The Study Of Mechanisms For Polι Overexpression In Esophageal Squamous Cancer And Its Effect On Chemo-radiosensitivity Of Esophageal Squamous Cancer Cell Lines

Part one:The mRNA expression levels of genes involved in the translesion DNA synthesis pathwayObjective To investigate the differential expression levels of genes involved in the translesion DNA synthesis (TLS) pathway between the normal and cancerous esophageal tissues.Methods The mRNA expression levels of TLS genes between the normal and cancerous esophageal tissues were analyzed with real-time PCR method, and overexpression of DNA polymerase iota (Polι) in the cancerous esophageal tissues was examined by immunohistochemistry.Results Statistical analysis revealed significant differences between normal and cancerous tissues in mRNA expression of REV3L, REV7L, RAD18, DNA polymerase kappa (Polκ) and Polι. The mRNA expression of the Polι gene was7.2-fold upregulated in tumor tissues, compared with normal controls. The increased expression of Polι in tumor samples was further confirmed by immunohistochemistry.Conclusions Gene expression for REV3L, REV7L, RAD18, Polκ and Polι was significantly elevated in the cancerous tissues compared with that of the normal tissues. The increase in the transcription of the TLS pathway genes, especially Polι, in esophageal carcinomas is a characteristic of this malignancy. Part two:Mechanisms for Polι overexpression in esophageal squamous cancer tissuesObjective To explore the potential mechanisms for the differential mRNA expression levels of the Polι gene between the normal and cancerous esophageal tissues in terms of the levels of DNA methylation and histone acetylation, and the binding affinity of transcription factors.Methods (1) The methylation levels of CpG sites in the promoter of the Polι gene in the normal and cancerous esophageal tissues were determined by bisulfite sequencing.(2) Expression levels of Polι mRNA in the human esophageal cancer cell lines Eca-109and TE-1, which were treated with DNA methyltransferase inhibitor and histone deacetylase inhibitor respectively, were detected by PCR method.(3) The recombinant luciferase reporter plasmids regulated by serially deleted Polι promoter were constructed and transfected into Eca-109and TE-1cells, to assess their transcriptional activity by measuring the luciferase activity. The above recombinant plasmids were then cotransfected with expression plasmids of transcription factors Spl and Oct-1to investigate whether the transcription factors studied can stimulate the expression of the Polι gene. Correlations between the mRNA expression levels of Polι and that of Sp1in the normal and cancerous esophageal tissues were explored by using real-time PCR. Binding affinitiy of Spl, Oct-1and RNA polymerase II at specific Polι promoter regions were quantified in the Eca-109and TE-1cells and the cancerous esophageal tissues by using chromatin immunoprecipitation (ChIP).Results (1) No significant differences of the methylation levels of the14CpG sites in the Polι promoter were found between the normal tissues and the cancerous esophageal tissues.(2) Polι expression levels in the Eca-109and TE-1cells were not affected by treatment with DNA methyltransferase inhibitor and histone deacetylase inhibitor respectively.(3) Cells transfected with the pGL3-1631vector had an increased luciferase activity, compared with Eca-109cells transfected with pGL3-541and pGL3-367vectors and TE-1cells with pGL3-794, pGL3-541and pGL3-367vectors.100ng pcDNA3.1-Spl,200ng pcDNA3.1-Sp1and200ng pcDNA3.1-Oct-1could further enhance the luciferase activity of TE-1cells transfected with pGL3-1631vectors. The mRNA levels of endogenous Polι in the cancerous esophageal tissues significantly correlated with that of Spl, but not with Oct-1. Binding affinitiy of Spl, Oct-1and RNA polymerase Ⅱ at specific Polι promoter regions were different, with a maximum binding affinity for Sp1at regionConclusions (1) Regulation of DNA methylation or histone acetylation does not contribute to the increased expression of Pol i in esophageal cancer tissues.(2) The Pol i promoter region between-1631and-794may contain cis-activating elements critical for transcription regulation of this gene. Spl plays a transactivating role in Pol i gene transcription and its binding between-1291and-1081of the promoter region may confer overexpression of Pol i in the esophageal cancer tissues.Part three:Effect of Polι overexpression on chemo-radiosensitivity of esophageal squamous carcinoma cell linesObjective:To explore effect of Polι overexpression on chemo-radiosenstivity of esophageal squmaous cancer cell lines.Methods:pcDNA-Polι and shRNA-Polι were constructed and transfected into esophageal squamous cancinoma cell line Eca-109. The ability of cell proliferation was assayed by Cell Counting Kit-8(CCK-8) and colony formation. Eca-109cell lines transfected with plasmid were treated with10μM cisplatin or irradiated using6MV X ray at different doses. Chemoresistance of Eca-109cell lines induced by Polι overexpression were evaluated using CCK-8method, and their radioresistance were determined with colony formation.Results:Up-regulated expression of Polι in Eca-109cell lines promoted cell proliferation and increased their chemo-radioresistance. Down-regulated expression of Pol ι in Eca-109cell lines slowed down cell proliferation and increased their chemo-radiosensitivity.Conclusions It may be an important strategy for increasing chemo-radiosenstivity of esophageal squamous cancer cells through inhibiting Polι overexpression.