LncRNA-ATB promotes autophagy by activating Yes-associated protein and inducing autophagy-related protein 5 expression in hepatocellular carcinoma

Figure 1 LncRNA-ATB is significantly up-regulated in human hepatocellular carcinoma tissues and predicts a poor prognosis. A: Relative expression levels of lncRNA-activated by transforming growth factor beta (lncRNA-ATB) in hepatocellular carcinoma (HCC) tissues and adjacent normal tissues; B: Relative expression levels of lncRNA-ATB in HCC patients with larger tumor sizes (> 5 cm) and smaller tumor sizes (≤ 5 cm); C: Relative expression levels of lncRNA-ATB in HCC patients with early stage (I/II) and advanced stage (III/IV) disease; D: Kaplan-Meier analysis of overall survival in 72 HCC patients according to median lncRNA-ATB expression. The log-rank test was used to calculate the P-value. Data are expressed as the mean ± SD of three independent experiments. Statistical significance is indicated at ^aP < 0.05. HCC: Hepatocellular carcinoma; LncRNA-ATB: LncRNA-activated by transforming growth factor beta.

Figure 2 Overexpression of lncRNA-ATB promotes the proliferation of hepatocellular carcinoma cells in vitro. A: LncRNA-activated by transforming growth factor beta (lncRNA-ATB) expression was determined by real-time PCR in hepatocellular carcinoma (HCC) cells transfected with pcDNA3.1-lncRNA-ATB; B: CCK-8 assays were performed to determine the proliferation of HCC cell lines (SMMC-7721 and HepG2) transfected with pcDNA3.1-lncRNA-ATB; C: Colony formation assays were performed to measure the proliferation of HCC cells transfected pcDNA3.1-lncRNA-ATB. Data are expressed as the mean ± SD of three independent experiments. Statistical significance is indicated at ^aP < 0.05 or ^bP < 0.01. HCC: Hepatocellular carcinoma; LncRNA-ATB: LncRNA-activated by transforming growth factor beta.

Figure 3 LncRNA-ATB promotes autophagy in hepatocellular carcinoma cells. A and B: Hepatocellular carcinoma (HCC) cells transfected with pcDNA3.1-lncRNA-ATB were subjected to Western blot analysis to detect LC3-II/LC3-I expression, with and without bafilomycin A1 treatment (20 μmol/L for 6 h); C and D: Immunofluorescence of LC3 puncta (green) was measured in HCC cells transfected with pcDNA3.1-lncRNA-ATB or treated with rapamycin; E and F: Electron microscopy identified highly visible autophagosomes and autolysosomes in HCC cells overexpressing lncRNA-ATB compared to negative control cells. HCC: Hepatocellular carcinoma; LncRNA-ATB: LncRNA-activated by transforming growth factor beta.

Figure 4 LncRNA-ATB promotes autophagy through modulation of Yes-associated protein activation. A and B: The protein levels of total Yes-associated protein (YAP) and p-YAP in hepatocellular carcinoma (HCC) cells transfected with pcDNA3.1-lncRNA-ATB were measured by Western blot; C and D: The nuclear localization of YAP was detected in transfected HCC cells by immunofluorescence; E and F: Knockdown of YAP in HCC cells transfected with si-YAP (si-YAP 1# and si-YAP 2#) was measured using Western blot; G and H: HCC cells were co-transfected with pcDNA3.1-lncRNA-ATB and si-YAP, and the protein levels of LC3 were determined by Western blot. Yes-associated protein: YAP; HCC: Hepatocellular carcinoma; LncRNA-ATB: LncRNA-activated by transforming growth factor beta.

Figure 5 LncRNA-ATB induces autophagy by regulating autophagy-related protein 5 expression. A and B: The mRNA expression of autophagy-related genes involved in autophagy was examined by real-time PCR in hepatocellular carcinoma (HCC) cells transfected with lncRNA-ATB; C and D: The protein levels of ATG5 were analyzed in HCC cells transfected with lncRNA-ATB by Western blot; E and F: RIP-derived RNA was evaluated by real-time PCR. Data are expressed as the mean ± SD of three independent experiments. Statistical significance is indicated at ^aP < 0.05 or ^bP < 0.01. ATG3: Autophagy-related protein 3; ATG5: Autophagy-related protein 5; ATG7: Autophagy-related protein 7; ATG10: Autophagy-related protein 10; ATG12: Autophagy-related protein 12; ATG16L: Autophagy-related protein 16L; HCC: Hepatocellular carcinoma; RIP: RNA immunoprecipitation; LncRNA-ATB: LncRNA-activated by transforming growth factor beta.

Figure 6 Yes-associated protein nuclear localization and autophagy-related protein 5 expression are positively correlated with lncRNA-ATB expression. A: Representative immunostaining for Yes-associated protein (YAP) in hepatocellular carcinoma (HCC) and adjacent normal tissues (magnification: ×200 or ×400); B: The expression levels of autophagy-related protein 5 (ATG5) in 72 pairs of HCC tumor and adjacent normal tissues were evaluated by real-time PCR; C: Pearson’s correlation analysis of the relationship between expression of lncRNA-ATB and ATG5 (r = 0.527, P < 0.001). Statistical significance is indicated at ^bP < 0.01. YAP: Yes-associated protein; ATG5: Autophagy-related protein 5; HCC: Hepatocellular carcinoma; LncRNA-ATB: LncRNA-activated by transforming growth factor beta.