Figure 4. TPT1-AS1 upregulated the expression of TPT1 by H3K4me3 modification. (A) Subcellular fractionation analysis showed that TPT1-AS1was mainly enriched in the nucleus of CRC cells. (B) FISH analysis the subcellular localization of TPT1-AS1 in tissues (magnification 100x). (C) qRT-PCR detected TPT1 expression in 72 CRC and 36 adjacent normal tissues. (D) The correlation between TPT1-AS1 and TPT1 expression was analyzed in 72 CRC tissues. (E) Western blot showed that our CRC specimen had a obviously increased TPT1 level compared with adjacent normal tissues. (F) TPT1 mRNA (upper)and protein (down) level were examined in TPT1-AS1 overexpression and knockdown cells. (G) ChIP assay showed that TPT1-AS1 depletion reduced the H3K4me3 modification in TPT1 transcription region. (H) ChIP analysis reveals that MLL1 mediated TPT1-AS1 regulation TPT1 transcription. (I) ChIP analysis demonstrated that H3K4me3 level was reduced in the TPT1 promoter region after MLL1 depletion. (J) RIP assays confirmed that TPT1-AS1 could physically bind to MLL1. (K) TPT1 expression was impeded when MLL1 was silenced by siRNA. 1,3 stand for Lv-con group; 2,4 stand for Lv-sh-TPT1-AS1 group; 5 stand for vector group; 6 stand for TPT1 overexpression group. *P<0.05.