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Next Generation Sequencing Facilitates Quantitative Analysis of Pressure Type and control Transcriptomes

ABSTRACT: With the aim to reveal the function of microRNAs in Hepatic stellate cells (HSCs) in response to portal hypertension, primary rat HSCs were exposed to pressure (10mmHg, 1 h) by using a pressure induced apparatus. Appling next-generation sequencing screened the pressurization induced miRNAs expression profile in HSCs. Among them miR-9a-5p was confirmed to be significantly increased after loading pressure in HSC by RT-PCR. It was found that inhibition of miR-9a-5p could significantly restrain HSCs proliferation and activation under pressure overload. Moreover, the results showed that the induction of miR-9a-5p upon pressure load might by increasing the phosphorylation of Akt but not FAK and Erk1/2. Luciferase reporter assay and western blot suggested that Sirt1 was a potential target gene of miR-9a-5p. Finally, we revealed that miR-9a-5p level was apparently higher in rat liver fibrotic model than in the normal control while Sirt1 level was decreased in fibrotic liver tissue. In conclusion, our results suggest that miR-9a-5p regulate HSCs proliferation through negative regulation of Sirt1 and suggest a potential biomarker for portal hypertension.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE63783 | GEO | 2017/08/29

SECONDARY ACCESSION(S): PRJNA269064

REPOSITORIES: GEO

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Project description:Liver fibrosis is a common pathological process of various chronic liver diseases that can develop into liver cancer. MicroRNAs (miRNAs) are a king of non-coding RNA which are closely related to liver diseases. Thus, this research hope to explore the effect of miR-484 on liver fibrosis and reveal its mechanism. The miRNAs profiles were screened by microRNA sequencing and the location of miR-484 was identified by fluorescence in situ hybridization (FISH) in human liver fibrotic tissues. MiR-484 expression was detected by qRT-PCR in rat primary hepatic stellate cells (HSCs). Bioinformatics analysis and dual-luciferase reporter assay were performed to determine the target gene of miR-484. Liver fibrosis specific signatures were analyzed by qRT-PCR and western blot after miR-484 mimic/inhibitor transfection. The cell apoptosis was detected by Annexin V-FITC/PI double staining. The effect of miR-484 silencing on fibrosis in vivo was investigated in thioacetamide (TAA) induced mice model using the adeno-associated virus carrying miR-484 inhibitor. Enrichment of miR-484 was observed in human liver fibrosis tissues and activated rat primary HSCs. FISH showed that miR-484 was prominently located at fibrotic region and the cytoplasm of HSCs in human liver tissues. Dual-luciferase reporter assay verified that the homeodomain-interacting protein kinases 1 (HIPK1) was the direct target of miR-484. After transfecting miR-484 inhibitor into HSC-T6, HIPK1 were significantly up-regulated, and α-SMA, col1a1, Wnt-3a, Wnt-5a, β-catenin and p-β-catenin were down-regulated, suggesting the restrain effect of miR-484 knockdown on HSCs activation. Conversely, the results were opposite with miR-484 mimic transfection. In addition, the apoptosis of HSC-T6 altered significantly after miR-484 modulation. Moreover, adeno-associated virus carrying miR-484 inhibitor alleviated mice liver fibrosis induced by TAA. In conclusion, miR-484 knockdown ameliorates liver fibrosis by promoting the apoptosis and suppressing HSCs activation via blocking Wnt/β-catenin signaling pathway. MiR-484 and its downstream gene HIPK1 might be selected as novel therapeutic targets of liver fibrosis.

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Next Generation Sequencing Facilitates Quantitative Analysis of Pressure Type and control Transcriptomes

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