HIF-1α-induced expression of m6A reader YTHDF1 drives hypoxia-induced autophagy and malignancy of hepatocellular carcinoma by promoting ATG2A and ATG14 translation

AbstractN6-methyladenosine (m6A), and its reader protein YTHDF1, play a pivotal role in human tumorigenesis by affecting nearly every stage of RNA metabolism. Autophagy activation is one of the ways by which cancer cells survive hypoxia. However, the possible involvement of m6A modification of mRNA in hypoxia-induced autophagy was unexplored in human hepatocellular carcinoma (HCC). In this study, specific variations in YTHDF1 expression were detected in YTHDF1-overexpressing, -knockout, and -knockdown HCC cells, HCC organoids, and HCC patient-derived xenograft (PDX) murine models. YTHDF1 expression and hypoxia-induced autophagy were significantly correlated in vitro; significant overexpression of YTHDF1 in HCC tissues was associated with poor prognosis. Multivariate cox regression analysis identified YTHDF1 expression as an independent prognostic factor in patients with HCC. Multiple HCC models confirmed that YTHDF1 deficiency inhibited HCC autophagy, growth, and metastasis. Luciferase reporter assays and chromatin immunoprecipitation demonstrated that HIF-1α regulated YTHDF1 transcription by directly binding to its promoter region under hypoxia. The results of methylated RNA immunoprecipitation sequencing, proteomics, and polysome profiling indicated that YTHDF1 contributed to the translation of autophagy-related genes ATG2A and ATG14 by binding to m6A-modified ATG2A and ATG14 mRNA, thus facilitating autophagy and autophagy-related malignancy of HCC. Taken together, HIF-1α-induced YTHDF1 expression was associated with hypoxia-induced autophagy and autophagy-related HCC progression via promoting translation of autophagy-related genes ATG2A and ATG14 in a m6A-dependent manner. Our findings suggest that YTHDF1 is a potential prognostic biomarker and therapeutic target for patients with HCC.

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CircCRIM1 Promotes Hepatocellular Carcinoma Proliferation and Angiogenesis by Sponging miR-378a-3p and Regulating SKP2 Expression

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.796686 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yang Ji ◽

Shikun Yang ◽

Xueqi Yan ◽

Li Zhu ◽

Wenjie Yang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Expression Level ◽

Luciferase Reporter ◽

Circular Rnas ◽

Clinicopathologic Characteristics ◽

Huh7 Cells ◽

Rna Immunoprecipitation ◽

Reporter Assays

Mounting evidence has demonstrated that circular RNAs have an important function in tumorigenesis and cancer evolvement. CircCRIM1 has been shown to be a poor prognostic element in multiple human malignancies. However, the clinical significance and mechanism of circCRIM1 in hepatocellular carcinoma (HCC) is still unclear. The present study confirmed the expression level of circCRIM1 using quantitative real-time PCR. In addition, circCRIM1 siRNA and overexpression vectors were used for transfection into LM3 or Huh7 cells to down- or up-regulate the expression of circCRIM1. In vitro and in vivo experiments were performed to explore the function of circCRIM1 in HCC. RNA pull-down, RNA immunoprecipitation, fluorescent in situ hybridization, and luciferase reporter assays were conducted to confirm the relationship between miR-378a-3p and circCRIM1 or S-phase kinase-associated protein 2 (SKP2) in HCC. Then, circCRIM1 was up-regulated in HCC and its expression level was significantly associated with poor prognosis and clinicopathologic characteristics. CircCRIM1 enhanced the proliferation and angiogenesis of HCC cells in vitro and promoted xenograft growth in vivo. Moreover, circCRIM1 upregulated the expression of SKP2 by functioning as a sponge for miR-378a-3p. These findings suggest that circCRIM1 boosts the HCC progression via the miR-378-3p/SKP2 axis and may act as a crucial epigenetic therapeutic molecule target in HCC.

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ALKBH5-Mediated m6A Modification of LncRNA KCNQ1OT1 Triggers the Development of LSCC via Upregulation of HOXA9

10.21203/rs.3.rs-637073/v1 ◽

2021 ◽

Author(s):

Yushan Li ◽

Bingrui Yan ◽

Xin Wang ◽

Qiuying Li ◽

Xuan Kan ◽

...

Keyword(s):

Epigenetic Modification ◽

Biological Effects ◽

Luciferase Reporter ◽

Dependent Manner ◽

Invasion And Metastasis ◽

Expression Levels ◽

Tumor Tissues ◽

Rna Immunoprecipitation ◽

Regulated Gene Expression

Abstract Background RNA epigenetic modification is the chemical basis of RNA-regulated gene expression, of which N6-methyladenosine (m6A) is one of the most common post-transcriptional modifications in mRNA. More and more studies show that m6A modification is involved in the development of complex human diseases, especially in the development of cancer. However, it is unclear whether the m6A modification of lncRNA modification plays an important role in the development of laryngeal squamous cell carcinoma (LSCC). Methods Microarray analysis was used to quantitatively detect the m6A apparent transcriptional modification level of lncRNA in LSCC tissue. Methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) was used to examine the m6A modification of KCNQ1OT1 transcript. Quantitative real-time PCR (qRT-PCR) was used to detect the expression levels of KCNQ1OT1, ALKBH5, HOXA9. Western blot was performed to measure the expression ALKBH5 and HOXA9 protein. The expression of KCNQ1OT1 in LSCC and non-tumor tissues was detected by In situ hybridization (ISH). The biological effects of KCNQ1OT1 in vivo and in vitro were tested, meanwhile, the effects of HOXA9 on LSCC cells were tested in vitro. The expression levels of ALKBH5 and HOXA9 in LSCC and non-tumor tissues, and the expression levels of Ki-67, MMP2 and MMP9 in xenograft tumors were detected by Immunohistochemistry(IHC). RNA immunoprecipitation (RIP) and RNA pull-down assay confirmed the interaction relationship between ALKBH5/YTHDF2 and KCNQ1OT1. Luciferase Reporter assay and rescue experiment verified that HOXA9 is the target of KCNQ1OT1. Results KCNQ1OT1 expression in LSCC tissues was low m6A-methylation and high expression than that in non-tumor tissues, KCNQ1OT1 knockdown inhibits the proliferation, invasion and metastasis of LSCC. Further studies showed that the N6-methyladenosine (m6A) demethylase ALKBH5 mediators KCNQ1OT1 expression via an m6A-YTHDF2-dependent manner. It was found that KCNQ1OT1 could directly bind to HOXA9 to further regulate the proliferation, invasion and metastasis of LSCC cells. Conclusions Our research indicates that ALKBH5 mediated m6A modification of LncRNA KCNQ1OT1 triggers the development of LSCC via up-regulation of HOXA9.

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Epigenetic regulation of ferroptosis via ETS1/miR-23a-3p/ACSL4 axis mediates sorafenib resistance in human hepatocellular carcinoma

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02208-x ◽

2022 ◽

Vol 41 (1) ◽

Author(s):

Yuanjun Lu ◽

Yau-Tuen Chan ◽

Hor-Yue Tan ◽

Cheng Zhang ◽

Wei Guo ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Drug Resistance ◽

Molecular Mechanisms ◽

Luciferase Reporter ◽

Dependent Manner ◽

Bioinformatics Analyses ◽

Sorafenib Treatment ◽

Hcc Cells

Abstract Background Drug resistance to sorafenib greatly limited the benefits of treatment in patients with hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) participate in the development of drug resistance. The key miRNA regulators related to the clinical outcome of sorafenib treatment and their molecular mechanisms remain to be identified. Methods The clinical significance of miRNA-related epigenetic changes in sorafenib-resistant HCC was evaluated by analyzing publicly available databases and in-house human HCC tissues. The biological functions of miR-23a-3p were investigated both in vitro and in vivo. Proteomics and bioinformatics analyses were conducted to identify the mechanisms that regulating miR-23a-3p. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were used to validate the binding relationship of miR-23a-3p and its targets. Results We found that miR-23a-3p was the most prominent miRNA in HCC, which was overexpressed in sorafenib non-responders and indicated poor survival and HCC relapse. Sorafenib-resistant cells exhibited increased miR-23a-3p transcription in an ETS Proto-Oncogene 1 (ETS1)-dependent manner. CRISPR-Cas9 knockout of miR-23a-3p improved sorafenib response in HCC cells as well as orthotopic HCC tumours. Proteomics analysis suggested that sorafenib-induced ferroptosis was the key pathway suppressed by miR-23a-3p with reduced cellular iron accumulation and lipid peroxidation. MiR-23a-3p directly targeted the 3′-untranslated regions (UTR) of ACSL4, the key positive regulator of ferroptosis. The miR-23a-3p inhibitor rescued ACSL4 expression and induced ferrotoptic cell death in sorafenib-treated HCC cells. The co-delivery of ACSL4 siRNA and miR-23a-3p inhibitor abolished sorafenib response. Conclusion Our study demonstrates that ETS1/miR-23a-3p/ACSL4 axis contributes to sorafenib resistance in HCC through regulating ferroptosis. Our findings suggest that miR-23a-3p could be a potential target to improve sorafenib responsiveness in HCC patients.

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The Long Non-coding RNA TMPO-AS1 Promotes Bladder Cancer Growth and Progression via OTUB1-induced E2F1 Deubiquitination

10.21203/rs.3.rs-74122/v1 ◽

2020 ◽

Author(s):

Yeyu Zhang ◽

Yuxing Zhu ◽

Mengqing Xiao ◽

Yaxin Cheng ◽

Dong He ◽

...

Keyword(s):

Bladder Cancer ◽

Molecular Mechanisms ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Dependent Manner ◽

E2f Transcription Factor ◽

Rna Immunoprecipitation ◽

Regulatory Loop

Abstract BackgroundBladder cancer (BC) is the most common malignant tumor of the urinary system. Increasing evidence indicates long non-coding RNAs (lncRNAs) play crucial roles in cancer tumorigenesis, development, and progression. However, the role of TMPO antisense RNA 1 (TMPO-AS1) is still need to be explored in BC.MethodsThe lncRNA TMPO-AS1 expression was evaluated by bioinformatics analysis and further validated by qRT-PCR. Loss- and gain-of- function assays were performed to determine the biological functions of TMPO-AS1 in BC proliferation, migration, and invasion. Chromatin immunoprecipitation, luciferase reporter assays, western blotting, RNA pull-down, RNA immunoprecipitation assays, and fluorescence in situ hybridization were conducted to explore the molecular mechanisms of TMPO-AS1/E2F transcription factor 1 (E2F1) loop. ResultsTMPO-AS1 is upregulated in bladder cancer and is associated with BC patients’ poor prognoses. Functional experiments demonstrated that TMPO-AS1 promotes bladder cancer cell proliferation, migration, invasion, and inhibits cell apoptosis in vivo and in vitro. Mechanically, E2F1 is responsible for the TMPO-AS1 upregulation. Additionally, TMPO-AS1 facilitates the interaction of E2F1 with OTU domain-containing ubiquitin aldehyde binding 1 (OTUB1), leading to E2F1 deubiquitination and stabilization, thereby promotes BC malignant phenotypes. Furthermore, rescue experiments showed that TMPO-AS1 promotes BC growth in an E2F1-dependent manner.ConclusionsOur study is the first to uncover a novel positive regulatory loop of TMPO-AS1/E2F1 important for the promotion of BC malignant behaviors. The TMPO-AS1/E2F1 loop should be considered in the quest for new BC therapeutic options.

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TCF4 and HuR Mediated-METTL14 Suppresses Dissemination of Colorectal Cancer via N6-Methyladenosine-Dependent Silencing of ARRDC4

10.21203/rs.3.rs-648817/v1 ◽

2021 ◽

Author(s):

Hao Wang ◽

Wei Wei ◽

Zhong-Yuan Zhang ◽

Yao Liu ◽

Bin Shi ◽

...

Keyword(s):

Colorectal Cancer ◽

Chromatin Immunoprecipitation ◽

Dependent Manner ◽

Dot Blot ◽

Downstream Target ◽

In Vivo Assays ◽

Rna Immunoprecipitation ◽

Underlying Mechanisms

Abstract Background: Metastasis remains the major obstacle to improved survival for colorectal cancer (CRC) patients. Dysregulation of N6-methyladenosine (m6A) is causally associated with the development of metastasis through poorly understood mechanisms. Methods: The expression of METTL14 and its correlation with clinicopathological features were evaluated by western blot and immunohistochemistry. The roles of METTL14 in CRC metastasis were determined through in vitro and in vivo assays. The underlying mechanisms of METTL14 regulation were explored using transcriptome-sequencing, m6A-seguencing, methylated RNA immunoprecipitation (MeRIP), m6A dot blot, RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP) assay.Results: METTL14 is functionally related to the inhibition of ARRDC4/ZEB1 signaling and to the consequent suppression of CRC metastasis. We unveil METTL14-mediated m6A modification profile and identify ARRDC4 as a direct downstream target of METTL14. Knockdown of METTL14 significantly enhances ARRDC4 mRNA stability relying on the “reader” protein YHTDF2 dependent manner. Moreover, TCF4 can induce METTL14 protein expression, and HuR suppresses METTL14 expression by directly binding to its promoter. Clinically, decreased METTL14 is correlated with poor prognosis and acts as an independent predictor of CRC survival. Conclusion: Our data suggest that TCF4 and HuR mediated-METTL14 can trigger the metastasis of CRC during cancer development via YHTDF2/ARRDC4/ZEB1 axis, which imposes great challenge that inhibition of METTL14 is a potential approach for cancer treatment.

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Long noncoding RNA RP11-757G1.5 sponges miR-139-5p and upregulates YAP1 thereby promoting the proliferation and liver, spleen metastasis of colorectal cancer

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-020-01717-5 ◽

2020 ◽

Vol 39 (1) ◽

Cited By ~ 1

Author(s):

Xiaojian Zhu ◽

Fanqin Bu ◽

Ting Tan ◽

Qilin Luo ◽

Jinfeng Zhu ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Proliferation ◽

In Situ Hybridization ◽

Cox Regression ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Cox Regression Analysis

Abstract Background Accumulating evidence indicates that long non-coding RNAs (lncRNAs) acting as crucial regulators in tumorigenesis. However, its biological functions of lncRNAs in colorectal cancer (CRC) have not been systematically clarified. Methods An unbiased screening was performed to identify disregulated lncRNAs revealed to be implicated in CRC carcinogenesis according to an online-available data dataset. In situ hybridization (ISH), RT-qPCR and RNA fluorescence in situ hybridization (RNA-FISH) were applied to detect RP11-757G1.5 expression in CRC tissues and cell lines. The associations of RP11-757G1.5 with clinicopathological characteristics were analyzed. Their effects on prognosis were analyzed by the Kaplan-Meier analysis, Log-rank test, Univariate and Multivariate Cox regression analysis. The potential biological function of RP11-757G1.5 in CRC was investigated by Colony formation, Edu cell proliferation, Flow cytometry, Wound healing and Transwell assays. Bioinformatics binding site analysis, Luciferase reporter assay, Ago2 immunoprecipitation assays, RNA pull-down assay, RT-qPCR and Western blotting were utilized to demonstrate the mechanism of RP11-757G1.5 acts as a molecular sponge of miR-139-5p to regulate the expression of YAP1. Finally, we further explore the potential role of RP11-757G1.5 in CRC orthotopic xenografts in vivo. Results We discovered a novel oncogenic lncRNA RP11-757G1.5, that was overexpressed in CRC tissues, especially in aggressive cases. Moreover, up-regulation of RP11-757G1.5 strongly correlated with poor clinical outcomes of patients with CRC. Functional analyses revealed that RP11-757G1.5 promoted cell proliferation in vitro and in vivo. Furthermore, RP11-757G1.5 stimulated cell migration and invasion in vitro and in vivo. Mechanistic studies illustrated that RP11-757G1.5 regulated the expression of YAP1 through sponging miR-139-5p and inhibiting its activity thereby promoting CRC progression and development. Conclusions Altogether, these results reveal a novel RP11-757G1.5/miR-139-5p/YAP1 regulatory axis that participates in CRC carcinogenesis and progression.

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microRNA-16 via Twist1 inhibits EMT induced by PM2.5 exposure in human hepatocellular carcinoma

Open Medicine ◽

10.1515/med-2019-0078 ◽

2019 ◽

Vol 14 (1) ◽

pp. 673-682 ◽

Cited By ~ 4

Author(s):

Hao Zhang ◽

Zhihu Li

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Line ◽

Epithelial Mesenchymal Transition ◽

Smooth Muscle Actin ◽

Human Hepatocellular Carcinoma ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Dependent Manner ◽

Mesenchymal Transition

AbstractEpidemiological study has confirmed that PM2.5 (particulate matter with an aerodynamic diameter less than 2.5 μm) is associated with the incidence and progression of human hepatocellular carcinoma (HCC). Accordingly, this study was undertaken to investigate the pro-metastatic effects of PM2.5 on human HCC cell line SMMC-7721 in vitro and to explore the underlying mechanisms. CCK-8 assay was performed to examine the effect of PM2.5 on the proliferation of SMMC-7721 cells; scratch wound assay and transwell matrigel system has been used to examine the effect of PM2.5 on the migration and invasion ability of SMMC-7721 cells; furthermore, effect of PM2.5 on epithelial mesenchymal transition (EMT) of SMMC-7721 cells were examined by examining the EMT markers vimentin, ɑ-smooth muscle actin (ɑ-SMA), and E-cadherin; furthermore, the roles of microRNA-16 (miR-16) and its target Twist1 in PM2.5 induced carcinogenic effects were also examined. Results of CCK-8 assay suggested that PM2.5 promoted the proliferation of SMMC-7721 cells in a dose and time dependent manner. PM2.5 also markedly promoted the migration and invasion ability of SMMC-7721 cells. Moreover, epithelial mesenchymal transition (EMT) was also triggered by PM2.5. On the other hand, microRNA-16 (miR-16) and its target Twist1 was found to be mediated by PM2.5, and miR-16 mimic could suppress the metastatic ability of SMMC-7721 cells exposure to PM2.5 via inversely regulating the expression of Twist1. Furthermore, dual Luciferase reporter assay confirmed the specifically binding of miR-16 to the predicted 3′-UTR of Twist1. The present study confirmed the pro-proliferative and pro-metastatic effect of PM2.5 on HCC cell line SMMC-7721. The possible mechanisms were EMT process induced by PM2.5 in SMMC-7721 cells, which was accompanied by a decrease in miR-16 and increase in Twist1 expression.

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Downregulation of miR-193a-3p is involved in the pathogenesis of hepatocellular carcinoma by targeting CCND1

PeerJ ◽

10.7717/peerj.8409 ◽

2020 ◽

Vol 8 ◽

pp. e8409 ◽

Cited By ~ 2

Author(s):

Shi-shuo Wang ◽

Zhi-guang Huang ◽

Hua-yu Wu ◽

Rong-quan He ◽

Li-hua Yang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Correlation Analysis ◽

Kegg Pathway ◽

Pearson Correlation ◽

Luciferase Reporter ◽

Kegg Pathway Analysis ◽

Reporter Assays ◽

Pearson Correlation Analysis ◽

Hcc Cells

Background Hepatocellular carcinoma (HCC) is the second-highest cause of malignancy-related death worldwide, and many physiological and pathological processes, including cancer, are regulated by microRNAs (miRNAs). miR-193a-3p is an anti-oncogene that plays an important part in health and disease biology by interacting with specific targets and signals. Methods In vitro assays were performed to explore the influences of miR-193a-3p on the propagation and apoptosis of HCC cells. The sequencing data for HCC were obtained from The Cancer Genome Atlas (TCGA), and the expression levels of miR-193a-3p in HCC and non-HCC tissues were calculated. The differential expression of miR-193a-3p in HCC was presented as standardized mean difference (SMD) with 95% confidence intervals (CIs) in Stata SE. The impact of miR-193a-3p on the prognoses of HCC patients was determined by survival analysis. The potential targets of miR-193a-3p were then predicted using miRWalk 2.0 and subjected to enrichment analyses, including Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and Protein-Protein Interaction (PPI) network analysis. The interaction between miR-193a-3p and one predicted target, Cyclin D1 (CCND1), was verified by dual luciferase reporter assays and Pearson correlation analysis. Results MiR-193a-3p inhibited the propagation and facilitated the apoptosis of HCC cells in vitro. The pooled SMD indicated that miR-193a-3p had a low level of expression in HCC (SMD: −0.88, 95% CI [−2.36 −0.59]). Also, HCC patients with a higher level of miR-193a-3p expression tended to have a favorable overall survival (OS: HR = 0.7, 95% CI [0.43–1.13], P = 0.14). For the KEGG pathway analysis, the most related pathway was “proteoglycans in cancer”, while the most enriched GO term was “protein binding”. The dual luciferase reporter assays demonstrated the direct interaction between miR-193a-3p and CCND1, and the Pearson correlation analysis suggested that miR-193a-3p was negatively correlated with CCND1 in HCC tissues (R = − 0.154, P = 0.002). Conclusion miR-193a-3p could suppress proliferation and promote apoptosis by targeting CCND1 in HCC cells. Further, miR-193a-3p can be used as a promising biomarker for the diagnosis and treatment of HCC in the future.

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Blocking siglec-10hi tumor-associated macrophages improves anti-tumor immunity and enhances immunotherapy for hepatocellular carcinoma

Experimental Hematology and Oncology ◽

10.1186/s40164-021-00230-5 ◽

2021 ◽

Vol 10 (1) ◽

Author(s):

Nan Xiao ◽

Xiaodong Zhu ◽

Kangshuai Li ◽

Yifan Chen ◽

Xuefeng Liu ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Function ◽

Cox Regression ◽

Human Tumor ◽

The Cancer Genome Atlas ◽

Sequencing Data ◽

Tumor Associated Macrophages ◽

Cox Regression Analysis ◽

Kaplan Meier

Abstract Background Tumor-associated macrophages (TAMs) promote key processes in the modulation of tumor microenvironment (TME). However, the clinical significance of heterogeneous subpopulations of TAMs in hepatocellular carcinoma (HCC) remains unknown. Methods HCC tissues from Zhongshan Hospital and data from The Cancer Genome Atlas were obtained and analyzed. Immunohistochemistry and flow cytometry were performed to detect the characteristics of sialic acid-binding immunoglobulin-like lectin 10high (Siglec-10hi) TAMs and explore their impact on the TME of HCC. The effect of Siglec-10 blockade was evaluated in vitro based on fresh human tumor tissues. Results Our data revealed that Siglec-10 was abundant in a large proportion of HCC specimens and prominently distributed on macrophages. Kaplan–Meier curves and Cox regression analysis showed that intratumoral Siglec-10+ cell enrichment was associated with unfavorable prognosis in patients with HCC. Notably, multiple anti-inflammatory cytokines and inhibitory receptors were enriched in Siglec-10hi TAMs. RNA sequencing data also revealed that numerous M2-like signaling pathways were significantly upregulated in Siglec-10hi TAMs. High infiltration of Siglec-10hi TAMs was associated with impaired CD8+ T cell function in HCC. Of note, blocking Siglec-10 with the competitive binding antibody Siglec-10 Fc led to decreased expression of immunosuppressive molecules and increased the cytotoxic effects of CD8+ T cells against HCC cells. Moreover, blocking Siglec-10 promoted the anti-tumor efficacy of the programmed cell death protein 1 (PD-1) inhibitor pembrolizumab. Conclusions Siglec-10hi TAMs are associated with immune suppression in the TME, and indicate poor prognosis in patients with HCC. Targeting Siglec-10hi TAMs may serve as a promising immunotherapy approach for HCC.

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Period 2 Regulates CYP2B10 Expression and Activity in Mouse Liver

Frontiers in Pharmacology ◽

10.3389/fphar.2021.764124 ◽

2021 ◽

Vol 12 ◽

Author(s):

MengLin Chen ◽

Min Chen ◽

Danyi Lu ◽

Yi Wang ◽

Li Zhang ◽

...

Keyword(s):

Mouse Liver ◽

Luciferase Reporter ◽

Specific Substrate ◽

Dependent Manner ◽

Hepatic Expression ◽

Period 2 ◽

Reporter Assays ◽

Expression And Activity

CYP2B10 is responsible for metabolism and detoxification of many clinical drugs. Here, we aimed to investigate a potential role of Period 2 (PER2) in regulating expression of hepatic CYP2B10. Regulatory effects of PER2 on hepatic expression of CYP2B10 and other enzymes were determined using Per2-deficient mice with exons 4-6 deleted (named Per2Del4-6 mice). In vitro and in vivo metabolic activities of CYP2B10 were probed using cyclophosphamide (CPA) as a specific substrate. Regulatory mechanism was investigated using luciferase reporter assays. Genotyping and Western blotting demonstrated loss of wild-type Per2 transcript and markedly reduced PER2 protein in Per2Del4-6 mice. Hepatic expression of a plenty of drug-metabolizing genes (including Cyp2a4/2a5, Cyp2b10, Ugt1a1, Ugt1a9, Ugt2b36, Sult1a1 and Sult1e1) were altered (and majority were down-regulated) in Per2Del4-6 mice. Of note, Cyp2b10, Ugt1a9 and Sult1a1 were three genes considerably affected with reduced expression. Decreased expression of CYP2B10 was translated to reduced metabolism and altered pharmacokinetics of CPA as well as attenuated CPA hepatotoxicity in Per2Del4-6 mice. Positive regulation of CYP2B10 by PER2 was further confirmed in both Hepa-1c1c7 and AML-12 cells. Based on luciferase reporter assays, it was shown that PER2 regulated Cyp2b10 transcription in a REV-ERBα-dependent manner. REV-ERBα was negatively regulated by PER2 (increased REV-ERBα expression in Per2Del4-6 mice) and itself was also a repressor of CYP2B10. In conclusion, PER2 positively regulates CYP2B10 expression and activity in mouse liver through inhibiting its repressor REV-ERBα.

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