Abstract Objective To investigate the effects of inhibition of MDC1 protein expression on xenografted tumors in nude mice, and to observe the histopathological and cellular changes in nude mice. Methods Three pairs of effective and control short hairpin RNA targeting MDC1 mRNA were designed and cloned into the pSIH1-H1-copGFP vector. Real-time PCR and Western blot were used to determine the mRNA and protein expression of MDC1. After selection by copGFP reporter gene, cells were divided into negative transfection group (ECA109-N) and MDC1 transfection group (ECA109-M). The transfected cells were injected into nude mice. The mice were divided into ECA109 group, ECA109-N group, and ECA109-M group. Each group was divided into irradiation subgroup and non-irradiation subgroup. The changes in tumor size after irradiation were evaluated in each group. Western blot was used to measure the expression of CHK1, CHK2, and CHK2T68 in xenografted tumors. Flow cytometry was used to analyze the cell cycle distribution and apoptosis of tumor cells in nude mice. The variance analysis was used to compare the mean of multiple groups, and the SNK-q test was used in the two two groups. Results The pMDC1-shRNA plasmid was successfully constructed and used to transfect ECA109 cells. ECA109-M cells were obtained by stable transfection with the recombinant plasmid. All inoculated nude mice survived with visible xenografted tumors at the underside of the paw in about one week. There was no swelling and wound in inoculation sites. There was no significant difference in tumor size between different groups (P>0.05). The tumor growth in the ECA109 group and the ECA109-N group significantly slowed down after irradiation with a dose of 15 Gy (P<0.05). Compared with the other two groups, the ECA109-M group had a significant smaller tumor size, significantly slower relative tumor growth, and significantly higher growth inhibition (all P<0.05). The q value of the ECA109-M group was 1.36. In the ECA109-M group, there were no significant changes in the protein expression of CHK1 and CHK2 after irradiation (P>0.05);however, the phosphorylation of CHK2T68 protein was significantly reduced after irradiation (P<0.05). There were no significant differences in cell cycle distribution or the proportion of apoptotic cells in tumor tissue between the three groups (P>0.05). Conclusions Inhibition of MDC1 protein expression by RNA interference can effectively inhibit the growth of xenografted tumors after irradiation in the nude mice by increasing their radiosensitivity.
Liu Zhikun,Zhu Shuchai,Su Jingwei et al. Lentivirus mediated RNAi silence esophageal MDC1 Eca109 cell gene expression of the influence of nude mouse transplantation tumor radiosensitivity[J]. Chinese Journal of Radiation Oncology, 2016, 25(7): 753-758.
Liu Zhikun,Zhu Shuchai,Su Jingwei et al. Lentivirus mediated RNAi silence esophageal MDC1 Eca109 cell gene expression of the influence of nude mouse transplantation tumor radiosensitivity[J]. Chinese Journal of Radiation Oncology, 2016, 25(7): 753-758.
[1]Ward IM,Minn K,van Deursen J,et al.p53 Binding protein 53BP1 is required for DNA damage responses and tumor suppression in mice[J].Mol Cell Biol,2003,23(7):2556-2563.DOI:10.1128/MCB.23.7.2556-2563.2003 [2]Sun ZX,Hsiao J,Fay DS,et al. Rad53 FHA domain associated with phosphorylated Rad9 in the DNA damage checkpoint[J].Science,1998,281(5374):272-274.DOI:10.1126/science.281.5374.272 [3]Sweeney FD,Yang F,Chi A,et al. Saccharomyces cerevisiae Rad9 acts as a Mec1 adaptor to allow Rad53 activation[J].Curr Biol,2005,15(15):1364-1375.DOI:10.1016/j.cub.2005.06.063 [4]Vialard JE,Gilbert CS,Green CM,et al. The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage[J].EMBO J,1998,17(19):5679-5688.DOI:10.1093/emboj/17.19.5679 [5]van den Bosch M,Lowndes NF.Remodelling the Rad9 checkpoint complex:preparing Rad53 for action[J].Cell Cycle,2004,3(2):117-120.DOI:10.4161/cc.3.2.652 [6]Anderson L,Henderson C,Adachi Y.Phosphorylation and rapid relocalization of 53BP1 to nuclear foci upon DNA damage[J].Mol Cell Biol,2001,21(5):1719-1729.DOI:10.1128/MCB.21.5.1719-1729.2001 [7]Ekblad CM,Friedler A,Veprintsev D,et al. Comparison of BRCT domains of BRCA1 and 53BP1:a biophysical analysis[J].Protein Sci,2004,13(3):617-625.DOI:10.1110/ps.03461404 [8]Schultz LB,Chehab NH,Malikzay A,et al.p53 binding protein 1(53BP1) is an early participant in the cellular response to DNA double-strand breaks[J].J Cell Biol,2000,151(7):1381-1390.DOI:10.1083/jcb.151.7.1381 [9]Rappold I,Iwabuchi K,Date T,et al. Tumor suppressor p53 binding protein 1(53BP1) is involved in DNA damage-signaling pathways[J].J Cell Biol,2001,153(3):613-620.DOI:10.1083/jcb.153.3.613 [10]Shang YL,Bodero AJ,Chen PL.NFBD1,a novel nuclear protein with signature motifs of FHA and BRCT,and an internal 41-amino acid repeat sequence,is an early participant in DNA damage response[J].J Biol Chem,2003,278(8):6323-6329.DOI:10.1074/jbc. M210749200 [11]Xu XZ,Stern DF.NFBD1/KIAA0170 is a chromatin-associated protein involved in DNA damage signaling pathways[J].J Biol Chem,2003,278(10):8795-8803.DOI:10.1074/jbc. M211392200 [12]Bi JP,Huang A,Liu T,et al. Expression of DNA damage checkpoint 53BP1 is correlated with prognosis,cell proliferation and apoptosis in colorectal cancer[J].Int J Clin Exp Pathol,2015,8(6):6070-6082 [13]Martin NT,Nahas SA,Tunuguntla R,et al. Assessing′Radiosensitivity′ with kinetic profiles of γ-H2AX,53BP1 and BRCA1 foci[J].Radiother Oncol,2011,101(1):35-38.DOI:10.1016/j.radonc.2011.05.065 [14]DiTullio Jr RA,Mochan TA,Venere M,et al.53BP1 functions in an ATM-dependent checkpoint pathway that is constitutively activated in human cancer[J].Nat Cell Biol,2002,4(12):998-1002.DOI:10.1038/ncb892 [15]Wilson KA,Stern DF.NFBD1/MDC1,53BP1 and BRCA1 have both redundant and unique roles in the ATM pathway[J].Cell Cycle,2008,7(22):3584-3594.DOI:10.4161/cc.7.22.7102.
2016, Vol. 25 
