ABSTRACT
ObjectiveTo identify the presence of side population (SP) cells in human ovarian cancer cell line OVCAR-3 and to investigate whether SP cells have the characteristics of cancer stem cells.MethodsSP and non-SP (NSP) cells from OVCAR-3 were isolated by fluorescence-activated cell sorting after being stained by DNA-binding dye Hoechst 33342.Limiting dilution transplantation assay,realtime PCR,and drug sensitivity assay were performed to compare the tumorigenic ability,differentiation ability in vivo,the mRNA expressiou of stemness marker (Oct-4,Klf4,and Nanog) and ATP-binding cassette (ABC) transporter (ABCG2,ABCB1,and ABCC2),and response to multiple drugs (cisplatin,paclitaxel,doxorubicin,and mitoxantrone )between SP and NSP cells.ResultsA few of SP cells [ ( 1.13 ±0.39) ％ ] which were sensitive to reserpine were identified in OVCAR-3 cells.The injection of as few as 102 SP cells initiated tumors in two of five mice.Tumor latency was 52 -61 days.However,the NSP cells did not generate any tumors in mice until 104 NSP cells were injected (two of five mice).Tumor latency was 64 - 98 days.Tumorigenicity of SP cells was enhanced by at least 100-fold than that of NSP cells.The SP cells regenerated both SP [ ( 2.09 ± 0.73 ) ％ ] and NSP populations in vivo with a fraction size that was comparable to the original population.The mRNA expression ofstemness genes Oct-4,Klf4 and ABC transporters ABCG2,ABCC2 genes were elevated in SP cells compared to NSP cells,the fold changes were 1.95±0.41 (P＜0.05),4.26 ±0.63 (P＜0.01),3.22±0.36 (P＜0.01),and 1.76±0.26 (P＜0.01 ),respectively.The relative activity of SP and NSP cells were 0.757 ± 0.105 versus 0.474 ± 0.035 (P＜0.01),0.521 ±0.092 versus 0.384 ±0.073 (P＜0.05),0.742 ±0.051 versus 0.526 ±0.088 (P ＜0.01 ),and 0.690 ± 0.096 versus 0.466 ± 0.112 ( P ＜ 0.01 ) when they exposed to 0.25 μg/ml cisplatin, 0.01μmol/Lpaclitaxel, 0.25μmol/Ldoxorubicin, and0.05μg/mlmitoxantrone,respectively.ConclusionsSP cells from OVCAR-3 have enhanced self-renewal,differentiation,and tumorinitiating capacity compared to NSP cells.The mRNA expression of stemness genes and ABC transporters are markedly elevated in SP cells,which showed resistance to multiple chemotherapeutic drugs and have characteristics of cancer stem-like cells.Therefore,SP phenotype could be used as a marker to isolate the cancer stem-like cells in ovarian cancer.
ABSTRACT
Objective To identify differentially expression of microRNAs associated with expression of estrogen receptor α(ERα)and progesterone receptor(PR)between type Ⅰ and type Ⅱ endometrial adenocarcinoma.Methods Two kinds of endometrial adenocarcinoma cell lines,Ishikawa and KLE,was transplanted into node mice and biopsied to identify the expression of ERα,PR and p53,and test their response to estrogen and progesterone.Cultured the two cell lines under the estrogen-free and progesteronefree circumstance,total RNA was isolated to identify the differentially expressed microRNAs by microarray for prediction the microRNAs which target ESR1 and PGR by software miRANDA and TargetScan,and then was validated by real-time PCR in two cell lines cultured both in vivo and in vitro and ten specimens from patients.Results Ishikawa cell line was confirmed from type Ⅰ endometrial adenocarcinoma.KLE cell line was confirmed from typeⅡendometrial adenocarcinoma.One hundred and twenty-six differentially expressed microRNAs between the two cell lines were identified by mieroRNA microarray,among of which may target ESR1 inchded hsa-miR-100,99a,and may tgrget PGR included hsa-miR-378,768-3p-The differential expression of hsa-miR-100,99a,378,768-3p identified by microarray between Ishikawa and KLE in vivo and in vitro was equal to that by real-time PCR,while Hsa-miR-100 was significantly down expressed in type Ⅰ group specimens compared to type Ⅱ group(P＜0.01).Conclusion Hsa-miR-100 is significantly down-expressed in type Ⅰ endometrial adenocarcinoma compared to type Ⅱ,which may be a great potential to target ESR1.