fig. 9a). Furthermore, from the results of K-M survival analysis, a low expression level of LATS2 was associated with poor survival time (Fig. 7b), which was consistent with the study of Jang SH et al . In addition, 15 drugs interacting with 29 DEGs against LUAD were further selected (Table 2). From this result, we found that TBXA2R interacted mostly with many types of drugs approved by the FDA as agonists or antagonists. However, none of these drugs have been reported to be related to the treatment of lung cancer through interactions with TBXA2R. Nevertheless, there is much evidence indicating that iloprost , acetaminophen , morphine , furosemide , and vinblastine  are broadly applied in chemotherapy for lung cancer patients. Nevertheless, this study still provides novel insights into LUAD pathogenesis and treatment. Moreover, the selected conventional drugs might find potentially innovative use in the future.
In addition, lasso-penalized Cox regression and multivariate Cox regression analyses were combined to assist with the construction of the risk score system. Among the remaining 4 DEGs, namely, PRKCE, DLC1, LATS2, and DPY19L1, three of them have been studied before. For example, deleted in liver cancer-1 (DLC1) is a tumour suppressor gene that has been reported to be involved in the genetic and epigenetic mechanisms of various human cancers, such as lung, colorectum, breast, and prostate carcinomas [45–47]. Xiaolan Qian et al. indicated that inactivation of DLC1 and downregulation of p15INK4b and p16Ink4a were combined to cause neoplastic transformation and poor prognosis in human cancer . The DLC1 gene encodes a Rho GTPase-activating protein (RhoGAP), and it is also an important tumour suppressor gene in lung cancer . Loss or downregulation of DLC1 expression always causes abnormal functions of Rho GTPases . In this study, we also found that deep deletion frequently occurred in DLC1 (Fig. 9a), and the survival analysis further validated that LUAD patients with lower expression levels of DLC1 were often related to poorer OS (P = 0.022) (Fig. S2). Specifically, we first identified DPY19L1 as the targeted mRNA that was associated with the tumourigenesis and prognosis of LUAD patients in this study. Furthermore, to validate the reliability of our risk score system, survival analysis and ROC curve analysis based on risk scores were conducted. Because the AUC values of the time-dependent ROC curves at 3 years and 5 years were both higher than 0.5, we considered our risk score system to be effective. After validation by univariate and multivariate Cox regression analyses based on the full clinical information, the risk score system derived from the expression levels of the 4 DEGs and the pathological stages might be treated as the only independent prognostic factors of OS in LUAD patients.
Then, based on the correlation results among the remaining 4 DEGs and their corresponding DELs, only two ceRNA networks were selected, namely, NAV2-AS2 – mir-31 – PRKCE and NAV2-SA2 – mir-31 – LATS2. Chang SH and Wang LH once suggested that miRNAs could cause mRNA degradation by binding to the 3’-untranslated region (3’-UTR) of the target genes . Surprisingly, in this study, the expression level of hsa-mir-31 was upregulated in LUAD patients, whereas the expression levels of PRKCE, LATS2, and NAV2-AS2 were downregulated (Fig. 8). On the basis of the results of survival analysis, lower expression levels of PRKCE, LATS2, and NAV2-AS2 were always correlated with poorer OS in LUAD patients (Fig. 7). All this evidence in our study improved the reliability of the ceRNA network.
In the above two ceRNA networks, NAV2-AS2, an upstream lncRNA, has never been studied; in contrast, the downstream mRNAs PRKCE and LATS2 related to LUAD have been studied extensively [51–53]. For example, PRKCE, a phorbol ester receptor, has been validated to be associated with various types of cell functions, such as cell cycle progression , ion channel control , cytokinesis , and regulation of transcription factor activity . The onset and progression of various types of chronic diseases have been indicated to be associated with PRKCE. These diseases include heart failure, obesity, diabetes, neurological diseases, and cancer . Regarding the mechanisms of NSCLC, Li Ding et al. indicated that PRKCE could keep lung cancer cells from undergoing apoptosis and further promote cell survival through the dysregulation of the mitochondrial caspase pathway . Furthermore, Junjie Wu et al. demonstrated that PRKCE regulated cell proliferation and was targeted by hsa-mir-129 . In the above section, we explored the alterations in LATS2. LATS2 is a presumed tumour suppressor gene that encodes a serine or threonine kinase . Dysregulation of the LATS2 functions has been validated to be related to a series of malignancies, including lung cancer , breast cancer , prostate cancer , and malignant mesothelioma . There are multiple mechanisms of action for LATS2 in various kinds of cancers, such as the regulation of the cell cycle by controlling G1/S and G2/M transition , induction of apoptosis by downregulating Bcl-2 and Bcl-X2 , and conservation of genetic stability by interacting with p53 . Furthermore, Susan Y. Luo et al. suggested that LATS2 might promote tumour growth via different signalling pathways, especially in EGFR mutant and wild-type LUAD . As the centre of the ceRNA network, hsa-mir-31 has been demonstrated to be a new diagnostic microRNA classifier for lung squamous cell carcinoma (LUSC) . In that research, Xiaogang Tan et al. also explored the functions between hsa-mir-31 and its potential targeted gene LATS2 in LUSC patients. However, he found that expression of hsa-mir-31 in the SK-MES-1 cell line did not regulate the activity of LATS2 . Nevertheless, more studies need to be conducted to explore the associations between hsa-mir-31 and the tumourigenesis and prognosis of LUAD.
The strength of our study is that we first the constructed NAV2-AS2 – mir-31 – PRKCE and NAV2-SA2 – mir-31 – LATS2 ceRNA networks based on a risk score system and explored their potential biological functions in the tumourigenesis and prognosis of LUAD patients. Considering the crucial roles of these genes, further studies might be focused on validating and testing the predicted ceRNA network and exploring their precise mechanisms of LUAD. Indeed, our study has several limitations. One is that all the data used in this study were derived from public databases and were not generated by the authors of this article. The clinical information did not include the family exposure history and other environmental factors, which will bias the results of Cox regression analysis. The other is that future experimental studies and verifications must be conducted to explore the mechanisms in depth. Despite these limitations, this study may provide novel insights into the molecular mechanisms of LUAD and assist with exploring potential and innovative targets and biomarkers of tumourigenesis, prognosis, diagnosis, and therapeutic drugs for LUAD patients.