Inhibition Of CD147 Expression By RNA Interference Reduces Proliferation,invasion And Increases Chemosensitivity In Cancer Stem Cell-like HT-29 Cells

Aim:To explore the biological functions of CD 147 in colorectal cancer stem cell-like cells,we use RNA interference for CD 147 knockdown.Methods:1)The human Oct4 promoter was amplified and cloned into the plasmid pEGFP-N1;2)The recombinant plasmid Oct4-EGFP was trasfected into human colorectal cancer cells line HT-29 and screened the stable cells with G418;3)EGFP+cells were pooled and sorted with a flow cytometry,and then assessed the expression of CSC surface marker CD44;4)The recombinant plasmid pYr-mir30-shRNA was transfected into HEK293 cell line with Lipo 2000,named Ad-shRNA and Ad-shRNA-control,respectively;5)The expression of CD 147 was tested by RT-PCR and western blot after infecting the CSC-like cells with Ad-shRNA and Ad-shRNA-control,respectively;6)The proliferation and chemosensitivity of CSC-like cells were detected by MTT assay;7)The invasion of CSC-like cells was assessed by Transwell assay.Results:1)The recombinant plasmid Oct4-EGFP was constructed successfully,and the positive cells were selected by G418;2)EGFP+cells were sorted with a flow cytometry,and the expression level of CD44 of these cells up to 99.0%;3)After adenovirus infecting,the results of RT-PCR and western blot showed that the expression of CD 147 was significantly reduced at both mRNA and protein levels;4)MTT assay showed that compared with control groups,the proliferation of Ad-shRNA group was inhibited to 42.4%?43.7%?59.3%and 61.1%at 24,48,72 and 96 h,respectively.shRNA mediated CD147 silencing increased the sensitivity of CSC-like cells to gemcitabine,docetaxel and cisplatin at 0.1,1 and 10 ?M(P<0.05).The results showed that there was no significant difference among the three groups to paclitaxel at 10 ?M(P=0.19).5)The invasion of Ad-shRNA group(31.5± 3.5)was obviously decreased compared with control groups(95.5±4.5).Conclusions:We successfully sorted the CSC-like cells from human colorectal cancer cell line HT-29.The results suggested that silencing the expression of CD 147 can significantly inhibited the proliferation and invasion of CSC-like HT-29 cells,but increased the chemosensitivity to gemcitabine,cisplatin,docetaxel and paclitaxel.A novel and promising therapeutic approach of targeted therapy may be developed based on CSC.