[Construction of a eukaryotic vector expressing E2F-1 Gene and its effect on proliferation of gastric cancer cell line MKN-45].

Abstract

BACKGROUND & 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).

CONCLUSION:

Overexpression of E2F-1 suppresses the proliferation of gastric cancer MKN-45 cells.

• Authors: Chang-Qing Wang, Qiang Xiao, Lei Li, Yu-Bo Xie, Ning Tan, Zhi-Wei Gong
• Journal: Ai zheng = Aizheng = Chinese journal of cancer (Ai Zheng) Vol. 26 Issue 10 Pg. 1064-8 (Oct 2007) China
• PMID: 17927874 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• E2F1 Transcription Factor
• E2F1 protein, human

Topics

• Animals
• COS Cells
• Cell Cycle
• Cell Line, Tumor
• Cell Proliferation
• Chlorocebus aethiops
• E2F1 Transcription Factor (genetics, metabolism)
• Gene Expression Regulation, Neoplastic
• Genetic Vectors
• Humans
• Plasmids
• Stomach Neoplasms (metabolism, pathology)
• Transfection
• Up-Regulation