Recently, the anti-cancer effect of nigericin has drawn increasing attentions, and its molecular mechanisms toward cancer cells were gradually discovered. A newly study by Yakisich et al. demonstrated that nigericin might be used in a co-therapy model of lung cancer in combination with other chemotherapeutic agents . Coincidentally, our lab also implied that Wnt/β-catenin signaling might have an essential role in colorectal cancer progression, and nigericin exerted anticancer effects on colorectal cancer cells by directly targeting the β-catenin destruction complex . Furthermore, our recent study has proved the potential toxicity of nigericin on human PC, and revealed the molecular mechanism of nigericin toward PC cells from the perspective of circRNA (31533620). However, the knowledge of nigericin needs to be further elucidated from multiple perspectives.
Along with the deepening of research on PC, numerous lncRNAs have shown to be essential for the tumorigenesis and progression by serving as tumor oncogenes or suppressors. In 2016, Li et al found that long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) could facilitate the advanced progression of PC by promoting autophagy in vitro . LncRNA myocardial infarction-associated transcript (MIAT) was found remarkably increased in PC tissues and cell lines, and PC patients with high MIAT level had poor prognosis than that with low MITA level . In contrast, Lnc-PCTST might exhibit as a potential tumor suppressor in PC, which inhibited cell proliferation, invasion, tumorigenesis and EMT by modulating TACC-3 . To further explore the anti-cancer mechanism of nigericin, we used high-throughput and bioinformatics methods to predict the changes of coding and non-coding RNAs when cells were exposed to the drug.
Firstly, the global expression profile of lncRNAs and mRNAs for 4 different nigericin treatment time points was determined by a custom sequencing platform. By venn analysis, our data confirmed that 76 common dys-regulated lncRNAs including 49 up-regulated and 27 down-regulated ones might participate in the process of nigericin damage. These lncRNAs were widely distributed on all chromosomes except for sex chromosome X. Meanwhile, the common differentially expressed mRNAs among the 3 compared groups were also found, in which 172 mRNAs were common up-regulated and 85 ones were down-regulated. Subsequently, we chose 5 random lncRNAs and 5 cancer-related genes for PCR detection between the 0 h and 32 h group, and the data were consistent well with our sequencing data, which demonstrated the high reliability and validity of the sequencing expression results. Of these common differentially expressed mRNAs, GADD45A was found to be variously expressed in cell lines derived from PC, and adenoviral-mediated expression of GADD45A (Ad-G45a) in these cells resulted in apoptosis via caspase activation and cell-cycle arrest in the G2/M phase . HMG-box transcription factor 1 (HBP1) had been described as a negative regulator of the Wnt/β-catenin signaling in many cancers, including breast cancer , osteosarcoma , glioma  and colorectal carcinoma . A recent study by Chan also indicated that HBP1 acted as a direct downstream target of FOXO1, and potently suppressed malignant phenotypes of oral cancer in oral cancer malignancy . Besides, other 3 validated genes (SESN2, SIK1 and KIF20A) were also proved to influence the proliferation, migration and invasion of PC cells [38–41]. These results might provide clues to the potential mechanisms of nigericin in PC.
Next, we conducted GO and KEGG pathway analyses to uncover the roles of these common differentially expressed mRNAs after nigericin treatment. The top 10 GO biological processes such as uridine catabolic process, nucleotide catabolic process and regulation of interleukin-6 biosynthetic process were found in the nigericin damage. Meanwhile, the differentially expressed mRNAs were significantly enriched in top 20 KEGG signaling pathways, including Aldosterone-regulated sodium reabsorption, Circadian rhythm, Mismatch repair, Drug metabolism-other enzymes, TNF signaling pathway, Transcriptional misregulation in cancers, TGF-beta signaling pathway, PI3K-Akt signaling pathway and so on. Moreover, the network between all KEGG pathways and their corresponding genes was also analyzed. These nigericin-related pathways have been also reported in PC. For example, the PI3K/Akt signaling pathway has long been related with PC metastasis. Tanno et al. showed that increased insulin-like growth factor I receptor expression induced by active Akt markedly enhanced the invasiveness of human PC cells . A recent review from Murthy et al also described the role of PI3K signaling in PC development and progression . In 2014, Zhu et al provided valuable baseline information regarding the TGF-β pathway in PC, which could be utilized in targeted therapy clinical trials . These involved non-coding RNAs (lncRNAs and mRNAs) and GO/KEGG analyses might partly explain the phenomena that nigericin had the anti-cancer properties.
To better understand the mechanisms of nigericin in PC cells, we built the co-expression network between lncRNAs and mRNAs. The network implied a complex relationship that one gene could correlate with multiple lncRNAs and one lncRNA might also regulate numerous mRNAs in different ways. For instance, up-regulated lnc-AGRN-2_9 was positively correlated with HBP1, GADD45A, SIK1 and SESN2, and negatively associated with TOP2A, CKAP2, while these mRNAs were implicated in tumorigenesis [32, 37–41, 45]. The co-expression network might imply the potential regulatory mechanisms between lncRNAs and mRNAs in the nigericin anti-cancer process.
It has been known that lncRNAs can cis-regulate the co-expressed and nearby coding genes . In this study, we constructed a cis-regulated network with the criterion that coding genes located at 100 kbp upstream and downstream of lncRNAs on the chromosome. Our results showed that 6 of 76 common differentially expressed lncRNAs possessed cis-regulated genes and each of the 6 lncRNAs only had one neighboring protein-coding gene. For example, we found that lnc-AGRN-2_3 and lnc-AGRN-2_9 shared the same cis-regulated gene MPV17L, which indicated that these two lncRNAs might play a similar role. Lnc-C9orf82-2_1 cis regulated ADAMTS1, and Masui et al. also suggested that ADAMTS1 was a potential biomarker to detect early-stage PCs . UNC50 has long been recognized as a Golgi apparatus protein in yeast, and is involved in nicotinic receptor trafficking in Caenorhabditis elegans. In 2015, Fang et al. found that UNC50 was correlated with G1/S transition and proliferation in hepatocellular carcinoma via the influencing epidermal growth factor receptor trafficking . Interestingly, our data showed that UNC50 was involved with the nigericin damage, which could be cis regulated by lnc-SLC25A3-3_1. These results revealed the prevalence of lncRNA-mediated cis regulation on nearby genes during the nigericin damage.
On the other hand, previous reports have indicated that lncRNAs are capable of binding to a specific site or sequence, including TFs, to achieve trans-regulation functions. We constructed a TF-lncRNA binary network combined by these common differentially expressed lncRNAs with TFs. The network showed that 44 up-regulated lncRNAs were found to correspond to 31 TFs, and 27 down-regulated lncRNAs corresponded to 12 TFs. Furthermore, we introduced target genes to build TF-lncRNA-gene ternary network. 10 up-regulated lncRNAs correspond to 3 TFs and 283 target genes, while 6 down-regulated lncRNAs were found to associate with 3 TFs and 125 target genes. Interestingly, up to 14 dys-regulated lncRNAs were regulated by 5 TFs, such as MYC, TAF1, E2F4, STAT1 and STAT2. Recent evidence strongly suggests that these 5 TFs potentially regulate the expression of target genes in PC or other cancers. For instance, Valenti et al. found that Mutp53 and E2F4 proteins formed a transcriptional repressive complex that assembled onto the regulatory regions of BRCA1 and RAD17 genes inhibiting their expressions in head and neck squamous cell carcinoma . Guerrero-Zotano et al. identified 18 of the 20 E2F4 target genes, and suggested a potential benefit of adjuvant CDK4/6 inhibitors in patients with ER+ breast cancer who failed to respond to preoperative estrogen deprivation . STAT1, which is a member of the family of signal transducers and transcription activators, corresponded to lymph node metastasis, advanced stage, tumor dedifferentiation and poor prognosis in patients with PC . A study from Seshacharyulu et al. also confirmed STAT1 as a key regulator through down-regualtion of MUC4 in PC . Thus, our cis- and trans-regulation predictions might provide a deep insight into the involved lncRNAs in nigericin treatment.
Finally, a PPI network with common differentially expressed genes, in which 12 hub proteins were identified, including TOP2A, MYC, ANAPC1, FBXW7, KIF20A, MTOR, CREB1, EXO1, MELK, NEDD4L, RACGAP1 and HERC2. The most significant hub proteins were TOP2A and MYC. TOP2A could induce tumor development and progression in many cancer types, including PC , prostate cancer  and breast cancer . In 2016, a phase II study by Tarpgaard et al. found that metastatic colorectal cancer (mCRC) patients, who were refractory to treatment with oxaliplatin-based chemotherapy, had TOP2A gene amplification in their tumor cells . Similarly, human estrogen receptor-positive breast cancer cells typically displayed elevated levels of Myc protein due to overexpression of MYC mRNA , and other studies had also identified the abnormal expression of MYC-binding protein (MYCBP) during tumorigenesis in multiple types of cancer, such as gastric cancer , colon cancer  and PC . Therefore, this core PPI network exhibited the associations between these interested genes, which might provide useful clues for the mechanism analysis of nigericin in PC.