CircC16orf62 promotes hepatocellular carcinoma progression through the miR-138-5p/PTK2/AKT axis

AbstractCircular RNA (circRNAs) functions vital in the pathogenesis and progression of hepatocellular carcinoma (HCC). However, the expressions and functions of certain circRNAs on metastasis and proliferation of that cancer is still unclear. Bioinformation analysis and qRT-PCR indicated that CircC16orf62 was prominent upregulated in HCC of which the expression level was positively associated to cancer’s malignant progression. Gain or loss-of-function studies indicated that the reduction of CircC16orf62 expression promotes the proliferation, invasion, and glycolysis of HCC in vitro and in vivo. The bioinformatic analysis found that miR-138-5p and PTK2 were the downstream target of CircC16or62. Then, the FISH(Fluorescence immunoin situ hybridization) and cell nucleoplasmic separation determined that CircC16orf62 located in the cell cytoplasm. Plasmid vectors or siRNAs were used to change the expression of CircC16orf62, miR-138-5p, and PTK2 in PC cell lines. CircC16orf62 functioned as a molecular sponge for miR-138-5p, and a competitive endogenous RNA for PTK2, promoting AKT/mTOR pathway activation. Our observations lead us to conclude that CircC16orf62 functions as an oncogene in HCC progression, behaving as a competitive endogenous RNA for miR-138-5p binding, thus activating the AKT/mTOR pathway. In conclusion, CircC16orf62 is an oncogene through the miR-138-5p/PTK2/Akt axis in HCC cells, indicating CircC16orf62 can be a therapeutic target with potentiality for liver cancer and a predictive marker for people with HCC.

Download Full-text

The Deubiquitinase OTUD3 Stabilizes ACTN4 to Activate NF-κB Signaling Pathway in Hepatocellular Carcinoma

10.21203/rs.3.rs-139689/v1 ◽

2021 ◽

Author(s):

Peiyi Xie ◽

Yanglin Chen ◽

Hongfei Zhang ◽

Guichao Zhou ◽

Qing Chao ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Signaling Pathway ◽

Protein Level ◽

Loss Of Function ◽

Downstream Target ◽

Gene And Protein Expression ◽

Hcc Cells

Abstract Background: OTUD3, a deubiquitinating enzyme, has emerged as important role in some cancer. It showed that OTUD3 plays suppressive role in breast cancer whereas oncogenic role in lung cancer. However, the function and mechanism of OTUD3 in hepatocellular carcinoma (HCC) progression remain elusive. Methods: Gene and protein expression of OTUD3 in HCC tissues were determined by qRT-PCR, western blot and immunohistochemistry. A series of gain- and loss-of-function assays were used to investigated the role of OTUD3 in HCC cells progression. Moreover, mass spectroscopic analysis and RNA-seq were used to identify the downstream targets of OTUD3 in HCC cells. The interaction between OTUD3 and ACTN4 was examined through co-IP experiment and in vitro ubiquitination assay.Results: In our research, OTUD3 was significantly overexpressed in HCC tissues and higher OTUD3 expression was correlated with bigger tumor size, more distant metastasis, and worse TNM stage. Additionally, OTUD3 promoted HCC cells growth and metastasis in vitro and in vivo. Furthermore, ACTN4 was identified as a downstream target of OTUD3 and ACTN4 protein level was significantly related to OTUD3 expression. Rescue experiments indicated that ACTN4 was essential for OTUD3-mediated HCC proliferation and metastasis in vitro and in vivo. Moreover, we identified that NF-κB signaling pathway was activated by OTUD3 through ACTN4 to promote HCC cells progression. Importantly, OTUD3 protein level was correlated with ACTN4 protein level and activity of NF-κB signaling pathway in HCC tissues. Conclusion: Our findings identify the oncogenic role of OTUD3 in HCC and suggest that OTUD3 can be considered as a pivotal prognostic biomarker and a potential therapeutic target.

Download Full-text

LINC00680 enhances hepatocellular carcinoma stemness behavior and chemoresistance by sponging miR-568 to upregulate AKT3

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01854-5 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Gege Shu ◽

Huizhao Su ◽

Zhiqian Wang ◽

Shihui Lai ◽

Yan Wang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Xenograft Model ◽

Luciferase Reporter ◽

Loss Of Function ◽

Mechanism Study ◽

Downstream Signaling ◽

Mouse Xenograft Model ◽

High Level

Abstract Background Hepatocellular carcinoma (HCC) has an extremely poor prognosis due to the development of chemoresistance, coupled with inherently increased stemness properties. Long non-coding RNAs (LncRNAs) are key regulators for tumor cell stemness and chemosensitivity. Currently the relevance between LINC00680 and tumor progression was still largely unknown, with only one study showing its significance in glioblastoma. The study herein was aimed at identifying the role of LINC00680 in the regulation HCC stemness and chemosensitivity. Methods QRT-PCR was used to detect the expression of LINC00680, miR-568 and AKT3 in tissue specimen and cell lines. Gain- or loss-of function assays were applied to access the function of LINC00680 in HCC cells, including cell proliferation and stemness properties. HCC stemness and chemosensitivity were determined by sphere formation, cell viability and colony formation. Luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays were performed to examine the interaction between LINC00680 and miR-568 as well as that between miR-568 and AKT3. A nude mouse xenograft model was established for the in vivo study. Results We found that LINC00680 was remarkably upregulated in HCC tissues. Patients with high level of LINC00680 had poorer prognosis. LINC00680 overexpression significantly enhanced HCC cell stemness and decreased in vitro and in vivo chemosensitivity to 5-fluorouracil (5-Fu), whereas LINC00680 knockdown led to opposite results. Mechanism study revealed that LINC00680 regulated HCC stemness and chemosensitivity through sponging miR-568, thereby expediting the expression of AKT3, which further activated its downstream signaling molecules, including mTOR, elF4EBP1, and p70S6K. Conclusion LINC00680 promotes HCC stemness properties and decreases chemosensitivity through sponging miR-568 to activate AKT3, suggesting that LINC00680 might be a potentially important HCC diagnosis marker and therapeutic target.

Download Full-text

CircRNA-SORE mediates sorafenib resistance in hepatocellular carcinoma by stabilizing YBX1

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-020-00375-5 ◽

2020 ◽

Vol 5 (1) ◽

Author(s):

Junjie Xu ◽

Lin Ji ◽

Yuelong Liang ◽

Zhe Wan ◽

Wei Zheng ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Nuclear Interaction ◽

Circular Rna ◽

Advanced Hepatocellular Carcinoma ◽

Proof Of Concept ◽

Potential Strategy ◽

Oncogenic Protein ◽

Hcc Cells

AbstractSorafenib is the first-line chemotherapeutic therapy for advanced hepatocellular carcinoma (HCC). However, sorafenib resistance significantly limits its therapeutic efficacy, and the mechanisms underlying resistance have not been fully clarified. Here we report that a circular RNA, circRNA-SORE (a circular RNA upregulated in sorafenib-resistant HCC cells), plays a significant role in sorafenib resistance in HCC. We found that circRNA-SORE is upregulated in sorafenib-resistant HCC cells and depletion of circRNA-SORE substantially increases the cell-killing ability of sorafenib. Further studies revealed that circRNA-SORE binds the master oncogenic protein YBX1 in the cytoplasm, which prevents YBX1 nuclear interaction with the E3 ubiquitin ligase PRP19 and thus blocks PRP19-mediated YBX1 degradation. Moreover, our in vitro and in vivo results suggest that circRNA-SORE is transported by exosomes to spread sorafenib resistance among HCC cells. Using different HCC mouse models, we demonstrated that silencing circRNA-SORE by injection of siRNA could substantially overcome sorafenib resistance. Our study provides a proof-of-concept demonstration for a potential strategy to overcome sorafenib resistance in HCC patients by targeting circRNA-SORE or YBX1.

Download Full-text

Upregulation of SGOL2 Predicts Poor Prognosis and Facilitates Hepatocellular Carcinoma Progression via Dysregulating the Cell Cycle by Directly Activating MAD2

10.21203/rs.3.rs-560609/v1 ◽

2021 ◽

Author(s):

Qingqing Hu ◽

Xiaochu Hu ◽

Yalei Zhao ◽

Lingjian Zhang ◽

Ya Yang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Cycle ◽

The Cancer Genome Atlas ◽

Loss Of Function ◽

Protein Levels ◽

Cancer Genome Atlas ◽

Centromeric Protein ◽

Hcc Cells

Abstract Background: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Shugoshin-like protein 2 (SGOL2) is a centromeric protein that ensures the correct and orderly process of mitosis by protecting and maintaining centripetal adhesions during meiosis and mitosis. However, the role of SGOL2 in cancer is not well understood. Methods: The mRNA and protein levels of SGOL2 and survival analysis were conducted in The Cancer Genome Atlas (TCGA) and further validated in 2 independent cohorts. Differential genes correlated with SGOL2 and mitotic arrest deficient 2 like 1 (MAD2) were obtained using LinkedOmics. Subsequently, loss-of-function and rescue assays were carried out in vitro and in vivo to assess the functions of SGOL2 in hepatic tumorigenisis. Findings: We found that SGOL2 was significantly overexpressed in HCC and predicted unfavorable overall survival in HCC patients. Next, we identified 47 differentially expressed genes positively correlated with both SGOL2 and MAD2 to be mainly involved in the cell cycle. In addition, SGOL2 downregulation suppressed the migration, invasion, proliferation, stemness and EMT of HCC cells and inhibited tumorigenesis in vivo. Furthermore, SGOL2 promoted tumor proliferation by activating MAD2-induced cell cycle dysregulation, which could be reversed by the MAD2 inhibitor M2I-1. We also proved that SGOL2 activated MAD2 by directly binding with MAD2. Conclusions: The results of this study showed that SGOL2 acts as an oncogene in HCC cells by directly activating MAD2 and then dysregulating the cell cycle, thereby providing a potential target for HCC patients in the future.

Download Full-text

Genetic evidence for the transcriptional-activating function of Homothorax during adult fly development

Development ◽

10.1242/dev.128.18.3405 ◽

2001 ◽

Vol 128 (18) ◽

pp. 3405-3413 ◽

Cited By ~ 4

Author(s):

Adi Inbal ◽

Naomi Halachmi ◽

Charna Dibner ◽

Dale Frank ◽

Adi Salzberg

Keyword(s):

Transcriptional Activity ◽

Target Genes ◽

Genetic Evidence ◽

In Vivo Studies ◽

Loss Of Function ◽

Native Form ◽

Downstream Target ◽

Activating Function

Homothorax (HTH) is a homeobox-containing protein, which plays multiple roles in the development of the embryo and the adult fly. HTH binds to the homeotic cofactor Extradenticle (EXD) and translocates it to the nucleus. Its function within the nucleus is less clear. It was shown, mainly by in vitro studies, that HTH can bind DNA as a part of ternary HTH/EXD/HOX complexes, but little is known about the transcription regulating function of HTH-containing complexes in the context of the developing fly. Here we present genetic evidence, from in vivo studies, for the transcriptional-activating function of HTH. The HTH protein was forced to act as a transcriptional repressor by fusing it to the Engrailed (EN) repression domain, or as a transcriptional activator, by fusing it to the VP16 activation domain, without perturbing its ability to translocate EXD to the nucleus. Expression of the repressing form of HTH in otherwise wild-type imaginal discs phenocopied hth loss of function. Thus, the repressing form was working as an antimorph, suggesting that normally HTH is required to activate the transcription of downstream target genes. This conclusion was further supported by the observation that the activating form of HTH caused typical hth gain-of-function phenotypes and could rescue hth loss-of-function phenotypes. Similar results were obtained with XMeis3, the Xenopus homologue of HTH, extending the known functional similarity between the two proteins. Competition experiments demonstrated that the repressing forms of HTH or XMeis3 worked as true antimorphs competing with the transcriptional activity of the native form of HTH. We also describe the phenotypic consequences of HTH antimorph activity in derivatives of the wing, labial and genital discs. Some of the described phenotypes, for example, a proboscis-to-leg transformation, were not previously associated with alterations in HTH activity. Observing the ability of HTH antimorphs to interfere with different developmental pathways may direct us to new targets of HTH. The HTH antimorph described in this work presents a new means by which the transcriptional activity of the endogenous HTH protein can be blocked in an inducible fashion in any desired cells or tissues without interfering with nuclear localization of EXD.

Download Full-text

circ-CBFB upregulates p66Shc to perturb mitochondrial dynamics in APAP-induced liver injury

Cell Death and Disease ◽

10.1038/s41419-020-03160-y ◽

2020 ◽

Vol 11 (11) ◽

Author(s):

Zhecheng Wang ◽

Yan Zhao ◽

Ruimin Sun ◽

Yu Sun ◽

Deshun Liu ◽

...

Keyword(s):

Liver Injury ◽

Mitochondrial Dynamics ◽

Circular Rna ◽

Protective Effects ◽

Bioinformatics Analyses ◽

Hepatocyte Injury ◽

Rna Immunoprecipitation ◽

Competitive Endogenous Rna

Abstract p66Shc, a master regulator of mitochondrial reactive oxygen species (mtROS), is a crucial mediator of hepatocyte oxidative stress. However, its functional contribution to acetaminophen (APAP)-induced liver injury and the mechanism by which it is modulated remain unknown. Here, we aimed to assess the effect of p66Shc on APAP-induced liver injury and to evaluate if circular RNA (circRNA) functions as a competitive endogenous RNA (ceRNA) to mediate p66Shc in APAP-induced liver injury. p66Shc-, miR-185-5p-, and circ-CBFB-silenced mice were injected with APAP. AML12 cells were transfected with p66Shc, miR-185-5p, and circ-CBFB silencing or overexpression plasmids or siRNAs prior to APAP stimulation. p66Shc was upregulated in liver tissues in response to APAP, and p66Shc silencing in vivo protected mice from APAP-induced mitochondrial dynamics perturbation and liver injury. p66Shc knockdown in vitro attenuated mitochondrial dynamics and APAP-induced hepatocyte injury. Mechanically, p66Shc perturbs mitochondrial dynamics partially by inhibiting OMA1 ubiquitination. miR-185-5p, which directly suppressed p66Shc translation, was identified by microarray and bioinformatics analyses, and its overexpression attenuated mitochondrial dynamics and hepatocyte injury in vitro. Furthermore, luciferase, pull-down and RNA immunoprecipitation assays demonstrated that circ-CBFB acts as a miRNA sponge of miR-185-5p to mediate p66Shc in APAP-induced liver injury. circ-CBFB knockdown also alleviated APAP-induced mitochondrial dynamics perturbation and hepatocyte injury. More importantly, we found that the protective effects of circ-CBFB knockdown on p66Shc, mitochondrial dynamics and liver injury were abolished by miR-185-5p inhibition both in vivo and in vitro. In conclusion, p66Shc is a key regulator of APAP-induced liver injury that acts by triggering mitochondrial dynamics perturbation. circ-CBFB functions as a ceRNA to regulate p66Shc during APAP-induced liver injury, which may provide a potential therapeutic target.

Download Full-text

CDK12 inhibition mediates DNA damage and is synergistic with sorafenib treatment in hepatocellular carcinoma

Gut ◽

10.1136/gutjnl-2019-318506 ◽

2019 ◽

Vol 69 (4) ◽

pp. 727-736 ◽

Cited By ~ 10

Author(s):

Cun Wang ◽

Hui Wang ◽

Cor Lieftink ◽

Aimee du Chatinier ◽

Dongmei Gao ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Dna Damage ◽

Cell Lines ◽

Synergistic Effects ◽

Loss Of Function ◽

Bioinformatics Analyses ◽

Sorafenib Treatment ◽

Hcc Cells

ObjectivesHepatocellular carcinoma (HCC) is one of the most frequent malignancies and a major leading cause of cancer-related deaths worldwide. Several therapeutic options like sorafenib and regorafenib provide only modest survival benefit to patients with HCC. This study aims to identify novel druggable candidate genes for patients with HCC.DesignA non-biased CRISPR (clustered regularly interspaced short palindromic repeats) loss-of-function genetic screen targeting all known human kinases was performed to identify vulnerabilities of HCC cells. Whole-transcriptome sequencing (RNA-Seq) and bioinformatics analyses were performed to explore the mechanisms of the action of a cyclin-dependent kinase 12 (CDK12) inhibitor in HCC cells. Multiple in vitro and in vivo assays were used to study the synergistic effects of the combination of CDK12 inhibition and sorafenib.ResultsWe identify CDK12 as critically required for most HCC cell lines. Suppression of CDK12 using short hairpin RNAs (shRNAs) or its inhibition by the covalent small molecule inhibitor THZ531 leads to robust proliferation inhibition. THZ531 preferentially suppresses the expression of DNA repair-related genes and induces strong DNA damage response in HCC cell lines. The combination of THZ531 and sorafenib shows striking synergy by inducing apoptosis or senescence in HCC cells. The synergy between THZ531 and sorafenib may derive from the notion that THZ531 impairs the adaptive responses of HCC cells induced by sorafenib treatment.ConclusionOur data highlight the potential of CDK12 as a drug target for patients with HCC. The striking synergy of THZ531 and sorafenib suggests a potential combination therapy for this difficult to treat cancer.

Download Full-text

The cerebral cavernous malformation proteins CCM2L and CCM2 prevent the activation of the MAP kinase MEKK3

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1510495112 ◽

2015 ◽

Vol 112 (46) ◽

pp. 14284-14289 ◽

Cited By ~ 35

Author(s):

Xavier Cullere ◽

Eva Plovie ◽

Paul M. Bennett ◽

Calum A. MacRae ◽

Tanya N. Mayadas

Keyword(s):

Endothelial Cells ◽

Cavernous Malformation ◽

Body Axis ◽

Cerebral Cavernous Malformations ◽

Loss Of Function ◽

Cavernous Malformations ◽

Downstream Target ◽

Density Flow

Three genes, CCM1, CCM2, and CCM3, interact genetically and biochemically and are mutated in cerebral cavernous malformations (CCM). A recently described member of this CCM family of proteins, CCM2-like (CCM2L), has high homology to CCM2. Here we show that its relative expression in different tissues differs from that of CCM2 and, unlike CCM2, the expression of CCM2L in endothelial cells is regulated by density, flow, and statins. In vitro, both CCM2L and CCM2 bind MEKK3 in a complex with CCM1. Both CCM2L and CCM2 interfere with MEKK3 activation and its ability to phosphorylate MEK5, a downstream target. The in vivo relevance of this regulation was investigated in zebrafish. A knockdown of ccm2l and ccm2 in zebrafish leads to a more severe “big heart” and circulation defects compared with loss of function of ccm2 alone, and also leads to substantial body axis abnormalities. Silencing of mekk3 rescues the big heart and body axis phenotype, suggesting cross-talk between the CCM proteins and MEKK3 in vivo. In endothelial cells, CCM2 deletion leads to activation of ERK5 and a transcriptional program that are downstream of MEKK3. These findings suggest that CCM2L and CCM2 cooperate to regulate the activity of MEKK3.

Download Full-text

JARID1B promotes colorectal cancer proliferation and Wnt/β-catenin signaling via decreasing CDX2 level

10.21203/rs.3.rs-17618/v1 ◽

2020 ◽

Author(s):

Da Huang ◽

Fan Xiao ◽

Fuzhou Hua ◽

Zhenzhong Luo ◽

Zhaoxia Huang ◽

...

Keyword(s):

Colorectal Cancer ◽

Signaling Pathway ◽

Critical Role ◽

Predictive Marker ◽

Cancer Proliferation ◽

Poor Overall Survival ◽

Downstream Target ◽

Cellular Analysis

Abstract Background: Jumonji AT-rich interactive domain 1B(JARID1B) has been shown to be upregulated in many human cancers and plays a critical role in the development of cancers cells. Nevertheless, its functional role in colorectal cancer (CRC) progression is not fully understood. Methods: Herein, JARID1B expression levels were detected in clinical CRC samples by western blotting and qRT-PCR. DLD-1 cells with JARID1B knockdown or overexpression by stably transfected plasmids were used in vitro and in vivo study. Colony formation, 5-ethynyl-20-deoxyuridine (EdU) and Real Time Cellular Analysis(RTCA) assays were used to detect cell proliferation and growth. Transcriptome and CHIP assays were used to examine the molecular biology changes and molecular interaction in these cells. Nude mice was utilized to study the correlation of JARID1B and tumor growth in vivo. Results: Here, we first observed that JARID1B was significantly upregulated in CRC tissue compared to adjacent normal tissues. In CRC patients, JARID1B high expression was positively relation with poor overall survival. Multivariate analyses revealed that high JARID1B expression was an independent predictive marker for the poor prognosis of CRC. In addition, we found that JARID1B promoted CRC cells proliferation by Wnt/β-catenin signaling pathway. Further studies demonstrated CDX2 as a downstream target of JARID1B, and our data demonstrated that CDX2 is crucial for JARID1B -mediated Wnt/β-catenin signaling pathway. Mechanistically, we demonstrated that JARID1B regulated CDX2 expression through demethylation of H3K4me3. Conclusions: CDX2 inhibited by JARID1B-derived H3K4me3 methylation promoted cells proliferation of CRC via Wnt/β-catenin signaling pathway. Therefore, our studies provided a novel insight into the role of JARID1B in CRC cells proliferation and potential new molecular target for treating CRC.

Download Full-text

BEZ235 Increases the Sensitivity of Hepatocellular Carcinoma to Sorafenib by Inhibiting PI3K/AKT/mTOR and Inducing Autophagy

BioMed Research International ◽

10.1155/2021/5556306 ◽

2021 ◽

Vol 2021 ◽

pp. 1-19

Author(s):

Weiya Cao ◽

Xueke Liu ◽

Yinci Zhang ◽

Amin Li ◽

Yinghai Xie ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Cycle ◽

Cell Lines ◽

Acquired Resistance ◽

Inhibitory Effect ◽

Mtor Pathway ◽

Protein Levels ◽

Huh7 Cells

Acquired resistance of hepatocellular carcinoma (HCC) to sorafenib (SFB) is the main reason for the failure of SFB treatment of the cancer. Abnormal activation of the PI3K/AKT/mTOR pathway is important in the acquired resistance of SFB. Therefore, we investigated whether BEZ235 (BEZ) could reverse acquired sorafenib resistance by targeting the PI3K/mTOR pathway. A sorafenib-resistant HCC cell line Huh7R was established. MTT assay, clone formation assay, flow cytometry, and immunofluorescence were used to analyze the effects of BEZ235 alone or combined with sorafenib on cell proliferation, cell cycle, apoptosis, and autophagy of Huh7 and Huh7R cells. The antitumor effect was evaluated in animal models of Huh7R xenografts in vivo. Western blot was used to detect protein levels of the PI3K/AKT/mTOR pathway and related effector molecules. In vitro results showed that the Huh7R had a stronger proliferation ability and antiapoptosis effect than did Huh7, and sorafenib had no inhibitory effect on Huh7R. SFB + BEZ inhibited the activation of the PI3K/AKT/mTOR pathway caused by sorafenib. Moreover, SFB + BEZ inhibited the proliferation and cloning ability, blocked the cell cycle in the G0/G1 phase, and promoted apoptosis in the two cell lines. The autophagy level in Huh7R cells was higher than in Huh7 cells, and BEZ or SFB + BEZ further promoted autophagy in the two cell lines. In vivo, SFB + BEZ inhibited tumor growth by inducing apoptosis and autophagy. We concluded that BEZ235 enhanced the sensitivity of sorafenib through suppressing the PI3K/AKT/mTOR pathway and inducing autophagy. These observations may provide the experimental basis for sorafenib combined with BEZ235 in trial treatment of HCC.

Download Full-text