Figure 3.

miRNA‐326 expression was regulated by SP1 and HDAC1. A, Using an open online Jaspar database (http://jaspar.genereg.net), bioinformatics analysis showed that SP1 was able to bind to the promoter of miRNA‐326 gene. B, miRNA‐326 expression was increased in MG63 and 143B cells 72 h after SP1 knockdown. C, miRNA‐326 expression was increased in MG63 and 143B cells 72 h after HDAC1 knockdown or treatment with HDAC inhibitor. D, ChIP assay was performed to determine the interaction between SP1, HDAC1 and acetylated histone 3 proteins and the promoter of miRNA‐326 gene. Both antibodies of HDAC1 and acetylated histone 3 proteins extracted the DNA sequence of miRNA‐326 gene promoter. Knockdown of SP1 decreased the DNA enrichment in HDAC1 protein complex, but increased the DNA enrichment in acetylated histone 3 protein complex. E, ChIP assay was also performed to detect the enrichment of miRNA‐326 gene promoter in SP1 and HDAC1 proteins in OS tissues and the matched adjacent normal tissues. F, In DAPA, WT and MT of miRNA‐326 promoter oligonucleotides were designed to pull down SP1 and HDAC1 proteins in MG63 and 143B cells. SP1 and HDAC1 proteins were pulled down by the WT oligonucleotides, but almost not by MT oligonucleotides. SP1 knockdown notably decreased the abundance of HDAC1 protein pulled down by the WT oligonucleotides. B and C: **P < .01, ***P < .001 vs WT group. D: ***P < .001 vs IgG group; ^## P < .01 vs WT group that did not undergo transfection. E: **P < .01 vs adjacent normal tissues. All above studies were repeated three times