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

Yanming Liu; Yue Cao; Wencan Cai; Liangyin Wu; Pingsen Zhao; Xin-Guang Liu

doi:10.7717/peerj.9100

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

PeerJ. 2020 Apr 29:8:e9100. doi: 10.7717/peerj.9100. eCollection 2020.

Authors

Yanming Liu^{1

2}, Yue Cao³, Wencan Cai², Liangyin Wu², Pingsen Zhao², Xin-Guang Liu¹

Affiliations

¹ Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Institute of Aging Research, Guangdong Medical University, Dongguan, Guangdong, China.
² Department of Clinical Laboratory, YueBei People's Hospital, Shaoguan, Guangdong, China.
³ Department of Medical Technology, Medical College of Shaoguan University, Shaogguan, Guangdong, China.

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: Bioinformatic analysis; HBV amplification; Hepatocellular carcinoma; Invasion; Proliferation; microRNA.

Grants and funding

This work was supported by the National Natural Science Foundation of China (Nos. 81671399 and 81971329), Guangdong Medical Research Fund (No. B2018262), Shaoguan Health Bureau Research Fund (No. Y19042), Shaoguan Science and Technology Fund (No. 2018sn095) and Outstanding Research Fund of YueBei People’s Hospital, Natural Science Foundation of Guangdong Province, China (No. 2016A030307031). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.