ZNF300 may influence the metastatic properties in pancreatic ductal adenocarcinoma.

摘要

Aberrant DNA methylation of promoter CpG islands (CGI) is a contributing.;factor that facilitates the dysregulation of gene expression to promote the.;tumorigenesis of pancreatic ductal adenocarcinoma (PDAC). Moreover, due to the.;overwhelming number of PDAC patients presented with metastasis at the time of.;diagnosis, it is necessary to better understand the process of metastasis. To understand.;the role of DNA methylation may in the metastasis of PDAC, two isogenic cells lines,;generated by Dr. Isaiah Fidler at the University of Texas MD Anderson Cancer.;Center, were used as a cell culture model. These cell lines were generated from.;orthotopic injections of an established pancreatic cancer cell line, colo357, into the.;pancreas of nude mice. Upon injection, metastasis formed in the liver. Metastatic cells.;from the metastases were isolated and grown in culture to yield a lowly metastatic cell.;line, fast growing (FG). This process was repeated using FG cells, and after three.;rounds of injection and isolation, the enriched metastatic cells were isolated to yield a.;highly metastatic variant, L3. 6. Preliminary work, performed by previous lab.;members, identified genes exhibiting promoter hypermethylation in a high metastatic.;cell line, L3.6pl, compared to the lowly metastatic isogenic variant cell line, FG. One.;of those genes, ZNF300, was chosen as a candidate gene for many reasons. First,;analysis of percent methylation within the ZNF300 CGI indicated increased.;methylation among L3.6pl cells compared to FG, specifically sixteen percent to one percent, respectively. In addition to increased methylation, the region of the ZNF300.;promoter that was hypermethylated was also shown to affect promoter activity.;according to luciferase promoter assays. Therefore, it was hypothesized that the.;observed hypermethylation in L3.6pl cells may lead to decreased ZNF300 expression.;Moreover, ZNF300 expression was identified in pancreatic tumor samples from ten.;PDAC patients via immunohistochemical staining; however, analysis from lymph.;node metastases from many of these patients showed diminished ZNF300 expression.;compared to the primary tumor. Taken together, this data supports our overall goal to.;determine if ZNF300 hypermethylation correlates to ZNF300 expression, and more.;importantly, if this correlation is related to the metastatic ability of L3.6pl cells.;To determine if ZNF300 is epigenetically regulated, methylation levels were.;quantified via COBRA and bisulfite sequencing in both FG and L3.6pl cells.;Additionally, ZNF300 expression was measured via qRT-PCR and western blotting.;analysis. To determine the functional role of ZNF300 in the metastatic process of.;L3.6pl, we attempted to overexpress ZNF300 using the p-RetroX-Tre3G Tet-ON.;inducible expression system. Following induction using doxycycline, both the.;expression and migratory ability were measured.;The data in this study concludes that, contrary to preliminary data, the ZNF300.;CpG island is not differentially methylated in L3.6pl cells compared to FG cells. Our.;expression data is in accordance with the methylation levels, and thus, indicated no.;significant difference in ZNF300 expression between the two isogenic variant cell.;lines. Moreover, we were unable to induce ZNF300 expression using the retroviral inducible system, but rather noted potentially off target effects of doxycycline in.;infected L3.6pl cells. Due to our inability to induce ZNF300 expression, we are unable.;to identify the role of ZNF300 in the metastatic process of PDAC.