Shenglin Huang

Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs

Circular RNAs (circRNAs) represent a class of widespread and diverse endogenous RNAs that may regulate gene expression in eukaryotes. However, the regulation and function of human circRNAs remain largely unknown. Here we generate ribosomal-depleted RNA sequencing data from six normal tissues and seven cancers, and detect at least 27,000 circRNA candidates. Many of these circRNAs are differently expressed between the normal and cancerous tissues. We further characterize one abundant circRNA derived from Exon2 of the HIPK3 gene, termed circHIPK3. The silencing of circHIPK3 but not HIPK3 mRNA significantly inhibits human cell growth. Via a luciferase screening assay, circHIPK3 is observed to sponge to 9 miRNAs with 18 potential binding sites. Specifically, we show that circHIPK3 directly binds to miR-124 and inhibits miR-124 activity. Our results provide evidence that circular RNA produced from precursor mRNA may have a regulatory role in human cells.

Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis

Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis

Circular RNA profile identifies circPVT1 as a proliferative factor and prognostic marker in gastric cancer

Circular RNAs (circRNAs) comprise a novel class of widespread non-coding RNAs that may regulate gene expression in eukaryotes. However, the characterization and function of circRNAs in human cancer remain elusive. Here we identified at least 5500 distinct circRNA candidates and a series of circRNAs that are differentially expressed in gastric cancer (GC) tissues compared with matched normal tissues. We further characterized one circRNA derived from the PVT1 gene and termed it as circPVT1. The expression of circPVT1 is often upregulated in GC tissues due to the amplification of its genomic locus. circPVT1 may promote cell proliferation by acting as a sponge for members of the miR-125 family. The level of circPVT1 was observed as an independent prognostic marker for overall survival and disease-free survival of patients with GC. Our findings suggest that circPVT1 is a novel proliferative factor and prognostic marker in GC.

MiR‐199a‐5p is negatively associated with malignancies and regulates glycolysis and lactate production by targeting hexokinase 2 in liver cancer

Cancer cells possess a unique metabolic phenotype that allows them to preferentially utilize glucose through aerobic glycolysis. This phenomenon is referred to as the “Warburg effect.” Accumulating evidence suggests that microRNAs (miRNAs), a class of small noncoding regulatory RNAs, interact with oncogenes/tumor suppressors and induce such metabolic reprograming in cancer cells. To systematically study the metabolic roles of miRNAs in cancer cells, we developed a gain‐of‐function miRNA screen in HeLa cells. Subsequent investigation of the characterized miRNAs indicated that miR‐199a‐5p acts as a suppressor for glucose metabolism. Furthermore, miR‐199a‐5p is often down‐regulated in human liver cancer, and its low expression level was correlated with a low survival rate, large tumor size, poor tumor differentiation status, high tumor‐node‐metastasis stage and the presence of tumor thrombus of patients. MicroRNA‐199a‐5p directly targets the 3′‐untranslated region of hexokinase 2 (HK2), an enzyme that catalyzes the irreversible first step of glycolysis, thereby suppressing glucose consumption, lactate production, cellular glucose‐6‐phosphate and adenosine triphosphate levels, cell proliferation, and tumorigenesis of liver cancer cells. Moreover, HK2 is frequently up‐regulated in liver cancer tissues and associated with poor patient outcomes. The up‐regulation of hypoxia‐inducible factor‐1α under hypoxic conditions suppresses the expression of miR‐199a‐5p and promotes glycolysis, whereas reintroduction of miR‐199a‐5p interferes with the expression of HK2, abrogating hypoxia‐enhanced glycolysis. Conclusion: miR‐199a‐5p/HK2 reprograms the metabolic process in liver cancer cells and provides potential prognostic predictors for liver cancer patients. (Hepatology 2015;62:1132‐1144)

MicroRNA-30d-5p inhibits tumour cell proliferation and motility by directly targeting CCNE2 in non-small cell lung cancer

MicroRNAs (miRNAs) are small, single-stranded, non-coding RNA molecules that are dysregulated in many types of human cancers, although their precise functions in driving non-small cell lung cancer (NSCLC) are incompletely understood. In the present study, we found that miR-30d-5p, often downregulated in NSCLC tissues, significantly inhibited the growth, cell cycle distribution, and motility of NSCLC cells. Furthermore, we demonstrated that cyclin E2 (CCNE2), which was often upregulated in NSCLC tissues, was a direct target of miR-30d-5p. CCNE2 expression promoted the proliferation, invasion, and migration of NSCLC cells. In addition, the re-introduction of CCNE2 expression antagonised the inhibitory effects of miR-30d-5p on the capacity of NSCLC cells for proliferation and motility. Together, these results suggest that the miR-30d-5p/CCNE2 axis may contribute to NSCLC cell proliferation and motility, indicating miR-30d-5p as a potential therapeutic target for the treatment of NSCLC.

Long noncoding RNA TSLNC8 is a tumor suppressor that inactivates the interleukin-6/STAT3 signaling pathway

Long noncoding RNAs can serve as oncogenes or tumor suppressors in human cancer; however, their biological functions and underlying mechanism in hepatocarcinogenesis are largely unknown. Here, we report a novel tumor suppressor long noncoding RNA on chromosome 8p12 (termed TSLNC8) that is frequently deleted and down‐regulated in hepatocellular carcinoma (HCC) tissues. The loss of TSLNC8 is highly associated with the malignant features of HCC and serves as a prognostic indicator for HCC patients. TSLNC8 significantly suppresses the proliferation and metastasis of HCC cells in vitro and in vivo. TSLNC8 exerts its tumor suppressive activity by competitively interacting with transketolase and signal transducer and activator of transcription 3 (STAT3) and modulating the STAT3‐Tyr705 and STAT3‐Ser727 phosphorylation levels and STAT3 transcriptional activity, thus resulting in inactivation of the interleukin‐6–STAT3 signaling pathway in HCC cells. Conclusion: TSLNC8 is a promising prognostic predictor for patients with HCC, and the TSLNC8–transketolase–STAT3 axis is a potential therapeutic target for HCC treatment. (Hepatology 2018;67:171‐187).

NF-κB signaling relieves negative regulation by miR-194 in hepatocellular carcinoma by suppressing the transcription factor HNF-1α

Activation of NF-κB signaling in liver cancer cells suppresses a microRNA that usually limits NF-κB activity. A cancerous inflammatory feedback loop Inflammation activates NF-κB signaling in cells, and chronic inflammation contributes to the development and progression of many cancers, including hepatocellular carcinoma (HCC). In HCC patient tumors, Bao et al. found that NF-κB activity was increased and that the abundance of a microRNA, miR-194, was decreased. Expressing miR-194 in HCC cells suppressed NF-κB activity and decreased both cell migration in culture and lung colonization after injection into mice. However, stimulating NF-κB signaling with the inflammatory cytokine TNFα reduced the abundance of a transcription factor (HNF-1α), leading to a decrease in the expression of miR-194 and promoting cell migration in culture. Thus, by shutting off the expression of a microRNA, proinflammatory cytokines trigger persistent NF-κB signaling that enables chronic inflammation to promote metastatic progression of liver cancer. Constitutive activation of the proinflammatory transcription factor nuclear factor κB (NF-κB) plays an important role in progression of hepatocellular carcinoma (HCC). Emerging modulators of NF-κB signaling are noncoding RNAs, especially microRNAs (miRNAs). We previously identified miRNAs that reduced the induction of NF-κB activity upon addition of tumor necrosis factor–α (TNFα) to HCC cells. We found that among these miRNAs, the abundance of liver-enriched miR-194 was decreased in HCC tissue and that low abundance of miR-194 correlated with a high occurrence of vascular invasion. Overexpressing miR-194 suppressed HCC cell migration and invasiveness in culture and metastatic seeding in mice. Transcripts encoding tripartite motif containing 23 (TRIM23), a ubiquitin ligase involved in NF-κB activation, and chromosome 21 open reading frame 91 (C21ORF91), a protein of unknown function, were identified as direct targets of miR-194 in HCC cells; knocking down either protein decreased the activity of a luciferase NF-κB reporter. Furthermore, the NF-κB pathway activator TNFα, an inflammatory cytokine, inhibited the transcription of miR-194 by decreasing the abundance of hepatocyte nuclear factor–1α (HNF-1α). The abundance of miR-194 positively correlated with that of HNF-1α and inversely correlated with that of TNFα in human HCC tissue. Thus, we identified a pathway in which TNFα–NF-κB signaling switches off negative regulation by suppressing HNF-1α–mediated expression of miR-194, revealing insight into the mechanisms linking inflammatory pathways, miRNA, and HCC metastasis.

MicroRNA-127-5p targets the biliverdin reductase B/nuclear factor-κB pathway to suppress cell growth in hepatocellular carcinoma cells.

Nuclear factor‐κB (NF‐κB) activation is one of the major mediators of inflammation‐induced cancer cell growth and progression. In previous studies, we screened a series of microRNAs (miRNAs) that targeted the NF‐κB signaling pathway. In this study, we showed that miR‐127‐5p suppressed NF‐κB activity through inhibition of p65 nuclear translocation. In addition, miR‐127‐5p also inhibited the transcription of downstream targets of the NF‐κB signaling pathway. While exploring the mechanism of the inhibition of NF‐κB activity by miR‐127‐5p, we found that miR‐127‐5p decreased the phosphorylation of p65. MicroRNA‐127‐5p inhibited the growth and colony formation of hepatocellular carcinoma (HCC) cells and decreased biliverdin reductase B (BLVRB) expression by directly binding to its 3′‐UTR. RNA interference of BLVRB suppressed HCC cell growth, whereas the overexpression of BLVRB promoted HCC cell growth. Furthermore, BLVRB blockade inhibited the phosphorylation of p65 protein and the expression of downstream targets of the NF‐κB signaling pathway, mimicking the function of miR‐127‐5p. The restoration of BLVRB in HCC cells overexpressing miR‐127‐5p impaired the suppression of HCC growth by miR‐127‐5p. Moreover, miR‐127‐5p was downregulated in 58% of HCC samples. In summary, we found that miR‐127‐5p suppressed NF‐κB activity by directly targeting BLVRB in HCC cells, and this finding improves our understanding of the molecular mechanism of inflammation‐induced HCC growth and proliferation and the successful inhibition of NF‐κB activity by cancer treatment.

Circular RNA: An emerging non-coding RNA as a regulator and biomarker in cancer

Circular RNA (circRNA) is a type of covalently closed non-coding RNA that may regulate gene expression in eukaryotes. The recent application of high-throughput RNA sequencing and bioinformatics approaches has revealed a large number of circRNAs in human cells. Emerging evidence indicates that many circRNAs have cell-type specific expression and are linked to physiological development and various diseases. Specially, circRNAs can either serve as oncogenic stimuli or tumor suppressors in cancer. circRNAs have also been shown to be enriched and stable in extracellular fluid, indicating the potential of circRNAs as cancer biomarkers. Here, we summarize the current knowledge of circRNAs, including their classification, biogenesis, properties, and databases, as well as their function and clinical implications in cancer.

exoRBase: a database of circRNA, lncRNA and mRNA in human blood exosomes

Abstract Exosomes, which are nanosized endocytic vesicles that are secreted by most cells, contain an abundant cargo of different RNA species that can modulate the behavior of recipient cells and may be used as circulating biomarkers for diseases. Here, we develop a web-accessible database (http://www.exoRBase.org), exoRBase, which is a repository of circular RNA (circRNA), long non-coding RNA (lncRNA) and messenger RNA (mRNA) derived from RNA-seq data analyses of human blood exosomes. Experimental validations from the published literature are also included. exoRBase features the integration and visualization of RNA expression profiles based on normalized RNA-seq data spanning both normal individuals and patients with different diseases. exoRBase aims to collect and characterize all long RNA species in human blood exosomes. The first release of exoRBase contains 58 330 circRNAs, 15 501 lncRNAs and 18 333 mRNAs. The annotation, expression level and possible original tissues are provided. exoRBase will aid researchers in identifying molecular signatures in blood exosomes and will trigger new exosomal biomarker discovery and functional implication for human diseases.