Therapeutic restoration of miR-126-3p as a multi-targeted strategy to modulate the liver tumor microenvironment.

BACKGROUND: Impaired natural killer (NK) cell-mediated antitumor responses contribute to the growth of liver tumors. Expression of a disintegrin and metalloprotease 9 (ADAM9) increases shedding of membrane-bound major histocompatibility complex class I chain-related protein A and results in evasion from NK cell-mediated cytolysis. ADAM9 is also involved in angiogenesis and tumor progression and is a target of miR-126-3p, a tumor suppressor that is downregulated and alters tumor cell behavior in the liver and other cancers. We evaluated the restoration of miR-126-3p and modulation of the miR-126-3p/ADAM9 axis as a therapeutic approach to simultaneously enhance NK cell-mediated cytolysis while targeting both tumor cells and their microenvironment. METHODS: Precursor miRNAs were loaded into milk-derived nanovesicles to generate therapeutic vesicles (therapeutic milk-derived nanovesicles) for the restoration of functional miR-126-3p in recipient cancer cells. RESULTS: Administration of therapeutic milk-derived nanovesicles increased miR-126-3p expression and reduced ADAM9 expression in target cells and was associated with an increase in membrane-bound major histocompatibility complex class I chain-related protein A. This enhanced NK cell cytolysis in adherent tumor cells and in multicellular tumor spheroids while also impairing angiogenesis and modulating macrophage chemotaxis. Moreover, IV administration of therapeutic milk-derived nanovesicles with adoptive transfer of NK cells reduced tumor burden in orthotopic hepatocellular cancer xenografts in mice. CONCLUSION: A directed RNA therapeutic approach can mitigate NK cell immune evasion, reduce angiogenesis, and alter the tumor cell phenotype through the restoration of miR-126-3p in liver tumor cells. The pleiotropic effects elicited by this multi-targeted approach to modulate the local tumor microenvironment support its use for the treatment of liver cancer.

Engineering Cell-Derived Nanovesicles for Targeted Immunomodulation.

Extracellular vesicles (EVs) show promise for targeted drug delivery but face production challenges with low yields. Cell-derived nanovesicles (CDNVs) made by reconstituting cell membranes could serve as EV substitutes. In this study, CDNVs were generated from mesenchymal stem cells by extrusion. Their proteomic composition, in vitro and in vivo toxicity, and capacity for loading RNA or proteins were assessed. Compared with EVs, CDNVs were produced at higher yields, were comprised of a broader range of proteins, and showed no detrimental effects on cell proliferation, DNA damage, or nitric oxide production in vitro or on developmental toxicity in vivo. CDNVs could be efficiently loaded with RNA and engineered to modify surface proteins. The feasibility of generating immunomodulatory CDNVs was demonstrated by preparing CDNVs with enhanced surface expression of PD1, which could bind to PD-L1 expressing tumor cells, enhance NK and T cell degranulation, and increase immune-mediated tumor cell death. These findings demonstrate the adaptability and therapeutic promise of CDNVs as promising substitutes for natural EVs that can be engineered to enhance immunomodulation.

Metabolic liver cancer: associations of rare and common germline variants in one-carbon metabolism and DNA methylation genes.

Animal studies implicate one-carbon metabolism and DNA methylation genes in hepatocellular carcinoma (HCC) development in the setting of metabolic perturbations. Using human samples, we investigated the associations between common and rare variants in these closely related biochemical pathways and risk for metabolic HCC development in a multicenter international study. We performed targeted exome sequencing of 64 genes among 556 metabolic HCC cases and 643 cancer-free controls with metabolic conditions. Multivariable logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs), adjusting for multiple comparisons. Gene-burden tests were used for rare variant associations. Analyses were performed in the overall sample and among non-Hispanic whites. The results show that among non-Hispanic whites, presence of rare functional variants in ABCC2 was associated with 7-fold higher risk of metabolic HCC (OR = 6.92, 95% CI: 2.38-20.15, P = 0.0004), and this association remained significant when analyses were restricted to functional rare variants observed in ≥2 participants (cases 3.2% versus controls 0.0%, P = 1.02 × 10-5). In the overall multiethnic sample, presence of rare functional variants in ABCC2 was nominally associated with metabolic HCC (OR = 3.60, 95% CI: 1.52-8.58, P = 0.004), with similar nominal association when analyses were restricted to functional rare variants observed in ≥2 participants (cases 2.9% versus controls 0.2%, P = 0.006). A common variant in PNPLA3 (rs738409[G]) was associated with higher HCC risk in the overall sample (P = 6.36 × 10-6) and in non-Hispanic whites (P = 0.0002). Our findings indicate that rare functional variants in ABCC2 are associated with susceptibility to metabolic HCC in non-Hispanic whites. PNPLA3-rs738409 is also associated with metabolic HCC risk.

RAB27B Drives a Cancer Stem Cell Phenotype in NSCLC Cells Through Enhanced Extracellular Vesicle Secretion.

Cancer stem cells (CSC) within non-small cell lung carcinoma (NSCLC) tumors drive NSCLC progression, metastasis, relapse, and intrinsic chemoresistance. Understanding the mechanisms that support the malignant phenotypes of NSCLC CSCs may provide insights for improved NSCLC therapeutic interventions. Here, we report that expression of RAB27B, a small GTPase, is significantly upregulated in NSCLC CSCs when compared with bulk cancer cells (BCC). Short hairpin RNA-mediated knockdown of RAB27B leads to a loss of stem cell marker gene expression and reduced NSCLC spheroid growth, clonal expansion, transformed growth, invasion, and tumorigenicity. We find that NSCLC CSCs secrete significantly more extracellular vesicles (EV) than BCCs, and that this is RAB27B-dependent. Furthermore, CSC-derived EVs, but not BCC-derived EVs, induce spheroid growth, clonal expansion, and invasion in BCCs. Finally, RAB27B is required for CSC-derived EV-induced stemness in BCCs. Taken together, our results indicate that RAB27B is required for maintenance of a highly tumorigenic, cancer-initiating, invasive stem-like cell population in NSCLC and RAB27B is involved in propagating EV-mediated communication from NSCLC CSCs to BCCs. Our findings further suggest that inhibition of RAB27B-dependent EV secretion may be a potential therapeutic strategy for NSCLC. Significance: Expression of RAB27B in CSCs leads to elevated levels of EVs that mediate communication between CSCs and BCCs that maintains a stem-like phenotype in NSCLC cells.

Extracellular vesicle­mediated miR­126­3p transfer contributes to inter­cellular communication in the liver tumor microenvironment.

Extracellular vesicles (EVs) and their contents are gaining recognition as important mediators of intercellular communication through the transfer of bioactive molecules, such as non­coding RNA. The present study comprehensively assessed the microRNA (miRNA/miR) content within EVs released from HepG2 liver cancer (LC) cells and LX2 hepatic stellate cells (HSCs) and determined the contribution of EV miRNA to intercellular communication. Using both transwell and spheroid co­cultures of LC cells and HSCs, miR­126­3p within EV was established as a mediator of HSC to LC cell communication that influenced tumor cell migration and invasion, as well as the growth of multicellular LC/HSC spheroids. Manipulation of miR­126­3p either by enforced expression using pre­miR­126­3p or by inhibition using antimiR­126­3p did not alter tumor cell viability, proliferation or sensitivity to either sorafenib or regorafenib. By contrast, enforced expression of miR­126­3p decreased tumor­cell migration. Knockdown of miR­126­3p in tumor cells increased disintegrin and metalloproteinase domain­containing protein 9 (ADAM9) expression and in HSCs increased collagen­1A1 accumulation with an increase in compactness of multicellular spheroids. Within LC/HSC spheroids, ADAM9 and vascular endothelial growth factor expression was increased by silencing of miR­126­3p but diminished with the restoration of miR­126­3p. These studies implicate miR­126­3p in functional effects on migration, invasion and spheroid growth of tumor cells in the presence of HSCs, and thereby demonstrate functional EV­RNA­based intercellular signaling between HSCs and LC cells that is directly relevant to tumor­cell behavior.

Development of a Lyophilized Off-the-Shelf Mesenchymal Stem Cell-Derived Acellular Therapeutic.

The therapeutic activities elicited by mesenchymal stem cells (MSC) are in part mediated through paracrine action by the release of extracellular vesicles (EV) and secreted proteins. Collectively, these MSC-derived factors, referred to as the secretome product (SP), are intrinsically therapeutic and represent an attractive alternative to cell-based therapies. Herein, we developed a lyopreservation protocol to extend the shelf-life of the MSC-SP without compromising the structural or functional integrity of the vesicular components. The SP isolated from normoxia- and anoxia-exposed MSC elicited protective effects in an in vitro model of oxidative injury and the bioactivity was retained in the lyophilized samples. Three separate formulations of MSC-SP were isolated by tangential flow filtration using sucrose, trehalose, and mannitol as lyoprotectant agents. The MSC-SPs were lyophilized using a manifold protocol and the structural and functional integrity were assessed. The trehalose formulation of SP exhibited the highest EV and protein recovery after manifold-based lyophilization. To facilitate development as a therapeutic, a shelf lyophilization protocol was developed which markedly enhanced the recovery of EV and proteins. In conclusion, lyophilization represents an efficient method to preserve the structural and functional integrity of the MSC-SP and can be used to develop an off-the-shelf therapeutic.

Extracellular RNA transfer from non-malignant human cholangiocytes can promote cholangiocarcinoma growth.

Extracellular vesicles (EV) within the cellular secretome are emerging as modulators of pathological processes involved in tumor growth through their ability to transfer donor-derived RNA into recipient cells. While the effects of tumor and stromal cell EVs within the tumor microenvironment have been studied, less is known about the contributions of normal, nontransformed cells. We examined the impact of EVs within the cellular secretome from nonmalignant cells on transformed cell growth and behavior in cholangiocarcinoma cells. These effects were enhanced in the presence of the pro-fibrogenic mediator TGF-ß. We identified miR-195 as a TGF-ß responsive miRNA in normal cells that can be transferred via EV to tumor cells and regulate cell growth, invasion, and migration. The effects of miR-195 involve modulation of the epithelial-mesenchymal transition through direct effects on the transcription factor Snail. These studies provide in vitro and in vivo evidence for the impact of normal cellular secretome on transformed cell growth, show the importance of EV RNA transfer, and identify mechanisms of EV-mediated transfer of miRNA as a contributor to tumor development, which may provide new therapeutic opportunities for targeting human cholangiocarcinoma.

Evaluation of In Vivo Toxicity of Biological Nanoparticles.

Biologically derived nanoparticles such as extracellular vesicles are promising candidates for therapeutic applications. In vivo toxicity of biological nanoparticles can result in tissue or organ damage, immunological perturbations, or developmental effects but cannot be readily predicted from in vitro studies. Therefore, an essential component of the preclinical assessment of these particles for their use as therapeutics requires screening for adverse effects and detailed characterization of their toxicity in vivo. However, there are no standardized, comprehensive methods to evaluate the toxicity profile of nanoparticle treatment in a preclinical model. Here, we first describe a method to prepare bovine milk-derived nanovesicles (MNVs). These MNVs are inexpensive to isolate, have a scalable production platform, and can be modified to achieve a desired biological effect. We also describe two vertebrate animal models, mice and zebrafish, that can be employed to evaluate the toxicity profile of biologically derived nanoparticles, using MNVs as an example. Treatment-induced organ toxicity and immunological effects can be assessed in mice receiving systemic injections of MNVs, and developmental toxicity can be assessed in zebrafish embryos exposed to MNVs in embryo water. Utilizing these animal models provides opportunities to analyze the toxicity profiles of therapeutic extracellular vesicles in vivo. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of milk-derived nanovesicles Basic Protocol 2: In vivo screening for organ toxicity and immune cell profiling using mice Basic Protocol 3: In vivo developmental toxicity screening using zebrafish.

Fabrication and Characterization of a Biomaterial Based on Extracellular-Vesicle Functionalized Graphene Oxide.

Mesenchymal stem cell (MSC) derived extracellular vesicles (EV) are emerging as acellular therapeutics for solid organ injury and as carriers for drug delivery. Graphene-based materials are novel two-dimensional crystal structure-based materials with unique characteristics of stiffness, strength and elasticity that are being explored for various structural and biological applications. We fabricated a biomaterial that would capture desirable properties of both graphene and stem cell derived EV. Metabolically engineered EV that express azide groups were cross-linked with alkyne-functionalized graphene oxide (GO) via a copper catalyzed alkyne-azide cycloaddition (CuAAC) reaction. The crosslinking between EV and GO was accomplished without the need for ligand expression on the metal. Scanning electron and fluorescence microscopy demonstrated excellent cross-linking between EV and GO. Biological effects were assessed by phagocytosis studies and cell viability studies. The uptake of GO or sonicated GO (sGO) resulted in a durable pro-inflammatory immune response. Cell studies further showed that crosslinked GO-EV scaffolds exhibited cell-type dependent cytotoxicity on liver cancer cells whereas there was minimal impact on healthy hepatocyte proliferation. In vitro, neither GO-EV nor sGO-EV induced DNA strand breaks. In vivo studies in zebrafish revealed gross developmental malformations but treatment-induced mortality was only seen with the highest doses of GO-EV and sGO-EV. With these advantages, this engineered biomaterial combining the versatility of graphene with the therapeutic effects of MSC-EV has potential for applications in tissue engineering and regenerative medicine.

Quantitation of Long Noncoding RNA Using Digital PCR.

Long noncoding RNAs (lncRNAs) are implicated in many physiological or disease processes and alterations in their expression may contribute to the development of various diseases. Accurate quantitation of lncRNA can be useful in measuring changes in expression in different settings such as in the circulation where the measurement of lncRNA may be useful as a biomarker of disease. However, the low levels of lncRNA expression require the use of highly sensitive detection technologies for accurate quantitation. Digital polymerase chain reaction (dPCR) is a sensitive method for absolute quantification of lncRNA and can be useful for measurement of gene expression when transcript levels are low. By providing a direct measurement without normalization, the use of dPCR may provide advantages for quantitation of low-abundance targets.

Detection of Circulating RNA Using Nanopore Sequencing.

RNA sequencing using nanopore sequencing is a powerful method for transcriptome analysis. The approach is appropriate for comprehensive profiling of the wide range of long noncoding RNAs. Use of nanopore-based sequencing can provide information on novel transcripts, sequence polymorphisms, and splicing variants, and thus has advantages over other gene expression profiling methods such as microarrays. Circulating extracellular long noncoding RNAs are of particular interest because of their potential use as biomarkers. Here, we describe a protocol for cDNA-PCR sequencing of circulating RNA for biomarker discovery in whole blood samples using commercially available kits and nanopore sequencing.

Biological Nanotherapeutics for Liver Disease.

Extracellular vesicles (EVs) are a heterogeneous group of biological nano-sized vesicles that are released from cells and contribute to intercellular communication. Emerging knowledge about their biogenesis, composition, release, and uptake has resulted in broad interest in elucidating their potential roles in disease pathophysiology. The distinct biological properties of these biological nanoparticles emphasize several appealing advantages for potential therapeutic applications compared with the use of synthetic nanoparticles. When administered systemically, EVs are taken up and sequestered within the liver, further emphasizing opportunities for therapeutic use. Consequently, there is growing interest in their use for liver diseases. EVs can be used directly as therapeutics, and several studies have highlighted the intrinsic therapeutic properties of mesenchymal stem cell-derived EVs for chronic and acute liver diseases. Alternatively, EVs can be modified to facilitate their use for the delivery of therapeutic cargo. In this review, we discuss the cellular sources of EV, provide a concise overview of their potential use in diverse processes, and outline several promising applications for the use of EV-based therapeutics for liver diseases. The use of EV-based therapeutics provides a viable approach to target hepatic pathophysiology.

Targeting Liver Cancer Stem Cells Using Engineered Biological Nanoparticles for the Treatment of Hepatocellular Cancer.

By exploiting their biological functions, the use of biological nanoparticles such as extracellular vesicles can provide an efficient and effective approach for hepatic delivery of RNA-based therapeutics for the treatment of liver cancers such as hepatocellular cancer (HCC). Targeting liver cancer stem cells (LCSC) within HCC provide an untapped opportunity to improve outcomes by enhancing therapeutic responses. Cells with tumor-initiating capabilities such as LCSC can be identified by expression of markers such as epithelial cell adhesion molecule (EpCAM) on their cell surface. EpCAM is a target of Wnt/ß-catenin signaling, a fundamental pathway in stem-cell growth. Moreover, mutations in the ß-catenin gene are frequently observed in HCC and can be associated with constitutive activation of the Wnt/ß-catenin pathway. However, targeting these pathways for the treatment of HCC has been challenging. Using RNA nanotechnology, we developed engineered biological nanoparticles capable of specific and effective delivery of RNA therapeutics targeting ß-catenin to LCSC. Extracellular vesicles isolated from milk were loaded with small interfering RNA to ß-catenin and decorated with RNA scaffolds to incorporate RNA aptamers capable of binding to EpCAM. Cellular uptake of these EpCAM-targeting therapeutic milk-derived nanovesicles in vitro resulted in loss of ß-catenin expression and decreased proliferation. The uptake and therapeutic efficacy of these engineered biological nanotherapeutics was demonstrated in vivo using tumor xenograft mouse models. Conclusion: ß-catenin can be targeted directly to control the proliferation of hepatic cancer stem cells using small interfering RNA delivered using target-specific biological nanoparticles. Application of this RNA nanotechnology-based approach to engineer biological nanotherapeutics provides a platform for developing cell-surface molecule-directed targeted therapeutics.

Safety of bovine milk derived extracellular vesicles used for delivery of RNA therapeutics in zebrafish and mice.

Extracellular vesicles are endogenous biological nanoparticles that have potential for use as therapeutic nanoparticles or as delivery vehicles for therapeutic agents. Milk nanovesicles (MNV) are extracellular vesicles isolated from bovine milk that have been explored for use as delivery vehicles for RNA therapeutics such as small interfering RNA (siRNA). We performed in vivo toxicological studies of MNV or therapeutic MNV (tMNV) loaded with siRNA as a prelude to their clinical use. Development toxicity was assessed in zebrafish embryos. Acute toxicity was assessed in both mice and zebrafish whereas safety, biochemical, histological and immune effects after multiple dosing were assessed in mice. Zebrafish embryo hatching was accelerated with MNV and tMNV. While acute toxicity or effects on mortality were not observed in zebrafish, developmental effects were observed at high concentrations of MNV. There was a lack of discernable toxicity, mortality and systemic inflammatory or immunological responses in mice following administration of either MNVs or tMNVs. The tolerability and lack of discernable developmental or systemic in vivo toxicity support their use as biological nano-therapeutics. Adoption of a standardized protocol for systematic analysis of in vivo safety and toxicity will facilitate preclinical assessment of EV based formulations for therapeutic use.

Isolation of Tissue Extracellular Vesicles from the Liver.

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

Extracellular Vesicle-Based Therapeutic Targeting of ß-Catenin to Modulate Anticancer Immune Responses in Hepatocellular Cancer.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Although HCC can respond to immune checkpoint inhibitors, such as monoclonal antibodies against programmed death 1 (PD-1), many patients fail to respond or develop secondary resistance. Activation of Wnt/ß-catenin signaling can contribute to immune evasion. Mutations in ß-catenin are among the most frequent mutations associated with HCC. Thus, our aim was to directly target ß-catenin to enhance the therapeutic response to immune checkpoint inhibition. A synthetic transgenic mouse model of experimental HCC induced by tyrosine-protein kinase Met/ß-catenin expression and extracellular vesicles (EVs) as a therapeutic delivery agent was used to evaluate the efficacy of directly targeting ß-catenin on the response to anti-PD-1. These studies showed that (1) oncogenic ß-catenin could be therapeutically targeted using a biological nanoparticle-based delivery approach, (2) targeting ß-catenin using small interfering RNA (siRNA) delivered within EVs can reduce tumor growth, and (3) the therapeutic response to anti-PD-1 can be enhanced by concomitantly targeting ß-catenin using therapeutic EVs. These preclinical studies establish the efficacy of the use of biological nanoparticles as an endogenous delivery vehicle for therapeutic RNA delivery and support the use of therapeutic strategies targeting tumor-intrinsic ß-catenin as an adjunct to anti-PD-1-based therapy. Conclusion: Combination therapy with anti-PD-1 and ß-catenin siRNA delivered using biological nanoparticles provides an effective strategy for the treatment of HCC. This strategy could be further exploited into targeted approaches for immune potentiation by countering oncogene-mediated resistance to immunotherapies.

Small RNA Sequencing across Diverse Biofluids Identifies Optimal Methods for exRNA Isolation.

Poor reproducibility within and across studies arising from lack of knowledge regarding the performance of extracellular RNA (exRNA) isolation methods has hindered progress in the exRNA field. A systematic comparison of 10 exRNA isolation methods across 5 biofluids revealed marked differences in the complexity and reproducibility of the resulting small RNA-seq profiles. The relative efficiency with which each method accessed different exRNA carrier subclasses was determined by estimating the proportions of extracellular vesicle (EV)-, ribonucleoprotein (RNP)-, and high-density lipoprotein (HDL)-specific miRNA signatures in each profile. An interactive web-based application (miRDaR) was developed to help investigators select the optimal exRNA isolation method for their studies. miRDar provides comparative statistics for all expressed miRNAs or a selected subset of miRNAs in the desired biofluid for each exRNA isolation method and returns a ranked list of exRNA isolation methods prioritized by complexity, expression level, and reproducibility. These results will improve reproducibility and stimulate further progress in exRNA biomarker development.

Therapeutic Efficacy of Vitamin D in Experimental c-MET-ß-Catenin-Driven Hepatocellular Cancer.

Aberrant activation of ß-catenin signaling is frequently observed in hepatocellular cancer. Although Wnt/ß-catenin signaling can be targeted by vitamin D, therapeutic use of vitamin D for this purpose is not currently established. We evaluated the therapeutic use of vitamin D or its analogs using a synthetic transgenic mouse of hepatocarcinogenesis induced by mutant ß-catenin, and MET overexpression in which 75% of mice develop well-differentiated HCC within 8 weeks in the absence of fibrosis. Vitamin D receptor expression was similar in both tumoral and nontumoral tissue. There was no significant difference in overall survival, or in tumor progression assessed by imaging, biochemical, or tumor cell burden assessments in mice receiving a vitamin D-supplemented diet containing 12.0 IU VD/g (HVD) compared with a standard diet (SD) containing 2.3 IU VD/g. Furthermore, systemic treatment with calcitriol [vitamin D analog 1α,25(OH)2D3] or EB1089 (synthetic vitamin D analog) by intraperitoneal injection for 4 weeks prolonged median survival but did not increase overall survival compared with controls. Although tumor formation was delayed in males compared with that in females, there was no difference in overall survival between males and females. In conclusion, although 1α,25(OH)2D3 is reported to inhibit ß-catenin signaling, as well as proliferation, migration, and differentiation in cancer cells, neither dietary supplementation with vitamin D nor treatment with vitamin D analogs altered the formation or growth of HCC associated with ß-catenin activation. These results conclusively demonstrate the lack of utility of targeting vitamin D for therapy of HCC in this setting.

Network analyses-based identification of circular ribonucleic acid-related pathways in intrahepatic cholangiocarcinoma.

Circular ribonucleic acids are non-coding ribonucleic acids that can be identified from genome sequencing studies. Although they can be readily detected, their regulation and functional role in human diseases such as cancer are unknown. Using a systematic approach, we analyzed ribonucleic acid-sequencing data from a well-characterized cohort of intrahepatic cholangiocarcinoma to identify genetic pathways related to circular ribonucleic acids. Although the expression of most circular ribonucleic acids was similar in both the cancer and non-cancer tissues, expression of circ2174 was significantly increased in cancer tissues. Network analysis of co-related genes identified several pathways associated with circ2174, and common regulatory mediators between genes in these pathways and circ2174. Among these, alterations in several genes involved in interleukin-16 signaling responses such Lck, interleukin-16, and macrophage inflammatory protein-1-beta were the most prominent. Octamer transcription factor (Oct)-2 was identified as a signal transducer that was common to both circ2174 and interleukin-16. Circ2174 has sequence complementarity to miR149 which can target Oct-2. These data suggest a mechanism whereby circ2174 can act as a sponge to regulate the expression of miR149, and thereby modulate Oct-2 and interleukin-16 signaling pathways in cholangiocarcinoma.