LINC01419 facilitates hepatocellular carcinoma growth and metastasis through targeting EZH2-regulated RECK

Abstract

Long non-coding RNAs (lncRNAs) have been reported to play significant roles in human tumorigenesis, for example, in hepatocellular carcinoma (HCC). This study explored the role of LINC01419, a new lncRNA, in HCC. In vitro study revealed that LINC01419 promotes growth and migration of HCC cells. Genes that affected cell proliferation and cell migration were identified using RNA-sequence. Subsequently, it was confirmed that LINC01419 binds to EZH2, leading to histone methylation of the RECK promoter. Interaction between LINC01419 and FUS stabilized EZH2 mRNA thereby enhancing EZH2 expression. Conclusively, the results of this study confirm that LINC01419 may serve as a potential target for HCC diagnosis and treatment.

Keywords: EZH2; LINC01419; RECK; hepatocellular carcinoma.

Figure 1

LINC01419 is highly expressed and associated with malignant phenotypes in HCC. (A) The expression level of LINC01419 in HCC tissues and normal liver tissues in the TCGA cohort has been indicated. (B) Showing relative LINC01419 expression in HCC cell lines and normal human liver cell line LO2. (C) HCC patients with high LINC01419expression level exhibited a low PFS rate than those with low LINC01419expression level. (D) HCC patients with high LINC01419expression levels had a low OS rate than those with low LINC01419expression levels as conformed using Kaplan-Meier assay.

Figure 2

Inhibiting LINC01419 decreases HCC cell proliferation and invasion. (A) Cell viability examination using MTT assay. (B) Showing impaired colony-forming ability in LINC01419-silenced cells. (C) Flow cytometry assay used to examine cell cycle distribution. (D) Examining HCC cell migration ability using transwell assay. (E) Protein levels of E-cadherin, N-cadherin, and Vimentin examination by western blot assay.

Figure 3

LINC01419 silences RECK epigenetically by binding to EZH2. (A) The different gene transcripts expression between si-ctrl cells and si- LINC01419 cells, demonstrated by hierarchical cluster. (B) The promoter regions of RECK showing EZH2 transcriptional sites, as indicated by UCSC. (C) LINC01419 interaction with EZH2, as revealed by the RIP experiments. (D) Desthiobiotinylation-LINC01419 bound EZH2 in HCC cells, as indicated by the pull-down assays.(E)Shows EZH2 down-regulation by si-RNA in HCC cells, and the knockdown efficiency examination using western blot assay. (F) qPCR assay examination of the mRNA expression level of RECK. (G) The western blot analysis of the RECK protein expression level. (H) Showing EZH2 and H3K27me3 enriched in the RECK promoter regions as indicated by CHIP assay. (I) Sowing increased EZH2 and H3K27me3levelsafter LINC01419 overexpression in HCC cell.

Figure 4

LINC01419 stabilizes EZH2 mRNA by recruiting FUS. (A, B) Showing RT-PCR and western blot assays used to examine EZH2 expression levels in HCC cells when LINC01419 was inhibited or overexpressed, respectively. (C) Luciferase reporter assay showing that LINC01419 did not affect EZH2 transcription. (D) FUS interaction with LINC01419 and EZH2-mRNA as validated by the RIP assay. (E) The estimated impact of LINC01419 down-regulation on FUS interaction with EZH2-mRNA using RIP assay. (F) The estimated impact of LINC01419 overexpression on FUS–interaction with EZH2 -mRNA using RIP assay. (G–H) A qRT-PCR assay used to examine the EZH2 expression level. (I) The degradation rate of EZH2-mRNA after treatment with actinomycin D. (J) Right panel: Correlation between LINC01419 expression level and EZH2 expression level examined by RT-PCR; Left panel: Correlation between RECK expression level and EZH2 expression level examined by RT-PCR.

Figure 5

RECK suppresses HCC cell proliferation and metastasis, and counteracts LINC01419 activity. (A) RT-PCR assay to examine the RECK expression level. (B) The mRNA level of RECK as examined by qRT-PCR. (C) The RECK protein level as examined by western blot assay. (D–F) Analysis of cells by MTT assays (D), colony formation (E), and transwell assays (F) (G) Western blot assay was performed to examine E-cadherin, N-cadherin and Vimentin expression levels.

Figure 6

c-jun elevated LINC01419 expression in HCC. (A) Indicates the promoter regions of LINC01419 with the putative c-jun TFBS. (B) RT-PCR assay used to examine LINC01419 expression levels. (C) Elevated luciferase activity in wild-type LINC01419 promoter caused by c-jun.