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.
Liu Zhikun,Zhu Shuchai,Su Jingwei et al. Effect of RNA interference for MDC1 gene on cell cycle and expression of related proteins in esophageal carcinoma cells after X-ray radiation[J]. Chinese Journal of Radiation Oncology, 2015, 24(6): 708-711.
[1] Raju U,Nakata E,Yang P,et al. In vitro enhancement of tumor cell radiosensitivity by a selective inhibitor of cyclooxygenase-2 enzyme:mechanistic considerations[J].Int J Radiat Oncol Biol Phys,2002,54(3):886-8894. [2] Strunz AM,PesCHKe P,Waldeck W,et al. Preferential radiosensitization in p53-mutated human tumour cell lines by pentoxifylline-mediated disruption of the G2/M checkpoint control[J].Int J Radiat Biol,2002,78(8):721-732. [3] Chen YH,Sanchez Y.CHK1 in the DNA damage response:conserved roles from yeasts to mammals. DNA Repair,2004,3(8－9):1025-1032. [4] Xu X,Stern DF.NFBD1/KIAA0170 Is a Chromatin-associated Protein Involved in DNA Damage Signaling Pathways[J].J Biol Chem,2003,278(10):8795-8803. [5] Bull EE,Dote H,Brady KJ,et al. Enhanced tumor cell radiosensitivity and abrogation of G2 and S phase arrest by the Hsp90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin[J].Clin Cancer Res,2004,10(23):8077-8084. [6] Benada J,Burdová K,Lidak T,et al. Polo-like kinase 1 inhibits DNA damage response during mitosis[J].Cell Cycle,2015,14(2):219-231.doi:10.4161/15384101.2014.977067. [7] Rybanska-Spaeder I, Ghosh R, Franco S.53BP1 mediates the fusion of mammalian telomeres rendered dysfunctional by DNA-PKcs loss or inhibition[J/OL].PLoS One,2014,9(9):e108731[2014-11-01].http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108731.DOI:10.1371/journal.pone.0108731. [8] Eliezer Y, Argaman L, Kornowski M,et al. Interplay between the DNA damage proteins MDC1 and ATM in the regulation of the spindle assembly checkpoint[J].J Biol Chem,2014,289(12):8182-8193.doi:10.1074/jbc. M113.532739. [9] Mok MT, Henderson BR.The in vivo dynamic interplay of MDC1 and 53BP1 at DNA damage-induced nuclear foci[J].Int J Biochem Cell Biol,2012,44(9):1398-1409.doi:10.1016/j.biocel.2012.05.025 [10] Wilson KA,Colavito SA,Schulz V.NFBD1/MDC1 regulates Cav1 and Cav2 independently of DNA damage and p53[J].Mol Cancer Res,2011,9(6):766-781.doi:10.1158/1541－7786.MCR-10-0317. [11] Bu Y, Suenaga Y, Okoshi R,et al. NFBD1/MDC1 participates in the regulation of G2/M transition in mammalian cells[J].Biochem Biophys Res Commun,2010,397(2):157-162. doi:10.1016/j.bbrc.2010.05.063. [12] Chen SJ,Wang G,Makrigiorgos GM,et al. Stable siRNA-mediated silencing of ATM alters the transcriptional profile of HeLa cells[J].Biochem Biophys Res Commun,2004,317(4):1037-1044. [13] Chen Y,Sanchez Y.The ATM-Chk2 and ATR-Chk1 pathways in DNA damage signaling and cancer[J].Adv Cancer Res,2010,108:73-112.doi:10.1016/B978-0-12－380888－2.00003-0. [14] Davari K, Frankenberger S, Schmidt A, et al. Checkpoint kinase 2 is required for efficient immunoglobulin diversification[J].Cell Cycle,2014,13(23):3659-3669.doi:10.4161/15384101.2014.964112. [15] Lou Z,Chini CCS,Minter-Dykhouse K,et al. Mediator of DNA Damage Checkpoint 1 regulates BRCA1 localization and phosphorylation in DNA damage checkpoint control[J].J Biol Chem,2003,278(16):13599－13602
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