Differentiation Antagonizing Non-protein Coding RNA Research Articles

Krüppel-like factor 5-induced overexpression of long non-coding RNA DANCR promotes the progression of cervical cancer via repressing microRNA-145-3p to target ZEB1

ABSTRACT Long non-coding RNA (lncRNA) differentiation antagonizing non-protein coding RNA (DANCR) participates in the development of diverse cancers. Nevertheless, the impact of DANCR on cervical cancer (CC) remains largely unknown. This study aims to explore the effects of DANCR sponging microRNA-145-3p (miR-145-3p) on CC. Expression of KLF5, DANCR, miR-145-3p, and zinc finger E-box binding homeobox 1 (ZEB1) in CC and adjacent normal tissues was determined. Human CC cell lines were, respectively, treated with silenced DANCR or miR145-3p mimic/inhibitor. Then, the viability, migration, invasion, and apoptosis of CC cells were measured. The cell growth in vivo was observed as well. Chromatin immunoprecipitation assay was performed to analyze the binding of KLF5 and DANCR promoter. Interaction among DANCR, miR-145-3p, and ZEB1 was assessed. KLF5, DANCR, and ZEB1 were upregulated but miR-145-3p was downregulated in CC tissues. KLF5 activated DANCR expression and the high DANCR expression was related to tumor staging, infiltrating muscle depth and lymphatic metastasis of CC patients. Reduced DANCR or elevated miR-145-3p repressed malignant behaviors of CC cells. The tumor diameter and weight were also repressed by DANCR silencing or miR-145-3p elevation. The effect of DANCR knockdown on CC cells could be reversed by miR-145-3p inhibitor. MiR-145-3p was targeted by DANCR and ZEB1 was targeted by miR-145-3p. KLF5-induced overexpression of DANCR promotes CC progression via suppressing miR-145-3p to target ZEB1. This study may provide potential targets for CC treatment.

LncRNA DANCR improves the dysfunction of the intestinal barrier and alleviates epithelial injury by targeting the miR-1306-5p/PLK1 axis in sepsis.

Intestinal barrier dysfunction often occurs in various acute or chronic pathological conditions and has been identified as an important clinical problem. Herein, we explored the biological role and molecular mechanism of Polo-like kinase 1 (PLK1) and differentiation antagonizing non-protein coding RNA (DANCR) in intestinal barrier dysfunction caused by sepsis. RT-qPCR analysis was used to examine PLK1, miR-1306-5p, and DANCR expression in NCM460 cells after LPS treatment. TUNEL assay and Western blot analysis were performed to explore PLK1 function in cell apoptosis and intestinal barrier in vitro. Hematoxylin and eosin staining, Western blot analysis, and TUNEL assay were used to investigate DANCR function in the intestinal barrier and cell apoptosis in vivo. The interaction between miR-1306-5p and PLK1 (or DANCR) was validated by luciferase reporter assay. As a result, PLK1 overexpression decreased cell apoptosis and promoted intestinal barrier function. Moreover, DANCR was validated as a sponge of miR-1306-5p to target PLK1. In addition, we found that DANCR overexpression decreased intestinal mucosal permeability and colon mucosa epithelial cell apoptosis in vivo. Conclusively, DANCR improved intestinal barrier dysfunction and alleviated epithelial injury by targeting the miR-1306-5p/PLK1 axis in sepsis.

Long noncoding RNA differentiation antagonizing nonprotein coding RNA promotes the proliferation, invasion and migration of neuroblastoma cells via targeting β-1, 4-galactosyltransferase III by sponging miR-338-3p.

Neuroblastoma is a common malignant tumor in children, and patients often have a poor prognosis. Long noncoding RNAs (lncRNAs) are involved in the regulation of neuroblastoma progression. However, the regulatory effect of lncRNA differentiation antagonizing nonprotein coding RNA (DANCR) on neuroblastoma is still not clear. The expression levels of DANCR, miR-338-3p and β-1, 4-galactosyltransferase III (B4GALT3) were determined by quantitative real-time PCR. 3-(4, 5-dimethyl-2 thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide, flow cytometry and transwell assays were used to evaluate the proliferation, apoptosis, migration and invasion abilities of neuroblastoma cells. Moreover, western blot analysis was performed to assess the levels of B4GALT3 and the proliferation, apoptosis and migration-related proteins. Besides, a dual-luciferase reporter assay was used to verify the interactions among DANCR, miR-338-3p and B4GALT3. Mice xenograft models were used to ascertain the effect of DANCR on neuroblastoma tumor growth in vivo. Our results revealed that DANCR was highly expressed in neuroblastoma tissues and cells, and its silencing impeded the progression of neuroblastoma cells. DANCR could interact with miR-338-3p. Knockdown of miR-338-3p recovered the inhibitory effect of DANCR knockdown on neuroblastoma progression. B4GALT3 was a target of miR-338-3p. B4GALT3 overexpression reversed the suppression effect of DANCR silencing on neuroblastoma progression. In-vivo experiments further confirmed that DANCR silencing inhibited neuroblastoma tumor growth. Our results indicated that DANCR promoted B4GALT3 expression to increase the proliferation, migration and invasion of neuroblastoma cells via sponging miR-338-3p, which provided a theoretical basis for the targeted therapy of neuroblastoma.

Long non-coding DANCR targets miR-185-5p to upregulate LIM and SH3 protein 1 promoting prostate cancer via the FAK/PI3K/AKT/GSK3β/snail pathway.

Long non-coding RNA differentiation antagonizing non-protein coding RNA (DANCR) acts as an oncogene in different cancers, although its roles in prostate cancer are not fully reported. We aimed to explore its mechanism in facilitating the malignancy of prostate cancer. The expression of DANCR, microRNA (miR)-185-5p and LIM and SH3 protein 1 (LASP1) in 40 pairs of prostate cancer tissues and normal tissues, five prostate cancer cell lines and one epithelial cell line was assessed by a quantitative real-time polymerase chain reaction, western blotting and immunohistochemistry, respectively. In transfected PC3 and C4-2 cells, cell proliferation, migration, invasion, cell cycle distribution and epithelial-mesenchymal transition (EMT) protein expression were tested via cell counting kit-8, wound healing, transwell, flow cytometry and western blot assays, respectively. The interactions between DANCR, miR-185-5p and LASP1 were verified by a dual-luciferase reporter assay. Rescue experiments were conducted to determine the roles of DANCR on the malignant properties of PC3 and C4-2 cells. The involvement of the signaling pathway was examined using a p-FAK inhibitor. DANCR and LASP1 expression was enhanced, whereas miR-185-5p expression was diminished in prostate cancer tissues and cell lines. Knockdown of DANCR suppressed cell proliferation, migration, invasion, G1-S transition and expression of EMT proteins of the transfected PC3 and C4-2 cells. DANCR sponged miR-185-5p to upregulate LASP1 expression. DANCR-miR-185-5p-LASP1 axis activates the FAK/PI3K/AKT/GSK3β/Snail pathway to promote the malignant properties of PC3 and C4-2 cells. These findings suggest that DANCR exerts oncogenic roles in prostate cancer via the miR-185-5p/LASP1 axis activating the FAK/PI3K/AKT/GSK3β/Snail pathway. It can be a potential biomarker in the diagnosis and monitoring of prostate cancer.

Analysis of crucial genes, pathways and construction of the molecular regulatory networks in vascular smooth muscle cell calcification.

Vascular calcification (VC) accompanies the trans-differentiation of vascular smooth muscle cells (VSMCs) into osteo/chondrocyte-like cells and resembles physiological bone mineralization. However, the molecular mechanisms underlying VC initiation and progression have remained largely elusive. The aim of the present study was to identify the genes and pathways common to VSMC and osteoblast calcification and construct a regulatory network of non-coding RNAs and transcription factors (TFs). To this end, the Gene Expression Omnibus dataset GSE37558 including mRNA microarray data of calcifying VSMCs (CVSMCs) and calcifying osteoblasts (COs) was analyzed. The differentially expressed genes (DEGs) were screened and functionally annotated and the microRNA (miRNA/mRNA)-mRNA, TF-miRNA and long non-coding RNA (lncRNA)-TF regulatory networks were constructed. A total of 318 DEGs were identified in the CVSMCs relative to the non-calcified VSMCs, of which 43 were shared with the COs. The CVSMC-related DEGs were mainly enriched in the functional terms cell cycle, extracellular matrix (ECM), inflammation and chemotaxis-mediated signaling pathways, of which ECM was enriched by the DEGs for the COs as well. The protein-protein interaction network of CVSMCs consisted of 281 genes and 3,650 edges. There were 30 hub genes in this network, including maternal embryonic leucine zipper kinase (MELK), which potentially regulates the differentially expressed TF (DETF) forkhead box (FOX)M1 and is a potential target gene of Homo sapiens miR-485-3p and miR-181d. The TF-miRNA network included 251 TFs and 60 miRNAs, including 10 DETFs such as FOXO1 and snail family transcriptional repressor 2 (SNAI2). Furthermore, the lncRNAs H19 imprinted maternally expressed transcript (H19) and differentiation antagonizing non-protein coding RNA (DANCR) were predicted as the upstream regulators of FOXO1 and SNAI2 in the lncRNA-TF regulatory network. DANCR, MELK and FOXM1 were downregulated, and H19, FOXO1 and SNAI2 were upregulated in the CVSMCs. Taken together, the CVSMCs and COs exhibited similar molecular changes in the ECM. In addition, the MELK-FOXM1, H19/DANCR-FOXO1 and SNAI2 regulatory pathways likely mediate VSMC calcification.

Long noncoding RNA DANCR confers cytarabine resistance in acute myeloid leukemia by activating autophagy via the miR-874-3P/ATG16L1 axis.

Autophagy is an important mechanism involved in the regulation of acute myeloid leukemia (AML) chemoresistance. The long noncoding RNA (lncRNA) differentiation antagonizing non‐protein coding RNA (DANCR) exhibits oncogenic activity in several types of human cancers, including AML, but it remains unclear whether it regulates autophagy and chemoresistance in AML. We report here that cytarabine (Ara‐C) treatment elevates DANCR expression in human AML cells. In addition, DANCR overexpression confers and its knockdown diminishes Ara‐C resistance in human AML cells, suggesting that DANCR positively regulates AML chemoresistance to Ara‐C. Moreover, DANCR promotes autophagy in Ara‐C‐treated human AML cells and acts as a sponge to decrease miR‐20a‐5p expression, thereby upregulating the expression of ATG16L1, a critical component of the autophagy machinery. Importantly, ATG16L1 silencing abrogates DANCR‐promoted autophagy and markedly restores DANCR‐conferred Ara‐C resistance, suggesting that DANCR promotes MIR‐874‐3P/ATG16L1 axis‐regulated autophagy to confer Ara‐C resistance in human AML cells. Together, this study identifies DANCR as a positive regulator of Ara‐C resistance in human AML cells, suggesting this lncRNA as a potential target for overcoming Ara‐C resistance in AML chemotherapy.

Long non-coding RNA DANCR modulates osteogenic differentiation by regulating the miR-1301-3p/PROX1 axis.

The balance of osteoblasts and marrow adipocytes from bone marrow mesenchymal stem cells (BM-MSCs) maintains bone health. Under aging or other pathological stimuli, BM-MSCs will preferentially differentiate into marrow adipocytes and reduce osteoblasts, leading to osteoporosis. Long non-coding RNA differentiation antagonizing non-protein coding RNA (DANCR) participates in the osteogenic differentiation of human BM-MSCs, but the mechanism by which DANCR regulates the osteogenic differentiation of human BM-MSCs has not been fully explained. We observed that DANCR and prospero homeobox 1 (PROX1) were downregulated during osteogenic differentiation of human BM-MSCs, while miR-1301-3p had an opposite trend. DANCR overexpression decreased the levels of alkaline phosphatase, RUNX2, osteocalcin, Osterix in BM-MSCs after osteogenic induction, but DANCR silencing had the opposite result. Moreover, DANCR sponged miR-1301-3p to regulate PROX1 expression. miR-1301-3p overexpression reversed the suppressive role of DANCR elevation on the osteogenic differentiation of human BM-MSCs. Also, PROX1 elevation abolished the promoting role of miR-1301-3p overexpression on the osteogenic differentiation of human BM-MSCs. In conclusion, DANCR suppressed the osteogenic differentiation of human BM-MSCs through the miR-1301-3p/PROX1 axis, offering a novel mechanismby which DANCR is responsible for the osteogenic differentiation of human BM-MSCs.

Emerging role of lncRNA DANCR in progenitor cells: beyond cancer.

Long noncoding RNAs (lncRNAs) are important participants in biological processes including cell proliferation, differentiation and death, as well as pathogenesis of various diseases. LncRNA differentiation antagonizing non-protein coding RNA (DANCR) is an emerging regulator in cell metabolism and many diseases besides cancers. DANCR is negative in epidermal, osteoblastic and endoderm differentiation, but positive in chondrogenic differentiation of progenitor cells. It is protective for calcification of the ligamentum flavum, stroke, acute myocardial infarction and arterial calcification, but a risk factor for bone loss, fracture healing and idiopathic pulmonary fibrosis. In addition, DANCR is a potential target for improving tissue regeneration. Mechanically, DANCR, a cytoplasmic lncRNA, sponges corresponding microRNAs or interacts with various proteins. This review aims to summarize the role of DANCR in progenitor cells and provide perspectives for further studies.

Long non-coding RNA DANCR promoted non-small cell lung cancer cells metastasis via modulating of miR-1225-3p/ErbB2 signal.

Currently, we aimed to illustrate the role of lncRNA differentiation antagonizing non-protein coding RNA (DANCR) and erb-b2 receptor tyrosine kinase 2 (ErbB2) in non-small cell lung cancer (NSCLC). Expression of DANCR, microRNA-1225-3p (miR-1225-3p) and ErbB2 mRNA was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) assays. The clinical value of DANCR was checked by a ROC curve analysis, a Kaplan-Meier analysis and a Pearson Chi-Square test. Transwell chamber assays were performed to determine the migration and invasion ability changes of SPCA1 and A549 cells. The protein expression of ErbB2 was tested by Western blot assays. The targeted binding effect between miR-1225-3p and DANCR or ErbB2 was confirmed by a Dual-Luciferase reporter assay and an RNA pull-down assay, respectively. In the current study, it was found that DANCR was upregulated and correlated with poor prognosis in patients with NSCLC. DANCR promoted NSCLC cells migration and invasion via upregulation of ErbB2. DANCR regulated ErbB2 at posttranscriptional level. Mechanically, it was illustrated that miR-1225-3p negatively regulated ErbbB2 and it-mediated migration and invasion via directly targeting in NSCLC cells. Meanwhile, it was showed that DANCR interacted with miR-1225-3p in a reciprocal suppression manner. Even further, through a RIP assay and a luciferase assay, we showed that DANCR interacted with miR-1225-3p through a microRNA response element (MRE-1225-3p) via directly binding. Finally, it was demonstrated that DANCR served as a miR-1225-3p sponge to promote ErbB2 expression and to facilitate ErbB2-mediated migration and invasion in NSCLC cells. In the current study, it was illustrated that DNACR promoted ErbB2-mediated migration and invasion via working as a ceRNA of miR-1225-3p in NSCLC cells.

Differentiation Antagonizing Non-protein Coding RNA Knockdown Alleviates Lipopolysaccharide-Induced Inflammatory Injury and Apoptosis in Human Chondrocyte Primary Chondrocyte Cells Through Upregulating miRNA-19a-3p.

ObjectiveTo confirm the role of long noncoding RNA differentiation antagonizing non‐protein coding RNA (DANCR) in chondrocyte inflammatory injury in osteoarthritis (OA) in vitro, as well as its molecular mechanism.MethodsHuman primary chondrocytes were treated with lipopolysaccharide (LPS) to construct a chondrocyte inflammatory injury in human OA cell model. Gene expression was detected using real‐time quantitative polymerase chain reaction. Cell inflammatory injury was evaluated by Cell Counting Kit‐8 assay, flow cytometry, and enzyme‐linked immunosorbent assay. The interplay between miRNA‐19a‐3p (miR‐19a) and DANCR was validated by dual‐luciferase reporter assay and RNA immunoprecipitation.ResultsExpression of DANCR was upregulated, and miR‐19a was downregulated in human OA cartilage and LPS‐treated primary chondrocytes in vitro. Moreover, DANCR expression was inversely correlated with miR‐19a in OA patients. LPS reduced cell viability and increased the apoptotic rate and secretion of interleukin (IL)‐1β, IL‐6, IL‐8, as well as tumor necrosis factor (TNF)‐α in primary chondrocyte cells in vitro, suggesting an inflammatory injury model of OA. Functionally, knockdown of DANCR could attenuate LPS‐induced apoptosis and inflammatory response, as evidenced by improved cell viability, and reduced apoptotic rate and products of IL‐1β, IL‐6, IL‐8, and TNF‐α. Notably, DANCR negatively regulated miR‐19a expression, presumably via sponging. Furthermore, miR‐19a deletion eliminated the effect of DANCR knockdown on apoptosis and the inflammatory response of primary chondrocytes under LPS stress.ConclusionDifferentiation antagonizing non‐protein coding RNA silencing could protect human chondrocyte cells against LPS‐induced inflammatory injury and apoptosis through targeting miR‐19a, suggesting a vital role of the DANCR/miR‐19a axis in OA.

Long Noncoding RNA DANCR Regulates Cell Proliferation by Stabilizing SOX2 mRNA in Nasopharyngeal Carcinoma

The long noncoding RNA DANCR (differentiation antagonizing non-protein coding RNA) displays aberrant expression in various cancers. However, its clinical value and functional mechanisms in nasopharyngeal carcinoma (NPC) remain poorly understood. We found that DANCR is dramatically up-regulated in human NPC, and that it is an indicator for poor survival prognosis. DANCR knockdown suppressed cell proliferation, colony formation invitro, and tumorigenicity invivo. Mechanistic analyses demonstrated that DANCR could bind to RNA-binding protein 3 (RBM3) protein and stabilize SOX2 mRNA, resulting in NPC cell proliferation. Our findings indicate that DANCR functions as an oncogene and a potential therapeutic target for NPC.

LncRNA DANCR promotes ATG7 expression to accelerate hepatocellular carcinoma cell proliferation and autophagy by sponging miR-222-3p.

LncRNA differentiation antagonizing non-protein coding RNA (DANCR) is an oncogene in various malignant cancers, including hepatocellular carcinoma (HCC). Autophagy is an intracellular self-digestion mechanism that accelerates the progression of HCC via promoting cell survival. However, the role of lncRNA DANCR in HCC, and the mechanism of lncRNA DANCR in the regulation of autophagy in HCC remains unknown. Therefore, the aims of this study are the investigation of the role of lncRNA DANCR in HCC, and the exploration of the molecular mechanism of lncRNA DANCR in regulating autophagy of HCC cells. In this study, the expression of lncRNA DANCR, miR-222-3p, and autophagy-related gene 7 (ATG7) was detected by qRT-PCR. The cell proliferation and colony formation were measured by Cell Counting Kit-8 (CCK-8) assay and colony formation assay. And the autophagic flux was evaluated by mRFP-GFP-LC3B reporter. The autophagy related proteins were analyzed by Western blotting. Besides, the relationship between lncRNA DANCR and miR-222-3p, as well as between miR-222-3p and ATG7, was determined by Dual-Luciferase reporter system. We found high expression of lncRNA DANCR and ATG7, and low expression of miR-222-3p in HCC tissues and cell lines. And lncRNA DANCR positively correlated with poor survival of HCC patients. Moreover, the knockdown of lncRNA DANCR inhibited cell proliferation and autophagy of HCC cells. And we predicted and proved that lncRNA DANCR induced cell proliferation, colony formation and autophagy by increasing ATG7 and suppressing miR-222-3p. Our study demonstrates the promoting role of lncRNA DANCR in HCC, and indicates the regulatory effects of lncRNA DANCR on regulating autophagy of HCC.

LncRNA DANCR Promotes Proliferation and Metastasis of Breast Cancer Cells Through Sponging miR-4319 and Upregulating VAPB.

Background: Breast cancer is one of the most prevalent cancers that often occur in females. Long noncoding RNA differentiation antagonizing nonprotein coding RNA (DANCR) has been involved in the pathogenesis of various tumors, including breast cancer. This study aimed to investigate the role and underlying mechanism of DANCR in breast cancer. Materials and Methods: The level of DANCR was detected in breast cancer tissues and cells by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability was evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Cell apoptosis was assessed using flow cytometry. Cell migration and invasion were estimated by the Transwell assay. The relationship between DANCR, miR-4319, and vesicle-associated membrane protein-associated protein B (VAPB) was confirmed by bioinformatic analysis and dual-luciferase reporter assay. The level of microRNA-4319 (miR-4319) was tested by qRT-PCR. The expression of VAPB was measured by qRT-PCR or Western blot assay. Results: DANCR and VAPB were upregulated, while miR-4319 was downregulated in breast cancer tissues and cells. Knockdown of DANCR hindered proliferation, migration, and invasion and promoted apoptosis of breast cancer cells. DANCR knockdown inhibited breast cancer development through regulating miR-4319. Inhibition of miR-4319 restrained breast cancer cell progression by targeting VAPB. Moreover, DANCR regulated VAPB expression by sponging miR-4319 in breast cancer cells. Conclusion: DANCR facilitated breast cancer cell progression through regulating the miR-4319/VAPB axis, indicating that DANCR might be a potential biomarker and therapeutic target for breast cancer treatment.

Exosome-Derived Differentiation Antagonizing Non-Protein Coding RNA Promotes Tumor Recurrences in Hepatitis C-Related Hepatocellular Carcinoma

Background & Aims: Differentiation antagonizing non-protein coding RNA (DANCR) associated with various types of neoplasms. Hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) (HCV-HCC) has a high risk of recurrence. We explored the role of DANCR in HCV-related hepatocarcinogenesis and identified potential therapeutic targets and non-invasive prognostic markers for long-term outcome of HCV-HCC after surgical resection. Methods: DANCR relevant to HCV-HCC were identified by comparative RNA-sequencing of tumor (T) and adjacent non-tumor (ANT) tissues in a screening set, validated using real-time PCR Target lncRNAs in tissues and serum exosomes were used to predict HCC recurrence of HCV-HCC after curative surgical resection in a large application cohort. Exosome-Derived DANCR was explored using JFH1-infected cells and macrophages. Results: We confirmed that DANCR was upregulated by HCV infection and identified as the lncRNA most relevant to HCV-HCC in tumor as compared to ANT tissues. In 183 HCV-HCC patients followed for 10 years after curative HCC resection, the expression level of circulating exosomal DANCR was positively associated with HCC recurrence and was the most predictive factor associated with HCC recurrence and mortality (hazard ratio/95% confidence intervals: 7.0/4.3–11.6 and 2.7/1.5–5.1, respectively). In vitro study further demonstrated that exosomal DANCR was positively associated with mesenchymal-associated β-catenin expression in HCV infectious hepatocyte and macrophages, which might consequently promote tumor cell migration and invasion abilities in the tumor microenvironment. Conclusions: The lncRNA DANCR is highly relevant to disease progression of HCV-HCC Furthermore, circulating exosomal DANCR might serve as a non-invasive prognostic biomarker for HCV-HCC. Funding Statement: This study supported by the grants from the Ministry of Science Technology (MOST) grant (108-2314-B-037-079-MY3) (106-2221-E-039-011-MY3); China medical University (CMU108-MF-93), (CMU108-Z-02), (CMU108-S-22); Kaohsiung Medical University (KMU-Q108009), (KMUQ109005), (108-CCH-KMU-001); partially by Kaohsiung Medical University Research Grant KMU-TC108A04 and KMU-TC108B06. This study was partially supported by of Liquid Biopsy and of Cancer Research, Kaohsiung Medical University, and the Center for Intelligent Drug Systems and Smart Bio-devices (IDS2B) from The Featured Areas Research Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. Declaration of Interests: All authors declare no support from any organization other than the below mentioned ones for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years; and no other relationships or activities that could appear to have influenced the submitted work. Hence, all authors declare themselves to be independent from funders with respect to this manuscript. Ethics Approval Statement: The study protocol was approved by the Institutional Review Board at each hospital and the TLCN User Committee and was carried out in accordance with the Declaration of Helsinki and the guidelines of the International Conference on Harmonization for Good Clinical Practice.

MicroRNA-33a-5p sponges to inhibit pancreatic \u03b2-cell function in gestational diabetes mellitus LncRNA DANCR

BackgroundGestational diabetes mellitus (GDM) is the most common medical complication associated with pregnancy, which may impose risks on both mother and fetus. Micro RNAs (miRNAs) and long noncoding RNAs (lncRNAs) are implied as vital regulators in GDM. A recent paper revealed dysregulation of miR-33a-5p in placental tissues of GDM patients. However, the biological function of miR-33a-5p in GDM remains elusive. This study focused on exploring the function and underlying mechanisms of miR-33a-5p in GDM.Methods12 GDM pregnancies and 12 healthy pregnancies were enrolled in the study. INS-1 cell line was applied in in vitro experiments. The expression levels of miR-33a-5p, lnc-DANCR (Differentiation Antagonizing Non-Protein Coding RNA), and ABCA1 (ATP-binding cassette transporter 1) mRNA were determined by RT-qPCR assay. Glucose and insulin levels were measured by ELISA assay. Luciferase reporter assay and western blot assay were applied to validate the target of miR-33a-5p.ResultsmiR-33a-5p was upregulated in the blood samples from GDM, and was positively correlated with blood glucose (p < 0.0001). Overexpression or inhibition of miR-33a-5p significantly inhibited or promoted cell growth and insulin production of INS-1 cells (p < 0.01). Furthermore, ABCA1 is a direct target of miR-33a-5p, and lnc-DANCR functions as a sponge for miR-33a-5p to antagonize the function of miR-33a-5p in INS-1 cells.ConclusionOur study demonstrated that lnc-DANCR-miR-33a-5p-ABCA1 signaling cascade plays a crucial role in the regulation of the cellular function of INS-1 cells.

Circulating exosome DANCR correlated to the recurrence of hepatitis C virus-related hepatocellular carcinoma.

e13688 Background: Long non-coding RNAs (lncRNAs) have been associated with various types of neoplasms. Hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) has high risk of recurrence. Methods: Differentially-expressed candidate lncRNAs relevant to HCV-HCC were identified by RNA-seq in Screening set, validated by qPCR in Validation set, and confirmed in external Test cohort. Target lncRNAs in tissue and serum exosome were applied to predict HCC recurrence of HCV-HCC after curative surgical resection in a large Application cohort. Pathogenetic mechanisms of target lncRNA were explored by using JFH1-infectious cells. Results: Of the top ten differentially-expressed lncRNAs relevant to HCV-related HCC identified in Screening set, differentiation antagonizing non-protein coding RNA (DANCR), which was upregulated by HCV infection and identified as the most relevant lncRNA to HCV-HCC, was overexpressed in tumor than in adjacent non-tumor (ANT) tissues. Of 183 HCV-HCC patients followed for 10 years after curative HCC resection, tumor DANCR, tumor/ANT DANCR ratio and circulating exosome DANCR levels were positively associated with HCC recurrence. Circulating exosome DANCR levels by droplet digital PCR provided the best predictive power of HCC recurrence with area under the curve of 0.88, when compared to tumor DANCR levels, tumor/ANT DANCR ratio, and alfa-fetoprotein. Exosome DANCR &gt; 50 copies/ml was the most predictive factor associated with HCC recurrence and mortality (hazard ratio/95% confidence intervals: 7.0/4.3-11.6 and 2.7/1.5-5.1 respectively, Cox regression model). Cell experiments showed that DANCR, positively associated with mesenchymal-associated β-catenin expression, promoted cell migration and invasion ability. Conclusions: lncRNA-DANCR was highly relevant to disease progression of HCV-HCC. Circulating exosome DANCR could serve as a non-invasive prognostic biomarker for HCV-HCC.

LncRNA DANCR-miR-758-3p-PAX6 Molecular Network Regulates Apoptosis and Autophagy of Breast Cancer Cells.

ObjectiveThis study set out to probe into the effects of long non-coding RNA (LncRNA) differentiation antagonizing non-protein coding RNA (DANCR) on apoptosis and autophagy of breast cancer (BC) cells.MethodsThe expression levels of DANCR, miR-758-3p and paired box 6 (PAX6) in BC tissues and cell lines were detected. The transcription and protein levels of PAX6, apoptosis-related factors (caspase-3, caspase-9, Bax/Bcl-2), and autophagy-related factors (LC3B, Atg5, Beclin-1) in BC cells were detected. The cell proliferation, apoptosis, autophagy and the regulatory relationship between genes and target genes were analyzed.ResultsDANCR and PAX6 were up-regulated in BC tissues and cell lines, while miR-758-3p was opposite. Down-regulating DANCR inhibited the malignant proliferation of BC cells and also promoted apoptosis and autophagy, which showed that caspase-3, caspase-9, Bax/Bcl-2, LC3B, Atg5 transcription and protein levels increased, while Beclin-1 transcription and protein levels decreased. DANCR regulated miR-758-3p in a targeted manner, and its over-expression could weaken the anti-cancer effect of miR-758-3p on BC cells. In addition, miR-758-3p also directly targeted PAX6, and knocking down its expression could weaken the inhibitory effect of down-regulating PAK6 on BC cell apoptosis and autophagy. We also found that DANCR acted as a competitive endogenous RNA sponge miR-758-3p, thus regulating the PAX6 expression.ConclusionDANCR-miR-758-3p-PAX6 molecular network plays a key regulatory role in BC cell apoptosis and autophagy, which may provide reference for treating patients.

Circulating lncRNA DANCR as a potential auxillary biomarker for the diagnosis and prognostic prediction of colorectal cancer

Studies have shown that long non-coding RNAs (lncRNAs) play vital roles in the development of cancer, including colorectal cancer (CRC). Our purpose is to validate the diagnostic value of serum differentiation antagonizing non-protein coding RNA (DANCR) in CRC by focusing on its expression and clinical application. lncRNA expression profiles of CRC patients were obtained and analyzed by repurposing the publically available microarray data. Tissue or serum specimens were obtained from 40 patients with primary CRC, 10 patients with recurrent CRC, 40 patients with colorectal polyps, and 40 healthy controls. It was found that DANCR level in the CRC tissue and serum was significantly increased, and serum DANCR expression was decreased in post-operative patients as compared with that in pre-treatment patients and recurrent patients. In addition, serum DANCR expression was significantly correlated with different TNM stages. Correlation analysis of DANCR and other diagnostic indicators showed that the serum DANCR expression level was significantly correlated with CA199 but not with CEA in CRC patients. As for diagnostic efficiency by ROC analysis, the area under the curve (AUC) of serum DANCR was higher than that of CEA and CA199 in CRC group vs. colorectal polyp group. Simultaneous detection of DANCR, CEA and CA199 yielded the highest sensitivity and AUC as compared with either of them alone. Taken together, serum DANCR was up-regulated in CRC patients and high expression of DANCR may prove to be a potential biomarker for the diagnosis of CRC.

Long Noncoding RNA DANCR Suppressed Lipopolysaccharide-Induced Septic Acute Kidney Injury by Regulating miR-214 in HK-2 Cells.

BackgroundAcute kidney injury (AKI) is one of the most important causes of death in sepsis patients. Here, we first measured the level of DANCR (differentiation antagonizing nonprotein coding RNA) expression in AKI, and the potential mechanism was further elucidated.Material/MethodsWe used qRT-PCR to examine the level of DANCR in patient blood serum samples and in HK-2 cells. In addition, DANCR overexpression was established using lentiviral transfection in HK-2 cells. Subsequently, Cell Counting Kit-8 (CCK-8) assay and flow cytometry were applied to evaluate the role of DANCR in HK-2 cells treated with lipopolysaccharide (LPS). Enzyme linked immunosorbent assay (ELISA), western blot and recovery experiments were performed to elucidate the further mechanism.ResultsWe found that DANCR was decreased in the serum of AKI patients and HK-2 cells treated with LPS. Additionally, DANCR promoted cell viability and suppressed cell apoptosis and cytokine production in LPS-treated HK-2 cells. Bioinformatics analysis showed that DANCR served as a sponge for miR-214. Furthermore, DANCR inhibited the expression of Krüppel-like factor 6 (KLF6).ConclusionsOur study suggests that AKI development could be alleviated by sponging miR-214 and regulating KLF6 expression, which provides a novel potential mechanism involved in the diagnosis and treatment of sepsis-induced AKI patients.

LncRNA DANCR Promotes Sorafenib Resistance via Activation of IL-6/STAT3 Signaling in Hepatocellular Carcinoma Cells.

BackgroundHepatocellular carcinoma (HCC) is one of the major malignancies and the second most common cause of cancer-related death worldwide. Sorafenib, an approved first-line systematic treatment agent for HCC, is capable to effectively improve the survival of patients with advanced HCC. The long-noncoding RNA (lncRNA) differentiation antagonizing non-protein coding RNA (DANCR) has been reported to exert oncogenic functions in several kinds of human cancers. However, the role of lncRNA DANCR in sorafenib resistance in HCC remains unknown.MethodsThe expression levels of DANCR in HCC tissues were detected by qRT-PCR. DANCR overexpression and knockdown models were established and utilized to investigate the functional role of DANCR on sorafenib resistance in HCC cells. The MS2-binding sequences-MS2-binding protein–based RNA immunoprecipitation assay, RNA pull-down and luciferase reporter assay was used to detect the association between DANCR and PSMD10 mRNA. The activation of DANCR transcription mediated by STAT3 was assessed by luciferase reporter and chromatin immunoprecipitation assays.ResultsWe found that DANCR was significantly overexpressed in HCC tissues and associated with prognosis of HCC patients. Overexpression and knockdown experiments demonstrated that DANCR promoted sorafenib resistance in HCC cells in vitro and in vivo. Mechanistically, the role of DANCR relied largely on the association with PSMD10. DANCR stabilized PSMD10 mRNA through blocking the repressing effect of several microRNAs on PSMD10. Besides, DANCR activated IL-6/STAT3 signaling via PSMD10. Furthermore, we revealed that DANCR transcription was enhanced by the activation of IL-6/STAT3 signaling, indicating a positive feedback loop of DANCR and IL-6/STAT3 signaling.ConclusionCollectively, our study is the first to elucidate the mechanism of DANCR-mediated sorafenib resistance via PSMD10-IL-6/STAT3 signaling axis, which provides a promising target for developing new therapeutic strategy for sorafenib tolerance of HCC.