The genes CNV and expression as a factors of HT-29 cells radioresistance.

Abstract

e15590 Background: The initial radioresistance of tumor cells associated with certain molecular genetic features has a great influence on the effectiveness of radiation therapy (RD). These features include the transcriptional activity of genes that regulate DNA repair, cell cycle and apoptosis, and their copy number variation (CNV). The aim of the study was to analyze the expression and CNV of genes that regulate DNA repair, cell cycle and apoptosis in HT-29 cells subjected to RD. Methods: HT-29 cell culture was used in the study. For the model experiment, doses of 5 and 7 Gy were used (irradiation was performed 5 times every 24 hours on a Novalis TX linear accelerator). Cell counting was carried out in Goryaev chamber (0.4% trypan blue solution). On the 5th day of irradiation, HT-29 was removed from the substrate with Trypsin / Versen solution. RNA was isolated according to the method of Chomczynski&Sacchi. For cDNA synthesis was used a "REVERTA-L". reagent. The phenol-chloroform method was used to DNA isolate. The RT-qPCR method was used to determine the CNV and expression of 32 genes: AKT, ATM, BRIP, BRCA1, BRCA2, CDK1, CDKN1B, CCND1, CCND3, EXO1, FGFR2, HIST1, H2AX, KU70, PTEN, RAD50, RAP80, RIF1, RNF8 TOPB1, TP53, XRCC4, BAX, CASP8, CASP3, CASP9, MDM2, BCL2, RBBP8, EP300, LIG4, C-FLIP. Statistical analysis was performed using ANOVA and Spearman's rank correlation coefficient (r). Results: After irradiation, only a specific pool of HT-29 cells remained viable: 32% for 5 Gy and 20% for 7 Gy of the initial number of cells. In HT-29 cells irradiated with 7 Gy, the CNV of the BRCA2, H2AX, CASP9 and RBBP8 genes was increased (p < 0.05) and the CNV of BCL2 gene was reduced (p < 0.05) relative to intact cells. In cells irradiated with 5 Gy only CASP9 and RBBP8 CNV was statistically significantly (p < 0.05) increased. In cells irradiated with 5 and 7 Gy, expression of BRCA2, H2AX, CASP9 and RBBP8 genes was statistically significantly (p < 0.05) increased and the expression of the BCL2 gene was reduced relative to intact cells. A strong positive correlation was observed between CNV and expression of the studied genes: r = 0.998 for control, r = 0.989 for cells irradiated at 5 Gy, and r = 0.993 for cells irradiated at 7 Gy. Conclusions: The study showed that 5 day RD at 5 and 7 Gy leads to selective survival of 32% and 20% of cells, respectively, with increased CNV and expression of BRCA2, H2AX, RBBP8 CASP9 genes and reduced CNV and expression of BCL2 gene.