摘要
AIM： TO identify differentially expressed microRNAs （miRNAs） in human colon cancer stem cells （SW1116csc） and study their function in SW1116csc proliferation. METHODS： SW1116csc were isolated from the human colon cancer cell line, SW1116 and cultured in serum- free medium. A miRNA microarray was used to detect differential expression profiles of rniRNAs in SW1116csc and SW1116 cells. Real-time quantitative polymerase chain reaction （PCR） was performed to verify the dif- ferential expression of candidate miRNAs obtained from the microarray. Target mRNAs of differentially expressed miRNAs were predicted with target predic- tion tools, miRNA expression plasmids were transfected into SW1116csc using Lipofectamine 2000 reagent. Cell proliferation curves were generated with trypan blue staining, and the colony formation rate of transfected cells was measured with the soft agar colony formation assay. Expression of target mRNAs and proteins from differentially expressed miRNAs were detected using reverse transcription （RT）-PCR and western blotting.RESULTS： Compared with expression in SW1116 cells, 35 miRNAs （including hsa-miR-192, hsa-miR-29b, hsa-miR-215, hsa-miR-194, hsa-miR-33a and hsa- miR-32） were upregulated more than 1.5-fold, and 11 miRNAs （including hsa-miR-93, hsa-miR-1231, hsa- miRPlus-F1080, hsa-miR-524-3p, hsa-miR-886-3p and hsa-miR-561） were downregulated in SW1116csc. The miRNA microarray results were further validated with quantitative RT-PCR. miR-93 was downregulated, and its predicted mRNA targets included BAMBI, CCND2, CDKNIA, HDACS, KIF23, MAP3K9, MAP3K11, MYCN, PPARD, TLE4 and ZDHHCl. Overexpressed miR-93 sig- nificantly inhibited cell proliferation and colony forma- tion by SW1116csc. Furthermore, miR-93 negatively regulated the mRNA and protein levels of HDAC8 and TLE4. CONCLUSION： Some miRNAs were differentially ex- pressed during differentiation of SW1116csc into SW1116 cells, miR-93 may inhibit SW1116csc proliferation and colony formation.
AIM:To identify differentially expressed microRNAs(miRNAs) in human colon cancer stem cells(SW1116csc) and study their function in SW1116csc proliferation.METHODS:SW1116csc were isolated from the humancolon cancer cell line,SW1116 and cultured in serum free medium. A miRNA microarray was used to detect differential expression profiles of miRNAs in SW1116cscand SW1116 cells. Real-time quantitative polymer asechain reaction(PCR) was performed to verify the differential expression of candidate miRNAs obtainedfrom the microarray. Target mRNAs of differentially expressed miRNAs were predicted with target prediction tools. miRNA expression plasmids were transfectedinto SW1116csc using Lipofectamine 2000 reagent. Cellproliferation curves were generated with trypan bluestaining,and the colony formation rate of transfectedcells was measured with the soft agar colony formationassay. Expression of target mRNAs and proteins from differentially expressed miRNAs were detected usingreverse transcription(RT) -PCR and western blotting.RESULTS:Compared with expression in SW1116 cells,35 miRNAs(including hsa-miR-192,hsa-miR-29b,hsa-miR-215,hsa-miR-194,hsa-miR-33a and hsa-miR-32) were upregulated more than 1.5-fold,and 11miRNAs(including hsa-miR-93,hsa-miR-1231,hsa-miRPlus-F1080,hsa-miR-524-3p,hsa-miR-886-3p andhsa-miR-561) were downregulated in SW1116csc. The miRNA microarray results were further validated with quantitative RT-PCR. miR-93 was downregulated,and its predicted mRNA targets included BAMBI,CCND2,CDKN1A,HDAC8,KIF23,MAP3K9,MAP3K11,MYCN,PPARD,TLE4 and ZDHHC1. Over expressed miR-93 sig-nificantly in hibited cell proliferation and colony formation by SW1116csc. Furthermore,miR-93 negatively regulated the mRNA and protein levels of HDAC8 and TLE4.CONCLUSION:Some miRNAs were differentially expressed during differentiation of SW1116csc into SW1116cells. miR-93 may inhibit SW1116csc proliferation andcolony formation.
基金
Supported by Medical guidance projects of Shanghai Science Committee,No.10411961800
Youth Science Foundation of Fudan University,No.08FQ49
