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RNA helicase p68 inhibits the transcription and post-transcription of Pkd1 in ADPKD.

ABSTRACT: Background: Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations of the PKD1 and PKD2 genes. Dysregulation of the expression of PKD genes, the abnormal activation of PKD associated signaling pathways, and the expression and maturation of miRNAs regulates cyst progression. However, the upstream factors regulating these abnormal processes in ADPKD remain elusive. Methods: To investigate the roles of an RNA helicase, p68, in ADPKD, we performed Western blot and qRT-PCR analysis, immunostaining and ChIP assay in cystic renal epithelium cells and tissues. Results: We found that p68 was upregulated in cystic renal epithelial cells and tissues. p68 represses Pkd1 gene expression via transcriptional and posttranscriptional mechanisms in renal epithelial cells, in that 1) p68 binds to the promoter of the Pkd1 gene together with p53 to repress transcription; and 2) p68 promotes the expression and maturation of miR-17, miR-200c and miR-182 and via these miRNAs, post-transcriptionally regulates the expression of Pkd1 mRNA. Drosha is involved in this process by forming a complex with p68. p68 also regulates the phosphorylation and activation of PKD proliferation associated signaling and the expression of fibrotic markers in Pkd1 mutant renal epithelial cells. Silence of p68 delays cyst formation in collecting duct cell mediated 3D cultures. In addition, the expression of p68 is induced by H₂O₂-dependent oxidative stress and DNA damage which causes downregulation of Pkd1 transcription in cystic renal epithelial cells and tissues. Conclusions: p68 plays a critical role in negatively regulating the expression of the PKD1 gene along with positively regulating the expression and maturation of miRNAs and activation of PKD associated signaling pathways to cause renal cyst progression and fibrosis in ADPKD.

SUBMITTER: Zhang L

PROVIDER: S-EPMC7381742 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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RNA helicase p68 inhibits the transcription and post-transcription of Pkd1 in ADPKD.

Zhang Lu L Li Linda Xiaoyan LX Zhou Julie Xia JX Harris Peter C PC Calvet James P JP Li Xiaogang X

Theranostics 20200709 18

Background: Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations of the PKD1 and PKD2 genes. Dysregulation of the expression of PKD genes, the abnormal activation of PKD associated signaling pathways, and the expression and maturation of miRNAs regulates cyst progression. However, the upstream factors regulating these abnormal processes in ADPKD remain elusive. Methods: To investigate the roles of an RNA helicase, p68, in ADPKD, we performed Wes ...[more]

PMID: 32724471

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Project description:BackgroundRelA/p65 a crucial member of NF-?B signaling pathway plays diverse role in mediating oncogenesis. Limited knowledge prevails on the mechanistic insights of RelA gene regulation. RNA helicase p68 apart from being a vital player of RNA metabolism acts as a transcriptional coactivator of several oncogenic transcription factors including ?-catenin and is highly implicated in cancer progression. In this study, we aim to discern the molecular mechanism of how an RNA helicase, p68 deploys a major oncogenic signaling pathway, Wnt/ ?-catenin to regulate the expression of RelA, an indispensable component of NF-?B signaling pathway towards driving colon carcinogenesis.MethodsImmunoblotting and quantitative RT-PCR was performed for determining the protein and mRNA expressions of the concerned genes respectively. Luciferase assay was employed for studying the promoter activity of RelA. Chromatin immunoprecipitation was used to evaluate the occupancy of transcription factors on the RelA promoter. Immunohistochemical analysis was conducted using FFPE sections derived from normal human colon and colon cancer patient samples. Finally, a syngeneic colorectal allograft mouse model was used to assess physiological significance of the in vitro findings.Resultsp68, ?-catenin and RelA proteins were found to bear strong positive correlation in normal and colon carcinoma patient samples. Both p68 and ?-catenin increased RelA mRNA and protein expression. p68, ?-catenin and Wnt3a elevated RelA promoter activity. Conversely, p68 and ?-catenin knockdown diminished RelA promoter activity and led to reduced RelA mRNA and protein expression. p68 was perceived to occupy RelA promoter with ?-catenin at the TCF4/LEF (TBE) sites thereby potentiating RelA transcription. p68 and ?-catenin alliance positively modulated the expression of signature NF-?B target genes. Enhanced NF-?B target gene expression by p68 was corroborated by findings in clinical samples. Tumors generated in mice colorectal allograft model, stably expressing p68 further reinforced our in vitro findings.ConclusionsWe report for the first time a novel mechanism of alliance between p68 and ?-catenin in regulating the expression of RelA and stimulating the NF-?B signaling axis towards driving colon carcinogenesis. This study unravels novel modes of p68-mediated colon carcinogenesis, marking it a potential target for therapy.

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RNA helicase p68 inhibits the transcription and post-transcription of Pkd1 in ADPKD.

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RNA helicase p68 inhibits the transcription and post-transcription of <i>Pkd1</i> in ADPKD.

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