The Mechanism and Clinical Significance of Circular RNAs in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignant tumors worldwide. In view of the lack of early obvious clinical symptoms and related early diagnostic biomarkers with high specificity and sensitivity, most HCC patients are already at the advanced stages at the time of diagnosis, and most of them are accompanied by distant metastasis. Furthermore, the unsatisfactory effect of the follow-up palliative care contributes to the poor overall survival of HCC patients. Therefore, it is urgent to identify effective early diagnosis and prognostic biomarkers and to explore novel therapeutic approaches to improve the prognosis of HCC patients. Circular RNA (CircRNA), a class of plentiful, stable, and highly conserved ncRNA subgroup with the covalent closed loop, is dysregulated in HCC. Increasingly, emerging evidence have confirmed that dysregulated circRNAs can regulate gene expression at the transcriptional or post-transcriptional level, mediating various malignant biological behaviors of HCC cells, including proliferation, invasion, metastasis, immune escape, stemness, and drug resistance, etc.; meanwhile, they are regarded as potential biomarkers for early diagnosis and prognostic evaluation of HCC. This article reviews the research progress of circRNAs in HCC, expounding the potential molecular mechanisms of dysregulated circRNAs in the carcinogenesis and development of HCC, and discusses those application prospects in the diagnosis and prognosis of HCC.

LncRNA DiGeorge syndrome critical region gene 5: A crucial regulator in malignant tumors.

Long non-coding RNA (lncRNA), a subgroup of ncRNA with a length of more than 200 nt without protein coding function, has been recognized by the academia for its mediating effects of dysregulated expression on the tumorigenesis and development of a variety of tumors. LncRNA DiGeorge syndrome critical region gene 5 (DGCR5), originally found to induce DiGeorge syndrome, has been confirmed to be extremely dysregulated in multiple tumors, which mediates the malignant phenotypes of hepatocellular carcinoma, pancreatic cancer, lung cancer, etc. through the regulation of Wnt/ß-catenin, MEK/ERK1/2 and other cancerous signaling pathways as a molecular sponge. Researches on the cancerous derivation-related pathways involved in DGCR5 can provide potential molecular intervention targets for tumor precision treatment. Moreover, liquid biopsy based on the detection of DGCR5 in body fluids is also expected to provide a non-invasive evaluation method for the early diagnosis and prognostic evaluation of malignant tumors.

Exosomal Non-Coding RNAs: Regulatory and Therapeutic Target of Hepatocellular Carcinoma.

Exosomes are small extracellular vesicles secreted by most somatic cells, which can carry a variety of biologically active substances to participate in intercellular communication and regulate the pathophysiological process of recipient cells. Recent studies have confirmed that non-coding RNAs (ncRNAs) carried by tumor cell/non-tumor cell-derived exosomes have the function of regulating the cancerous derivation of target cells and remodeling the tumor microenvironment (TME). In addition, due to the unique low immunogenicity and high stability, exosomes can be used as natural vehicles for the delivery of therapeutic ncRNAs in vivo. This article aims to review the potential regulatory mechanism and the therapeutic value of exosomal ncRNAs in hepatocellular carcinoma (HCC), in order to provide promising targets for early diagnosis and precise therapy of HCC.

Circ-LAMP1 contributes to the growth and metastasis of cholangiocarcinoma via miR-556-5p and miR-567 mediated YY1 activation.

Dysregulation of circular RNAs (circRNAs) executes important regulatory roles in carcinogenesis. Nonetheless, few studies focused on the mechanisms of circRNAs in cholangiocarcinoma (CCA). qRT-PCR was applied to verify the dysregulated circRNAs in CCA. Fisher's exact test, Kaplan-Meier analysis and Cox regression model were utilized to investigate the clinical implications of circ-LAMP1 in the patients with CCA. The viability, apoptosis, migration and invasion of CCA cells were detected after silencing/overexpression of circ-LAMP1. Xenograft and lung metastasis assays were performed to verify the in vitro results. The regulatory networks of circ-LAMP1 were unveiled by bioinformatic analysis, RNA immunoprecipitation (RIP), RNA pulldown and luciferase reporter assays. Up-regulation of circ-LAMP1 was found in CCA tissue samples and cell lines. Enhanced level of circ-LAMP1 was linked to clinical severity, high post-operative recurrence and poor prognosis for the patients with CCA. Gain/loss-of-function assays confirmed the oncogenic role of circ-LAMP1 in mediating cell growth, apoptosis, migration and invasion. Nevertheless, the level of circ-LAMP1 had no effect on normal biliary epithelium proliferation and apoptosis. Animal study further verified the in vitro data. Mechanistically, circ-LAMP1 directly sponged miR-556-5p and miR-567, thereby releasing their suppression on YY1 at post-transcriptional level. Rescue assay indicated that the oncogenic role of circ-LAMP1 is partially dependent on its modulation of YY1 in CCA. In summary, this study suggested that circ-LAMP1 might be used as a promising biomarker/therapeutic target for CCA.

Yin Yang 1-induced LINC00667 up-regulates pyruvate dehydrogenase kinase 1 to promote proliferation, migration and invasion of cholangiocarcinoma cells by sponging miR-200c-3p.

Cholangiocarcinoma (CCA) is one of the most aggressive and lethal malignancies. Long noncoding RNAs (lncRNAs) are being found to play crucial roles in CCA progression. This work aims to investigate the roles of long intergenic non-protein coding RNA 667 (LINC00667) in progression of CCA. RT-qPCR and western blot were applied to detect gene expression. Clinical correlation and survival were analyzed by statistical methods. Overexpression and RNA interference approaches were used to investigate the effects of LINC00667 on CCA cells. Tumor xenograft assay was performed to detect the function of LINC00667 in vivo. Transcriptional regulation and competing endogenous RNA (ceRNA) mechanism were predicted via bioinformatics analysis. ChIP, luciferase reporter, and Ago2 RIP assays further confirmed the predicted results. Our data indicated that LINC00667 was highly expressed in CCA tissues and cells, and transcription factor Yin Yang 1 (YY1) induced LINC00667 expression in CCA cells. Up-regulated LINC00667 was significantly associated with lymph node metastasis, advanced TNM stage, and poor prognosis. Knockdown of LINC00667 suppressed the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of CCA cells, while overexpression of LINC00667 acquired opposite effects. Moreover, knockdown of LINC00667 inhibited tumor growth in vivo. In addition, LINC00667 was demonstrated to function as a ceRNA for miR-200c-3p, and then LINC00667 up-regulated pyruvate dehydrogenase kinase 1 (PDK1) to promote CCA development by inhibiting miR-200c-3p. These findings identified a pivotal role of LINC00667 in tumorigenesis and development of CCA. Targeting the YY1/LINC00667/miR-200c-3p/PDK1 axis may provide a new therapeutic strategy for CCA treatment.

A Circular RNA, Cholangiocarcinoma-Associated Circular RNA 1, Contributes to Cholangiocarcinoma Progression, Induces Angiogenesis, and Disrupts Vascular Endothelial Barriers.

BACKGROUND AND AIMS: Circular RNAs (circRNAs) and extracellular vesicles (EVs) are involved in various malignancies. We aimed to clarify the functions and mechanisms of dysregulated circRNAs in the cells and EVs of cholangiocarcinoma (CCA). APPROACH AND RESULTS: CircRNA microarray was used to identify circRNA expression profiles in CCA tissues and bile-derived EVs (BEVs). CCA-associated circRNA 1 (circ-CCAC1) expression was measured by quantitative real-time PCR. The clinical importance of circ-CCAC1 was analyzed by receiver operating characteristic curves, Fisher's exact test, Kaplan-Meier plots, and Cox regression model. The functions of circ-CCAC1 and exosomal circ-CCAC1 were explored in CCA cells and human umbilical vein endothelial cells (HUVECs), respectively. Different animal models were used to verify the in vitro results. RNA sequencing, bioinformatics, RNA immunoprecipitation, RNA pulldown, chromatin immunoprecipitation followed by sequencing, and luciferase reporter assays were used to determine the regulatory networks of circ-CCAC1 in CCA cells and HUVECs. Circ-CCAC1 levels were increased in cancerous bile-resident EVs and tissues. The diagnostic and prognostic values of circ-CCAC1 were identified in patients with CCA. For CCA cells, circ-CCAC1 increased cell progression by sponging miR-514a-5p to up-regulate Yin Yang 1 (YY1). Meanwhile, YY1 directly bound to the promoter of calcium modulating ligand to activate its transcription. Moreover, circ-CCAC1 from CCA-derived EVs was transferred to endothelial monolayer cells, disrupting endothelial barrier integrity and inducing angiogenesis. Mechanistically, circ-CCAC1 increased cell leakiness by sequestering enhancer of zeste homolog 2 in the cytoplasm, thus elevating SH3 domain-containing GRB2-like protein 2 expression to reduce the levels of intercellular junction proteins. In vivo studies further showed that increased circ-CCAC1 levels in circulating EVs and cells accelerated both CCA tumorigenesis and metastasis. CONCLUSIONS: Circ-CCAC1 plays a vital role in CCA tumorigenesis and metastasis and may be an important biomarker/therapeutic target for CCA.

Elevation of circular RNA circ_0005230 facilitates cell growth and metastasis via sponging miR-1238 and miR-1299 in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a highly malignant carcinoma with high mortality rate worldwide. Emerging evidence indicates that aberrantly expressed circular RNAs (circRNAs) functions crucial roles in tumor progression. In this work, we focused on a novel circRNA, circ_0005230, in carcinogenesis and development of CCA. Circ_0005230 levels in CCA specimens and cells were measured by qRT-PCR. The clinical implication of circ_0005230 was analyzed by fisher's exact test. Gain/loss of-function assays were conducted to reveal the effects of circ_0005230 on the cell proliferation, apoptosis, migration and invasion of CCA cells. Xenograft and lung metastatic models were constructed to confirm the in vitro data. Dual luciferase reporter and rescue assays were carried out to illuminate the mechanism behind the regulatory actions. As data showed, circ_0005230 was elevated in tumors and CCA cells. Its expression in tumor samples was related to clinical severity. Functionally, circ_0005230 significantly facilitated cell growth, clone-forming ability and metastatic properties and inhibit cell apoptosis in CCA cells. The in vivo study further validated the in vitro results. However, knockdown of circ_0005230 did not affect normal biliary epithelial (HIBEC) cell growth and apoptosis. For the mechanism investigation, circ_0005230 could directly sponge miR-1238 and miR-1299 to exert its oncogenic functions. Overall, this work showed that circ_0005230 might act as an effective therapeutic target for CCA.

Altered expression of long non-coding RNA GAS5 in digestive tumors.

Cancer has become one of the most important diseases that affect human health and life. The effects of cancer in the digestive system are particularly prominent. Recently, long non-coding RNA (lncRNA) has attracted the attention of more and more researchers and has become an emerging field of gene research. The lncRNA growth arrest-specific 5 (GAS5) is a novel lncRNA that has attracted the attention of researchers in recent years and plays an important role in the development of tumors, especially in digestive system tumors. GAS5 was first identified in a mouse cDNA library. It was generally considered that it has the role of tumor suppressor genes, but there are still studies that have a certain ability to promote cancer. Furthermore, the 5-bp indel polymorphism (rs145204276) in the GAS5 promoter region also has a carcinogenic effect. The discovery of GAS5 and in-depth study of single nucleotide polymorphism (SNP) mechanism can provide a new way for the prevention and treatment of digestive system tumors.

miR-124 Suppresses Pancreatic Ductal Adenocarcinoma Growth by Regulating Monocarboxylate Transporter 1-Mediated Cancer Lactate Metabolism.

BACKGROUND/AIMS: Increasing evidence shows that reprogramming of energy metabolism is a hallmark of cancer. Considering the emergence of microRNAs as crucial modulators of cancer, this study aimed to better understand the molecular mechanisms of miR-124 in regulating glycolysis in human pancreatic cancer. METHODS: RT-PCR was used to investigate the expression of monocarboxylate transporters (MCTs) in pancreatic ductal adenocarcinoma (PDAC) patient samples and the PANC-1 cell line. A public database and immunochemistry were used for comprehensive analysis of MCT1 expression. The targeting of MCT1 by miR-124 was predicted by software and validated for the MCT1 3'-UTR by dual-luciferase reporter analysis. Cell proliferation, apoptosis, migration, xenografting, and the intracellular pH and L-lactate levels were assessed. Hypoxia-inducible factor-α (HIF-1α) and lactate dehydrogenase A (LDH-A) expression levels were determined by RT-PCR and western blotting. RESULTS: MCT1 expression was higher in PDAC tissue than in normal tissue. Inhibition of MCT1 affected lactate metabolism, resulting in a higher intracellular pH and less proliferation of PANC-1 cells. MCT1 was the target gene of miR-124. In in vitro experiments, miR-124 inhibited the glycolytic activity of PANC-1 cells by targeting MCT1, further decreasing the tumor phenotype by increasing the intracellular pH through LDH-A and HIF-1α. In in vivo experiments, overexpression of miR-124 and silencing of MCT1 significantly inhibited tumor growth. CONCLUSION: miR-124 inhibits the progression of PANC-1 by targeting MCT1 in the lactate metabolic pathway. Our findings provide novel evidence for further functional studies of miR-124, which might be useful for future therapeutic approaches to PDAC.

Hepatitis B virus-X protein regulates high mobility group box 1 to promote the formation of hepatocellular carcinoma.

Hepatitis B virus (HBV) infection is a risk factor for hepatocellular carcinoma (HCC). HBV X protein (HBx) is an important carcinogen for HBV-induced HCC. When the HBx gene is integrated into the host cell genome, it is difficult to eradicate. The identification of an effective target to inhibit the oncogenic function of HBx is therefore critically important. The present study demonstrated that HBx, particularly truncated HBx, was expressed in several HBV-derived cell lines (e.g., Hep3B and SNU423). By analyzing data from The Cancer Genome Atlas, it was revealed that high expression of high mobility group box 1 (HMGB1) was associated with the process and prognosis of HCC. In vitro experiments confirmed that HBx could regulate the expression of HMGB1 and knockdown of HMGB1 could decrease the ability of HBx to promote cellular proliferation. HBx could also upregulate six transcription factors (GATA binding protein 3, Erb-B2 receptor tyrosine kinase 3, heat shock transcription factor 1, nuclear factor κB subunit 1, TATA-box binding protein and Kruppel-like factor 4), which could directly regulate HMGB1. By analyzing genes that are co-expressed with HMGB1, several signaling pathways associated with the development of HCC were identified. HBx and HMGB1 were revealed to be involved in these pathways, which may be the mechanism by which HBx promotes HCC by regulating HMGB1. These findings suggested that HMGB1 may be an effective target for inhibiting HBV-induced HCC.

The role of the long non-coding RNA HOXA11-AS in promoting proliferation and metastasis of malignant tumors.

Long non-coding RNA (lncRNA) is one of the focuses and hotspots of biological research in recent years. At the same time, tumors have become the main disease that endangers human health. In recent years, a large number of researchers have explored the relationship between lncRNA and tumors. HOXA11-AS is one of these lncRNAs. The long non-coding RNA HOXA11 antisense RNA (HOXA11-AS) is a novel lncRNA recently discovered. It is found in a variety of tumors such as ovarian cancer, glioma, and gastric cancer (GC) and so on, and is defined as an oncogene. It promotes tumor proliferation and metastasis by interacting with proteins such as miRNA and EZH2. In this paper, we review the mechanism of interaction between HOXA11-AS and various tumors in recent years, and believe that it can be a potential tumor marker and therapeutic target in the future prevention and treatment of tumors.

Current insight into a cancer-implicated long noncoding RNA ZFAS1 and correlative functional mechanisms involved.

OBJECTIVES: A vast of transcripts have been found aberrantly expressed in cancers functioning as mechanical factors. The association between the deregulation of long noncoding RNAs (lncRNAs) and clinicopathologic features is one of the most investigated in this field, and many lncRNAs have already been revealed to be potential biomarkers or therapeutic targets. Zinc finger antisense 1 (ZFAS1) has been found one promising lncRNA, initially discovered downregulated in human breast cancer and could regulate alveolar development and epithelial cell differentiation in mice mammary gland, however the subsequent investigation provided inverse outcomes as overexpressed in other human cancers. Further excavation of this transcript indicated that ZFAS1 could function as oncogenic factor via many approaches to contribute to the advance of cancers, including induction of epithelial-mesenchymal transition, sponging microRNAs, destabilization p53 gene and many others. MATERIALS AND METHODS: In this work, we summarized current evidence regarding the biological functions and mechanisms of ZFAS1 in human cancers. The related studies were obtained through a systematic search of PubMed, Embase and Cochrane Library. RESULTS: LncRNA ZFAS1 is one cancer-implicated product with multiple functional mechanisms, and its potential clinical values is gradually unfolded in many types of cancers, including breast cancer, hepatocellular carcinoma, colorectal cancer, gastric cancer, glioma, ovarian cancer and many others. CONCLUSION: The multi-mechanisms of lncRNA ZFAS1 fertilizes its role of potential clinical biomarkers and therapeutic targets.

LncRNA HOXA-AS2 and its molecular mechanisms in human cancer.

Long non-coding RNAs (lncRNAs), a novel class of noncoding RNAs, are commonly defined as RNA molecules more than 200 nucleotides in length. Emerging research indicated that lncRNA played a vital role in human tumorigenesis and progression by serving as tumor oncogenes or suppressors. LncRNA has been shown to get involved in participate various biological processes, such as cell growth, anti-apoptosis, migration and invasion. LncRNA HOXA cluster antisense RNA2 (HOXA-AS2) is a novel cancer-related lncRNA. It was recently found to exhibit aberrant expression in a variety of malignancies, including breast cancer, gastric cancer, gallbladder carcinoma, hepatocellular carcinoma and pancreatic cancer. The oncogenicity of lncRNA HOXA-AS2 mainly inhibits or promotes the expression of related genes through direct or indirect pathways, suggesting that HOXA-AS2 likely represents a feasible biomarker or therapeutic target in human cancers. In this review, we summarize current evidences concerning the biological functions and mechanisms of HOXA-AS2 during tumor development.