Dataset Information

MicroRNA (miR)-433 and miR-22 dysregulations induce histone-deacetylase-6 overexpression and ciliary loss in cholangiocarcinoma.

ABSTRACT: Cholangiocytes normally express primary cilia, a multisensory organelle that detects signals from the cellular environment. Cilia are significantly reduced in cholangiocarcinoma (CCA) by a mechanism involving overexpression of histone deacetylase 6 (HDAC6). Despite HDAC6 overexpression in CCA, we found no differences in its mRNA level, suggesting a posttranscriptional regulation, possibly involving microRNAs (miRNAs). Here, we describe that at least two HDAC6-targeting miRNAs, miR-433 and miR-22, are down-regulated in CCA both in vitro and in vivo. Experimental restoration of these miRNAs in CCA cells reduced HDAC6 expression, induced ciliary restoration, and decreased the malignant phenotype. Furthermore, in contrast to the mature forms, levels of precursor forms of these miRNAs were higher in CCA compared to normal cholangiocytes and accumulated in the nuclei, suggesting a defective nuclear export. We assessed the expression of Exportin-5, the protein responsible for transporting miRNA precursors out of the nucleus, and found it to be reduced by 50% in CCA compared to normal cholangiocytes. Experimental overexpression of Exportin-5 in CCA cells restored precursor and mature forms of these miRNAs to normal levels, inducing a decrease in the expression of HDAC6 and a decrease in the malignant phenotype. Conversely, short hairpin RNA (shRNA) depletion of Exportin-5 in normal cholangiocytes resulted in increased nuclear retention of precursor miRNAs, decreased mature miRNAs, increased cell proliferation, and shorter cilia. CONCLUSION:These data suggest that down-regulated Exportin-5 impairs the nuclear export of miR-433 and miR-22 precursor forms, causing a decrease in levels of mature miR-433 and miR-22 forms, and leading to overexpression of HDAC6 and ciliary loss in CCA. (Hepatology 2018).

SUBMITTER: Mansini AP

PROVIDER: S-EPMC6078832 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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MicroRNA (miR)-433 and miR-22 dysregulations induce histone-deacetylase-6 overexpression and ciliary loss in cholangiocarcinoma.

Mansini Adrian P AP Lorenzo Pisarello Maria J MJ Thelen Kristen M KM Cruz-Reyes Maetzin M Peixoto Estanislao E Jin Sujeong S Howard Brynn N BN Trussoni Christy E CE Gajdos Gabriella B GB LaRusso Nicholas F NF Perugorria Maria J MJ Banales Jesus M JM Gradilone Sergio A SA

Hepatology (Baltimore, Md.) 20180521 2

Cholangiocytes normally express primary cilia, a multisensory organelle that detects signals from the cellular environment. Cilia are significantly reduced in cholangiocarcinoma (CCA) by a mechanism involving overexpression of histone deacetylase 6 (HDAC6). Despite HDAC6 overexpression in CCA, we found no differences in its mRNA level, suggesting a posttranscriptional regulation, possibly involving microRNAs (miRNAs). Here, we describe that at least two HDAC6-targeting miRNAs, miR-433 and miR-22 ...[more]

PMID: 29406621

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Project description:Background: MicroRNAs (miRNAs) have an increasing functional role in some neurodegenerative diseases. Autophagy, the degradation of bulk protein in the cytoplasm, is the quality control function of protein and has a protective role in the survival of neural cells. miR-433 may play a regulatory role in neurodegenerative diseases. Many aspects underlying the mechanism of miR-433 in neural development and neurodegeneration are not clear. Methods: In this study, we established stable cell lines expressing miR-433 by infecting mouse hippocampal neural cell line (HT-22) cells with rLV-miR-433 and the control rLV-miR. Pre-miR-433 expression was analyzed using polymerase chain reaction (PCR). Mature miR-433 expression was measured using quantitative PCR (qPCR). The effect of miR-433 overexpression on cell proliferation was determined using a CCK-8 assay and flow cytometry. RNA interference was used to analyze the function of Cdk12 in mediating the effect of miR-433 on cell proliferation. The effect of miR-433 overexpression on cell apoptosis was determined by flow cytometry. Autophagy-related genes Atg4a, LC3B, and Beclin-1 were determined using qPCR, Western blot, or immunofluorescence. In addition, RNA interference was used to analyze the effect of Atg4a on the induction of autophagy. TargetScan 7.2 was used to predict the target genes of miR-433, and Smad9 was determined using qPCR. Results: Our results indicated that miR-433 increased the expression of Atg4a and induced autophagy by increasing the expression of LC3B-Ⅱ and Beclin-1 in an Atg4a-dependent manner. In addition, miR-433 upregulated the expression of Cdk12 and inhibited cell proliferation in a Cdk12-dependent manner and promoted apoptosis in HT-22 cells under the treatment of 10-hydroxycamptothecin. Conclusion: The results of our study suggest that miR-433 may regulate neuronal growth by promoting autophagy and attenuating cell proliferation. This might be a potential therapeutic intervention in neurodegenerative diseases.

Project description:BACKGROUND & AIMS:Disruption of circadian rhythm is associated with cancer development and progression. MicroRNAs (miRNAs) are a class of small non-coding RNAs that trigger mRNA translation inhibition. We aimed to evaluate the role of Per1 and related miRNAs in cholangiocarcinoma growth. METHODS:The expression of clock genes was evaluated in human cholangiocarcinoma tissue arrays and cholangiocarcinoma lines. The rhythmic expression of clock genes was evaluated in cholangiocarcinoma cells and H69 (non-malignant cholangiocytes) by qPCR. We measured cell proliferation, cell cycle and apoptosis in Mz-ChA-1 cells after Per1 overexpression. We examined tumor growth in vivo after injection of Per1 overexpressing cells. We verified miRNAs that targets Per1. The circadian rhythm of miR-34a was evaluated in cholangiocarcinoma and H69 cells. We evaluated cell proliferation, apoptosis and invasion after inhibition of miR-34a in vitro, and the potential molecular mechanisms by mRNA profiling after overexpression of Per1. RESULTS:Expression of Per1 was decreased in cholangiocarcinoma. The circadian rhythm of Per1 expression was lost in cholangiocarcinoma cells. Decreased cell proliferation, lower G2/M arrest, and enhanced apoptosis were shown in Per1 overexpressing cells. An in vivo study revealed decreased tumor growth, decreased proliferation, angiogenesis and metastasis after overexpressing Per1. Per1 was verified as a target of miR-34a. miR-34a was rhythmically expressed in cholangiocarcinoma cells and H69. The inhibition of miR-34a decreased proliferation, migration and invasion in cholangiocarcinoma cells. mRNA profiling has shown that overexpression of Per1 inhibits cell growth through regulation of multiple cancer-related pathways, such as cell cycle, cell growth and apoptosis pathways. CONCLUSIONS:Disruption of circadian rhythms of clock genes contribute to the malignant phenotypes of human cholangiocarcinoma. LAY SUMMARY:The current study is about how biological clock and its regulators affect the bile duct tumor growth. The disruption of biological clock has a negative impact in different cancers. Per1 is a gene that is involved in maintaining the biological clock and show 24h oscillation. Reduced levels of Per1 and disruption of 24h circadian rhythm was found in bile duct cancer cells. Therefore, a genetic modified bile duct cancer cells was created. It has a higher level of Per1 expression and partially recovered circadian rhythm. Those genetic modified cells also displayed slower cell growth or higher rate of cell death. We also used mice model that lack of immune system to show that our genetic modified bile duct cells form smaller tumor. In addition, we tried to see how Per1 is communicating with other genes in regarding of controlling the tumor growth. We found Per1 is regulated by microRNA-34a, a small non-coding RNA that directly binds to genes and inhibit gene expression. Decreased level of miR-34a has also significantly reduced tumor growth through controlling the cell growth and cell death balance. Therefore bile duct cancer patients may be treated with miR-34a inhibitor or Per1 stimulator in the future.

Dataset Information

MicroRNA (miR)-433 and miR-22 dysregulations induce histone-deacetylase-6 overexpression and ciliary loss in cholangiocarcinoma.

Publications

MicroRNA (miR)-433 and miR-22 dysregulations induce histone-deacetylase-6 overexpression and ciliary loss in cholangiocarcinoma.

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