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CASC15 promotes epithelial to mesenchymal transition and facilitates malignancy of hepatocellular carcinoma cells by increasing TWIST1 gene expression via miR-33a-5p sponging

https://doi.org/10.1016/j.ejphar.2019.172589Get rights and content

Abstract

Long noncoding RNA cancer susceptibility candidate 15 (CASC15) facilitates progression of hepatocellular carcinoma (HCC) cells, but the molecular mechanisms remain unknown. CASC15 co-expressing genes were explored in the Cancer Genome Atlas Liver Hepatocellular Carcinoma dataset. Putative co-expressing genes were analyzed by Gene Ontology and biological pathway enrichment analysis. CASC15 overexpression or knockdown and TWIST1 overexpression or knockdown in HCC cells was achieved by lentiviral transduction or plasmid transfection. Interaction between CASC15 and microRNA-33a-5p (miR-33a-5p) was verified by argonaute 2-RNA Immunoprecipitation (AGO2-RIP) and luciferase reporter assays. HCC cell malignancy was determined by cell proliferation, apoptosis, migration and invasion assays. Tumorigenicity was evaluated by xenograft assay. Epithelial-to-mesenchymal transition (EMT) of HCC cells was assessed by Western blot. TWIST1, sex-determining region Y-related high-mobility group box 4 (Sox4) and Versican were found as putative CASC15 co-expressing genes. CASC15 positively regulated TWIST1 gene expression as well as protein level of Sox4 and Versican in HCC cells. Positive correlation in expression between CASC15 and TWIST1 mRNA was verified in 42 pairs of HCC pathologic and adjacent tissue specimens. CASC15 upregulated TWIST1 gene expression in HCC cells by sponging miR-33a-5p. CASC15 promoted EMT in HCC cells by increasing N-cadherin and Vimentin protein level while decreasing that of E-cadherin through TWIST1. CASC15 facilitated HCC cell proliferation, migration and invasion while reducing cell apoptosis through TWIST1. CASC15 facilitated the tumorigenicity of HCC cells in vivo. a CASC15 could promote EMT and facilitate malignancy of HCC cells by increasing TWIST1 gene expression via miR-33a-5p sponging.

Section snippets

Introductions

As the most frequently diagnosed primary liver cancer, hepatocellular carcinoma (HCC) is currently the third leading cause of cancer-related death (Forner et al., 2018; Llovet et al., 2016). Although the molecular mechanisms underlying HCC pathogenesis and progression are not fully under stood, non-coding RNAs such as microRNAs (miRNAs) and long noncoding RNAs (lncRNA) have been recognized as important regulators involved in these processes (Klingenberg et al., 2017; Wong et al., 2018).

LncRNA

Bioinformatic analysis

Transcriptome sequencing data in the Cancer Genome Atlas (TCGA) Liver Hepatocellular Carcinoma dataset was accessed through www.cbioportal.org/study?id=lihc_tcga#summary (Cerami et al., 2012). Data of 429 HCC samples were subject to the analysis. Expression of CASC15 was found positively correlated with that of a total of 477 genes with Spearman's rho >0.45, among which 455 were annotated in FunRich database. These putative CASC15 co-expressing genes in HCC were further analyzed by Gene

Bioinformatic analysis on putative CASC15 co-expressing genes in HCC

LncRNAs have been demonstrated to regulate tumor development through various of mechanisms, including functioning as so-called “competitive endogenous RNAs (ceRNAs)” that interact with miRNAs and inhibit their gene-silencing functions, thus liberating the expression of miRNA target genes. We first explored genes whose expression positively correlated with that of CASC15 in TCGA liver hepatocellular carcinoma data using Cbioportal online platform. Top 15 genes with highest or lowest Spearman's

Discussions

In the present research, we investigated the molecular mechanism underlying the tumor-promoting role of CASC15 in HCC. We first explored potential CASC15 co-expressing genes in TCGA Liver Hepatocellular Carcinoma dataset to see if CASC15 expression was positively correlated with that of any oncogene or tumor promoting gene in HCC. Sox4 and Versican were two of the 10 genes with highest Spearman's rho we found. Although we later chose TWIST1 for functional study, we propose that the possibility

Author agreement

All the author agree and approve the final manuscript.

Conflicts of interest

The co-authors declare that there are no financial and/or non-financial conflicts of interest existing in this research.

Funding

None.

Acknowledgements

Not applicable.

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