Gamma-aminobutyric acid promotes human hepatocellular carcinoma growth through overexpressed gamma-aminobutyric acid A receptor α3 subunit

Figure 1 RT-PCR confirmed the knockdown effect on GABRA3 expression by Si-1, but not by Si-2, Si-3 and a negative control Si-Mock in HepG2 cells. β-actin was used to quantify RNAs.

Figure 2 Effect of GABRA3-siRNA on the growth of HCC cells. A: MTT assay of HepG2 cells transfected with Si-1 vectors to GABRA3 and a negative control vector (Si-Mock). Y-axis: Average value of absorbance (ABS) at 570 nm, measured with a microplate reader (n = 6, P < 0.05); B and C: Soft agar colony formation assay of HepG2/Si-1 and HepG2/Si-Mock. Same cells were incubated in low-melting agarose as described in Materials and Methods. Two weeks later, colonies were photographed and numbers of colonies were counted. (n = 3, P < 0.05); D: Cell cycle of HepG2/Si-1 and HepG2/Si-Mock measured by flow cytometry; E: Tumor volume in nude mice with HepG2/Si-1 or HepG2/Si-Mock injected (mm³).

Figure 3 Effect of GABA on the growth of HCC cells. HepG2 cells were treated with GABA at serial concentrations (0, 1, 10, 20, 40 and 60 μmol/L). A: MTT assay. Y-axis: Average value of absorbance (ABS) at 570 nm, measured with a microplate reader (n = 6, P < 0.05); B and C: Soft agar colony formation assay. Same cells were incubated in low-melting agarose as described in Materials and Methods. Two weeks later, colonies were photographed and numbers of colonies were counted. (n = 3, P < 0.05); D: Cell cycle measured by flow cytometry; E: Tumor volume in nude mice with HepG2 and GABA (0 μmol/L and 20 μmol/L) injected (mm³).

Figure 4 GABA stimulated HCC cell proliferation through GABRA3. HepG2/Si-1 and HepG2/Si-Mock cells were treated with or without GABA (20 μmol/L). A: MTT assay of HepG2/Si-Mock cells treated with or without GABA. (n = 6, P < 0.05); B: MTT assay of HepG2/Si-1 cells treated with or without GABA. (n = 6); C and D: Soft agar colony formation assay. (n = 6, P < 0.05 for HepG2/Si-Mock vs HepG2/Si-Mock + GABA); E: Cell cycle measured by flow cytometry; F: Tumor volume in nude mice (mm³).