免疫印迹
分子生物学
癌症研究
生物
小RNA
基因
生物化学
作者
Jingyuan Wen,Zhao Huang,Yi Wei,Lin Xue,Yufei Wang,Jinli Liao,Jintai Liang,Xiaoping Chen,Liang Chu,Bixiang Zhang
标识
DOI:10.1186/s11658-022-00370-4
摘要
Abstract Background MicroRNAs (miRNAs) play crucial roles in the development of hepatocellular carcinoma (HCC). Hsa-microRNA-27b-3p ( hsa-miR-27b ) is involved in the formation and progression of various cancers, but its role and clinical value in HCC remain unclear. Methods The expression of hsa-miR-27b in HCC was examined by quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH) assays of clinical samples. Cell Counting Kit-8 assays (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU) incorporation assays, Transwell assays, filamentous actin ( F-actin ) staining and western blot analyses were used to determine the effects of hsa-miR-27b on HCC cells in vitro. Subcutaneous xenograft and lung metastatic animal experiments were conducted to verify the role of hsa-miR-27b in HCC in vivo. In silico prediction, qRT-PCR, western blot, anti-Argonaute 2 ( AGO2 ) RNA immunoprecipitation (RIP) and dual luciferase reporter assays were applied to identify the target genes of hsa-miR-27b . To detect the impacts of hsa-miR-27b on nuclear factor kappa B ( NF-кB ) signalling cascades mediated by transforming growth factor-activated kinase-binding protein 3 ( TAB3 ), we performed qRT-PCR, western blot assays, immunofluorescence staining, immunohistochemistry (IHC) and dual-luciferase reporter assays. Recombinant oncolytic adenovirus (Onco Ad ) overexpressing hsa-miR-27b was constructed to detect their therapeutic value in HCC. Results The expression of hsa-miR-27b was lower in HCC than in adjacent non-tumourous tissues (ANTs), and the reduced expression of hsa-miR-27b was associated with worse outcomes in patients with HCC. Hsa-miR-27b significantly inhibited the proliferation, migration, invasion, subcutaneous tumour growth and lung metastasis of HCC cells. The suppression of hsa-miR-27b promoted the nuclear translocation of NF-κB by upregulating TAB3 expression. TAB3 was highly expressed in HCC compared with ANTs and was negatively correlated with the expression of hsa-miR-27b . The impaired cell proliferation, migration and invasion by hsa-miR-27b overexpression were recovered by ectopic expression of TAB3 . Recombinant Onco Ad with overexpression of hsa-miR-27b induced anti-tumour activity compared with that induced by negative control (NC) Onco Ad in vivo and in vitro. Conclusions By targeting TAB3 , hsa-miR-27b acted as a tumour suppressor by inactivating the NF-кB pathway in HCC in vitro and in vivo, indicating its therapeutic value against HCC. Graphical Abstract
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