Cisplatin can act through lncRNAs that regulate disease progression in lung cancer by sponging miRNAs, whose interaction with lncRNAs modulates a variety of genes in cancer apoptosis, migration, proliferation and resistance [37–42]. As the role of lncRNAs in the anticancer effect of monofunctional platinum(II) complexes had not yet been assessed, we used microarray analysis to identify regulated lncRNAs in A549 and IMR90 cells after treatment with cisplatin or phenanthriplatin. To validate our microarray results, we performed qRT-PCR on total RNA extracts from cisplatin and phenanthriplatin treated cell preparations and found that the relative fold change expression of regulated lncRNAs calculated via PCR amplification generally corroborated the microarray data (Fig. 2). Next, we applied bioinformatics methods to our microarray results and discovered that a set of miRNAs predicted to target the three most upregulated lncRNAs by cisplatin treatment also had high sequence homology to the three most downregulated lncRNAs, AGO2-1, COX7A1-2 and SLC26A3-1, after phenanthriplatin treatment (Table 1, 2). These results suggest that cisplatin might upregulate a set of lncRNAs that sponge and nullify the gene regulatory effect of these miRNAs; whereas, phenanthriplatin, by reducing the expression of lncRNAs that could prevent the action of the same miRNAs, might promote miRNA gene silencing.
Several of the miRNAs detected in our analysis are associated with Wnt/β-catenin signaling. Expression of miR-25-5p may be targeted by two lncRNAs regulated by phenanthriplatin, e.g., lnc-COX7A1-2, that the monofunctional complex reduces, and lnc-MRPL39-10, which phenanthriplatin increases (Table 2). The sponging of miR-25-5p via increased lnc-MRPL39-10 expression could promote anticancer effect through the Wnt pathway as the reduction of this miRNA in A549 cells is associated with decreased cell viability, invasion and migration by decreasing Wnt signaling (Fig. 3) .
As phenanthriplatin reduced the expression of the lncRNAs, AGO2-1, COX7A1-2, and SLC26A3-1 (Table 2), this could promote the action of miRNAs that might be sponged by these lncRNAs. Increased miR-149-3p and miR-185-5p expression downregulates Wnt/β-catenin signaling reducing cancer proliferation in A549 and lung adenocarcinoma cells [33–34]. Overexpression of miR-608 induced cell death in A549 cells via signaling that integrates the Wnt pathway . MiR-708-5p expression also suppressed DNA methylation decreasing Wnt/β-catenin signaling and impairing the stemness characteristics of NSCLC cells . Therefore, phenanthriplatin could act to suppress NSCLC by repressing lncRNAs that regulate miR-25-5p, -185-5p, -608, and − 708-5p, through the Wnt/β-catenin pathway (Fig. 3).
However, increasing the expression of some of the miRNAs may also promote cancer through the Wnt/β-catenin pathway. Suppression of miR-378 in A549 cells inactivates Wnt/β-catenin signaling leading to reduced cell proliferation . MiR-650 can be upregulated in A549 cells where it promoted proliferation and invasion through the Wnt-1/β‑catenin pathway by acting on inhibitor of growth 4 . Increased expression of miR-1253, which directly targets Wnt5A, is associated with the proliferation, migration, and invasion of NSCLC cells . Similarly, miR-1254 targets the Wnt/β-catenin pathway antagonist, secreted frizzled related protein 1 (SFRP1), and was upregulated in lung cancer tissues and cells promoting proliferation . Our results suggest that the monofunctional complex might act against NSCLC via Wnt/β-catenin signaling if the miRNAs, miR-149-3p, -185-5p, -608, and − 708-5p were not sponged by the phenanthriplatin downregulated lncRNAs, AGO2-1, COX7A1-2 and SLC26A3-1. Nonetheless, increased expression of the miRs, -25-5p, -378, -650, -1253, and − 1254, when these lncRNAs are suppressed by the monofunctional complex, could promote cancer by modulating the Wnt/β-catenin pathway possibly in conjunction with other pathway signaling (Fig. 3).
LncRNAs are also able to act in NSCLC through the TGF-β pathway and integrated MAPK/ERK and PI3K/AKT signaling [43–44]. We identified several miRNAs that might integrate TGF-β signaling components through lncRNAs regulated by phenanthriplatin (Fig. 3). Overexpression of miR-608 induced cell death in A549 cells through the MAPK/ERK pathway . As the three lncRNAs, AGO2-1, COX7A1-2, and SLC26A3-1, which are predicted to sponge miR-608, were downregulated by the monofunctional complex (Table 2), we would expect that expression of this miRNA would lead to NSCLC cell death via ERK signaling. However, suppression of AGO2-1, COX7A1-2, and SLC26A3-1, could also have an opposite effect in NSCLC cells by upregulating the miRs, -25-5p, -30a-3p and − 378, which act via MAPK/ERK, and are projected targets of these lncRNAs (Table 2), and have reduced anti-cancer efficacy when their expression is increased [25, 28, 30]. As miR-25-5p may be sponged via phenanthriplatin-mediated upregulation of lnc-MRPL39-10 (Table 2), this miRNA could be downregulated in A549 cells, which is associated with anti-cancer effect via PI3K signaling . Reduction of AGO2-1, COX7A1-2, and SLC26A3-1 by phenanthriplatin would also be expected to lead to increased miR-138-5p and − 608, whose expression modulates PTEN/PI3K/AKT signaling and is associated in A549 cells with anticancer effect [23, 31, 45–47]. In retinoblastoma tumors, increased miR-4516 expression promotes tumor growth through the PTEN/AKT pathway .
We also examined whether cisplatin and phenanthriplatin might regulate Wnt/β-catenin and TGF-β signaling differently in IMR90 compared to A549 cells through lncRNA regulation of miRNAs. Interestingly, a majority of the miRNAs, e.g., miR-149-3p, -185-5p, -608, -650, -708-5p, and − 4516, identified as potential targets of lncRNAs in A549 cells, were predicted to be regulated identically in IMR90 cells albeit by different lncRNAs (Table 4, Fig. 4). Specifically, cisplatin upregulates lncRNAs, e.g., ATAD2B-5, GNG11-3, and MRPS5-1, that collectively should sponge all of these miRNAs, and phenanthriplatin downregulates lncRNAs, e.g., AGO2-1, ARCN1-1, and HYPM-1, that should allow expression of these miRNAs. Cisplatin also downregulates HYPM-1, which should allow expression of miR-608; whereas, the monofunctional complex upregulates TMEM243-1, a lncRNA with high sequence homology to this miRNA. Therefore, regulation of Wnt/β-catenin and TGF-β signaling through miR-149-3p, -185-5p, -608, -650, -708-5p, and − 4516 would be predicted to be similar in both IMR90 and A549 cells.
However, we also identified a potential miRNA target, miR-4458, unique to IMR90 cells. Cisplatin treatment upregulated lncRNA, ATAD2B-5, which might sponge miR-4458, while phenanthriplatin downregulated both AGO2-1 and HYPM-1, lncRNAs with targeting homology to this miRNA (Table 4). As increased expression of miR-4458 prevents proliferation and migration in NSCLC cell lines via the PI3K/AKT pathway , cisplatin treatment might suppress miR-4458 with ATAD2B-5 allowing cancer progression, while phenanthriplatin, by decreasing the expression of AGO2-1 and HYPM-1, could allow miR-4458 expression through modulating the PI3K/AKT pathway to promote anti-cancer activity.
As in A549 cells, phenanthriplatin regulated a distinct set of lncRNAs compared to cisplatin in IMR90 cells (Table 1, 3) and might modulate Wnt/β-catenin and TGF-β signaling differently in normal lung fibroblast cells than in lung cancer cells (Fig. 3, 4). In A549 cells, miR-25-5p expression may be sponged by upregulated lnc-MRPL39-10, but we found in IMR90 cells that this miRNA would be potentially decoyed by lnc-HYPM-1, which is downregulated by phenanthriplatin (Table 2, 3, 4). The absence of suppression of miR-25-5p by lnc-HYPM-1 in IMR90 cells could promote cell proliferation and viability via Wnt/β-catenin and/or TGF-β signaling integrating MAPK/ERK as found in A549 cells . Similarly, miR-138-5p and miR-608 are potentially not subject to sponging in A549 cells treated with the monofunctional complex as their lncRNA targets are downregulated, but in IMR90 cells, these miRNA could be sponged by upregulated lnc-TMEM243-1 (Table 2, 3, 4). Therefore, reduced expression of miR-138-5p and − 608 due to TMEM243-1 decoying in IMR90 cells could lead to reduced cytotoxicity via TGF-β signaling (miR-138-5p) or Wnt/β-catenin and TGF-β signaling (miR-608) as suggested by the effects of suppressing these miRNAs in A549 cells [23, 46–49].