Long non-coding RNA highly up-regulated in liver cancer promotes exosome secretion

Figure 1 Identification of the exosomes. A: Transmission electronc microscopy (TEM) of exosomes extracted from the serum of patients with hepatocellular carcinoma (HCC); B: TEM of exosomes isolated from HCC cell medium; C: Western blot analysis of exosomal markers (CD63 and TSG101) in HCC serum; D: Western blot analysis of exosomal markers (CD63 and TSG101) in HCC cell medium; E: Nanoparticle tracking analysis (NTA) for detection of exosomes extracted from the HCC serum; F: NTA detection of exosomes extracted from HCC cell medium.

Figure 2 Detection of long non-coding RNA highly up-regulated in liver cancer in hepatocellular carcinoma. A: Expression of HULC in the serum exosomes of patients with HCC; B: Expression of HULC in liver cancer tissues; C: Correlation between HULC expression in HCC serum exosomes and tissue. Error bars stand for the mean ± SD of at least triplicate experiments. HULC: Highly upregulated in liver cancer; HCC: Hepatocellular carcinoma.

Figure 3 Detection of long non-coding RNA highly up-regulated in liver cancer in hepatocellular carcinoma cells. A:Expression of HULC in HepG2, SMMC7721, and LO2 cells; B: Expression of HULC in HepG2 transfected with HULC inhibitors or plasmids causing HULC overexpression; C: Expression of HULC in SMMC7721 transfected with HULC inhibitors or plasmids causing HULC overexpression. Error bars stand for the mean ± SD of at least triplicate experiments. ^aP < 0.05 vs LO2 group; ^bP < 0.05 vs LO2 group; ^cP < 0.01 vs NC group; ^dP < 0.01 vs HULC-NC group; ^eP < 0.05 vs NC group; ^fP < 0.05 vs HULC-siRNA-NC group. HULC: Highly upregulated in liver cancer; NC: Normal control group; HULC: HULC over-expression plasmid group; HULC-NC: HULC over-expression normal control plasmid group; HULC-siRNA: HULC low-expression plasmid group; HULC-siRNA-NC: HULC low-expression control plasmid group.

Figure 4 Long non-coding RNA highly up-regulated in liver cancer induces hepatocellular carcinoma cell proliferation and invasion and inhibits apoptosis in vitro. A: Proliferation of hepatocellular carcinoma (HCC) cells transfected with HULC inhibitors or plasmids causing HULC overexpression; B: Apoptotic pictures of HCC cells (×100). The blue fluorescence is DAPI nuclear staining and green fluorescence is the apoptotic cell. The index of apoptosis is the number of apoptotic cells/total number of visual field × 100%; C: Column chart of index of apoptosis in different groups; D: Effect of HULC on invasion of HCC cells evaluated bTranswell assay (×100); E: Column chart of number of invasive HCC cells in different groups. Error bars stand for the mean ± SD of at least triplicate experiments. ^aP < 0.05 vs HULC-NC 24 h group; ^bP < 0.05 vs HULC-NC 36 h group; ^cP < 0.05 vs HULC-NC 48 h group; ^dP < 0.05 vs HULC-NC 72 h group; ^eP < 0.05 vs NC group; ^fP < 0.05 vs HULC-NC group; ^gP < 0.01 vs NC group; ^hP < 0.01 vs HULC-siRNA-NC group; ⁱP < 0.05 vs NC group; ^jP < 0.05 vs HULC-NC group; ^kP < 0.05 vs NC group; ^lP < 0.05 vs HULC-siRNA-NC group. HULC: Highly upregulated in liver cancer; NC: Normal control group; HULC: HULC over-expression plasmid group; HULC-NC: HULC over-expression normal control plasmid group; HULC-siRNA: HULC low-expression plasmid group; HULC-siRNA-NC: HULC low-expression control plasmid group.

Figure 5 Long non-coding RNA highly up-regulated in liver cancer mediates secretion of exosomes from hepatocellular carcinoma cells via miR-372-3p /Rab11a. A: Sequence of binding sites between HULC and miR-372-3p; B: Dual-luciferase reporter assay for miR-372-3p and HULC; C: Relative expression of miR-372-3p after HCC cells were transfected with HULC inhibitors or plasmids causing HULC overexpression; D: Sequence of binding sites with Rab11a and miR-372-3p; E: Dual-luciferase reporter assay for miR-372-3p and Rab11a; F: Relative expression of Rab11a after HCC cells were transfected with miR-372-3p-siRNA or plasmids causing miR-372-3p overexpression. Error bars stand for the mean ± SD of at least triplicate experiments. ^aP < 0.05 vs WT-HULC-NC group; ^bP < 0.05 vs NC group; ^cP < 0.01 vs NC group; ^dP < 0.05 vs WT-Rab11a -NC group; ^eP < 0.05 vs NC group; ^fP < 0.01 vs NC group. HULC: Highly upregulated in liver cancer; NC: Normal control group; HULC: HULC over-expression plasmid group; HULC-siRNA: HULC low-expression plasmid group; miRNA-372-3p: miRNA-372-3p over-expression plasmid group; miR-372-3p-siRNA: miR-372-3p low-expression plasmid group; WT-HULC: HULC wild type luciferase reporter plasmid group; MUT-HULC: HULC mutant luciferase reporter plasmids group; WT-Rab11a: Rab11a wild type luciferase reporter plasmid group; MUT-Rab11a: Rab11a mutant luciferase reporter plasmids group.

Figure 6 Expression of Rab11a in hepatocellular carcinoma cells transfected with highly up-regulated in liver cancer-siRNA and miR-372-3p-siRNA. Silencing HULC inhibited Rab11a expression, while silencing HULC and miR-372-3p increased Rab11a expression in vitro. Error bars stand for the mean ± SD of at least triplicate experiments. ^aP < 0.05 vs NC group; ^bP < 0.05 vs NC group. HULC: Highly upregulated in liver cancer; NC: Normal control group; HULC-siRNA: HULC low-expression plasmid group; miR-372-3p-siRNA: miR-372-3p low-expression plasmid group.

Figure 7 Increased long non-coding RNA highly up-regulated in liver cancer and Rab11a and decreased miR-372-3p promote the secretion of exosomes from hepatocellular carcinoma cells. A: Quantification of exosomes after transfecting HULC over-expression plasmid into HCC cells; B: Quantification of exosomes after transfecting miR-372-3p low-expression plasmid into HCC cells; C: Quantification of exosomes after transfecting Rab11a over-expression plasmid into HCC cells. Error bars stand for the mean ± SD of at least triplicate experiments. ^aP < 0.01 vs NC group; ^bP < 0.01 vs HULC-NC group; ^cP < 0.01 vs NC group; ^dP < 0.01 vs miRNA-372-3p-siRNA-NC group; ^eP < 0.01 vs NC group; ^fP < 0.01 vs Rab11a-NC group. HULC: Highly upregulated in liver cancer; HULC: HULC over-expression group; HULC-NC: HULC (over-expression plasmid normal control group); NC: Normal control; miR-372-3p-siRNA: miR-372-3p low-expression group; miR-372-3p-siRNA-NC: miR-372-3p low-expression plasmid normal control group; Rab11a: Rab11a over-expression group; Rab11a-NC: Rab11a over-expression plasmid normal control group.