We next evaluated the potential regulatory effect of MTF1 and YY2 on GTSE1. Western blot and quantitative real-time PCR showed that both MTF1 and YY2 overexpression increased the mRNA and protein expression levels of GTSE1, whereas simultaneous knockdown of MTF1 and YY2 reduced GTSE1 levels (Figures 6F–6I). The bioinformatics databases DataBase of Transcriptional Start Sites (DBTSS) and JASPAR were used to identify the putative binding site in the region 2,000 bp upstream of the GTSE1 transcription start site, indicating that MTF1 could directly bind to the GTSE1 promoter region through putative binding site 1, and this interaction was confirmed with a chromatin immunoprecipitation (ChIP) assay (Figure 7A). Since LINC00665 and MTF1 showed opposite functions in regulating the malignant progression of glioma, we hypothesized that downregulation of MTF1 expression might rescue the LINC00665-mediated reduction in GTSE1 expression. In line with this expectation, the LINC00665+ + MTF1− group showed stronger reductions in GTSE1 mRNA and protein expression levels compared with the LINC00665+ + MTF1+ group, and their expression levels were significantly higher in the LINC00665− + MTF1+ group than those in the LINC00665− + MTF1− group (Figures 7B and 7C).
