Long noncoding RNA NEAT1 suppresses hepatocyte proliferation in fulminant hepatic failure through increased recruitment of EZH2 to the LATS2 promoter region and promotion of H3K27me3 methylation

Fig. 1. Apoptosis and lncRNA NEAT1 expression increased in D-GalN/LPS-treated cells, whereas LATS2 expression decreased. ALT, AST, and LDH expression patterns in HL-7702 cells with or without D-GalN/LPS treatment (a–c). The proliferation of HL-7702 cells was determined by EdU assay (×200) (d). The apoptosis of HL-7702 cells was determined by TUNEL staining (×200) (e). The lncRNA NEAT1 expression patterns in HL-7702 cells determined by RT-qPCR (f). The mRNA expression patterns of LATS2 in HL-7702 cells determined by RT-qPCR (g). Control, HL-7702 cells without treatment; D-GalN/LPS, HL-7702 cells treated with D-GalN/LPS; *p < 0.05 vs. control. Statistical data are described as mean ± standard deviation. Independent sample t test was used for comparisons between two groups. The experiment was repeated three times independently. RT-qPCR reverse transcription quantitative polymerase chain reaction, lncRNA NEAT1 long noncoding RNA nuclear-enriched abundant transcript 1, LATS2 large tumor-suppressor kinase 2, D-GalN/LPS D-galactosamine/lipopolysaccharide, AST aspartate aminotransferase, ALT alanine aminotransferase, LDH lactate dehydrogenase, ELISA enzyme-linked immunosorbent assay, EdU…

Fig. 2. Knockdown of lncRNA NEAT1 inhibits apoptosis and induces cell proliferation by modulating LATS2. The expression patterns of lncRNA NEAT1 and LATS2 in cells treated with oe-NEAT1, oe-LATS2, sh-NEAT1, or sh-LATS2 evaluated by RT-qPCR (a). The protein expression patterns of LATS2 in cells treated with oe-NEAT1, oe-LATS2, sh-NEAT1, or sh-LATS2 evaluated by western blot analysis (b). Quantification of protein expression from b (c). *p < 0.05 vs. the oe-NC group; #p < 0.05 vs. the sh-NC group. The proliferation of HL-7702 cells treated with sh-NEAT1, sh-LATS2, oe-NEAT1, and/or oe-LATS2 measured by the EdU assay (×200) (d). The apoptosis of HL-7702 cells treated with sh-NEAT1, sh-LATS2, oe-NEAT1, and/or oe-LATS2 measured by TUNEL staining (×200) (e). *p < 0.05 vs. the oe-NC group; #p < 0.05 vs. the oe-LATS2 group; &p < 0.05 vs. the sh-NC group. oe-NC group, HL-7702 cells treated with oe-NC; oe-NEAT1 group, HL-7702 cells treated with oe-NEAT1; oe-LATS2 group, HL-7702 cells treated with oe-LATS2; sh-NEAT1 group, HL-7702 cells treated with sh-NEAT1; sh-LATS2 group, HL-7702 cells treated with sh-LATS2; oe-NEAT1+oe-LATS2 group, HL-7702 cells treated with both oe-NEAT1 and oe-LATS2.…

Fig. 3. Downregulation of lncRNA NEAT1 restores LATS2 expression in FHF rats. ALT, AST, and LDH expression patterns in the FHF rat model measured by ELISA (a–c). Liver pathology in FHF rats tested by HE staining (d). The lncRNA NEAT1 and LATS2 expression patterns in liver tissues of FHF rats evaluated by RT-qPCR (e). Immunohistochemical staining of LATS2 expression in liver tissues of FHF rats (×400) (f). Quantification of the results of immunohistochemical staining (g). *p < 0.05 vs. the normal group; #p < 0.05 vs. the FHF+oe-NC group; &p < 0.05 vs. the FHF+sh-NC group. Normal group, normal rats without injection; PBS group, normal rats injected with PBS; FHF group, FHF rats induced by D-GalN/LPS injection; FHF+oe-NC group, FHF rats treated with oe-NC; FHF+oe-LATS2 group, FHF rats treated with oe-LATS2; FHF+sh-NC group, FHF rats treated with sh-NC; FHF+sh-NEAT1 group, FHF rats treated with sh-NEAT1. Statistical data are described as mean ± standard deviation. One-way analysis of variance was used for comparisons among multiple groups, followed by Tukey’s post hoc test. n = 6. RT-qPCR reverse transcription quantitative polymerase chain reaction, lncRNA NEAT1 long noncoding RNA…

Fig. 4. Downregulation of lncRNA NEAT1 or overexpression of LATS2 inhibits the progression of FHF in vivo. The protein expression patterns of YAP1 in the nucleus/cytoplasm of rat liver determined by western blot analysis (a, b). Immunohistochemical staining of Ki67 expression in the livers of FHF rats (×400) (c, d). Apoptosis in the livers of FHF rats observed by TUNEL staining (×400) (e, f). The pathological changes in the livers of FHF rats detected by HE staining (×400) (g). *p < 0.05 vs. the FHF+oe-NC group; #p < 0.05 vs. the FHF+sh-NC group. FHF group, FHF rats induced by D-GalN/LPS injection; FHF+oe-NC group, FHF rats treated with oe-NC; FHF+oe-LATS2 group, FHF rats treated with oe-LATS2; FHF+sh-NC group, FHF rats treated with sh-NC; FHF+sh-NEAT1 group, FHF rats treated with sh-NEAT1. Statistical data are described as mean ± standard deviation. One-way analysis of variance was used for comparisons among multiple groups, followed by Tukey’s post hoc test. n = 6 in each group. D-GalN/LPS D-galactosamine/lipopolysaccharide, lncRNA NEAT1 long noncoding RNA nuclear-enriched abundant transcript 1, LATS2 large tumor-suppressor kinase 2, FHF fulminant hepatic failure, TUNEL terminal…

Fig. 5. LncRNA NEAT1 recruits EZH2 to the LATS2 promoter and represses LATS2 expression. The interaction between lncRNA NEAT1 and EZH2 was verified by RIP assay. *p < 0.05 vs. the IgG group (a). The enrichment of EZH2 and H3K27me3 on the LATS2 promoter region determined by ChIP (b). The protein expression patterns of EZH2, H3K27me3, and LATS2 in response to overexpression or knockdown of EZH2 measured by western blot analysis (c). Quantification of protein expression in c (d). Control group, HL-7702 cells without treatment; oe-NC group, HL-7702 cells treated with oe-NC; sh-NC group, HL-7702 cells treated with sh-NC; oe-EZH2 group, HL-7702 cells treated with oe-EZH2; sh-EZH2 group, HL-7702 cells treated with sh-EZH2. Statistical data are described as mean ± standard deviation. One-way analysis of variance was used for comparisons among multiple groups, followed by Tukey’s post hoc test. The experiment was repeated three times independently. lncRNA NEAT1 long noncoding RNA nuclear-enriched abundant transcript 1, LATS2 large tumor-suppressor kinase 2, EZH2 enhancer of zeste homolog 2, NC negative control, RIP RNA immunoprecipitation, ChIP chromatin immunoprecipitation.

Fig. 6. LncRNA NEAT1/EZH2/LATS2 activates the Hippo/YAP signaling pathway. The protein expression patterns of YAP1 in HL-7702 cells treated with D-GalN/LPS quantified by western blot analysis (a). Quantification of protein expression in a (b). The binding relationship between YAP1 and P73, assessed by coIP (c). *p < 0.05 vs. the D-GalN/LPS+oe-NC group; #p < 0.05 vs. the D-GalN/LPS+sh-NC group. D-GalN/LPS+oe-NC group, HL-7702 cells treated with oe-NC in the presence of D-GalN/LPS treatment; D-GalN/LPS+sh-NC group, HL-7702 cells treated with sh-NC in the presence of D-GalN/LPS treatment; D-GalN/LPS+oe-NEAT1 group, HL-7702 cells treated with oe-NEAT1 in the presence of D-GalN/LPS treatment; D-GalN/LPS+oe-LATS2 group, HL-7702 cells treated with oe-LATS2 in the presence of D-GalN/LPS treatment; D-GalN/LPS+sh-NEAT1 group, HL-7702 cells treated with sh-NEAT1 in the presence of D-GalN/LPS treatment; D-GalN/LPS+sh-LATS2 group, HL-7702 cells treated with sh-LATS2 in the presence of D-GalN/LPS treatment. Statistical data are described as mean ± standard deviation. One-way analysis of variance was used for comparisons among multiple groups, followed by Tukey’s post hoc test. The experiment was…

Fig. 7. Proposed underlying mechanism of action of lncRNA NEAT1 in the apoptosis and proliferation of hepatocytes in FHF. In FHF hepatocytes, lncRNA NEAT1 recruits EZH2 to the promoter region of LATS2 and induces the production of H3K27me3, thus repressing the expression of LATS2. Upon inhibition of the LATS2 expression, YAP1 could not be activated and enter the nucleus, which results in binding to P73 and induces the apoptosis mediated by P73.