Long noncoding RNA HULC promotes hepatocellular carcinoma progression

Figures

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  Figure 1. HULC is overexpressed in HCC. (A–C) HULC is upregulated in HCC tissues compared with adjacent non-tumor liver control tissues in two GEO data sets (GSE39791 and GSE76427) and a TCGA cohort. HULC expression in (D) HCC tissues (n=42) and (E) HCC cells analyzed by qPCR. (F) The overall survival of HCC patients from the TCGA cohort with high and low HULC expression. *P < 0.05, **P < 0.01.

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  Figure 2. HULC promotes proliferation, migration, and invasion of HCC cells in vitro. (A–D) Viability of HCC cells transfected with siHULC or pcDNA-HULC as measured by CCK8 assays. (E–H) Transwell migration and invasion assays in cells with HULC silencing or overexpression. *P < 0.05, **P < 0.01.

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  Figure 3. HULC is a ceRNA and acts as a sponge for miR-2052 in HCC cells. (A) The predicted binding sites of HULC and miR-2052. (B, C) Quantitative PCR analysis of miR-2052 after mimic and inhibitor transfection. (D, E) Relative luciferase activities of HULC-WT and HULC-MUT reporter measured in presence of miR-2052 mimic cotransfection. (F, G) qPCR analysis of miR-2052 expression in HCC cells transfected with siHULC or siNC, and Vec or pcDNA-HULC. (H, I) RIP assay was used to explore the enrichment of miR-2052 by HULC. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 4. miR-2052 inhibits the proliferation, migration, and invasion of HCC cells in vitro and in vivo. (A) miR-2052 expression in HCC tissues analyzed by qPCR (n=42). (B) Correlation between HULC and miR-2052 expression in paired HCC tissues (n=42). (C–E) Tumor volume and weight measured in tissues from nude mice injected with miR-2052 stable HLF cells. (F) Ki67 levels measured by immunohistochemistry. (G, H) The expression of miR-2052 and HULC in xenograft tumors measured by qPCR. *P < 0.05, ***P < 0.001.

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  Figure 5. MET is a direct target of miR-2052. (A) Schematic view of miR-2052 putative binding site in the WT and MUT 3′ UTR of MET. (B) Luciferase activity assays in HCC cells transfected with WT and MUT 3′ UTR of MET luciferase reporter plasmids with miR-2052 mimics. (C, D) Relative mRNA and protein levels of MET in HLF and 97H cells after miR-2052 mimics and inhibitors transfection. (E) MET protein levels in HLF and 97H cells after HULC knockdown or overexpression. (F) MET protein levels in HCC cells after HULC knockdown with or without miR-2052 inhibition. (G) MET protein levels in HCC tissues (n=42). *P < 0.05, **P < 0.01.

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  Figure 6. HULC promotes HCC progression through the miR-2052/MET axis in vitro. (A–D) CCK8 assays for HLF and 97H cells cotransfected with siHULC and miR-2052 mimic or inhibitor or MET. (E–H) Transwell assays for HLF and 97H cells cotransfected with siHULC and miR-2052 mimic or inhibitor or MET. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 7. HULC promotes HCC growth through the miR-2052/MET axis in vivo. (A–C) Volume and weight measurements for tumors from nude mice injected with HLF cells stably overexpressing HULC. (D–F) Expression of HULC, miR-2052, and MET in tumor xenografts measured by qPCR. (G) Ki67 and MET levels measured by immunohistochemistry. (H) MET levels measured by western blot and gray level analysis. *P < 0.05, **P < 0.01, ***P < 0.001.