Effect of RNA interference for MDC1 gene on cell cycle and expression of related proteins in esophageal carcinoma cells after X-ray radiation

Abstract

Objective To apply RNA interference technique for reducing the expression of MDC1 gene in esophageal carcinoma cell line ECA109, observe the changes in cell cycle and radiosensitivity after radiation, and discuss related mechanisms. Methods Three pairs of effective interference sequences and negative control sequences were synthesized for MDC1 mRNA sequence, and a recombinant plasmid was constructed with the vector pSIH1-H1-copGFP. RT-PCR and Western blot were used to determine the expression levels of MDC1 mRNA and protein. Colony-forming assay was applied to measure radiosensitivity, flow cytometry to determine cell cycle, Western blot to determine the expression of CHK1 and CHK2 proteins, and laser scanning confocal microscope to observe the number of MDC1 blotches inside the nucleus. One-way analysis of variance was used to analyze the differences between groups. Results The pSIH1-H1-copGFP plasmid was constructed successfully and ECA109 cells were infected to obtain ECA109M cells with stable transfection. The expression levels of MDC1 mRNA and protein in ECA109M cells were lower than those in ECA109N and ECA109 cells (P=0.032 and 0.041, respectively). After 5-Gy radiation, ECA109M cells had a lower proportion of G2+ M cells than ECA109N and ECA109 cells (P=0.026). After 5-Gy radiation, ECA109, ECA109N, and ECA109M cells had similar expression levels of CHK1 and CHK2 proteins (P=0.345 and 0.451, respectively), and ECA109M cells had a lower expression level of CHK2 T68 protein than ECA109 and ECA109N cells (P=0.012). ECA109 cells had a D0 value of 3.06 Gy and an SF2 value of 0.91; the D0 values for ECA109N and ECA109M cells were 2.90 Gy and 1.88 Gy, respectively, and the SF2 values for them were 0.89 and 0.84, respectively (P=0.021 and 0.037, respectively). Conclusions RNA interference can reduce the expression levels of MDC1 protein and cell cycle-related proteins, release cell cycle arrest, and enhance radiosensitivity in esophageal carcinoma ECA109 cells. Key words: Esophageal carcinoma; RNA interference; cell cycle protein; MDC1; radiosensitivity