Aberrant expression of two miRNAs promotes proliferation, hepatitis B virus amplification, migration and invasion of hepatocellular carcinoma cells: evidence from bioinformatic analysis and experimental validation.
As key negative regulators of gene expression, microRNAs (miRNAs) play an important role in the onset and progression of hepatocellular carcinoma (HCC). This study aimed to identify the miRNAs involved in HCC carcinogenesis and their regulated genes.
The Gene Expression Omnibus (GEO) dataset (GSE108724) was chosen and explored to identify differentially expressed miRNAs using GEO2R. For the prediction of potential miRNA target genes, the miRTarBase was explored. Enrichment analysis of Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed by the DAVID online tool. The hub genes were screened out using the CytoHubba plug-in ranked by degrees. The networks between miRNAs and hub genes were constructed by Cytoscape software. MiRNA mimics and negative control were transfected into HCC cell lines and their effects on proliferation, hepatitis B virus DNA (HBV-DNA) replication, TP53 expression, migration, and invasion were investigated. The following methods were employed: MTT assay, quantitative PCR (qPCR) assay, western blotting, wound healing assay, and transwell assay.
A total of 50 differentially expressed miRNAs were identified, including 20 upregulated and 30 downregulated miRNAs, in HCC tumor tissues compared to matched adjacent tumor-free tissues. The top three upregulated (miR-221-3p, miR-222-3p, and miR-18-5p) and downregulated (miR-375, miR-214-3p and miR-378d) miRNAs, ranked by |log2 fold change (log2FC)|, were chosen and their potential target genes were predicted. Two gene sets, targeted by the upregulated and the downregulated miRNAs, were identified respectively. GO and KEGG pathway analysis showed that the predicted target genes of upregulated and downregulated miRNAs were mainly enriched in the cell cycle and cancer-related pathways. The top ten hub nodes of gene sets ranked by degrees were identified as hub genes. Analysis of miRNA-hub gene network showed that miR-221-3p and miR-375 modulated most of the hub genes, especially involving regulation of TP53. The q-PCR results showed that miR-221-3p and miR-375 were markedly upregulated and downregulated, respectively, in HCC cells and HCC clinical tissue samples compared to non-tumoral tissues. Furthermore, miR-221-3p overexpression significantly enhanced proliferation, HBV-DNA replication, as well as the migration and invasion of HCC cells, whereas miR-375 overexpression resulted in opposite effects. Western blotting analysis showed that the overexpression of miR-221-3p and miR-375 reduced and increased TP53 expression, respectively.
The present study revealed that miR-211-3p and miR-375 may exert vital effects on cell proliferation, HBV-DNA replication, cell migration, and invasion through the regulation of TP53 expression in HCC.
Liu Y
，Cao Y
，Cai W
，Wu L
，Zhao P
，Liu XG
... -
《PeerJ》
Upregulation of microRNA-140-3p inhibits epithelial-mesenchymal transition, invasion, and metastasis of hepatocellular carcinoma through inactivation of the MAPK signaling pathway by targeting GRN.
Invasion and metastasis in hepatocellular carcinoma (HCC) results in poor prognosis. Human intervention in these pathological processes may benefit the treatment of HCC. The aim of the present study is to elucidate the mechanism of miR-140-3p affecting epithelial-mesenchymal transition (EMT), invasion, and metastasis in HCC. Microarray analysis was performed for differentially expressed genes screening. The target relationship between miR-140-3p and GRN was analyzed. Small interfering RNA (siRNA) against granulin (GRN) was synthesized. EMT markers were detected, and invasion and migration were evaluated in HCC cells introduced with a miR-140-3p inhibitor or mimic, or siRNA against GRN. A mechanistic investigation was conducted for the determination of mitogen-activated protein kinase (MAPK) signaling pathway-related genes and EMT markers (E-cadherin, N-cadherin, and Vimentin). GRN was highlighted as an upregulated gene in HCC. GRN was a target gene of miR-140-3p. Elevation of miR-140-3p or inhibition of GRN restrained the EMT process and suppressed the HCC cell migration and invasion. HCC cells treated with the miR-140-3p mimic or siRNA-GRN exhibited decreased GRN expression and downregulated the expressions of the MAPK signaling pathway-related genes, N-cadherin, and Vimentin but upregulated the expression of E-cadherin. GRN silencing can reverse the activation of the MAPK signaling pathway and induction of EMT mediated by miR-140-3p inhibition. Taken together, the results show that miR-140-3p confers suppression of the MAPK signaling pathway by targeting GRN, thus inhibiting EMT, invasion, and metastasis in HCC.
Zhang QY
，Men CJ
，Ding XW
《-》
ResearchGate
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钛学术
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