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DDX3 Represses Stemness by Epigenetically Modulating Tumor-suppressive miRNAs in Hepatocellular Carcinoma.

ABSTRACT: Studies indicate that the presence of cancer stem cells (CSCs) is responsible for poor prognosis of hepatocellular carcinoma (HCC) patients. In this study, the functional role of DDX3 in regulation of hepatic CSCs was investigated. Our results demonstrated that reduced DDX3 expression was not only inversely associated with tumor grade, but also predicted poor prognosis of HCC patients. Knockdown of DDX3 in HCC cell line HepG2 induced stemness gene signature followed by occurrence of self-renewal, chemoreisistance, EMT, migration as well as CSC expansion, and most importantly, DDX3 knockdown promotes tumorigenesis. Moreover, we found positive correlations between DDX3 level and expressions of tumor-suppressive miR-200b, miR-200c, miR-122 and miR-145, but not miR-10b and miR-519a, implying their involvement in DDX3 knockdown-induced CSC phenotypes. In addition, DDX3 reduction promoted up-regulation of DNA methyltransferase 3A (DNMT3A), while neither DNMT3B nor DNMT1 expression was affected. Enriched DNMT3A binding along with hypermethylation on promoters of these tumor-suppressive miRNAs reflected their transcriptional repressions in DDX3-knockdown cells. Furthermore, individual restoration of these tumor-suppressive miRNAs represses DDX3 knockdown-induced CSC phenotypes. In conclusion, our study suggested that DDX3 prevents generation of CSCs through epigenetically regulating a subset of tumor-suppressive miRNAs expressions, which strengthens tumor suppressor role of DDX3 in HCC.

SUBMITTER: Li HK

PROVIDER: S-EPMC4921922 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Long non-coding RNAs (lncRNAs) play important regulatory roles in hepatocellular carcinoma (HCC). However, the function of LOC107985656 in HCC progression remains unclear. The lncRNA, mRNA and miRNA levels in HCC tissues or cells were measured using real-time quantitative polymerase chain reaction (RT-qPCR). The proliferation of cancer cells was evaluated using 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) viability and colony formation assays. Bioinformatics prediction, dual luciferase assay and RNA pull-down assay were performed to analyze the relationships between LOC107985656 and miR-106b-5p, or miR-106b-5p and large tumor suppressor 1 (LATS1). The protein expression levels were detected using Western blot. Results showed that LncRNA LOC107985656 was downregulated in HCC tissues and cells. Upregulation of LOC107985656 inhibited the proliferation of HCC cells, whereas its knockdown promoted this phenomenon. LOC107985656 could activate the tumor-suppressive Hippo pathway by repressing yes association protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1, also known as TAZ) (two homologs of Yki) protein expression in HCC. Further investigation suggested that LOC107985656 regulated the expression of LATS1 by acting as a sponge for absorbing miR-106b-5p in HCC cells. In conclusion, this study unraveled the role of LOC107985656 following a ceRNA (competing endogenous RNAs) mechanism for the miR-106b-5p/LATS1 axis in HCC. The results indicate potential diagnostic and therapeutic applications of LOC107985656 in HCC.Abbreviations:HCC: hepatocellular carcinoma; LncRNA: long non-coding RNA; LATS1: large tumor suppressor 1; MTT: 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; YAP: yes association protein; WWTR1: WW domain-containing transcription regulator protein 1; cDNA: single-stranded complementary DNA; RT-qPCR: real-time quantitative polymerase chain reaction; Radio-Immunoprecipitation Assay (RIPA); BCA: bicinchoninic acid; ASO: antisense oligonucleotide; MST1/2: Ste20-like kinases 1/2; TEAD: TEA domain transcription factor; ceRNA: competing endogenous RNAs.

Project description:BackgroundChronic alcohol consumption is more frequently associated with advanced, aggressive hepatocellular carcinoma (HCC) tumors. Alcohol adversely impacts ER/Golgi membrane trafficking and Golgi protein N-glycosylation in hepatocytes; these effects have been attributed (in part) to dysregulated adenosine diphosphate-ribosylation factor (ARF) GTPase signaling. Here, we investigated the role of the ARF GTPase guanine exchange factor PSD4 in HCC progression.MethodsR-based bioinformatics analysis was performed on publicly available array data. Modulating gene expression was accomplished via lentiviral vectors. Gene expression was analyzed using quantitative real-time PCR and immunoblotting. PSD4 promoter methylation was assessed using quantitative methylation-specific PCR. Phospho-p65(S276)/DNMT1 binding to the PSD4 promoter was analyzed via chromatin immunoprecipitation. We constructed ethanol/DEN-induced and DEN only-induced transgenic murine models of HCC.ResultsWe identified PSD4 as a hypermethylated, suppressed gene in alcohol-related HCC tumors; however, PSD4 was not dysregulated in all-cause HCC tumors. Certain HCC cell lines also displayed varying degrees of PSD4 downregulation. PSD4 overexpression or knockdown decreased and increased cell migration and invasiveness, respectively. Mechanistically, PSD4 transcription was repressed by TNF-α-induced phospho-p65(S276)'s recruitment of DNA methyltransferase 1 (DNMT1), resulting in PSD4 promoter methylation. PSD4 inhibited pro-EMT CDC42 activity, resulting in downregulation of E-cadherin and upregulation of N-cadherin and vimentin. Hepatocyte-specific PSD4 overexpression reduced ethanol/DEN-induced HCC tumor progression and EMT marker expression in vivo.ConclusionsPSD4 is a hypermethylated, suppressed gene in alcohol-related HCC tumors that negatively modulated pro-EMT CDC42 activity. Furthermore, we present a novel phospho-NF-κB p65(S276)/DNMT1-mediated promoter methylation mechanism by which TNF-α/NF-κB signaling represses PSD4 transcription in HCC cells.

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DDX3 Represses Stemness by Epigenetically Modulating Tumor-suppressive miRNAs in Hepatocellular Carcinoma.

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