Long Noncoding RNA AC007639.1 Promotes the Pathogenesis and Progression of Hepatocellular Carcinoma Through Inhibiting Apoptosis and Stimulating Chemotherapeutic Resistance

Figure 1. LncRNA AC007639.1 is highly expressed in hepatocellular carcinoma tissues and implies a poor prognosis. (A) Heatmap of transcriptome (left) and volcano plot (middle) of different genes, and lncRNA AC007639.1 expressions in TCGA-LIHC cases (right, n=420, https://gdc.cancer.gov/resources-tcga-users/tcga-code-tables/data-levels); (B) Heatmap of transcriptome (left) and volcano plot (middle) of different genes, and lncRNA AC007639.1 expressions in SRP069212 cases (right, n=355); (C, D) Kaplan-Meier survival curves of TCGA-LIHC dataset analyzed by TCGAbiolinks or SRP069212 dataset analyzed by GEPIA, respectively. ****p < 0.001 (Mann-Whitney test).

Figure 2. The role of LncRNA AC007639.1 in liver cancer. (A) Differential gene expressions between the high- and low- expression of lncRNA AC007639.1 groups (TCGA-LIHC). (B) LncRNA AC007639.1 functions analyzed by GSVA. The up-regulated genes in proliferative liver cancer were highly enriched in the AC007639.1 high expression group (p<0.001), while the down-regulated genes in proliferative liver cancer is highly enriched in the AC007639.1 low expression group (p<0.001). (C) GO analysis and (D) KEGG and cluster network analysis using Clusterprofiler Package of R language, screening criteria p < 0.05, correction p < 0.05.

Figure 3. LncRNA AC007639.1 regulates the susceptibility of liver cancer to doxorubicin by inhibiting the p53 pathway, analyzed by GSVA. (A) The effect of lncRNA AC007639.1 on cell cycle (p < 0.001); (B) The effects of lncRNA AC007639.1 on doxorubicin resistance (p < 0.001); (C) The effects of lncRNA AC007639.1 on p53 target genes (p < 0.001).

Figure 4. Knockdown of lncRNA AC007639.1 inhibits the growth of hepatocellular carcinoma cells and promotes apoptosis. (A) qRT-PCR on the relative AC007639.1 expression in HepG2, Hep3B, and SMMC-7721 cells transfected with two lncRNA AC007639.1 or control siRNAs (Lnc-S1, Lnc-S3, or Lnc-NC, respectively); (B) CCK-8 assay showing inhibition of HepG2, Hep3B, and SMMC-7721 cells at different concentrations of doxorubicin after siRNAs transfection; (C) EdU analysis showing cell proliferation after transfection of scrambled shRNA (NC) or shRNA for lncRNA (shRNA-Lnc) (field of view: 200x) in HepG2 or Hep3B cells treated with 2.5 µM doxorubicin for 48 hours; (D) Western blots of p53 in the three cells lines after exposure to different concentrations of doxorubicin. NC: scrambled control siRNA or shRNA. S1, siRNA1; S3, siRNA3. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. Knockdown of lncRNA AC007639.1 inhibits the growth of tumor xenografts in nude mice. (A) The size of HepG2 xenografts using HepG2-LNC-KD or HepG2-LNC-NC cells at 14 days; (B) Weight of xenografts at collection; (C) Xenografts growth monitored in mm3; (D) Relative AC007639.1 and p53 expressions (normalized to β-actin) in xenografts with HepG2-LNC-KD or HepG2-LNC-NC cells; (E) p53 and PCNA immunohistochemistry in xenograft tissues (field of view: 200x). Brown colored cells were positive for P53 or PCNA, respectively. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not statistically significant.

Figure 6. The involvement of lncRNA AC007639.1 in different pathways. (A) Heatmap showing different gene expressions after the knock down of lncRNA AC007639.1 in HepG2 cells using siRNA3; (B) Volcano plot of different gene expressions after the knock down of lncRNA AC007639.1 in HepG2 cell line; (C) Pathways enrichment after the knock down of lncRNA AC007639.1 in HepG2 cell line.

Figure 7. The role of ANGPTL4 in hepatocellular carcinoma cells. (A) The protein expression of ANGPTL4 in HepG2 cells after transfection of NC siRNA or siRNA3 (n=5). Protein (B) or RNA (C) expression of ANGPTL4 in HepG2 xenografts. (D) Knocking down of ANGPTL4 using ANGPTL4-siRNA 1 (AN1) or ANGPTL4-siRNA 2 (AN2) increased the inhibition of Doxorubicin in HepG2, Hep3B, and SMMC-7721 cells. (E) EdU analysis showing cell proliferation after transfection of scrambled shRNA (shRNA-NC) or shRNA-ANGPTL4 (field of view: 200x) in HepG2 or Hep3B cells treated with 2.5 µM doxorubicin for 48 hours. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not statistically significant.