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Deubiquitinase ZRANB1 drives hepatocellular carcinoma progression through SP1-LOXL2 axis

ABSTRACT: Deubiquitinase (DUB) zinc finger RANBP2-type containing 1 (ZRANB1) has been reported to have a close relationship with cancers. However, its underlying role and molecular mechanisms in hepatocellular carcinoma (HCC) remain elusive. In this study, we demonstrated that ZRANB1 was highly expressed in HCC tissues. Additionally, ZRANB1 overexpression was correlated with poorer survival and ZRANB1 could be an independent predictor of poor prognosis for HCC patients. Through gain- and loss-of-function assays, we examined the oncogenic role of ZRANB1 in regulating HCC cell growth and metastasis in vitro and in vivo. To identify the downstream targets of ZRANB1 in regulating HCC tumorigenesis, we performed RNA-seq and demonstrated that Lysyl oxidase-like 2 (LOXL2) was the most significantly downregulated gene after ZRANB1 knockdown. Furthermore, the scatter plots indicated a significant positive correlation between ZRANB1 and LOXL2 expression in clinical HCC specimens. We also demonstrated that ZRANB1 knockdown downregulated the expression of LOXL2 and suppressed HCC growth and metastasis in vitro and in vivo. The effects of ZRANB1 knockdown were reversed by LOXL2 overexpression. More importantly, ZRANB1 regulated LOXL2 through specificity protein 1 (SP1) and SP1 overexpression rescued the suppression of HCC growth and metastasis induced by ZRANB1 knockdown. Mechanistically, ZRANB1 bound with SP1 directly and stabilized the SP1 protein by deubiquitinating it. The expression patterns of ZRANB1, SP1 and LOXL2 were evaluated in HCC patients. In summary, our research highlights a novel role of ZRANB1 in the tumorigenesis of HCC and suggests a new candidate prognostic biomarker for HCC treatment.

SUBMITTER: Li Q

PROVIDER: S-EPMC8569347 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The clinical outcomes of hepatocellular carcinoma (HCC) remain dismal. Elucidating the molecular mechanisms for the progression of aggressive HCC holds the promise for developing novel intervention strategies. The transactivation response element RNA-binding protein (TRBP/TARBP2), a key component of microRNA (miRNA) processing and maturation machinery has been shown to play conflicting roles in tumor development and progression. We sought to investigate the expression of TARBP2 in HCC using well-characterized HCC cell lines, patient-derived tissues and blood samples. Additionally, the potential prognostic and diagnostic value of TARBP2 in HCC were analyzed using Kaplan-Meier plots and ROC curve. Cell counting kit-8 (CCK-8), wound healing and transwell assays examined the ability of TARBP2 to induce cell proliferation, migration, and invasion in HCC cell lines. RNA sequencing was applied to identify the downstream elements of TARBP2. The interaction of potential targets of TARBP2, miR-145 and serpin family E member 1 (SERPINE1), was assessed using luciferase reporter assay. TARBP2 expression was down-regulated in HCC cell lines relative to normal hepatocyte cells, with a similar pattern further confirmed in tissue and blood samples. Notably, the loss of TARBP2 was demonstrated to promote proliferation, migration, and invasion in HCC cell lines. Interestingly, the reduction of TARBP2 was shown to result in the upregulation of SERPINE1, also known as plasminogen activator inhibitor (PAI-1), which is a vital gene of the HIF-1 signaling pathway. Knockdown of SERPINE1 rescued the TARBP2-lost phenotype. Moreover, TARBP2 depletion induced the upregulation of SERPINE1 through reducing the processing of miR-145, which directly targets SERPINE1. Finally, overexpression of miR-145 repressed SERPINE1 and rescued the functions in sh-TARBP2 HCC cells. Our findings underscore a linear TARBP2-miR-145-SERPINE1 pathway that drives HCC progression, with the potential as a novel intervention target for aggressive HCC.

Project description:BackgroundEmerging evidence suggests that circular RNAs play critical roles in disease development especially in cancers. Previous genome-wide RNA-seq studies found that a circular RNA derived from SOD2 gene was highly upregulated in hepatocellular carcinoma (HCC), however, the role of circSOD2 in HCC remains largely unknown.MethodsThe expression profiling of circSOD2 and microRNA in HCC patients were assessed by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). SiRNA or CRISPR-CAS9 were used to silence gene expression. The biological function of circSOD2 in HCC was investigated using in vitro and in vivo studies including, trans-well cell migration, cell apoptosis, cell cycle, CCK8, siRNA interference, western blots, and xenograft mouse model. The underlying molecular mechanism was determined by Chromatin Immunoprecipitation quantitative real time PCR (ChIP-qPCR), bioinformatic analysis, biotin-pull down, RNA immunoprecipitation, 5-mc DNA pulldown and luciferase assays.ResultsIn accordance with previous sequencing results, here, we demonstrated that circSOD2 was highly expressed in HCC tumor tissues compared with normal liver tissues. Mechanically, we showed that histone writer EP300 and WDR5 bind to circSOD2 promoter and trigger its promoter H3K27ac and H3K4me3 modification, respectively, which further activates circSOD2 expression. SiRNA mediated circSOD2 suppression impaired liver cancer cell growth, cell migration, prohibited cell cycle progression and in vivo tumor growth. By acting as a sponge, circSOD2 inhibits miR-502-5p expression and rescues miR-502-5p target gene DNMT3a expression. As a DNA methyltransferase, upregulated DNMA3a suppresses SOCS3 expression by increasing SOCS3 promoter DNA methylation. This event further accelerates SOCS3 downstream JAK2/STAT3 signaling pathway activation. In addition, we also found that activated STAT3 regulates circSOD2 expression in a feedback way.ConclusionThe novel signaling axis circSOD2/miR-502-5p/DNMT3a/JAK2/STAT3/circSOD2 provides a better understanding of HCC tumorigenesis. The molecular mechanism underlying this signaling axis offers new prevention and treatment of HCC.

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Deubiquitinase ZRANB1 drives hepatocellular carcinoma progression through SP1-LOXL2 axis

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