[Effect on retardation of G2/M phase in esophageal carcinoma cells transfected with CHK1 and CHK2 shRNA after irradiation].
OBJECTIVE
To observe the effect of RNA interference on CHK1 and CHK2 expression and change of G2/M phase arrest in esophageal carcinoma cells after irradiation.


METHODS
Four sequences short hairhip RNA (shRNA) of each CHK1 and CHK2 genes were constructed and connected with vector of pENTR/U6 plasmid, respectively, and then transfected into Eca109 cells with lipofectamine 2000 reagent. Protein and mRNA expression of CHK1 and CHK2 genes were detected with Western blotting and RT-PCR, respectively. Cell cycling was measured by flow cytometry after 5 Gy irradiation. Cell survival rate after 5 Gy irradiation was evaluated by clonegenetic assay.


RESULTS
Four shRNA vector each of CHK1 and CHK2 genes were successfully constructed and transfected into Ecal09 cells, respectively. Protein expression of CHK1 and CHK2 were obviously decreased. Their mRNA expressions were also decreased after transfected with shRNA of CHK1 and CHK2. Arrest of G2/M stage in Eca109 cells were obviously decreased only in cells transfected with CHK1 shRNA but not with CHK2 shRNA at 12 h after 5 Gy irradiation. In first progeny Eca109 cells transfected with CHK1 and CHK2 shRNA, expression of CHK1 and CHK2 protein was also decreased. The level of phosphorylated CHK2-T68 expression was decreased at 1 h after 5 Gy irradiation, and at 72 h only transfected with CHK2 shRNA but not with CHK1 shRNA. Phosphorylation level of CHK1-S345 was not increased after transfected with CHK1 or CHK2 shRNA, but arrest of G2/M stage still remained at 12 h after 5 Gy irradiation and at 72 h accordingly. The cell survival rate was decreased in Eca109 cells transfected with CHK1 or CHK2 shRNA after 5 Gy irradiation.


CONCLUSIONS
After transfected with shRNA of CHK1 or CHK2, their expressions of mRNA and protein in Ecal09 cells are markedly inhibited and this inhibition effect can be observed in their first progeny cells and at least hold for 3 days. Arrest of G2/M phase can be reduced after irradiation when teansfected with shRNA of CHK1 and the radiosensitivity of Ec109 cells can be increased.
Wang
Yu-xiang
YX
Department of Radiation Oncology, the 4th Hospital of Hebei Medical University, Shijiangzhuang 050011, China.
Zhu
Shu-chai
SC
Feng
Wei
W
Li
Juan
J
Su
Jing-wei
JW
Li
Ren
R
D015153:Blotting, Western; D002455:Cell Division / Q000235:genetics / Q000502:physiology / Q000528:radiation effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics / Q000502:physiology / Q000528:radiation effects; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D004938:Esophageal Neoplasms / Q000235:genetics / Q000473:pathology / Q000503:physiopathology; D016195:G2 Phase / Q000235:genetics / Q000502:physiology / Q000528:radiation effects; D005720:Gamma Rays; D005822:Genetic Vectors; D006801:Humans; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
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