[Down-regulation of Smoothened gene expression inhibits proliferation of breast cancer stem cells].
OBJECTIVE
To investigate the influence of down-regulating Smoothened (SMO) gene expression through short hairpin RNA (shRNA) on the proliferation of breast cancer stem cells.


METHODS
Human SMO shRNA was designed, synthesized chemically, and transfected into MCF-7 cells to down-regulate SMO gene. By using G418, stable cells with down-regulated SMO were selected. In vitro proliferation of these cells was measured by CCK8 assay. The proportion of CD44(+)/CD24(-) cells was detected by flow cytometry and the mammospheres formation was determined by suspension sphere culture. The expression of SMO, GLI1 and Oct4 was detected by Western blot. In vivo, the volume of tumor was measured every 3 days and the expression of SMO, GLI1 and Oct4 detected by Western blot.


RESULTS
In vitro, the cells were transfected with SMO-shRNA and selected by G418 after 21 days. SMO-shRNA effectively down-regulated the expression of SMO gene and protein, and inhibited the proliferation of MCF-7 and markedly reduced the proportion of CD44(+)/CD24(-) cells and mammospheres. In vivo, SMO-shRNA treatment of MCF-7 significantly inhibited the volume of tumor. The positive rate of SMO in negative control and SMO-shRNA group was 5/5 and 2/5, respectively. The expression of SMO, GLI1 and Oct4 in different groups were 0.72 ± 0.17 and 0.21 ± 0.09, 1.21 ± 0.21 and 0.47 ± 0.12, 0.83 ± 0.13 and 0.25 ± 0.07. SMO, GLI1 and Oct4 down-regulation significantly suppressed at protein levels (P < 0.05).


CONCLUSIONS
The shRNA by chemical synthesis can effectively down-regulate SMO gene expression and inhibit the proliferation of breast cancer stem cells.
The Center of Clinical Pathology, Dalian Medical University, Dalian 116044, China.
Jun
J
Mao
Pan-hong
PH
Fan
Wei
W
Ma
Qing-qing
QQ
Zhang
Bo
B
Wang
Shu-jun
SJ
Fan
Lian-hong
LH
Li
D000818:Animals; D049109:Cell Proliferation; D015536:Down-Regulation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D061986:MCF-7 Cells; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D014411:Neoplastic Stem Cells / Q000473:pathology*; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D000072086:Smoothened Receptor; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D047368:Tumor Burden; D000071676:Zinc Finger Protein GLI1
C400551:GLI1 protein, human; D018960:Hyaluronan Receptors; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D034741:RNA, Small Interfering; D043562:Receptors, G-Protein-Coupled; C491023:SMO protein, human; D000072086:Smoothened Receptor; D014157:Transcription Factors; D000071676:Zinc Finger Protein GLI1