Background
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in developed countries and is likely to rise to second place within the next decade worldwide [
1‐
3]. Despite survival being greatly improved in other cancers, the 5-year survival rate for PDAC remains < 3%, and the median survival time of patients with PDAC is usually less than 7.8 months in China [
4,
5]. The highly aggressive phenotype, which is characterized by rapid invasion and high risk of recurrence and metastasis, is one of the most important causes of the high mortality in PDAC [
6,
7]. Despite considerable advances in the prevention, diagnosis, and treatment of PDAC, no effective biomarkers or notably better therapeutic strategies have emerged [
8,
9]. Thus, it is crucial to further explore the biological and molecular mechanism of PDAC progression and develop a molecule-oriented method for early diagnosis and targeted therapy.
Circular RNAs (circRNAs) are non-coding RNAs with a covalently closed loop structure and arise from the back-splicing of pre-mRNA transcripts [
10,
11]. Unlike linear RNAs, circRNAs contain neither 5′-3′ polarities nor polyadenylated tails [
12]. Moreover, as a novel type of endogenous non-coding RNA, circRNAs are widespread, conserved, stable, and tissue specific [
11,
13]. Emerging evidence shows that circRNAs mainly function as a miRNA sponge to regulate the expression of the downstream target gene [
14]. In addition, some circRNAs have been found to interact with RNA binding proteins (RBPs) or function as templates for protein translation [
15,
16]. Moreover, previous studies have demonstrated that circRNAs exert impact on biological processes of cancers, including proliferation, migration, invasion, and apoptosis [
17]. However, the potential correlation between circRNAs and PDAC progression and the underlying mechanism remains unclear.
The mesenchymal-epithelial transition factor (MET), identified as a pivotal tyrosine kinase, plays critical roles in biological processes such as cell proliferation, morphogenesis, survival, and the initialization and progression of cancer [
18,
19]. Previous studies have demonstrated that MET was frequently overexpressed and played an important role in the progression of PDAC. For example, Neesse A.et al. found that MET is involved in the stromal biology of PDAC and promotes cancer progression [
20]. Logan-Collins.et al. revealed that MET plays critical roles in chemotherapy and radiation resistant of PDAC [
21]. Recently, increasing evidence has confirmed that circRNAs participate in tumorigenesis and the progression of a variety of cancers by regulating their downstream target genes [
22]. Thus, exploring the association between circRNAs and MET is of great significance for the development of effective therapeutic strategies in PDAC.
In the present study, we identified an oncogenic circRNA generated from exon 2 of the BFAR gene, termed circBFAR, which was overexpressed in a 208-case cohort of patients with PDAC. We demonstrated that circBFAR was aberrantly upregulated in PDAC cells, induced the proliferation and invasiveness phenotype in vitro, and promoted tumorigenesis and metastasis in vivo. Moreover, circBFAR overexpression correlated positively with progression and was related to poorer prognosis of patients with PDAC. Importantly, we revealed that circBFAR sponged miR-34b-5p to upregulate MET expression and therefore promoted PDAC progression. Administration of a MET inhibitor could effectively attenuate circBFAR-mediated tumorigenicity of PDAC cells in vivo. Collectively, our study revealed that the circBFAR/miR-34b-5p/MET axis played a crucial role in PDAC progression and in particular, identified circBFAR as a potential biomarker and therapeutic target in PDAC.
Discussion
As novel RNA molecules, numerous recent investigations have demonstrated that circRNAs play important roles in pleiotropically modulated cellular function [
32]. For instance, several circRNAs can function as miRNA sponges to regulate gene expression, combine with different proteins to influence function of associated proteins, or encode polypeptides that might have similar functions with their mRNA-encoded proteins [
15,
33‐
35]. Although circRNAs have been demonstrated to be involved in the progression of multiple cancers, the precise mechanism of their impact on the biological processes of PDAC is largely unknown. In the present study, we screened for differentially expressed circRNAs in six pairs of PDAC and matched non-tumorous tissues and identified an uncharacterized circRNA, termed as circBFAR, which was highly expressed in PDAC. Gain and loss function experiments demonstrated that circBFAR promoted the tumorigenesis and aggressiveness of PDAC in vitro and in vivo. Moreover, circBFAR overexpression was closely related with poor prognosis of patients with PDAC. Mechanistically, we found that circBFAR upregulated MET expression via sponging miR-34b-5p; thereby further activating the MET signaling pathway to induce the proliferation and migration of PDAC. In addition, we demonstrated that inhibition of MET significantly inhibited circBFAR-induced tumorigenicity of PDAC in vivo. Thus, our findings identify a novel regulatory mechanism by which a circRNA promotes PDAC proliferation and metastasis, and provide a new strategy for PDAC treatment.
Accumulating evidence have revealed that circRNAs regulate cellular function as miRNA sponges. Thomas. et al. found that ciRS-7 functioned as a sponge of miR-7, resulting in increased levels of miR-7 targets [
14]. Zheng. et al. reported that circHIPK3 acted as an miRNA sponge and bound to a host of miRNAs in human cancers [
23]. Herein, RNA pull-down assays showed that circBFAR interacted with miR-34b-5p. Luciferase reporter assays validated the sponge effect of circBFAR on miR-34b-5p and further confirmed the binding sites on circBFAR. In addition, rescue experiments showed that the circBFAR knockdown-induced suppression of colony formation, migration, and invasion could be rescued using an miR-34b-5p inhibitor. Our results provided evidence to support the view that circBFAR binds to miR-34b-5p, acting as “miRNA sponge”, which is essential to the progression of PDAC.
Previous studies have reported that the miR-34 family mediates its antitumor effects in a variety of cancers [
36]. Qu. et al. found that miR-34a was involved in long noncoding RNA lncARSR-mediated sunitinib drug resistance in renal cancer [
37]. Chen. et al. provided direct evidence that mir-34 regulates the stem cell compartment by downregulating MET expression in prostate cancer [
38]. Although miR-34 has been characterized as a vital tumor suppresser in tumor progression, the role and molecular mechanism underlying the regulation of miR-34b-5p in PDAC remains unclear. In the present study, we identified that miR-34b-5p directly bound to MET 3′ UTR to downregulate its expression. Moreover, MET depletion led to the inhibition of the MET signaling pathway, which ultimately resulted in the repression of PDAC progression. Importantly, we found that miR-34b-5p was sponged by circBFAR, which suppressed the targeting effect of miR-34b-5p on MET, leading to increased MET expression and the proliferation, migration, and invasion of PDAC. Therefore, the identification of the circBFAR/miR-34b-5p/MET axis expands our knowledge of the regulatory mechanism underlying PDAC progression.
Dysregulation of the MET signaling pathway occurs in a wide range of human cancers, including breast, colorectal, lung, pancreatic, hepatic, and ovarian cancers [
39‐
42]. Inhibition of MET signaling has emerged as a promising approach for cancer therapy. Martinez-Marti et al. found that the combination of the MET inhibitor, capmatinib, and Erb-B2 receptor tyrosine kinases, ErbB-1/2/4, inhibitors successfully inhibited tumor growth in NSCLC-bearing mice [
43]. Huang et al. reported that the combination of a MET inhibitor and an autophagy suppressor efficiently treated liver cancer in mice [
44]. Despite the remarkable successes in animal experiments, most approved agents have proven insufficient to cure human patients. A lack of appropriate indicators is one of the important causes for this failure; therefore, developing biomarkers for MET-targeting therapy might represent an effective approach to improve its therapeutic efficiency in PDAC [
45,
46]. In the present study, we found that overexpression of circBFAR significantly increased the expression of MET in PDAC. Moreover, silencing MET reversed circBFAR-induced progression of PDAC cells. Importantly, blockage of MET signaling using PHA dramatically inhibited the tumorigenesis in circBFAR-transduced PDAC-bearing mice in vivo
. Our findings provide evidence to support circBFAR as a potential biomarker for clinical MET-targeting therapy in PDAC.
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