Non-coding RNAs (ncRNAs), which occupy the vast majority of human transcripts are known for their inability to encode proteins. NcRNAs consist of a diverse range of RNA species, including long non-coding RNAs (lncRNAs), which have significant meaning for epigenetic modification, post-transcriptional regulation of target genes, molecular interference, etc. The dysregulation of ncRNAs will mediate the pathogenesis of diverse human diseases, like cancer. Pancreatic cancer, as one of the most lethal malignancies in the digestive system that is hard to make a definite diagnosis at an early clinicopathological stage with a miserable prognosis. Therefore, the identification of potential and clinically applicable biomarker is momentous to improve the overall survival rate and positively ameliorate the prognosis of patients with pancreatic carcinoma. LncRNAs as one kind of ncRNAs exert multitudinous biological functions, and act as molecular sponges, relying on microRNA response elements (MREs) to competitively target microRNAs (miRNAs), thereby attenuating the degradation or inhibition of miRNAs to their own downstream protein-coding target genes, also thus regulating the initiation and progression of neoplasms. LncRNAs, which emerge aforementioned function are called competing endogenous RNAs (ceRNAs). Consequently, abundant research of lncRNAs as potential biomarkers is of critical significance for the molecular diagnosis, targeted therapy, as well as prognosis monitoring of pancreatic cancer.

Background: Accumulation evidence indicates the vital role of long non-coding RNAs (lncRNAs) in tumorigenesis and the progression of malignant tumors, including pancreatic cancer (PC). However, the role and the molecular mechanism of long non-coding RNA 00976 is unclear in pancreatic cancer. Methods: In situ hybridization (ISH) and qRT-PCR was performed to investigate the association between linc00976 expression and the clinicopathological characteristics and prognosis of patients with PC. Subsequently, linc00976 over-expression vector and shRNAs were transfected into PC cells to up-regulate or down-regulate linc00976 expression. Loss- and gain-of function assays were performed to investigate the role of linc00976 in proliferation and metastasis in vitro and vivo. ITRAQ, bioinformatic analysis and rescue assay were used to illustrate the ceRNA mechanism network of linc00976/miR-137/OTUD7B and its downstream EGFR/MAPK signaling pathway. Results: linc00976 expression was overexpressed in PC tissues and cell lines and was positively associated with poorer survival in patients with PC. Function studies revealed that linc00976 knockdown significantly suppressed cell proliferation, migration and invasion in vivo and in vitro, whereas its overexpression reversed these effects. Based on Itraq results and online database prediction, Ovarian tumor proteases OTUD7B was found as a downstream gene of linc00976, which deubiquitinated EGFR mediates MAPK signaling activation. Furthermore, Bioinformatics analysis and luciferase assays and rescue experiments revealed that linc00976/miR137/OTUD7B established the ceRNA network modulating PC cell proliferation and tumor growth. Conclusion: The present study demonstrates that linc00976 enhances the proliferation and invasion ability of PC cells by upregulating OTUD7B expression, which was a target of miR-137. Ultimately, OTUD7B mediates EGFR and MAPK signaling pathway, suggesting that linc00976/miR-137/OTUD7B/EGFR axis may act as a potential biomarker and therapeutic target for PC.

Hypoxia contributes to pancreatic cancer progression and promotes its growth and invasion. Previous research principally focused on hypoxia-inducible factor-1 alpha (HIF-1α) and HIF-2α (HIF1A and EPAS1) as the major hypoxia-associated transcription factors in pancreatic cancer. However, the role of HIF-3α (HIF3A) has not been investigated. Therefore, HIF-1α, HIF-2α, and HIF-3α expression levels were measured under normoxic and hypoxic conditions. In addition, HIF-3α expression was measured in human pancreatic cancer tissue specimens and the impact of altered HIF-3α expression on cell invasion and migration was investigated in vitro and in vivo, as well as the underlying mechanisms. Under hypoxic conditions, HIF-3α expression was stimulated in pancreatic cancer cells to a greater degree than HIF-1α and HIF-2α expression. HIF-3α protein levels were also elevated in pancreatic cancer tissues and correlated with reduced survival and greater local invasion and distant metastasis, whereas knockdown of HIF-3α, under hypoxic conditions, suppressed pancreatic cancer cell invasion and migration. Under normoxia, HIF-3α overexpression promoted pancreatic cancer cell invasion and migration and stimulated F-actin polymerization. In summary, HIF-3α promotes pancreatic cancer cell invasion and metastasis in vivo and promotes pancreatic cancer cell invasion and metastasis by transcriptionally activating the RhoC–ROCK1 signaling pathway. Implications: HIF3α is overexpressed in pancreatic cancer, and targeting the HIF3α/RhoC–ROCK1 signaling pathway may be a novel therapeutic approach for the treatment of pancreatic cancer invasion and metastasis. Mol Cancer Res; 16(1); 124–34. ©2017 AACR.

Recently, long non-coding RNAs (lncRNA) have been proven to regulate pancreatic cancer (PC) progression. We aimed to explore the pathogenesis of LINC00941 in PC regarding protein binding. By using PCR analysis, we found that LINC00941 was overexpressed in PC tissues and was higher in patients with liver metastasis than in patients without liver metastasis. In addition, high LINC00941 expression was associated with a poor prognosis. Functional experiments and mice models were respectively used to evaluate PC cell proliferation and migration in vitro and in vivo. The results suggested that LINC00941 overexpression promoted PC proliferation and metastasis. Subsequently, RNA pull-down, mass spectrometry (MS), and RNA-binding protein immunoprecipitation (RIP) were performed to identify LINC00941-interacting proteins. The results suggested that ANXA2 was the potential LINC00941-interacting protein. Nucleotides 500–1390 of LINC00941 could bind to the Annexin 1 domain of ANXA2. LINC00941-mediated malignant phenotype of PC was reversed by ANXA2 depletion. Co-immunoprecipitation (Co-IP) followed by MS was conducted to determine the potential interacting protein of LINC00941. The results illustrated that NEDD4L, an E3 ligase involved in ubiquitin-mediated protein degradation, bound to the Annexin 1 domain of ANXA2 and promoted its degradation. Mechanically, LINC00941 functioned as a decoy to bind to ANXA2 and suppressed its degradation by enclosing the domain that binds to NEDD4L. Eventually, LINC00941 upregulated ANXA2 and activated FAK/AKT signaling, increasing PC cell proliferation and metastasis. This study indicates that LINC00941 promotes PC proliferation and metastasis by binding ANXA2 and potentiating its stability, leading to the activation of FAK/AKT signaling. Our data demonstrate that LINC00941 may serve as a novel target for prognosis and therapy.

Cancer stem cells (CSCs) play an important role in the development of pancreatic cancer. We previously showed that the microRNA miR-137 is downregulated in clinical samples of pancreatic cancer, and its expression negatively regulates the proliferation and invasiveness of pancreatic cancer cells. The stemness features of pancreatic cancer cells was detected by flow cytometry, immunofluorescence and sphere formation assay. Xenograft mouse models were used to assess the role of miR-137 in stemness features of pancreatic cancer cells in vivo. Dual-luciferase reporter assays were used to determine how miR-137 regulates KLF12. Bioinformatics and Chromatin immunoprecipitation analysis of KLF12 recruitment to the DVL2 promoters. Involvement of the Wnt/β-catenin pathways was investigated by western blot and Immunohistochemistry. miR-137 inhibits pancreatic cancer cell stemness in vitro and vivo. KLF12 as miR-137 target inhibits CSC phenotype in pancreatic cancer cells. Suppression of KLF12 by miR-137 inhibits Wnt/β-catenin signalling. KLF12 expression correlates with DVL2 and canonical Wnt pathway in clinical pancreatic cancer. Our results suggest that miR-137 reduces stemness features of pancreatic cancer cells by Targeting KLF12-associated Wnt/β-catenin pathways and may identify new diagnostic and therapeutic targets in pancreatic cancer.