The noncanonical function of borealin, a component of chromosome passenger complex, promotes glycolysis via stabilization of survivin in squamous cell carcinoma cells.

The chromosome passenger complex (CPC) is a kinase complex formed by Aurora B, borealin, survivin and inner centromere protein (INCENP). The CPC is active during mitosis and contributes to proper chromosome segregation via the phosphorylation of various substrates. Overexpression of each CPC component has been reported in most cancers. However, its significance remains unclear, as only survivin is known to confer chemoresistance. This study showed that the overexpression of borealin, a CPC component, stabilized survivin protein depending on its interaction with survivin. Unexpectedly, the accumulation of survivin by borealin overexpression did not affect the well-characterized functions of survivin, such as chemoresistance and cell proliferation. Interestingly, the overexpression of borealin promoted lactate production but not the overexpression of the deletion mutant that lacks the ability to bind to survivin. Consistent with these findings, the expression levels of glycolysis-related genes were enhanced in borealin-overexpressing cancer cells. Meanwhile, the overexpression of survivin alone did not promote lactate production. Overall, the accumulation of the borealin-survivin complex promoted glycolysis in squamous cell carcinoma cells. This mechanism may contribute to cancer progression via excessive lactate production.

Involvement of the OTUB1-YAP1 axis in driving malignant behaviors of head and neck squamous cell carcinoma.

BACKGROUND: Comprehending the molecular mechanisms underlying head and neck squamous cell carcinoma (HNSCC) is vital for the development of effective treatment strategies. Deubiquitinating enzymes (DUBs), which regulate ubiquitin-dependent pathways, are potential targets for cancer therapy because of their structural advantages. Here we aimed to identify a potential target for HNSCC treatment among DUBs. METHODS: A screening process was conducted using RNA sequencing data and clinical information from HNSCC patients in the TCGA database. A panel of 88 DUBs was analyzed to identify those associated with poor prognosis. Subsequently, HNSCC cells were modified to overexpress specific DUBs, and their effects on cell proliferation and invasion were evaluated. In vivo experiments were performed to validate the findings. RESULTS: In HNSCC patients, USP10, USP14, OTUB1, and STAMBP among the screened DUBs were associated with a poor prognosis. Among them, OTUB1 showed the most aggressive characteristics in both in vitro and in vivo experiments. Additionally, OTUB1 regulated the stability and nuclear localization of YAP1, a substrate involved in cell proliferation and invasion. Notably, OTUB1 expression exhibited a positive correlation with the HNSCC-YAP score in HNSCC cells. CONCLUSIONS: This study highlights the critical role of OTUB1 in HNSCC progression via modulating YAP1. Targeting the OTUB1-YAP1 axis holds promise as a potential therapeutic strategy for HNSCC treatment.

CD4+ T-cell-dependent differentiation of CD23+ follicular B cells contributes to the pulmonary pathology in a primary Sjögren's syndrome mouse model.

Introduction: Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease that affects the function of exocrine glands, such as the lacrimal and the salivary glands. Extraglandular lesions and malignant lymphoma also occur during the progressive stage of pSS. We have, herein, focused on the pulmonary lesions of pSS and have aimed clarifying their pathophysiological mechanism by comparing the glandular with the extraglandular lesions observed in a mouse model of pSS. Results: The histopathological analysis of lung tissues obtained from NFS/sld mice that have undergone neonatal thymectomy was performed. Moreover, in vivo and in vitro experiments were conducted along with immunological analyses in order to characterize the unique phenotypes of the pulmonary lesions identified in these pSS model mice. Inflammatory lesions with a bronchus-associated lymphoid tissue-like structure were identified in the lungs of pSS model mice. In addition, relative to salivary gland lesions, pulmonary lesions showed increased CD23+ follicular B (FB) cells. In vitro and pulmonary B cells were more readily driven to CD23+ FB cell phenotype than salivary gland B cells in pSS model mice. Furthermore, the CD23+ FB cell differentiation was found to be enhanced in a CD4+ T-cell-dependent manner under a Th2-type condition in the lungs of herein examined pSS model mice. Discussion: A Th2-type response in the pSS lung may promote the progression of autoimmune lesions through an enhanced abnormal differentiation of B cells.

Cancer cell-derived novel periostin isoform promotes invasion in head and neck squamous cell carcinoma.

It recently has been reported that partial-epithelial-mesenchymal transition (p-EMT) program is associated with metastasis in head and neck squamous cell carcinoma (HNSCC). We previously have identified POSTN (which encodes periostin) as an invasion-promoting molecule in HNSCC. Interestingly, POSTN expression is frequently observed in cancer cells with higher p-EMT score by using a previous single-cell transcriptomic data of HNSCC cases. Although it is known that POSTN has 11 splicing variants, the role of them has not been determined in HNSCC. Here, we found that HNSCC cells with EMT features expressed POSTN isoforms, Iso3 (lacking exon 17 and 21) and Iso5 (lacking exon 17), whereas fibroblast expressed Iso3 and Iso4 (lacking exon 17, 18, and 21). The expression of POSTN Iso3 and Iso4 are known to be widely observed in various cell types including stromal cells. Therefore, we focused on the role of novel cancer cell-derived POSTN isoform, Iso5, in HNSCC. Single overexpression of POSTN Iso5 as well as Iso3 promoted invasion. Surprisingly, Iso5 synergistically promoted invasion together with Iso3. Notably, Iso5 as well as Iso3 upregulated p-EMT-related genes. We suggest that a novel cancer-specific POSTN isoform lacking exon 17 (Iso5) can be a useful marker for detecting cancer cells undergoing p-EMT. Moreover, a POSTN Iso5 can be a novel target for diagnosis and therapy in HNSCC.

Disturbed natural killer cell homeostasis in the salivary gland enhances autoimmune pathology via IFN-γ in a mouse model of primary Sjögren's syndrome.

Objective: Innate lymphoid cells (ILCs), including natural killer (NK) cells, ILC1, ILC2, lymphoid tissue-inducer (LTi) cells, and ILC3 cell, play a key role in various immune responses. Primary Sjögren's syndrome (pSS) is an autoimmune disease characterized by chronic inflammation of exocrine glands, such as the lacrimal and salivary glands (SGs). The role of NK cells among ILCs in the pathogenesis of pSS is still unclear. In this study, the characteristics and subsets of NK cells in the salivary gland (SG) tissue were analyzed using a murine model of pSS. Methods: Multiple phenotypes and cytotoxic signature of the SG NK cells in control and pSS model mice were evaluated by flow cytometric analysis. Intracellular expression of interferon-γ (IFN-γ) among T cells and NK cells from the SG tissues was compared by in vitro experiments. In addition, pathological analysis was performed using anti-asialo-GM1 (ASGM1) antibody (Ab)-injected pSS model mice. Results: The number of conventional NK (cNK) cells in the SG of pSS model mice significantly increased compared with that in control mice at 6 weeks of age. The production level of IFN-γ was significantly higher in SG NK cells than in SG T cells. The depletion of NK cells by ASGM1 Ab altered the ratio of tissue resident NK (rNK) cells to cNK cells, which inhibited the injury to SG cells with the recovery of saliva secretion in pSS model mice. Conclusion: The results indicate that SG cNK cells may enhance the autoreactive response in the target organ by upregulating of IFN-γ, whereas SG rNK cells protect target cells against T cell cytotoxicity. Therefore, the activation process and multiple functions of NK cells in the target organ could be helpful to develop potential markers for determining autoimmune disease activity and target molecules for incurable immune disorders.

Exposure to Multiwall Carbon Nanotubes Promotes Fibrous Proliferation by Production of Matrix Metalloproteinase-12 via NF-κB Activation in Chronic Peritonitis.

The toxicologic effects of nanomaterials, such as carbon nanotubes (CNTs), on the immune system are understood well. However, the precise relationship between long-term exposure to CNTs and chronic inflammation remains unclear. In this study, a mouse model of chronic peritonitis was established using i.p. injection of multiwalled CNTs treated by the Taquann method with high dispersion efficiency. Chronic peritonitis with fibrosis was observed in Taquann-treated multiwalled CNT (T-CNT)-injected mice, but not in Taquann-treated titanium dioxide-injected mice. In vivo and in vitro experiments showed that matrix metalloproteinase-12 (MMP-12) of macrophages was up-regulated by T-CNT to enhance fibroblast activation and profibrotic molecule expression in fibroblasts. In addition, T-CNT-induced peritonitis reduced MMP-12 expression in Nfκb1-/- mice, suggesting that MMP-12-producing macrophages play a key role in chronic inflammation due to T-CNT exposure through NF-κB activation. The results of this study could be helpful in understanding the molecular toxicity of nanomaterial and chronic inflammation.

Virus Infections Play Crucial Roles in the Pathogenesis of Sjögren's Syndrome.

Sjögren's syndrome (SS) is an autoimmune disease especially targeting exocrine glands, such as the salivary and lacrimal glands. A radical therapy for SS based on its etiology has not been established because of the complex pathogenesis of the disease. Several studies have demonstrated a relationship between virus infection and SS pathogenesis. In particular, infection with the Epstein-Barr (EB) virus among others is a potent factor associated with the onset or development of SS. Specifically, virus infection in the target organs of SS triggers or promotes autoreactive responses involving the process of autoantigen formation, antigen-presenting function, or T-cell response. Our review of recent research highlights the crucial roles of virus infection in the pathogenesis of SS and discusses the critical association between virus infection and the etiology of autoimmunity in SS.

Protective effects of resveratrol against 5-fluorouracil-induced oxidative stress and inflammatory responses in human keratinocytes.

Although 5-fluorouracil (5-FU) is currently used as an anti-cancer chemotherapy, adverse effects such as oral mucositis potentially limit its clinical application. Additionally, the prevention of 5-FU-induced side effects are scarce. Resveratrol is known to decrease oxidative damage and inflammation. In this study, we examined the protective effects of resveratrol on 5-FU-induced oxidative stress and inflammatory responses in normal human keratinocytes (HaCaT cell) as in vitro oral mucositis model. HaCaT cells were exposed to 5-FU and simultaneously treated with resveratrol. The effects of resveratrol on 5-FU-induced cytotoxicity were evaluated using cell viability assay. The production of reactive oxygen species (ROS) was measured using a fluorescence spectrophotometer. The effects of resveratrol on nuclear factor erythroid 2-related factor 2 (Nrf2), silent information regulator transcript-1 (SIRT-1), and nuclear factor kappa B (NF-κB) signaling and inflammatory cytokine expression were examined. Resveratrol suppressed 5-FU-induced overproduction of ROS by upregulating anti-oxidant defense genes through Nrf2 activation and SIRT-1 expression. Concerning inflammatory responses, resveratrol suppressed the 5-FU-induced expression of pro-inflammatory cytokines via NF-κB nuclear translocation. Conversely, N-acetylcysteine reduced ROS levels without affecting the expression of pro-inflammatory cytokines. Resveratrol might be useful for preventing 5-FU-induced adverse effects by activating anti-oxidant and anti-inflammatory responses.

Formation of Autoimmune Lesions Is Independent of Antibiotic Treatment in NOD Mice.

The relationship between autoimmunity and changes in intestinal microbiota is not yet fully understood. In this study, the role of intestinal microbiota in the onset and progression of autoimmune lesions in non-obese diabetic (NOD) mice was evaluated by administering antibiotics to alter their intestinal microenvironment. Flow cytometric analysis of spleen cells showed that antibiotic administration did not change the proportion or number of T and B cells in NOD mice, and pathological analysis demonstrated that autoimmune lesions in the salivary glands and in the pancreas were also not affected by antibiotic administration. These results suggest that the onset and progression of autoimmunity may be independent of enteral microbiota changes. Our findings may be useful for determining the appropriate use of antibiotics in patients with autoimmune diseases who are prescribed drugs to maintain systemic immune function.

APC/CCdh1 is required for the termination of chromosomal passenger complex activity upon mitotic exit.

During mitosis, the chromosomal passenger complex (CPC) ensures the faithful transmission of the genome. The CPC is composed of the enzymatic component Aurora B (AURKB) and the three regulatory and targeting components borealin, INCENP, and survivin (also known as BIRC5). Although the CPC is known to be involved in diverse mitotic events, it is still unclear how CPC function terminates after mitosis. Here we show that borealin is ubiquitylated by the anaphase promoting complex/cyclosome (APC/C) and its cofactor Cdh1 (also known as FZR1) and is subsequently degraded in G1 phase. Cdh1 binds to regions within the N terminus of borealin that act as a non-canonical degron. Aurora B has also been shown previously to be degraded by the APC/CCdh1 from late mitosis to G1. Indeed, Cdh1 depletion sustains an Aurora B activity with stable levels of borealin and Aurora B throughout the cell cycle, and causes reduced efficiency of DNA replication after release from serum starvation. Notably, inhibition of Aurora B kinase activity improves the efficiency of DNA replication in Cdh1-depleted cells. We thus propose that APC/CCdh1 terminates CPC activity upon mitotic exit and thereby contributes to proper control of DNA replication.

Prognostic value of partial EMT-related genes in head and neck squamous cell carcinoma by a bioinformatic analysis.

OBJECTIVE: Recent studies have revealed that the ability of cancer cells to undergo intermediate state of epithelial-to-mesenchymal transition (EMT), partial EMT (p-EMT), poses a higher metastatic risk rather than complete EMT. Here, we examined the prognostic value of p-EMT-related genes in head and neck squamous cell carcinoma (HNSCC) by bioinformatic approaches. MATERIALS AND METHODS: We used RNA-seq data of 519 primary HNSCC cases obtained from TCGA database. We compared the expression of p-EMT-related genes in HNSCC tissues with normal tissues. We evaluated the prognostic value of p-EMT-related genes in HNSCC cases by log-rank test. We examined the expression of p-EMT-, EMT-, and epithelial differentiation-related genes by qPCR. RESULTS: Among p-EMT-related genes that were highly expressed in HNSCC cases, high expression of SERPINE1, ITGA5, TGFBI, P4HA2, CDH13, and LAMC2 was significantly correlated with poor survival of HNSCC patients. By gene expression pattern, HNSCC cell lines were classified into three groups: epithelial phenotype, EMT phenotype, and p-EMT phenotype. CONCLUSIONS: Our findings suggest that p-EMT program may be involved in poor prognosis of HNSCC. SERPINE1, ITGA5, TGFBI, P4HA2, CDH13, and LAMC2 can be used for a prognostic marker. Moreover, HNSCC cells with p-EMT phenotype can be a useful model for investigating a nature of p-EMT.

The pan-cancer landscape of prognostic germline variants in 10,582 patients.

BACKGROUND: While clinical factors such as age, grade, stage, and histological subtype provide physicians with information about patient prognosis, genomic data can further improve these predictions. Previous studies have shown that germline variants in known cancer driver genes are predictive of patient outcome, but no study has systematically analyzed multiple cancers in an unbiased way to identify genetic loci that can improve patient outcome predictions made using clinical factors. METHODS: We analyzed sequencing data from the over 10,000 cancer patients available through The Cancer Genome Atlas to identify germline variants associated with patient outcome using multivariate Cox regression models. RESULTS: We identified 79 prognostic germline variants in individual cancers and 112 prognostic germline variants in groups of cancers. The germline variants identified in individual cancers provide additional predictive power about patient outcomes beyond clinical information currently in use and may therefore augment clinical decisions based on expected tumor aggressiveness. Molecularly, at least 12 of the germline variants are likely associated with patient outcome through perturbation of protein structure and at least five through association with gene expression differences. Almost half of these germline variants are in previously reported tumor suppressors, oncogenes or cancer driver genes with the other half pointing to genomic loci that should be further investigated for their roles in cancers. CONCLUSIONS: Germline variants are predictive of outcome in cancer patients and specific germline variants can improve patient outcome predictions beyond predictions made using clinical factors alone. The germline variants also implicate new means by which known oncogenes, tumor suppressor genes, and driver genes are perturbed in cancer and suggest roles in cancer for other genes that have not been extensively studied in oncology. Further studies in other cancer cohorts are necessary to confirm that germline variation is associated with outcome in cancer patients as this is a proof-of-principle study.

Achaete-Scute Homologue 2-Regulated Follicular Helper T Cells Promote Autoimmunity in a Murine Model for Sjögren Syndrome.

Follicular helper T (Tfh) cells contribute to various immune responses as well as to the pathogenesis of several immune diseases. However, the precise mechanism underlying the onset or development of autoimmunity via Tfh cells remains unclear. Herein, the detailed relationship between autoimmune disease and Tfh cells was analyzed using a murine model for Sjögren syndrome (SS) wherein the mice underwent neonatal thymectomy. Germinal center (GC) development was promoted in this SS model along with an increase of Tfh cells and GC B cells. The severity of the autoimmune lesions was correlated with the number of Tfh cells detected in the spleen of the SS model mice. In addition, treatment with an anti-CD20 monoclonal antibody effectively suppressed the autoimmune lesions with a reduction of Tfh cells and GC B cells. Comprehensive gene analysis revealed that several genes associated with Tfh cell differentiation, including achaete-scute homologue 2 (Ascl2), were up-regulated in peripheral CD25- CD4+ T cells in SS model mice compared with those in control mice. Moreover, an experiment using CD4CreBcl6fl/fl mice that received neonatal thymectomy treatment demonstrated that Ascl2 contributes to the Tfh cell differentiation associated with autoimmunity during the early stages, independent of Bcl6. In conclusion, our results indicate that abnormal Tfh cell differentiation via Ascl2 regulation might contribute to the pathogenesis of autoimmunity.

CCL22-Producing Resident Macrophages Enhance T Cell Response in Sjögren's Syndrome.

Macrophages (MΦs) are critical regulators of immune response and serve as a link between innate and acquired immunity. The precise mechanism of involvement of tissue-resident MΦs in the pathogenesis of autoimmune diseases is not clear. Here, using a murine model for Sjögren's syndrome (SS), we investigated the role of tissue-resident MΦs in the onset and development of autoimmunity. Two unique populations of CD11bhigh and CD11blow resident MΦs were observed in the target tissue of the SS model. Comprehensive gene expression analysis of chemokines revealed effective production of CCL22 by the CD11bhigh MΦs. CCL22 upregulated the migratory activity of CD4+ T cells by increasing CCR4, a receptor of CCL22, on T cells in the SS model. In addition, CCL22 enhanced IFN-γ production of T cells of the SS model, thereby suggesting that CCL22 may impair the local immune tolerance in the target organ of the SS model. Moreover, administration of anti-CCL22 antibody suppressed autoimmune lesions in the SS model. Finally, histopathological analysis revealed numerous CCL22-producing MΦs in the minor salivary gland tissue specimens of the SS patients. CCL22-producing tissue-resident MΦs may control autoimmune lesions by enhancing T cell response in the SS model. These results suggest that specific chemokines and their receptors may serve as novel therapeutic or diagnostic targets for SS.

Effect of coffee or coffee components on gut microbiome and short-chain fatty acids in a mouse model of metabolic syndrome.

We previously showed that male Tsumura Suzuki obese diabetes (TSOD) mice, a spontaneous mouse model of metabolic syndrome, manifested gut dysbiosis and subsequent disruption of the type and quantity of plasma short-chain fatty acids (SCFAs), and daily coffee intake prevented nonalcoholic steatohepatitis in this mouse model. Here, we present a preliminary study on whether coffee and its major components, caffeine and chlorogenic acid, would affect the gut dysbiosis and the disrupted plasma SCFA profile of TSOD mice, which could lead to improvement in the liver pathology of these mice. Three mice per group were used. Daily intake of coffee or its components for 16 wk prevented liver lobular inflammation without improving obesity in TSOD mice. Coffee and its components did not repair the altered levels of Gram-positive and Gram-negative bacteria and an increased abundance of Firmicutes in TSOD mice but rather caused additional changes in bacteria in six genera. However, caffeine and chlorogenic acid partially improved the disrupted plasma SCFA profile in TSOD mice, although coffee had no effects. Whether these alterations in the gut microbiome and the plasma SCFA profile might affect the liver pathology of TSOD mice may deserve further investigation.

Long-term polarization of alveolar macrophages to a profibrotic phenotype after inhalation exposure to multi-wall carbon nanotubes.

BACKGROUND: Nanomaterials are widely used in various fields. Although the toxicity of carbon nanotubes (CNTs) in pulmonary tissues has been demonstrated, the toxicological effect of CNTs on the immune system in the lung remains unclear. METHODS AND FINDING: In this study, exposure to Taquann-treated multi-walled CNTs (T-CNTs) was performed using aerosols generated in an inhalation chamber. At 12 months after T-CNT exposure, alveolar inflammation with macrophage accumulation and hypertrophy of the alveolar walls were observed. In addition, fibrotic lesions were enhanced by T-CNT exposure. The macrophages in the bronchoalveolar lavage fluid of T-CNT-exposed mice were not largely shifted to any particular population, and were a mixed phenotype with M1 and M2 polarization. Moreover, the alveolar macrophages of T-CNT-exposed mice produced matrix metalloprotinase-12. CONCLUSIONS: These results suggest that T-CNT exposure promoted chronic inflammation and fibrotic lesion formation in profibrotic macrophages for prolonged periods.

Neutrophils induce smooth muscle hyperplasia via neutrophil elastase-induced FGF-2 in a mouse model of asthma with mixed inflammation.

BACKGROUND: Bronchial asthma is traditionally characterized by chronic allergic inflammation, including eosinophilia and elevated Th2 cytokines. Recently, IL-17-derived neutrophil infiltration was shown to correlate with asthma severity and airway remodelling. OBJECTIVE: To investigate the role of IL-17-derived neutrophils in airway remodelling in chronic bronchial asthma. METHODS: We utilized house dust mite antigen-induced mouse models of asthma. Intranasal sensitization and chronic antigen challenge caused a mixed allergic inflammation that included eosinophils and neutrophils (Mix-in group). We neutralized IL-17 and fibroblast growth factor (FGF-2) and investigated the mechanism of airway remodelling in the Mix-in group. RESULTS: The Mix-in group displayed neutrophilic infiltration and high levels of IL-17 in lung tissue. The Mix-in group also exhibited more bronchial smooth muscle hyperplasia. IL-17 neutralization decreased the magnitude of all of these effects in the Mix-in group. Antibody arrays revealed an increase in FGF-2 in the Mix-in Group relative to the Eo-ip group, and FGF-2 elevation was associated with smooth muscle hypertrophy/hyperplasia. High concentrations of neutrophil elastase enhanced E-cadherin/ß-catenin signalling in bronchial epithelial cells. Neutrophil elastase inhibitor treatment decreased FGF-2 production and E-cadherin/ß-catenin signalling, which inhibited smooth muscle hyperplasia. CONCLUSION: The IL-17/neutrophil axis may play an important role in airway remodelling by contributing to smooth muscle hypertrophy/hyperplasia in mixed allergic inflammation and accordingly represents an attractive therapeutic target for severe asthma.

Invasion-Related Factors as Potential Diagnostic and Therapeutic Targets in Oral Squamous Cell Carcinoma-A Review.

It is well recognized that the presence of cervical lymph node metastasis is the most important prognostic factor in oral squamous cell carcinoma (OSCC). In solid epithelial cancer, the first step during the process of metastasis is the invasion of cancer cells into the underlying stroma, breaching the basement membrane (BM)-the natural barrier between epithelium and the underlying extracellular matrix (ECM). The ability to invade and metastasize is a key hallmark of cancer progression, and the most complicated and least understood. These topics continue to be very active fields of cancer research. A number of processes, factors, and signaling pathways are involved in regulating invasion and metastasis. However, appropriate clinical trials for anti-cancer drugs targeting the invasion of OSCC are incomplete. In this review, we summarize the recent progress on invasion-related factors and emerging molecular determinants which can be used as potential for diagnostic and therapeutic targets in OSCC.

Aurora Kinase Inhibitors in Head and Neck Cancer.

Aurora kinases are a group of serine/threonine kinases responsible for the regulation of mitosis. In recent years, with the increase in Aurora kinase-related research, the important role of Aurora kinases in tumorigenesis has been gradually recognized. Aurora kinases have been regarded as a new target for cancer therapy, resulting in the development of Aurora kinase inhibitors. The study and application of these small-molecule inhibitors, especially in combination with chemotherapy drugs, represent a new direction in cancer treatment. This paper reviews studies on Aurora kinases from recent years, including studies of their biological function, their relationship with tumor progression, and their inhibitors.

The microRNA-15a-PAI-2 axis in cholangiocarcinoma-associated fibroblasts promotes migration of cancer cells.

BACKGROUND: Cholangiocarcinoma (CCA) has an abundance of tumor stroma which plays an important role in cancer progression via tumor-promoting signals. This study aims to explore the microRNA (miRNA) profile of CCA-associated fibroblasts (CCFs) and the roles of any identified miRNAs in CCA progression. METHODS: miRNA expression profiles of CCFs and normal skin fibroblasts were compared by microarray. Identified downregulated miRNAs and their target genes were confirmed by real-time PCR. Their binding was confirmed by a luciferase reporter assay. The effects of conditioned-media (CM) of miRNA mimic- and antagonist-transfected CCFs were tested in CCA migration in wound healing assays. Finally, the levels of miRNA and their target genes were examined by real-time PCR and immunohistochemistry in clinical CCA samples. RESULTS: miR-15a was identified as a downregulated miRNA in CCFs. Moreover, PAI-2 was identified as a novel target gene of miR-15a. Recombinant PAI-2 promoted migration of CCA cells. Moreover, CM from miR-15a mimic-transfected CCFs suppressed migration of CCA cells. Lower expression of miR-15a and higher expression of PAI-2 were observed in human CCA samples compared with normal liver tissues. Importantly, PAI-2 expression correlated with poor prognosis in CCA patients. CONCLUSIONS: These findings highlight the miR-15a/PAI-2 axis as a potential therapeutic target in CCA patients.