Investigation of INSIG1 gene in differentiated panc-1 cells

PANC-1 cell line is mainly used in studies explaining pancreatic cancer. Since this cell line was shown to have proliferative response to insulin, its use as a tool to explain metabolic diseases was planned. In this respect, our previous study involved creation of an experimental model utilizing PANC-1 cells. In this study, PANC-1 cells were differentiated by a special 14 day protocol and microarray analyses were carried out that revealed 4-fold significant change in expression of INSIG1 gene, which is involved in cholesterol metabolism, lipogenesis and regulation of glucose homeostasis. In this thesis, INSIG1 gene was sequenced in the PANC-1 cell line to understand if this differentiation protocol had any effect at the DNA level. In addition, INSIG1 mRNA expression levels were determined via quantitative RT-PCR to confirm the microarray results of the previous study. In line with these aims, INSIG1 gene was amplified utilizing primer pairs that encompass different transcripts of the gene and that sequencing was perfomed on PANC-1 cells at basal level and day 14 of the differentation protocol. For the expression analysis, cells were collected on day 0 and 14 of the differentation protocol, followed by RNA isolation and cDNA synthesis. SYBR Green and UPL prob techniques were utilized in quantitative RT-PCR to detect relative expression of INSIG1 in these cells. As a result of the sequence analysis, we have detected 1 single nucleotide deletion and 5 single nucleotide changes in different intronic regions. Moreoever, quantitative RT-PCR showed 4 fold decrease of INSIG1 expression on day 14 samples, validating microarray results. As a result, INSIG1 was characterized first time in PANC-1 cell line. It was observed that the differentiation of the cells did not cause a change in INSIG1 gene sequence. Moreover, INSIG1 mRNA expression analysis confirmed microarray results. Key Words: PANC-1, INSIG1, Differentitation, Sanger Sequencing, Metabolic Pathway