FIGURE 2.

KDM3A binds with the promoter region of HOXA1 gene and increases the expression of HOXA1 by erasing the H3K9me2 in HepG2 cells. (A) Methylation-specific binding sites of HOXA1 gene by the JASPAR database (http://jaspar.genereg.net/). (B) Expression box of KDM3A between 81 HCC and normal liver tissue specimens after differentially analyzing the GSE62232 dataset (p = 3.006E-03). (C) Positive correlation between KDM3A and HOXA1 expression by the StarBase database analysis (r = 0.252, p = 8.15E-07). (D) Co-expression of KDM3A and HOXA1 by the MEM analysis (p = 0.0123), wherein the abscissa represents different samples; the squares corresponding to the samples and KDM3A represent co-expression intensity of KDM3A and HOXA1; the color of the squares was shown on the upper right, and the deeper the color, the stronger the co-expression intensity of the two; the right side of the squares represents the significance of overall intensity of KDM3A and HOXA1. (E,F) ChIP analysis was performed using anti-H3K9me2, anti-H3K9me3, anti-H3K4me3, anti-H3K27ac, and anti-H3K27me3 antibodies in HepG2 cells treated with 3xFLAG-KDM3A or KDM3A siRNA. * indicates p < 0.05 compared with Mock or scramble siRNA by paired t-test. (G,H) Western blots and quantification of KDM3A in HepG2 cells treated with KDM3A siRNA and/or HOXA1 recombinant lentiviral expression vectors. (I) HepG2 cell viability was evaluated by MTT assay at indicated time points. (J) Representative view (×200) of HepG2 cells migrating from upper transwell chambers without Matrigel into lower ones and statistics of migrating cells; representative view (×200) of HepG2 cells invading from Matrigel-coated upper transwell chambers into lower ones and statistics of invading cells. * (compared with scramble siRNA + oe-NC) and # (compared with KDM3A siRNA + oe-NC) indicate p < 0.05 by Tukey’s test-corrected one-way ANOVA for (G,H,J) and by Bonferroni-corrected repeated measures ANOVA for (I).