[Effects of RNA interference on CHK1 and CHK2 expression and G2/M arrest in esophageal carcinoma cell line TE13 after irradiation].
OBJECTIVE
Cell cycle of tumor cells often changes after irradiation. This study was to observe the effect of RNA interference (RNAi) on mRNA and protein expression of human checkpoint kinases CHK1 and CHK2 and on cell cycle of esophageal carcinoma cell line TE13 after irradiation.


METHODS
Short hairpin RNAs (shRNAs) of CHK1 and CHK2 genes were designed and constructed, and then transfected into TE13 cells. The expression of CHK1 and CHK2 were detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Cell cycle was measured by flow cytometry after 5-Gy radiation. Cell survival after irradiation was evaluated with colony forming assay.


RESULTS
After transfection of CHK1 and CHK2 shRNAs, the mRNA and protein expression of CHK1 and CHK2 in TE13 cells were inhibited obviously. At 24 h after 5-Gy radiation, G2/M phase proportion was higher in irradiated TE13 cells than in control TE13 cells (61.47% vs. 32.17%), and significantly lower in CHK1 shRNA-and CHK2 shRNA-transfected TE13 cells than in untransfected TE13 cells (28.13% and 42.80% vs. 61.47%, P<0.05)û cell survival rates were 27% in simplex irradiation group, 14% in CHK1 shRNA transfection plus irradiation group, and 21% in CHK2 shRNA transfection plus irradiation group. In the first generation of transfected TE13 cells, the inhibition of CHK1 and CHK2 expression was almost diminished at 120 h after transfection. At 120 h after transfection and 24 h after 5-Gy irradiation, G2/M phase proportion was significantly higher in CHK1 shRNA-and CHK2 shRNA-transfected groups than in simplex irradiation group (P<0.05)û but at 144 h after transfection and 48 h after 5-Gy irradiation, the difference between transfection groups and simplex irradiation group was not significant (P>0.05).


CONCLUSIONS
RNAi could inhibit the expression of CHK1 and CHK2, release G2/M phase arrest after irradiation, and enhance radiosensitivity of TE-13 cells. G2/M phase of TE-13 cells after irradiation may be regulated by CHK1 and CHK2.
Zhu
Shu-Chai
SC
Department of Radiation Oncology, The Fourth Affiliated Hospital, Hebei Medical University, Shijiangzhuang, Hebei, PR China. sczhu@heinfo.net
Wang
Yu-Xiang
YX
Li
Ren
R
Han
Chun
C
Qiu
Rong
R
Shen
Wen-Bin
WB
D002453:Cell Cycle / Q000528:radiation effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000528:radiation effects; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D003037:Cobalt Radioisotopes; D004938:Esophageal Neoplasms / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D011494:Protein Kinases / Q000096:biosynthesis* / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection
D003037:Cobalt Radioisotopes; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases