Regulation of MIR100HG by KLF7 transcription factor in hepatocellular carcinoma

The MIR100HG gene, located on the q arm of chromosome 11 in humans, is a long non-coding RNA (lncRNA). MIR100HG is abnormally expressed in various cancers and plays an oncogenic or tumor-suppressive role by participating in multiple pathways associated with tumor cell biology and cancer. Although there are few studies on MIR100HG in the literature, it is a gene expressed in colorectal cancer, laryngeal squamous cell carcinoma, gastric cancer, and hepatocellular carcinoma. In this thesis, the transcriptional regulation of MIR100HG by the KLF7 transcription factor has been elucidated. Five different deletion constructs of the human MIR100HG promoter region (1244 bp -1013/+231, 1046 bp -815/+231, 909 bp -678/+231, 628 bp -397/+231, and 419 bp -188/+231) were prepared and cloned. Among these, the cloning of the 1046 bp (-815/+231) and 909 bp (-678/+231) promoter constructs was accomplished within this thesis study. In transient transfection experiments using the human hepatocellular carcinoma cell model (Hep3B), it was determined that all promoter constructs were active, and the promoter fragments of 628 bp (-397/+231), 1046 bp (-815/+231), and 1244 bp (-1013/+231) showed the highest basal activity. Bioinformatic analyses identified potential KLF7 binding sites within the MIR100HG promoter region. Co-transfection of promoter fragments with the KLF7 transcription factor increased the transcriptional level of MIR100HG. Additionally, qRT-PCR demonstrated that KLF7 overexpression increased MIR100HG expression at the mRNA level under both normoxic and hypoxic conditions. Chromatin immunoprecipitation (ChIP) analysis confirmed that KLF7 functionally binds to different regions of the MIR100HG promoter. The validation of KLF7 overexpression, under normoxic and hypoxic conditions, was shown at the mRNA level through qRT-PCR, MTT, colony formation, scratch (migration), and IFC assays.