[Construction of a eukaryotic vector expressing E2F-1 Gene and its effect on proliferation of gastric cancer cell line MKN-45].
OBJECTIVE
E2F-1 is overexpressed in many tumors and functions as an oncogene or tumor suppressor gene. This study was to construct the recombinant eukaryotic vector expressing E2F-1 cDNA, and explore the effect of E2F-1 overexpression on the proliferation of human gastric carcinoma cell line MKN-45.


METHODS
The fragment of E2F-1 gene was amplified by polymerase chain reaction (PCR). A eukaryotic vector expressing E2F-1 (pCMV-E2F1-HA) was constructed with pCMV-HA, and transfected into COS-7 cells to identify whether E2F-1 was successfully subcloned into the eukaryotic vector. pCMV-E2F1-HA was transfected into MKN-45 cells. The expression of E2F-1 in MKN-45 cells was detected by Western blot. The proliferation of MKN-45 cells was observed by colony formation assay and MTS method. Cell cycle was analyzed by flow cytometry (FCM).


RESULTS
Recombinant plasmid pCMV-E2F1-HA was successfully constructed and transfected into COS-7 and MKN-45 cells. MKN-45 cell clones were significantly fewer in pCMV-E2F1-HA group than in pCMV-HA group (4.7+/-1.8 vs. 30.2+/-6.7, P<0.01). The proliferation rate of MKN-45 cells was significantly lower in pCMV-E2F1-HA group (73.5% on Day 5, 63.5% on Day 6, 56.1% on Day 7) than in pCMV-HA group (100%, P<0.01). The G0/G1 phase proportion of MKN-45 cells was significantly higher in pCMV-E2F1-HA group than in pCMV-HA group (71.4% vs. 59.7%, P<0.05); the S phase proportion was significantly lower in pCMV-E2F1-HA group than in pCMV-HA group (18.7% vs. 30.7%, P<0.05).


CONCLUSIONS
Overexpression of E2F-1 suppresses the proliferation of gastric cancer MKN-45 cells.
Wang
Chang-Qing
CQ
Department of Gastrointestinal and Gland Surgery, The First Affiliated Hospital, Guangxi Medical University, Nanning, Guangxi, 530027, P. R. China.
Xiao
Qiang
Q
Li
Lei
L
Xie
Yu-Bo
YB
Tan
Ning
N
Gong
Zhi-Wei
ZW
D000818:Animals; D019556:COS Cells; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002522:Chlorocebus aethiops; D050687:E2F1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D013274:Stomach Neoplasms / Q000378:metabolism / Q000473:pathology*; D014162:Transfection; D015854:Up-Regulation
D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human