ZNF460-mediated circRPPH1 promotes TNBC progression through ITGA5-induced FAK/PI3K/AKT activation in a ceRNA manner.

BACKGROUND: Circular RNAs are highly stable regulatory RNAs that have been increasingly associated with tumorigenesis and progression. However, the role of many circRNAs in triple-negative breast cancer (TNBC) and the related mechanisms have not been elucidated. METHODS: In this study, we screened circRNAs with significant expression differences in the RNA sequencing datasets of TNBC and normal breast tissues and then detected the expression level of circRPPH1 by qRT‒PCR. The biological role of circRPPH1 in TNBC was then verified by in vivo and in vitro experiments. Mechanistically, we verified the regulatory effects between circRPPH1 and ZNF460 and between circRPPH1 and miR-326 by chromatin immunoprecipitation (ChIP), fluorescence in situ hybridization assay, dual luciferase reporter gene assay and RNA pull-down assay. In addition, to determine the expression of associated proteins, we performed immunohistochemistry, immunofluorescence, and western blotting. RESULTS: The upregulation of circRPPH1 in TNBC was positively linked with a poor prognosis. Additionally, both in vivo and in vitro, circRPPH1 promoted the biologically malignant behavior of TNBC cells. Additionally, circRPPH1 may function as a molecular sponge for miR-326 to control integrin subunit alpha 5 (ITGA5) expression and activate the focal adhesion kinase (FAK)/PI3K/AKT pathway. CONCLUSION: Our research showed that ZNF460 could promote circRPPH1 expression and that the circRPPH1/miR-326/ITGA5 axis could activate the FAK/PI3K/AKT pathway to promote the progression of TNBC. Therefore, circRPPH1 can be used as a therapeutic or diagnostic target for TNBC.

MiR-326-mediated overexpression of NFIB offsets TGF-ß induced epithelial to mesenchymal transition and reverses lung fibrosis.

Idiopathic Pulmonary Fibrosis (IPF) is a progressively fatal and incurable disease characterized by the loss of alveolar structures, increased epithelial-mesenchymal transition (EMT), and aberrant tissue repair. In this study, we investigated the role of Nuclear Factor I-B (NFIB), a transcription factor critical for lung development and maturation, in IPF. Using both human lung tissue samples from patients with IPF, and a mouse model of lung fibrosis induced by bleomycin, we showed that there was a significant reduction of NFIB both in the lungs of patients and mice with IPF. Furthermore, our in vitro experiments using cultured human lung cells demonstrated that the loss of NFIB was associated with the induction of EMT by transforming growth factor beta (TGF-ß). Knockdown of NFIB promoted EMT, while overexpression of NFIB suppressed EMT and attenuated the severity of bleomycin-induced lung fibrosis in mice. Mechanistically, we identified post-translational regulation of NFIB by miR-326, a miRNA with anti-fibrotic effects that is diminished in IPF. Specifically, we showed that miR-326 stabilized and increased the expression of NFIB through its 3'UTR target sites for Human antigen R (HuR). Moreover, treatment of mice with either NFIB plasmid or miR-326 reversed airway collagen deposition and fibrosis. In conclusion, our study emphasizes the critical role of NFIB in lung development and maturation, and its reduction in IPF leading to EMT and loss of alveolar structures. Our study highlights the potential of miR-326 as a therapeutic intervention for IPF. The schema shows the role of NFIB in maintaining the normal epithelial cell characteristics in the lungs and how its reduction leads to a shift towards mesenchymal cell-like features and pulmonary fibrosis. A In normal lungs, NFIB is expressed abundantly in the epithelial cells, which helps in maintaining their shape, cell polarity and adhesion molecules. However, when the lungs are exposed to factors that induce pulmonary fibrosis, such as bleomycin, or TGF-ß, the epithelial cells undergo epithelial to mesenchymal transition (EMT), which leads to a decrease in NFIB. B The mesenchymal cells that arise from EMT appear as spindle-shaped with loss of cell junctions, increased cell migration, loss of polarity and expression of markers associated with mesenchymal cells/fibroblasts. C We designed a therapeutic approach that involves exogenous administration of NFIB in the form of overexpression plasmid or microRNA-326. This therapeutic approach decreases the mesenchymal cell phenotype and restores the epithelial cell phenotype, thus preventing the development or progression of pulmonary fibrosis.

MiR-326 sponges TET2 triggering imbalance of Th17/Treg differentiation to exacerbate pyroptosis of hepatocytes in concanavalin A-induced autoimmune hepatitis.

INTRODUCTION AND OBJECTIVES: MicroRNA-326 is abnormally expressed in autoimmune diseases, but its roles in autoimmune hepatitis (AIH) are unknown. In this study, we aimed to investigate the effect of miR-326 on AIH and the underlying mechanism. MATERIALS AND METHODS: Concanavalin A was administrated to induce AIH in mice and the expression levels of miR-326 and TET2 was evaluated by qRT-PCR and western blot, respectively. The percentages of Th17 and Treg cells were evaluated by flow cytometry and their marker proteins were determined by western blot and ELISA. The mitochondrial membrane potential (MMP) and ROS level were tested with the JC-1 kit and DCFH-DA assay. The binding relationships between miR-326 and TET2 were verified by dual-luciferase reporter assay. The liver tissues were stained by the HE staining. In vitro, AML12 cells were cocultured with mouse CD4+T cells. The expression levels of pyroptosis-related proteins were assessed by western blot. RESULTS: Concanavalin A triggered AIH and enhanced the expression level of miR-326 in mice. It increased both Th17/Treg ratio and the levels of their marker proteins. The expression of TET2 was decreased in AIH mice. Knockdown of miR-326 could decrease the levels of pyroptosis-related proteins, the ROS level and increase MMP. In mouse CD4+T cells, miR-326 sponged TET2 to release IL-17A. Coculture of AML12 cells with isolated CD4+T cells from miR-326 knockdown AIH mice could relieve pyroptosis. CONCLUSIONS: Knockdown of miR-326 exerted anti-pyroptosis effects via suppressing TET2 and downstream NF-κB signaling to dampen AIH. We highlighted a therapeutic target in AIH.

Silencing of circRPPH1 Inhibits the Progression of Non-Small-Cell Lung Cancer Through miR-326/ERBB4 Signal Axis.

Circular RNAs (circRNAs) play critical roles in the recurrence and progression of non-small-cell lung cancer (NSCLC). This study aimed to investigate the function and underlying mechanism of a novel circRNA (circRPPH1) in NSCLC. Localization of circRPPH1 was determined via FISH assay, while cell proliferation was assessed via CCK8 and colony formation assay. Cell migration and invasion were studied using transwell assay, while binding sites between miR-326 and circRPPH1 or ERBB4 were verified by luciferase reporter, RIP, and RNA pull-down assays. Moreover, xenograft assay was performed to verify the in vivo roles of circRPPH1. Results indicated that circRPPH1 was highly expressed in NSCLC tissues and cells, where circRPPH1 levels were predictive of poor prognosis. The malignant behavior of NSCLC cells was exacerbated by overexpressing circRPPH1, while opposite effects were observed when it was knocked down. Direct interaction between miR-326 and circRPPH1 or ERBB4 was confirmed in NSCLC cells, while rescue experiment results showed that circRPPH1 exerted an oncogenic role via miR-326-ERBB4 signal axis. Moreover, in vitro, growth of NSCLC cells was significantly attenuated following circRPPH1 depletion. The study concluded that circRPPH1 was involved in promoting NSCLC progression via the miR-326/ERBB4 axis, which provided a novel potential target for the diagnosis or treatment of NSCLC.

MicroRNA-326 negatively regulates CD155 expression in lung adenocarcinoma.

Treatment with immune checkpoint inhibitors induces a durable response in some patients with non-small-cell lung cancer, but eventually gives rise to drug resistance. Upregulation of CD155 expression is implicated as one mechanism of resistance to programmed death receptor-1 (PD-1)/PD-1 ligand (PD-L1) inhibitors, and it is therefore important to characterize the mechanisms underlying regulation of CD155 expression in tumor cells. The aim of this study was to identify microRNAs (miRNAs) that might regulate CD155 expression at the posttranscriptional level in lung cancer. Comprehensive miRNA screening with target prediction programs and a dual-luciferase reporter assay identified miR-346, miR-328-3p, miR-326, and miR-330-5p as miRNAs that bind to the 3'-UTR of CD155 mRNA. Forced expression of these miRNAs suppressed CD155 expression in lung cancer cell lines. Immunohistochemical staining of CD155 in tissue specimens from 57 patients with lung adenocarcinoma revealed the median tumor proportion score for CD155 to be 68%. The abundance of miR-326 in these specimens with a low level of CD155 expression was significantly greater than in specimens with a high level (p < 0.005). Our results thus suggest that miR-326 negatively regulates CD155 expression in lung adenocarcinoma and might therefore play a role in the development of resistance to PD-1/PD-L1 inhibitors.

Extracellular vesicles derived from PM2.5-exposed alveolar epithelial cells mediate endothelial adhesion and atherosclerosis in ApoE-/- mice.

Epidemiological studies suggested that PM2.5 (particle matters with an aerodynamic diameter ≤2.5 µm) exposure is associated with atherosclerosis. Extracellular vesicles (EVs) are messengers between intracellular communications which are important in diseases procession. At present, whether EVs derived from PM2.5-exposed alveolar epithelial cells (P-EVs) involve in atherosclerosis has not been clearly understood. This study is performed to investigate the effects of P-EVs on the development of endothelium adhesion and atherosclerosis. Here, ApoE-/- mice were randomized into different groups receiving one of the following treatments, filtered air (FA), PM2.5, PBS, PBS-treated alveolar epithelial cells-derived EVs (EVs), or P-EVs. Then the atherosclerosis level in aortas or aorta sections was evaluated by oil red O staining. The results indicated that ApoE-/- mice treated with P-EVs or PM2.5 showed more obvious atherosclerosis plaques in aortas and aortic arches than those treated with EVs or PBS. Endothelial cells (ECs) were treated with PBS, EVs, P-EVs, or PM2.5. The adhesion property, miRNAs level and expressions of IκBα, phosphorylated IκBα, NF-κB p65, phosphorylated NF-κB p65, and VCAM1 in ECs were determined. It was found that P-EVs activated IκBα-NF-κB-VCAM1 signaling and increased adhesion of ECs, and such effects could be reversed by adalimumab (the TNF-α inhibitor) or miR-326-3p inhibitor. Further study suggested that P-EVs induced upregulation of TNF-α and miR-326-3p in recipient ECs and contributed to the phosphorylation of NF-κB p65. Collectively, EVs derived from PM2.5-exposed alveolar epithelial cells played an important role in the development of atherosclerosis via activating IκBα-NF-κB-VCAM1 signaling.

Circ_0082182 upregulates the NFIB level via sponging miR-326 to promote oxaliplatin resistance and malignant progression of colorectal cancer cells.

Circular RNAs (circRNAs) are key regulators in tumor metastasis and drug resistance. This study was designed to investigate circ_0082182 function and mechanism in oxaliplatin (OXA) resistance and cancer progression of colorectal cancer (CRC). The circ_0082182, microRNA-326 (miR-326), and nuclear factor I B (NFIB) levels were quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell sensitization was analyzed by Cell Counting Kit-8 assay. The proliferation ability was determined via EdU assay, and apoptosis was measured by flow cytometry. Transwell assay and wound healing assay were performed to assess cell invasion and migration. The protein level was examined through Western blot. The binding interaction was conducted via dual-luciferase reporter assay. Xenograft tumor assay was used to explore the circ_0082182 function in vivo. The circ_0082182 level was upregulated in OXA-resistant CRC samples and cells. Downregulation of circ_0082182 suppressed OXA resistance, proliferation, invasion, and migration but promoted apoptosis of OXA-resistant CRC cells. Circ_0082182 acted as a sponge for miR-326. The regulatory role of circ_0082182 was ascribed to the miR-326 sponging function. MiR-326 directly targeted NFIB to impede OXA resistance and cancer progression in CRC cells. NFIB level was regulated by circ_0082182 via sponging miR-326. Circ_0082182 promoted tumor growth in OXA-resistant xenograft tumor model through mediating the miR-326/NFIB axis. These data suggested that circ_0082182 elevated the NFIB expression to regulate OXA resistance and CRC progression by absorbing miR-326.

Regulation of tumorigenesis and ferroptosis in non-small cell lung cancer by a novel BBOX1-AS1/miR-326/PROM2 axis.

Dysregulation of long non-coding RNAs (lncRNAs) is associated with the tumorigenesis and ferroptosis of non-small cell lung cancer (NSCLC). BBOX1 antisense RNA 1 (BBOX1-AS1) functions as an oncogenic driver in NSCLC. Here, we aim to investigate the regulation effect and underlying mechanism of BBOX1-AS1 in NSCLC progression and ferroptosis. RNA expression was detected by quantitative real-time PCR (qRT-PCR), and protein expression was measured by immunoblotting. Cell growth was assessed by CCK-8 and colony formation assays. Transwell assay was applied to evaluate cell invasion and migration. RNA pull-down and dual-luciferase reporter assays were applied to verify the relationship between miR-326 and BBOX1-AS1 or prominin 2 (PROM2). The role of BBOX1-AS1 in NSCLC tumorigenicity was also analyzed by xenograft assays. Silencing BBOX1-AS1 or PROM2 impeded NSCLC cell growth, migration, and invasion. Silencing BBOX1-AS1 induced cell apoptosis and ferroptosis. BBOX1-AS1 up-regulated PROM2 expression, and re-expression of PROM2 reversed the effects of BBOX1-AS1 depletion on cell malignant phenotypes and ferroptosis. BBOX1-AS1 post-transcriptionally modulated PROM2 expression by sponging miR-326. MiR-326 was validated as a mediator of BBOX1-AS1 in regulating NSCLC cell malignant phenotypes and ferroptosis. Additionally, BBOX1-AS1 deficiency in vivo resulted in the suppression of xenograft tumor growth. Together, our study defines a novel BBOX1-AS1/miR-326/PROM2 axis in regulating NSCLC malignant progression and ferroptosis, offering new evidence for the oncogenic role of BBOX1-AS1 in NSCLC. These findings may provide a basis for the future usage of targeting BBOX1-AS1 in NSCLC treatment.

N6-methyladenosine-modified circular RNA QSOX1 promotes colorectal cancer resistance to anti-CTLA-4 therapy through induction of intratumoral regulatory T cells.

BACKGROUND: Colorectal cancer (CRC) is the 3rd most common cancer worldwide. CircRNAs are promising novel biomarkers for CRC. T regulatory (Treg) cells express the immune checkpoint receptor of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and promote tumor immunological tolerance. We therefore investigate the biological functions and mechanisms of circQSOX1 in CRC tumorigenesis; involvement of circQSOX1 in promoting Treg cell-mediated CRC immune escape in anti-CTLA-4 therapy. METHODS: Bioinformatics analyses were performed for circQSOX1expressions, specific binding sites, and N6-methyladenosine (m6A) motifs of circQSOX1, thatwere further validated with a series of experiments. Functions of circQSOX1 in promoting CRC development, Treg cells-based immune escape, and anti-CTLA-4 therapy response were investigated both in vitro and in vivo. RESULTS: High circQSOX1 expression was associated with carcinogenesis and poor clinical outcome of CRC patients. METTL3-mediated RNA m6A modification on circQSOX1 could be read by IGF2BP2 in CRC cells. CircQSOX1 promoted CRC development by regulating miR-326/miR-330-5p/PGAM1 axis. CircQSOX1 regulated glycolysis and promoted immune escape of CRC cells, and inhibits anti-CTLA-4 therapy response in CRC patients. CONCLUSION: m6A-modified circQSOX1 facilitated CRC tumorigenesis by sponging miR-326 and miR-330-5p to promotes PGAM1 expression, which further promoted CRC immune escape by activating glycolysis and inactivating the anti-CTLA-4 therapy response of CRC. Combined treatment with sh-circQSOX1 and anti-CTLA-4 could be a strategy to overcome Treg cell-mediated CRC immune therapy resistance.

Circ_0008717 Sponges miR-326 to Elevate GATA6 Expression to Promote Breast Cancer Tumorigenicity.

Circular RNAs (circRNAs) have been reported to paly roles in the progression and management of breast cancers (BC). This work aimed to detect the role and mechanism of circ_0008717 in BC tumorigenesis. Expression levels of genes and proteins were evaluated by quantitative real-time polymerase chain reaction and western blot. In vitro assays were conducted using cell counting kit-8, colony formation, transwell, tube formation, and flow cytometry assays, respectively. The interaction between miR-326 and circ_0008717 or GATA6 (GATA Binding Protein six) was confirmed by bioinformatics analysis, and dual-luciferase reporter assay and RNA immunoprecipitation assay. The murine xenograft models were established to perform in vivo assay. Circ_0008717 and GATA6 were highly expressed, while miR-326 was lowly expressed in BC tissues and cells. Functionally, knockdown of circ_0008717 not only suppressed breast cancer cell proliferation, angiogenesis, migration, invasion and epithelial-mesenchymal transition (EMT) in vitro, but also hindered tumor growth and EMT process in vivo. Mechanistically, Circ_0008717 directly bound to miR-326, which targeted GATA6. Rescue experiments showed that miR-326 reversed the anticancer action of circ_0008717 knockdown on BC cells. Moreover, miR-326 restoration repressed BC cell growth and metastasis, which were attenuated by GATA6 overexpression. In addition, we also observed that circ_0008717 could regulate GATA6 expression by sponging miR-326. Circ_0008717 promoted breast cancer growth and metastasis through miR-326/GATA6 axis, revealing a potential therapeutic target for breast cancer treatment.

Adhesion G Protein-Coupled Receptor G2 Promotes Hepatocellular Carcinoma Progression and Serves as a Neutrophil-Related Prognostic Biomarker.

Adhesion G protein-coupled receptor G2 (ADGRG2) is an orphan adhesion G protein-coupled receptor (GPCR), which performs a tumor-promoting role in certain cancers; however, it has not been systematically investigated in hepatocellular carcinoma (HCC). In the current study, we utilized multiple databases to analyze the expression and diagnostic and prognostic value of ADGRG2 in HCC and its correlation with immune infiltration and inflammatory factors. The function and upstream regulatory miRNA of ADGRG2 were validated through qPCR, Western blot, CCK8, wound healing, and dual luciferase assays. It turned out that ADGRG2 was significantly higher in HCC and had a poor survival rate, especially in AFP ≤ 400 ng/mL subgroups. Functional enrichment analysis suggested that ADGRG2 may be involved in cancer pathways and immune-related pathways. In vitro, siRNA-mediated ADGRG2 silencing could inhibit the proliferation and migration of Huh7 and HepG2 cells. There was a highly significant positive correlation between ADGRG2 and neutrophils. Moreover, NET-related genes were filtered and confirmed, such as ENO1 and S100A9. Meanwhile, the high expression of ADGRG2 was also accompanied by the highest number of inflammatory cytokines, chemokines, and chemokine receptors and good immunotherapy efficacy. Finally, AGDGR2 may be sensitive to two drugs (PIK-93 and NPK76-II-72-1) and can be targeted by miR-326. In conclusion, ADGRG2 may serve as a novel biomarker and drug target for HCC diagnosis, immunotherapy, and prognosis and was related to neutrophils and the inflammatory process of liver cancer development.

Downregulation of NHE-3 (SLC9A3) expression by MicroRNAs in intestinal epithelial cells.

Na+/H+ exchanger-3 (NHE-3) is the major apical membrane transporter involved in vectorial Na+ absorption in the intestine. Dysregulation of NHE-3 expression and/or function has been implicated in pathophysiology of diarrhea associated with gut inflammation and infections. Therefore, it is critical to understand the mechanisms involved in the regulation of NHE-3 expression. MicroRNAs (miRNAs) are highly conserved small RNAs that can regulate gene expression at the posttranscriptional level. To date, however, very little is known about the regulation of NHE-3 expression by microRNAs. Therefore, current studies were undertaken to examine the potential miRNA candidates that can regulate the expression of NHE-3 in intestinal epithelial cells. In silico analysis, using different algorithms, predicted several miRNAs that target NHE-3. MicroRNAs with highest context and target score, miR-326, miR-744-5p, and miR-330-5p, were selected for the current study. Human NHE-3 gene 3' untranslated region [3'UTR; 160 base pair (bp)] was cloned into pmirGLO vector upstream of luciferase reporter and transiently transfected with mimics of miR-326, miR-744-5p, and miR-330-5p into Caco-2, HT-29, and SK-CO15 cells. Cotransfection of NHE-3 3' UTR with miR-326 and -miR-330-5p mimics resulted in a significant decrease in relative luciferase activity. Transfection of miR-326 and -330-5p mimics into SK-CO15 cells significantly decreased the NHE-3 protein expression, with no change in NHE-3 messenger ribonucleic acid (mRNA) levels. Our findings demonstrate a novel mechanism for posttranscriptional regulation of NHE-3 by miR-326 and -330-5p by translational repression. We speculate that miR-326 and -330-5p dependent pathways may be involved in modulating NHE-3 expression under physiological and pathophysiological conditions.

Long noncoding RNA SNHG9 facilitates growth of glioma stem-like cells via miR-326/SOX9 axis.

BACKGROUND: Glioma stem-like cells (GSCs) are greatly responsible for the progression of glioma. Long noncoding RNAs (lncRNAs) play an important role in glioma tumor progression. This study aims to explore the role and underlying mechanism of lncRNA SNHG9 in regulating GSC cell growth. METHODS: GSCs were obtained from glioma cells (U87 and U251) and referred to as GSC-87 and GSC-251, respectively. The interactions between miR-326 and SNHG9 or SOX9 were analyzed using luciferase reporter assay. Cell growth of GSCs was evaluated by EdU assay and sphere formation assay. RESULTS: SNHG9 expression was significantly higher in GSC-87 and GSC-251 cells than in U87 and U251 cells. SNHG9 overexpression promoted GSC cell growth, whereas SNHG9 knockdown inhibited GSC cell growth. Mechanistically, SNHG9 acted as a competitive endogenous RNA of miR-326 to elevate the expression of SOX9, a direct target of miR-326. Moreover, transfection with miR-326 inhibitor counteracted SNHG9 knockdown-mediated inhibition of GSC cell growth. CONCLUSIONS: SNHG9 facilitates growth of GSCs via the miR-326/SOX9 axis. This study provides a promising therapeutic target for glioma treatment.

Long non-coding RNA SNHG1 activates glycolysis to promote hepatocellular cancer progression through the miR-326/PKM2 axis.

BACKGROUND: Hepatocellular cancer (HCC) is a lethal malignancy with extremely poor prognosis. In the present study, we aimed to investigate the role and underlying mechanism of SNHG1 in HCC progression. METHODS: Combined with bioinformatics and experimental validation, we explored the clinical significance of SNHG1 in HCC. A Cell Counting Kit-8 assay, cell colony formation assay, and subcutaneous tumorigenesis experiments of nude mice were conducted to evaluate the pro-proliferative capacity of SNHG1. Glucose consumption and lactate production were measured to explore the regulatory role of SNHG1 in glycolysis. Nuclear-cytoplasmic separation, quantitative real-time polymerase chain reaction and Western blot assays, chromatin immunoprecipitation, and luciferase reporter and RNA immunoprecipitation assays were performed to investigate the molecular mechanisms of SNHG1 in HCC. RESULTS: SNHG1 expression was dramatically increased in HCC and positively correlated with poor prognosis. E2F1 bound to the SNHG1 promoter region to activate SNHG1 transcription. Furthermore, SNHG1 served as a molecular sponge for miR-326 to sequester the interaction of miR-326 and pyruvate kinase M2 (PKM2), facilitating the expression of PKM2. Activating PKM2 expression was revealed to be one of mechanisms of SNHG1 with respect to promoting glycolysis and the proliferation of HCC cells. CONCLUSIONS: E2F1-activated SNHG1 modulates the miR-326/PKM2 axis to facilitate glycolysis and the proliferation of HCC cells. Targeting SNHG1 could be a promising therapeutic option for HCC.

Circ_0000591 served as endogenous RNA for miR-326 to promote progression of cholangiocarcinoma via the TLR4/MyD88/IL6 axis.

BACKGROUND: Cholangiocarcinoma (CCA) is the second most common liver malignancy with poor prognosis after hepatocellular carcinoma (HCC). Emerging evidences have proved that circular RNAs (circRNAs) are involved in the progression of multiple cancers, while their roles in tumorigenesis of CCA remains largely unclear. In the present study, we found circ_0000591 was upregulated in CCA cells and tissues. The highly expressed circ_0000591 could promote the proliferation, migration, invasion and carcinogenesis of CCA cells in vitro and in vivo. Functionally, circ_0000591 induced the proliferation, migration and invasion of CCA cells through sponging miR-326. Moreover, silencing of circ_0000591 attenuated LPS-induced anti-apoptosis of CCA cells through the TLR4/MyD88/IL6 pathway. In conclusion, this study revealed a novel regulatory mechanism that circ_0000591 contributed to the progression of CCA via miR-326/TLR4/MyD88/IL6 axis. These findings enhanced our knowledge of potential molecular mechanism involved in the malignant progression of CCA.

Targeting HIF1-alpha/miR-326/ITGA5 axis potentiates chemotherapy response in triple-negative breast cancer.

PURPOSE: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer that is frequently treated with chemotherapy. However, many patients exhibit either de novo chemoresistance or ultimately develop resistance to chemotherapy, leading to significantly high mortality rates. Therefore, increasing the efficacy of chemotherapy has potential to improve patient outcomes. METHODS: Here, we performed whole transcriptome sequencing (both RNA and small RNA-sequencing), coupled with network simulations and patient survival data analyses to build a novel miRNA-mRNA interaction network governing chemoresistance in TNBC. We performed cell proliferation assay, Western blotting, RNAi/miRNA mimic experiments, FN coating, 3D cultures, and ChIP assays to validate the interactions in the network, and their functional roles in chemoresistance. We developed xenograft models to test the therapeutic potential of the identified key miRNA/proteins in potentiating chemoresponse in vivo. We also analyzed several patient datasets to evaluate the clinical relevance of our findings. RESULTS: We identified fibronectin (FN1) as a central chemoresistance driver gene. Overexpressing miR-326 reversed FN1-driven chemoresistance by targeting FN1 receptor, ITGA5. miR-326 was downregulated by increased hypoxia/HIF1A and ECM stiffness in chemoresistant tumors, leading to upregulation of ITGA5 and activation of the downstream FAK/Src signaling pathways. Overexpression of miR-326 or inhibition of ITGA5 overcame FN1-driven chemotherapy resistance in vitro by inhibiting FAK/Src pathway and potentiated the efficacy of chemotherapy in vivo. Importantly, lower expression of miR-326 or higher levels of predicted miR-326 target genes was significantly associated with worse overall survival in chemotherapy-treated TNBC patients. CONCLUSION: FN1 is central in chemoresistance. In chemoresistant tumors, hypoxia and resulting ECM stiffness repress the expression of the tumor suppressor miRNA, miR-326. Hence, re-expression of miR-326 or inhibition of its target ITGA5 reverses FN1-driven chemoresistance making them attractive therapeutic approaches to enhance chemotherapy response in TNBCs.

MiR-326 inhibits trophoblast growth, migration, and invasion by targeting PAX8 via Hippo pathway.

Preeclampsia (PE), a pregnancy disorder that affects 5-7% of pregnant women, is among the primary causes for maternal and perinatal mortality. PE is believed to be associated with insufficient invasion of villous and extravillous trophoblasts (EVTs), which hampers uterine spiral artery remodeling and finally induces PE. But the mechanism responsible for reduction of trophoblast invasion remains unclear. In this study, placental tissues taken from healthy donors and PE patients were used to evaluate the miR-326 expression; CCK8 and colony formation assays were used to confirm the effect of miR-326 on cell proliferation; transwell assay was used to demonstrate the effect of miR-326 on cell invasion capability; western blot was used to investigate the underlying mechanism; and luciferase assay was used to detect the effect of miR-326 on YAP/TAZ-mediated transcription activity. It was revealed the miR-326 expression was higher in placentas from PE patients than from healthy donors. After transfection of miR-326 mimics, trophoblast proliferation and invasion were impaired. Using TargetScan, we speculated that PAX8 was a target of miR-326, which was later confirmed by western blot. The YAP/TAZ expression was also downregulated after transfection with miR-326. Luciferase assay demonstrated that overexpression of miR-326 suppressed YAP/TAZ-mediated transcription activity by targeting PAX8. Overexpression of PAX8 could partly rescue miR-326-induced suppression of trophoblast proliferation and invasion. Taken together, our result indicated that miR-326 suppresses trophoblast growth, invasion, and migration by means of targeting PAX8 via the Hippo pathway.

CKAP2L, a crucial target of miR-326, promotes prostate cancer progression.

BACKGROUND: The overexpression of aberrant cell cycle signaling pathway associated protein has been implicated in multiple malignancies and the identification of all-important one among is the crux of the precise targeted therapy. CKAP2L (Cytoskeleton Associated Protein 2 Like) plays a newish role in cancer progression through activation of the process of cell cycle and mitosis. In this study, we aim to delineate the prominent dysregulated expression of CKAP2L and comprehensively reveal its deregulation in prostate cancer.  METHOD: CKAP2L expression was examined in the normal and tumor tissues of prostate cancer patients with RT-QPCR and Western blot. IHC showed the different expression in normal prostate tissue, tissue of BPH, low Gleason Score and high Gleason Score prostate cancer patients. Transwell, colony formation, MTT and flow cytometry were performed to detected the changes in cellular function in vitro. The xenograft model was conducted for the changes in vivo. Dual luciferase and RIP proved the binding relation between CKAP2L and miR-326. RESULTS: In multiple datasets, CKAP2L was found upregulated and positively associated with Gleason grade and poor clinical outcomes of patients. shRNA mediated silence of CKAP2L suppressed cell proliferation, impaired monolayer formation, inhibited cell invasion. CKAP2L was confirmed to be the direct target of miR-326, which had a carcinostatic effect by binding the 3'untranslated regions (3'UTRs) of CKAP2L mRNA. The deletion of CKAP2L resulted in reduced expression of genes involved in the mitotic cell cycle such as multiple cyclin-dependent kinases and cyclins, but also several genes encoding proteins involved in chromosome segregation and spindle assembly. CONCLUSION: Taken together, CKAP2L plays a carcinogenic role in prostate cancer by regulates the expression of cycle-associated proteins.

The Effect of Interferon-Beta Therapy on T-Helper 17/miR-326 and T-Helper 1/miR-29b-3p Axis in Relapsing-Remitting Multiple Sclerosis Patients.

BACKGROUND: We aimed to evaluate the therapeutic effects of interferon-beta (IFN-ß) on hsa-miR29b-3p and hsa-miR326 in isolated T-helper (Th)1 and Th17 cells expressed by relapsing-remitting multiple sclerosis (RRMS) patients before and after 1 year of treatment with IFN-ß. METHODS: The study was done on 19 RRMS patients pre- and posttreatment with IFN-ß to evaluate the frequency of Th1 and Th17 cells by flow cytometry. The expression level of hsa-miR-29b-3p and hsa-miR-326 in isolated Th1 and Th17 cells was assessed by quantitative polymerase chain reaction. Enzyme-linked immunosorbent assay was also used to measure the plasma levels of I interferon -gamma and interleukin (IL)-17A. RESULTS: Th17 cells and plasma levels of IL-17A decreased in RRMS patients after IFN-ß therapy but hsa-miR-29b-3p and hsa-miR-326 expression had no significant change in treated RRMS patients versus baseline. MxA gene expression was significantly induced upon IFN-ß therapy in patients with RRMS. CONCLUSION: IFN-ß therapy is more effective on Th17 than Th1, but it does not reform altered expression of hsa-miR-326 and hsa-miR-29b-3p in Th17 and Th1, respectively.

Hsa_circ_0020850 promotes the malignant behaviors of lung adenocarcinoma by regulating miR-326/BECN1 axis.

BACKGROUND: Circular RNAs (circRNAs) are a novel type of endogenous RNAs and play vital roles in lung adenocarcinoma. However, the function and underlying mechanism of circ_0020850 in lung adenocarcinoma remain unknown. METHODS: The levels of circ_0020850, microRNA-326 (miR-326), and Beclin1 (BECN1) were analyzed by real-time quantitative polymerase chain reaction and western blot analyses. The migration and invasion were determined by wound healing and transwell assays, respectively. Colony formation assay was used to assess cell proliferation ability. The angiogenic ability was analyzed by Matrigel angiogenesis assay. The apoptosis rate was calculated by flow cytometry assay. Dual-luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays were conducted to confirm the interaction relationship among circ_0020850, miR-326, and BECN1. A xenograft mice model was established to assess the role of circ_0020850 in vivo. RESULTS: We found that circ_0020850 was obviously overexpressed in lung adenocarcinoma tissues and cells. Knockdown of circ_0020850 inhibited migration, invasion, proliferation, and angiogenesis but induced apoptosis in lung adenocarcinoma cells in vitro, as well as curbed tumor growth in vivo. MiR-326 was a target of circ_0020850, and knockdown of miR-326 abolished the suppression effect of circ_0020850 on the malignant behaviors of lung adenocarcinoma cells. Additionally, miR-326 could negatively regulate BECN1 expression, thereby regulating lung adenocarcinoma cell phenotypes. Importantly, circ_0020850 could directly bind to miR-326 and thus relieve miR-326-mediated inhibition on BECN1. CONCLUSION: Circ_0020850 promoted the malignant development of lung adenocarcinoma by regulating miR-326/BECN1 axis, indicating that circ_0020850 might serve as a promising target for the diagnosis and treatment of lung adenocarcinoma patients.