Transcriptional regulation of MIR100HG, an important LncRNA, in hepatocellular cancer

Recent studies have revealed the significant role of long non-coding RNAs (lncRNAs) in various diseases. MIR100HG was initially identified through its over-expression in neuronal cells in a human transcriptome analysis. Bioinformatics and expression studies have elucidated the role of MIR100HG as a tumor promoter in human cancers including neuroblastoma, leukemia, and breast cancer. However, limited information exists in the literature regarding the role of the MIR100HG gene in hepatocellular carcinoma (HCC). This thesis aims to investigate the expression levels of MIR100HG in different hepatocellular cancer cell lines under normoxic and hypoxic conditions. Epithelial cell lines (Hep3B and HUH-7), mesenchymal cell lines (SNU-398 and SNU-475), and a healthy liver model (Clone-9) were selected for this purpose. Cobalt chloride was used to induce a hypoxic environment. Real-time PCR analyses were performed using specific MIR100HG primers. The results of the thesis study showed that MIR100HG was most highly expressed in the Hep3B cell line under both normoxic and hypoxic conditions, followed by the SNU-398 cell line. Hypoxia induction was confirmed by examining the mRNA and protein levels of HIF-1α. The transcriptional regulation of the MIR100HG promoter was investigated under hypoxic and normoxic conditions through the transient transfection of five different lengths of promoter fragments into Hep3B cells. The most active promoter fragments were found to be -397/+231, -815/+231, and -1031/+231, with increased activity observed under hypoxic conditions. The functional binding of HIF-1α to the HRE regions of the MIR100HG promoter in Hep3B cells under hypoxic conditions was confirmed by chromatin immunoprecipitation (ChIP) analysis. The importance and necessity of MIR100HG in cell characterization were determined for the first time in this thesis study through the silencing and over-expression of MIR100HG in Clone-9 cells. Real-time analysis, MTT assay, colony formation assay, scratch assay, and Matrigel assay performed after over-expression and silencing experiments demonstrated the role of MIR100HG in cellular processes such as cell survival, proliferation, and migration in Clone-9 cells. It is anticipated that the data obtained in this thesis will guide researchers in further cancer research.