Transcriptional factor III A promotes colorectal cancer progression by upregulating cystatin A

Figure 4 Transcriptional factor III A regulates cystatin A by binding to the promoter of cystatin A gene. A: Heat map of RNA-sequencing (RNA-Seq) for transcriptional factor III A gene (Gtf3a)-knockdown and scramble control cells; B: Volcano plot of RNA-Seq for Gtf3a-knockdown and scramble control cells; C: mRNA expression of Gtf3a, cystatin A (Csta), cystatin SN gene (Cst1), and cystatin S genes (Cst4) in short hairpin (sh)scramble-SW480 and shGTF3A#1 and #4-SW480 cells were detected using real-time quantitative polymerase chain reaction; D: GTF3A, CSTA, CST1, and CST4 in shscramble-SW480 and shGTF3A#1 and #4-SW480 cells were detected using Western blot; E: RNA fluorescence in situ hybridization was performed to verify the locations of the Csta promoter probe (lncRNA) and GTF3A in SW480 cells; F: Electrophoretic mobility shift assay (EMSA) was used to test the interaction of the Csta promoter and GTF3A. The Csta promoter plus GTF3A antibody as the super shift in EMSA; G: Luciferase activity assay was used to detect the interaction of GTF3A with the Csta promoter and the transcription of Csta. Group 1 (Csta promoter-luc blank vector plus GTF3A blank plasmid) and group 2 (Csta promoter-luc blank vector plus GTF3A-plasmid) served as the control groups, and group 3 (Csta promoter-luc plus GTF3A) and group 4 (Csta promoter-luc plus GTF3A blank vector) as experimental groups. Csta expression is expressed as the mean ± SEM of three independent experiments. ^cP < 0.001. GTF3A: Transcriptional factor III A; GAPDH: Glyceraldehydes-3-phosphate dehydrogenase; CSTA: Cystatin A; CST1: Cystatin SN; CST4: Cystatin S.