[Effect of RNA interference inhibition to expression of CD133 on tumor cell biological characteristics in KATO-III CD133(+) cells of human gastric cancer].
OBJECTIVE
To investigate the changes in proliferation, invasiveness, clone sphere formation and chemosensitivity of human gastric cancer cell lines of KATO-III CD133(+) cells transfected with small interfering RNA (siRNA) against CD133 gene.


METHODS
CD133(+) cells of KATO-III cell lines were isolated by magnetic activated cell sorting (MACS). CD133 siRNA was designed and synthesized, and then transfected into KATO-III CD133(+) cells. Cell fluorescence counting under confocal laser scanning microscope was used to determine the transfection efficiency after transfection with the CD133 FITC-siRNA. The knock-down effect of the CD133 gene and expression of epithelial-mesenchymal transition (EMT)-related factors were detected by RT-PCR and Western blotting. Cell counting kit-8 assay (CCK-8), transwell chamber and colony sphere forming assay were performed to measure the variation of cell proliferative, invasive, colony formation viability and chemosensitivity to 5-FU after the above-mentioned treatment.


RESULTS
The transfection efficiency was (87.7±8.1)%. The CD133 mRNA and protein expression levels in the interference group were lower than those in negative control group. Twenty-four, 48 and 72 hours after transfection, cells proliferation activity was significantly inhibited in the interference group compared with negative control group, (all P<0.01). Seventy-two hours after transfection, compared with negative control group, cells proliferation activity was reduced by (52.1±8.0)%. The invasive cell number reduced (41.7±6.0 vs. 130.3±11.0, P<0.05) and clone formation rate decreased significantly [(24.3±4.3)% vs. (45.1±6.4)%, P<0.01] in the interference group. EMT-related gene E-cadherin protein expression increased, while the Snail and N-cadherin protein expression reduced in the interference group (all P<0.01). The cells sensitivity to 5-FU was significantly enhanced in the interference group, and the cell inhibition rate of 5-Fu was (62.4±3.3)%, higher than that in negative control group [(21.5±2.2)%, P<0.01].


CONCLUSIONS
The expression of CD133 gene plays an important role in cell proliferation, invasiveness, colony formation and resistance to chemotherapy of KATO-III CD133(+) gastric cancer cells. It suggests that CD133 can be used as one of surface markers for detection of gastric cancer stem cells. Inhibition of CD133 expression may be a promising way for gastric cancer biotherapy.
The First Department of General Surgery, The Third People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 201900, China. Jiang_md@hotmail.com.
Shou-lian
SL
Wang
Ji-wei
JW
Yu
Cheng
C
Cai
Rui-qi
RQ
Lu
Ju-gang
JG
Wu
Xiao-chun
XC
Ni
Bo-jian
BJ
Jiang
D000071916:AC133 Antigen; D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D005472:Fluorouracil / Q000494:pharmacology; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D014162:Transfection
D000071916:AC133 Antigen; D015703:Antigens, CD; D006023:Glycoproteins; C000606023:PROM1 protein, human; D010455:Peptides; D034741:RNA, Small Interfering; D005472:Fluorouracil