Long non-coding RNA00364 represses hepatocellular carcinoma cell proliferation via modulating p-STAT3-IFIT2 signaling axis

Figures

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  Figure 1. Interferon-γ stimulated leads to LncRNA00364 upregulation in HCC (A) Heat map showing all the upregulated genes including LncRNA00364 induced by interferon –γ treatment in HepG2 cells based on microarray analysis. (B, C) Relative LncRNA00364 expression in 57 paired human HCC tissues and para-tumor tissues by qRT-PCR analysis, **p<0.01. (D) LncRNA00364 mRNA expression in HCC cell lines including HepG2, PLC, Hep3B, 7721 and LM3 by using qRT-PCR. (E) LncRNA00364 mRNA expression in HCC cell lines treated with mock control or IFN-γ. **p<0.01. Results were triplicate experiments with mean ± SD. *P<0.05, **P<0.01.

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  Figure 2. LncRNA00364 inhibits proliferation and promotes apoptosis of HCC cells in vitro and in vivo (A) The expression of LncRNA00364 in HepG2 and LM3 cell lines, **p<0.01. (B) Cell proliferation detected by CCK-8 assay in each line as indicated, **p<0.01. (C) Cell apoptosis detected by Annexin-V-FITC assay in each cell line as indicated, **p<0.01. (D) Cell cycle detected by PI staining assay in each cell line as indicated. (E) RNA FISH analysis of LncRNA00364 localization in HepG2 cells. (F) The average volume of tumors between the LncRNA00364 overexpression and control groups from six independent experiments, **p<0.01 (n=6) compared with vector group. (G) Expression of LncRNA00364 was analyzed in the tumor xenografts tissues from mice by qRT-PCR, *p<0.01. (H) The apoptosis expression of the tumor xenografts tissues from mice was detected by TUNEL assay, **p<0.01. Results were triplicate experiments with mean ± SD. *P<0.05, **P<0.01.

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  Figure 3. LncRNA00364 suppresses STAT3 phosphorylation on Tyr705 (A, B) The protein level of STAT3, pSTAT3 (Tyr705), STAT1, pSTAT1 (Lys310), AKT and pAKT with LncRNA00364 overexpression or deficiency were analyzed by western blot assays. (C) Diagram showing the conserved STAT3 transcriptional response element of LncRNA00364. (D) RNA pull-down analysis to determine the interaction of phosphorylated STAT3 with mutated LncRNA00364 in HepG2 cells. (E) The distribution of pSTAT3 and STAT3 in cytoplasm and nucleus in HCC cells transfected with LncRNA00364 overexpression or vector by nucleocytoplasmic separation.

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  Figure 4. LncRNA00364 increases apoptosis through upregulation of IFIT2 in vitro and in vivo (A) The protein levels of IFIT2, ISG15 and OAS1 with LncRNA00364 overexpression analyzed by western blot assays. (B) The p-STAT3 and IFIT2 protein level with LncRNA00364 overexpression or STAT3 inhibitor Stattic analyzed by western blot assays. (C) The mRNA levels of IFIT2, ISG15 and OAS1 with ectopic expression analyzed by Real-time PCR assays. (D) Luciferase reporter assay (RLA) in 293T cells co-transfected with the different length of IFIT2 promoter reporter gene, STAT3 and Renilla 24 h, treated with interferon-γ for 12 h. RLA is shown as the mean of three independent experiments ± SD. *P <0.05, IFIT2 1.0Kb and 1.5Kb group compared with pGL3-basic group. (E) Luciferase reporter assay (RLA) in 293T cells co-transfected with the IFIT2 promoter reporter gene, mutation of STAT3 or WT, and Renilla 24 h, treated with interferon-γ for 12 h. RLA is shown as the mean of three independent experiments ± SD. *P <0.05, WT group compared with T705F group. (F) IFIT2 expression in datasets of Kaiser, Hong and Gaedcke from the Oncomine database. **p<0.01. (G) Expression of IFIT2 in 57 paired human HCC tissues…

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  Figure 5. LncRNA00364 positively correlates with IFIT2 and its lower expression predicts worse patient prognosis in HCC (A) Significantly positive correlation between LncRNA00364 and IFIT2 in HCC samples. (**p<0.01, n=57). (B, C) The correlation between LncRNA00364 and OS, TTR in HCC samples. (OS *p=0.015, n=157; TTR *p=0.017, n=157).

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  Figure 6. Working model shows that LncRNA00364 may contribute to the development of HCC