The DNA methylation profile of non-coding RNAs improves prognosis prediction for pancreatic adenocarcinoma

Pancreatic adenocarcinoma (PAC) is one of the most prevalent type of highly lethal malignancy in the digestive system. It is the fourth most dominant cancer-associated death, with a 5-year survival rate less than 5% [1]. Ttreatment of PAC, including surgical resection, radiotherapy and chemotherapy, has been improved in the recent years [2]. However, even with these disease management options, the overall survival (OS) rates of PAC patients are still far from satisfactory. The poor effectiveness of treatment therapies results from the high aggressiveness of PAC, its resistance to chemotherapy drugs, and a lack of early diagnosis [3]. Therefore, to reduce mortality and improve the management of PAC patients, identification of prognosis biomarkers during the early stage of PAC is extremely important.

Non-coding RNAs (ncRNAs) have been widely found in complex eukaryotic organisms, and have gradually emerged as a new research focus because of their unique biological roles in tumour progression [4]. Although they cannot be translated into proteins, ncRNAs play critical roles in chromosomal modification, transcriptional interference, post-transcriptional modification and translational regulation via epigenetic modifications in human cells [5]. Emerging evidence has revealed that ncRNAs with dysregulated expression profiles are involved in the pathogenesis of multiple cancers, including colorectal cancer [6], breast cancer [7], as well as gastric cancer [8]. Furthermore, epigenetic modifications, especially DNA methylation, have been determined to play pivotal roles in the regulation of protein-encoding genes, miRNAs and lncRNAs [9]. Zhang et al. [10] demonstrated that the tumour suppressor microRNA-596 (miR-596) was silenced by hypermethylation at its relevant promoter in gastric cancer but not in normal tissues. Furthermore, the miR-596 expression of gastric cancer cell lines was reduced, accompanied by an increment of miR-596 methylation, which is consistent with previous study. miR-770 has also been reported to be significantly decreased in gastric cardia adenocarcinoma (GCA) as a result of the hypermethylation of the lncRNA coded by MEG3. Additionally, the aberrant methylated CpG sites of MEG3 and dysregulated miR-770 content were closely related to a poor prognosis for patients [11]. These studies indicated that abnormal DNA methylation could be a vital biomarker in the pathophysiology and evaluation of cancers. Hence, we hypothesize that the methylation of ncRNAs can predict the prognosis of PAC.

To date, the prognostic values assessed by the methylation of ncRNAs and the association between miRNAs and lncRNAs in PAC remain poorly elucidated. In our present study, differential methylation patterns of miRNA and lncRNA were analysed in PAC using the 485,577 sites identified by the HumanMethylation450 BeadChip, which is annotated by The Cancer Genome Atlas (TCGA). Using the Least Absolute Shrinkage and Selection Operator (LASSO) regression model, we successfully developed miRNA and lncRNA methylation-based classifiers to assess OS of patients. Functional enrichment analysis of differently methylated ncRNAs was performed to analyse the target genes and the association between miRNAs and lncRNAs.

PAC is one of the most invasive and lethal carcinomas worldwide, with multiple types of molecular and cellular heterogeneity [14]. Due to limited treatment strategies, the incidence of PAC is increasing yearly and most patients died within 1 year. Therefore, revealing the pathogenic mechanism of PAC will help to advance new treatment strategies. In recent decades, researches have shown that ncRNAs play crucial roles in various cancers with the great improvements in RNA-seq technologies [15]. Although several studies have focused on the functional effect of ncRNA methylation [16], the interrelation between the methylation of ncRNAs and PAC remains still elusive. DNA methylation has been proved to be critical for the regulation of protein-coding gene expressions. Accumulating evidence has illustrated that abnormal methylation at promoter CpG islands of ncRNAs may be involved in the pathogenesis and prognosis of cancers [11], and this information may contribute to prognostic prediction as well as individualized therapy in these patients.

In this study, we conducted a comprehensive and detailed analysis of the DNA methylation profile of ncRNAs in a cohort of PAC specimens from the TCGA database to investigate the altered DNA methylation patterns in PAC. Furthermore, we identified several important clinically relevant observations. Firstly, according to the 485,577 CpG sites 2 kb upstream of the TSS in ncRNAs (miRNA or lncRNA), 511 CpG sites of miRNA and 1441 CpG sites of lncRNA were filtered out using different methylation degrees between PAC and the normal neighbouring tissues. The LASSO regression method was used to further determine the ncRNA signature, which identified 8 CpG sites of miRNA and 7 CpG sites of lncRNA with regression coefficients: miR-200a, miR-935, miR-124-3, miR-129-2, miR-1249, miR-4479, miR-615, miR-429, LINC00421, KB-1732A1.1, LINC00900, RP11-175E9.1, RP11-465B22.8, RP11-676J15.1, and MIR4500HG. Reports have indicated that most of the 8 miRNAs are closely related to tumorigenesis [17‐21] while none of the lncRNAs have been studied. Among the 8 miRNAs, miR-200a plays a vital part in the mesenchymal-to-epithelial transition process of pancreatic cancer stem cells by altering cancer migration and invasion [22]. Additionally, miR-935 participates in cell proliferation, migration and apoptosis by reacting with its target, INPP4A, in pancreatic cancer [18]. Emerging evidence illustrated that many ncRNAs are targets of methylation-associated silencing in several diseases, and hypermethylation of the CpG sites could lead to decreased expression of ncRNA [23]. In our study, we confirmed that the methylation levels of CpG sites were negatively corrected with the expression of miRNA or lncRNA when we screened out the CpG sites of miRNAs and lncRNAs, and this outcome is consistent with other studies. It is reported that promoter hypermethylation of miR-766-3p downregulated the expression of miR-766-3p, indicating the poor prognosis in renal cell carcinoma [24]. Therefore, we speculated that these two methylation-based classifiers of ncRNA can be used to determine the prognosis of pancreatic cancer, possibly by altering the expression of ncRNA to indirectly affect the progression of pancreatic cancer. In addition, the expression pattern of the typical ncRNAs, which were correlated with the CpG sites in the methylation-based classifiers may also could be used for the prognosis of PAC patients. Secondly, the developed methylation-based classifiers of the 8 miRNAs or the 7 lncRNAs can predict the prognosis of PAC precisely, which was the highlight of this study. And tdROC analysis also confirmed the predictive accuracy of the classifiers for OS in PAC patients and in PAC patients with DFS, which is conducive to guiding the implementation of treatment strategies at different stages of the disease. Additionally, the patients can be divided into two groups by this new method based on the median risk score cut-off point. We found that the OS of the patients in the low-risk groups outperformed that of the high-risk group. Consistently, the classifiers could also distinguish difference in OS between the two risk groups according to the clinicopathological risk factors based on the Kaplan–Meier curves, which could help to determine credible individual measures for the patients. Notably, it is urgent to develope additional treatments to improve preoperative risk assessment for PAC patients in low-risk groups. Meanwhile, the subgroup of PAC patients with high-risk scores identified with these two classifiers might be candidates for more aggressive treatment strategies. There is absolutely no doubt that our miRNA and lncRNA classifiers possessed their own unique prediction compared with the clinicopathological risk factor. And when the two classifiers combination with clinicopathological risk factors, it would provide a more accurate prediction for OS at different times for PAC patients. Surprisingly, we found that the methylation-based classifier of miRNA exhibited better predictive power than that of lncRNA, which is partially due to the indirect effect of lncRNA. Because lncRNA could interact with miRNA as a competitive endogenous RNA and affect mRNA expression [25]. Therefore, the two methylation-based classifier signatures have shown a favourable effect on survival prediction, which will contribute to therapeutic decision-making.

Tumorigenesis is a complex biological process including multifarious epigenetic alterations. Methylation of miRNAs and lncRNAs may participate in the development of carcinoma and play a crucial role in the disease progression. To evaluate the biological function of the ncRNAs in PAC, DAVID performed functional enrichment analysis of the target genes and co-expression genes. And 2442 target genes and 2524 co-expressed genes with abnormal methylation of miRNA and lncRNA, respectively, were identified. The GO results showed the major differences in tumour biology in terms of cellular component, biological process and molecular function. Based on the KEGG pathway analysis results, insulin secretion and the MAPK, Ras, and calcium signalling pathways were significantly enriched, which have previously been validated as vital to the tumorigenesis of PAC [26‐29]. Notably, the MAPK and Ras signalling pathways are frequently affected in PAC. Yang et al. [30] reported that, in PAC, the tumour suppressor RKIP is regulated by the Ras coded protein, named KRAS, probably via the MAPK-ERK signalling pathway. Moreover, several miRNAs were demonstrated to function as regulators of oncogenic KRAS signalling in PAC [27], which provides novel therapeutic evidence for targeting the Ras signalling pathway in patients. Therefore, these two candidate prognostic classifiers will be beneficial for improving prognosis prediction accuracy and guiding individualized treatment in PAC.

However, there are several limitations in this study. Although we identified aberrantly methylated sites of miRNAs and lncRNAs, whether the alterations represent a cause or an outcome of the cancer process remains unknown. Actually, methylation can occur in both the intergenic regions and repetitive sequences of the human genome and gene promoters. And aberrant hypermethylation of CpG sites in gene promoters may silence the gene expression that is critical to cell homeostasis, DNA integrity, or genome stability [31]. On the other hand, hypomethylation of CpG sites in the intergenic regions and repetitive sequences may lead to genomic instability and the loss of gene imprinting [32]. Furthermore, gene expression is related to the degree of methylation [33], which may be discrepant in different genes. Regrettably, this study mainly focused on the association between the methylation level of ncRNA and the prognosis of pancreatic cancer. It will be interesting to study the correlation of these different methylation styles in the future. In addition, no additional experimental data analysis on the underlying mechanisms of miRNAs and lncRNAs was performed in this study. It is urgent to conduct further experimental studies to address these issues.

In summary, our study identified two methylation-based classifiers of ncRNA associated with OS in PAC. Notably, the altered DNA methylation patterns could accurately predict the prognosis of PAC patients. These patterns are also involved in major cancer signalling pathways known to be crucial in carcinogenesis. Although more experimental studies are required for further confirmation, this is the first study to examine the relationship between aberrant DNA methylation of ncRNAs and the prognosis of PAC patients, and the results may contribute to the development of individual therapy.