Changes in gene-expression profiles of colon carcinoma cells induced by wild type K-ras2.
OBJECTIVE
To further elucidate the possible molecular biological activity of wild type K-ras2 gene by detecting changes in wild type K-ras2 gene-induced gene-expression profiles of colon carcinoma cells using cDNA microarray techniques.


METHODS
Total RNA was isolated from peripheral blood of health volunteers. Reverse transcription of RNA and polymerase chain reaction were used to synthesize wild type K-ras2 cDNA. K-ras2 cDNA fragment was cloned into a T easy vector and sequenced. A eukaryotic expression vector pCI-neo-K-ras2 was constructed and transfected to Caco2 cell line using the liposome method. Finally, mRNA was isolated, reverse-transcribed to cDNA from pCI-neo-K-ras2 or pCI-neo blank vector-transfected Caco cells, and analyzed by cDNA microarray assay.


RESULTS
Restriction enzyme analysis and DNA sequencing verified that the constructed expression vector was accurate. High-quality RNA was extracted and reverse transcribed to cDNA for microarray assay. Among the 135 genes, the expression was up-regulated in 24 and down-regulated in 121. All these differentially expressed genes were related to cell proliferation, differentiation, apoptosis and signal transduction.


CONCLUSIONS
Differentially expressed genes can be successfully screened from wild type K-ras2-transfected colon carcinoma cells using microarray techniques. The results of our study suggest that wild type K-ras2 is related to the negative regulation of cell proliferation, metabolism and transcriptional control, and provide new clues to the further elucidation of its possible biological activity.
Li
Hong
H
Inpatient Department of Special Need Treatment, General Hospital of Chinese PLA, Beijing, China.
Cao
Hou-Fa
HF
Li
Yuan
Y
Zhu
Mei-Ling
ML
Wan
Jun
J
D017209:Apoptosis / Q000235:genetics; D018938:Caco-2 Cells; D002454:Cell Differentiation / Q000235:genetics; D049109:Cell Proliferation; D003110:Colonic Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras); D012333:RNA, Messenger / Q000378:metabolism; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000235:genetics; D014162:Transfection; D018631:ras Proteins / Q000235:genetics* / Q000378:metabolism
C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins