Circular RNA circTRIM33–12 acts as the sponge of MicroRNA-191 to suppress hepatocellular carcinoma progression
TL;DR: The important role of circTRIM33–12 in the proliferation, migration, invasion and immune evasion abilities of HCC cells is revealed and a new perspective on circRNAs in HCC progression is provided.
Abstract: Recently, the dysregulation of circular RNA (circRNA) have been shown to have important regulatory roles in cancer development and progression, including hepatocellular carcinoma (HCC). However, the roles of most circRNAs in HCC are still unknown. The expression of circular tripartite motif containing 33–12 (circTRIM33–12) in HCC tissues and cell lines was detected by qRT-PCR. The role of circTRIM33–12 in HCC progression was assessed by western blotting, CCK-8, flow cytometry, transwell and a subcutaneous tumor mouse assays both in vitro and in vivo. In vivo circRNA precipitation, RNA immunoprecipitation, luciferase reporter assays were performed to evaluate the interaction between circTRIM33–12 and miR-191. Here, we found that circTRIM33–12, is downregulated in HCC tissues and cell lines. The downregulation of circTRIM33–12 in HCC was significantly correlated with malignant characteristics and served as an independent risk factor for the overall survival (OS) and recurrence-free survival (RFS) of patients with HCC after surgery. The reduced expression of circTRIM33–12 in HCC cells increases tumor proliferation, migration, invasion and immune evasion. Mechanistically, we demonstrated that circTRIM33–12 upregulated TET1 expression by sponging miR-191, resulting in significantly reduced 5-hydroxymethylcytosine (5hmC) levels in HCC cells. These results reveal the important role of circTRIM33–12 in the proliferation, migration, invasion and immune evasion abilities of HCC cells and provide a new perspective on circRNAs in HCC progression.
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TL;DR: The expression of circUHRF1 is higher in human HCC tissues than in matched adjacent nontumor tissues and may drive resistance to anti-PD1 immunotherapy in HCC patients, indicating poor clinical prognosis and NK cell dysfunction in patients with HCC.
Abstract: Natural killer (NK) cells play a critical role in the innate antitumor immune response Recently, NK cell dysfunction has been verified in various malignant tumors, including hepatocellular carcinoma (HCC) However, the molecular biological mechanisms of NK cell dysfunction in human HCC are still obscure The expression of circular ubiquitin-like with PHD and ring finger domain 1 RNA (circUHRF1) in HCC tissues, exosomes, and cell lines was detected by qRT-PCR Exosomes were isolated from the culture medium of HCC cells and plasma of HCC patients using an ultracentrifugation method and the ExoQuick Exosome Precipitation Solution kit and then characterized by transmission electronic microscopy, NanoSight and western blotting The role of circUHRF1 in NK cell dysfunction was assessed by ELISA In vivo circRNA precipitation, RNA immunoprecipitation, and luciferase reporter assays were performed to explore the molecular mechanisms of circUHRF1 in NK cells In a retrospective study, the clinical characteristics and prognostic significance of circUHRF1 were determined in HCC tissues Here, we report that the expression of circUHRF1 is higher in human HCC tissues than in matched adjacent nontumor tissues Increased levels of circUHRF1 indicate poor clinical prognosis and NK cell dysfunction in patients with HCC In HCC patient plasma, circUHRF1 is predominantly secreted by HCC cells in an exosomal manner, and circUHRF1 inhibits NK cell-derived IFN-γ and TNF-α secretion A high level of plasma exosomal circUHRF1 is associated with a decreased NK cell proportion and decreased NK cell tumor infiltration Moreover, circUHRF1 inhibits NK cell function by upregulating the expression of TIM-3 via degradation of miR-449c-5p Finally, we show that circUHRF1 may drive resistance to anti-PD1 immunotherapy in HCC patients Exosomal circUHRF1 is predominantly secreted by HCC cells and contributes to immunosuppression by inducing NK cell dysfunction in HCC CircUHRF1 may drive resistance to anti-PD1 immunotherapy, providing a potential therapeutic strategy for patients with HCC
271 citations
Cites background or methods from "Circular RNA circTRIM33–12 acts as ..."
...The circRIP assay was performed as described previously [18]....
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...Methods: The expression of circular ubiquitin-like with PHD and ring finger domain 1 RNA (circUHRF1) in HCC tissues, exosomes, and cell lines was detected by qRT-PCR....
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...Given that circRNAs act as miRNA sponges [18, 28, 29], we wondered whether circUHRF1 can interact with certain miRNAs in NK cells....
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...The in vivo metastasis assays are described in our previous studies [21] and in the Supplementary Materials and Methods....
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...Immunohistochemistry was performed, and the intensity of the positive staining was measured as described in our previous study [18]....
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TL;DR: The results suggest the critical role of circFGFR1 in the proliferation, migration, invasion, and immune evasion abilities of NSCLC cells and provide a new perspective on circRNAs duringNSCLC progression.
Abstract: Immune system evasion, distance tumor metastases, and increased cell proliferation are the main reasons for the progression of non-small cell lung cancer (NSCLC) and the death of NSCLC patients. Dysregulation of circular RNAs plays a critical role in the progression of NSCLC; therefore, further understanding the biological mechanisms of abnormally expressed circRNAs is critical to discovering novel, promising therapeutic targets for NSCLC treatment. The expression of circular RNA fibroblast growth factor receptor 1 (circFGFR1) in NSCLC tissues, paired nontumor tissues, and cell lines was detected by RT-qPCR. The role of circFGFR1 in NSCLC progression was assessed both in vitro by CCK-8, clonal formation, wound healing, and Matrigel Transwell assays and in vivo by a subcutaneous tumor mouse assay. In vivo circRNA precipitation, RNA immunoprecipitation, and luciferase reporter assays were performed to explore the interaction between circFGFR1 and miR-381-3p. Here, we report that circFGFR1 is upregulated in NSCLC tissues, and circFGFR1 expression is associated with deleterious clinicopathological characteristics and poor prognoses for NSCLC patients. Forced circFGFR1 expression promoted the migration, invasion, proliferation, and immune evasion of NSCLC cells. Mechanistically, circFGFR1 could directly interact with miR-381-3p and subsequently act as a miRNA sponge to upregulate the expression of the miR-381-3p target gene C-X-C motif chemokine receptor 4 (CXCR4), which promoted NSCLC progression and resistance to anti-programmed cell death 1 (PD-1)- based therapy. Taken together, our results suggest the critical role of circFGFR1 in the proliferation, migration, invasion, and immune evasion abilities of NSCLC cells and provide a new perspective on circRNAs during NSCLC progression.
156 citations
Cites background or methods from "Circular RNA circTRIM33–12 acts as ..."
..., Chicago, IL) as in our previous study [7] and described in Additional file 1: Supplementary Materials and Methods....
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...In vivo circRNA precipitation (circRIP), RIP, and luciferase reporter assays were performed as described in previous studies [7, 20, 22]....
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...RT-qPCR, western blotting analysis, IHC, and FISH assays were performed as described in previous studies [7, 20] and described in Additional file 1: Supplementary Materials and Methods....
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...Cell proliferation, clonal formation, wound-healing cell migration, and Matrigel invasion assays were performed as described in our previous studies [7, 21] and in Additional file 1: Supplementary Materials and Methods....
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...In addition, we have reported that circTRIM33–12 inhibits hepatocellular carcinoma proliferation, invasion, and immune system evasion by inhibiting oncogenic miR-191 and promoting tet methylcytosine dioxygenase 1 (TET1) expression [7]....
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TL;DR: In this paper, a review of strategies that can be used to overexpress or knockdown circRNAs as a therapeutic approach is presented and major challenges and future directions for the development of circRNA-based therapeutics are discussed.
Abstract: Significant progress has been made in circular RNA (circRNA) research in recent years. Increasing evidence suggests that circRNAs play important roles in many cellular processes, and their dysregulation is implicated in the pathogenesis of various diseases. CircRNAs are highly stable and usually expressed in a tissue- or cell type-specific manner. Therefore, they are currently being explored as potential therapeutic targets. Gain-of-function and loss-of-function approaches are typically performed using circRNA expression plasmids and RNA interference-based strategies, respectively. These strategies have limitations that can be mitigated using nanoparticle and exosome delivery systems. Furthermore, recent developments show that the cre-lox system can be used to knockdown circRNAs in a cell-specific manner. While still in the early stages of development, the CRISPR/Cas13 system has shown promise in knocking down circRNAs with high specificity and efficiency. In this review, we describe circRNA properties and functions and highlight their significance in disease. We summarize strategies that can be used to overexpress or knockdown circRNAs as a therapeutic approach. Lastly, we discuss major challenges and propose future directions for the development of circRNA-based therapeutics.
151 citations
01 Jan 2013
TL;DR: 5 hmC may be served as a prognostic marker for HCC and the decreased expression of TET1 is likely one of the mechanisms underlying 5hmC loss in HCC, according toincopathological analysis and data showed that expression ofTET1, but not TET2 and TET3, was downregulated in H CC.
Abstract: DNA methylation is an important epigenetic modification and is frequently altered in cancer. Convert of 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5 hmC) by ten-eleven translocation (TET) family enzymes plays important biological functions in embryonic stem cells, development, aging and disease. Recent reports showed that level of 5 hmC was altered in various types of cancers. However, the change of 5 hmC level in hepatocellular carcinoma (HCC) and association with clinical outcome were not well defined. Here, we reported that level of 5 hmC was decreased in HCC tissues, as compared with non-tumor tissues. Clincopathological analysis showed the decreased level of 5 hmC in HCC was associated with tumor size, AFP level and poor overall survival. We also found that the decreased level of 5 hmC in non-tumor tissues was associated with tumor recurrence in the first year after surgical resection. In an animal model with carcinogen DEN-induced HCC, we found that the level of 5 hmC was gradually decreased in the livers during the period of induction. There was further reduction of 5 hmC in tumor tissues when tumors were developed. In contrast, level of 5 mC was increased in HCC tissues and the increased 5 mC level was associated with capsular invasion, vascular thrombosis, tumor recurrence and overall survival. Furthermore, our data showed that expression of TET1, but not TET2 and TET3, was downregulated in HCC. Taken together, our data indicated 5 hmC may be served as a prognostic marker for HCC and the decreased expression of TET1 is likely one of the mechanisms underlying 5 hmC loss in HCC.
136 citations
TL;DR: It is found that circMET overexpression promoted HCC development by inducing an epithelial to mesenchymal transition and enhancing the immunosuppressive tumor microenvironment and sitagliptin may enhance the efficacy of anti-PD1 therapy in a subgroup of patients with HCC.
Abstract: Amplification of chromosome 7q21-7q31 is associated with tumor recurrence and multidrug resistance, and several genes in this region are powerful drivers of hepatocellular carcinoma (HCC). We aimed to investigate the key circular RNAs (circRNAs) in this region that regulate the initiation and development of HCC. We used qRT-PCR to assess the expression of 43 putative circRNAs in this chromosomal region in human HCC and matched nontumor tissues. In addition, we used cultured HCC cells to modify circRNA expression and assessed the effects in several cell-based assays as well as gene expression analyses via RNA-seq. Modified cells were implanted into immunocompetent mice to assess the effects on tumor development. We performed additional experiments to determine the mechanism of action of these effects. circMET (hsa_circ_0082002) was overexpressed in HCC tumors, and circMET expression was associated with survival and recurrence in HCC patients. By modifying the expression of circMET in HCC cells in vitro, we found that circMET overexpression promoted HCC development by inducing an epithelial to mesenchymal transition and enhancing the immunosuppressive tumor microenvironment. Mechanistically, circMET induced this microenvironment through the miR-30-5p/Snail/ dipeptidyl peptidase 4(DPP4)/CXCL10 axis. In addition, the combination of the DPP4 inhibitor sitagliptin and anti-PD1 antibody improved antitumor immunity in immunocompetent mice. Clinically, HCC tissues from diabetic patients receiving sitagliptin showed higher CD8+ T cell infiltration than those from HCC patients with diabetes without sitagliptin treatment. circMET is an onco-circRNA that induces HCC development and immune tolerance via the Snail/DPP4/CXCL10 axis. Furthermore, sitagliptin may enhance the efficacy of anti-PD1 therapy in a subgroup of patients with HCC.
128 citations
References
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TL;DR: It is found that a human circRNA, antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), is densely bound by microRNA (miRNA) effector complexes and harbours 63 conserved binding sites for the ancient miRNA miR-7.
Abstract: Circular RNAs (circRNAs) in animals are an enigmatic class of RNA with unknown function. To explore circRNAs systematically, we sequenced and computationally analysed human, mouse and nematode RNA. We detected thousands of well-expressed, stable circRNAs, often showing tissue/developmental-stage-specific expression. Sequence analysis indicated important regulatory functions for circRNAs. We found that a human circRNA, antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), is densely bound by microRNA (miRNA) effector complexes and harbours 63 conserved binding sites for the ancient miRNA miR-7. Further analyses indicated that CDR1as functions to bind miR-7 in neuronal tissues. Human CDR1as expression in zebrafish impaired midbrain development, similar to knocking down miR-7, suggesting that CDR1as is a miRNA antagonist with a miRNA-binding capacity ten times higher than any other known transcript. Together, our data provide evidence that circRNAs form a large class of post-transcriptional regulators. Numerous circRNAs form by head-to-tail splicing of exons, suggesting previously unrecognized regulatory potential of coding sequences.
5,922 citations
TL;DR: This study serves as the first functional analysis of a naturally expressed circular RNA, ciRS-7, which contains more than 70 selectively conserved miRNA target sites, and is highly and widely associated with Argonaute proteins in a miR-7-dependent manner.
Abstract: MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by direct base pairing to target sites within untranslated regions of messenger RNAs. Recently, miRNA activity has been shown to be affected by the presence of miRNA sponge transcripts, the so-called competing endogenous RNA in humans and target mimicry in plants. We previously identified a highly expressed circular RNA (circRNA) in human and mouse brain. Here we show that this circRNA acts as a miR-7 sponge; we term this circular transcript ciRS-7 (circular RNA sponge for miR-7). ciRS-7 contains more than 70 selectively conserved miRNA target sites, and it is highly and widely associated with Argonaute (AGO) proteins in a miR-7-dependent manner. Although the circRNA is completely resistant to miRNA-mediated target destabilization, it strongly suppresses miR-7 activity, resulting in increased levels of miR-7 targets. In the mouse brain, we observe overlapping co-expression of ciRS-7 and miR-7, particularly in neocortical and hippocampal neurons, suggesting a high degree of endogenous interaction. We further show that the testis-specific circRNA, sex-determining region Y (Sry), serves as a miR-138 sponge, suggesting that miRNA sponge effects achieved by circRNA formation are a general phenomenon. This study serves as the first, to our knowledge, functional analysis of a naturally expressed circRNA.
5,885 citations
TL;DR: It is shown here that TET1, a fusion partner of the MLL gene in acute myeloid leukemia, is a 2-oxoglutarate (2OG)- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro.
Abstract: DNA cytosine methylation is crucial for retrotransposon silencing and mammalian development. In a computational search for enzymes that could modify 5-methylcytosine (5mC), we identified TET proteins as mammalian homologs of the trypanosome proteins JBP1 and JBP2, which have been proposed to oxidize the 5-methyl group of thymine. We show here that TET1, a fusion partner of the MLL gene in acute myeloid leukemia, is a 2-oxoglutarate (2OG)- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro. hmC is present in the genome of mouse embryonic stem cells, and hmC levels decrease upon RNA interference–mediated depletion of TET1. Thus, TET proteins have potential roles in epigenetic regulation through modification of 5mC to hmC.
5,155 citations
TL;DR: An activating immunoreceptor-MHC ligand interaction that may promote antitumor NK and T cell responses is defined.
Abstract: Stress-inducible MICA, a distant homolog of major histocompatibility complex (MHC) class I, functions as an antigen for gammadelta T cells and is frequently expressed in epithelial tumors. A receptor for MICA was detected on most gammadelta T cells, CD8+ alphabeta T cells, and natural killer (NK) cells and was identified as NKG2D. Effector cells from all these subsets could be stimulated by ligation of NKG2D. Engagement of NKG2D activated cytolytic responses of gammadelta T cells and NK cells against transfectants and epithelial tumor cells expressing MICA. These results define an activating immunoreceptor-MHC ligand interaction that may promote antitumor NK and T cell responses.
2,916 citations
TL;DR: It is demonstrated that all three mouse Tet proteins (Tet1, Tet2 and Tet3) can also catalyse a similar reaction, uncover the enzymatic activity of the Tet proteins, and demonstrate a role for Tet1 in ES cell maintenance and inner cell mass cell specification.
Abstract: DNA methylation is one of the best-characterized epigenetic modifications. Although the enzymes that catalyse DNA methylation have been characterized, enzymes responsible for demethylation have been elusive. A recent study indicates that the human TET1 protein could catalyse the conversion of 5-methylcytosine (5mC) of DNA to 5-hydroxymethylcytosine (5hmC), raising the possibility that DNA demethylation may be a Tet1-mediated process. Here we extend this study by demonstrating that all three mouse Tet proteins (Tet1, Tet2 and Tet3) can also catalyse a similar reaction. Tet1 has an important role in mouse embryonic stem (ES) cell maintenance through maintaining the expression of Nanog in ES cells. Downregulation of Nanog via Tet1 knockdown correlates with methylation of the Nanog promoter, supporting a role for Tet1 in regulating DNA methylation status. Furthermore, knockdown of Tet1 in pre-implantation embryos results in a bias towards trophectoderm differentiation. Thus, our studies not only uncover the enzymatic activity of the Tet proteins, but also demonstrate a role for Tet1 in ES cell maintenance and inner cell mass cell specification.
2,364 citations