Apoptosis induced by short hairpin RNA-mediated STAT6 gene silencing in human colon cancer cells.
BACKGROUND
The relationship between signal transduction and tumors has become one of the foci in cancer research. Signal transducer and activator of the transcription 6 (STAT6) signaling pathway is found to be activated in some cancer cells. But the function of the pathway in cancer cells is unknown. This study was undertaken to investigate the effect of the Stat6 signaling pathway on apoptosis in human colon cancer cells (HT-29 cells) and the possible mechanism of Stat6 by RNA interference techniques.


METHODS
Four eukaryotic expression plasmid vectors of short hairpin RNA (shRNA) specific for the STAT6 gene were designed and generated by molecular biological technology. The plasmid vectors were transfected into HT-29 cells by cation liposomes to block the Stat6 signaling pathway. The expressions of STAT6 mRNA and phosph-Stat6 protein were detected by the reverse transcriptase polymerase chain reaction (RT-PCR) method and flow cytometry respectively to screen the most effective shRNA at 72 hours after transfection. The apoptosis condition of the cells in which the expression of the STAT6 gene had been interfered was analyzed by flow cytometry and confocal microscopy. Both mRNA and protein expression of B cell lymphoma-2 (Bcl-2) and Bax were detected by RT-PCR and western blotting.


RESULTS
Two effective eukaryotic expression plasmid vectors of shRNA specific for the STAT6 gene were generated successfully. One can reduce the expression of the STAT6 gene by 82.4% and the other by 56.8% (P < 0.01). The apoptotic rate of colon cancer cells in which STAT6 gene expression had been interfered was significantly higher than that in controlled colon cancer cells (P < 0.01). In the cells in which the Stat6 signaling pathway was blocked, the levels of mRNA and protein Bcl-2 were significantly decreased, whereas those of Bax were significantly increased (P < 0.01).


CONCLUSIONS
The Stat6 signaling pathway can inhibit apoptosis in human colon cancer cells. The subsequent disorder of Bcl-2/Bax expression may play an important part in that process. The STAT6 gene may serve as a potential target in cancer therapy.
Zhang
Ming-sheng
MS
Department of Cancer Radio-Chemotherapy, Zhongnan Hospital, Wuhan University, Wuhan 430071, China. zms75@163.com
Zhou
Yun-feng
YF
Zhang
Wen-jie
WJ
Zhang
Xiao-lian
XL
Pan
Qin
Q
Ji
Xue-mei
XM
Luo
Zhi-guo
ZG
Wu
Jian-ping
JP
D017209:Apoptosis; D020868:Gene Silencing; D019073:HT29 Cells; D006801:Humans; D010957:Plasmids; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D034741:RNA, Small Interfering / Q000494:pharmacology*; D050800:STAT6 Transcription Factor / Q000037:antagonists & inhibitors* / Q000235:genetics; D015398:Signal Transduction; D051028:bcl-2-Associated X Protein / Q000032:analysis / Q000235:genetics
D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human; D051028:bcl-2-Associated X Protein