| BackgroundOvarian cancer is one of the most common malignant tumors in women, whichis ranking the first among the leading gynecologic cancer. About70%percent ofpatients have local or distant metastases before they seek medical attention due tolack of typical symptom and effective tests to make a diagnosis, as a result, five-yearsurvival rate of patients with an advanced disease is still dissatisfied. Relapse andmetastasis always result in poor clinical outcome. Recent studies indicate that thereis a small subpopulation of cancer cells with stem cell-like characteristics, namelytumor stem cells(TSCs), which have the ability to self-renew and to differentiate intothe heterogeneous non-tumorigenic cancer cell type and to form tumor in thedistance, leading to the generation, progression, relapse and metastasis of the tumor.Strategies targeting to restrain the recurrence of TSCs would develop if the themolecular mechanism of TSCs is clear,we can inhibit TSCs proliferation andpromote its apoptosis.miRNAs are non-coding, single-stranded RNAs about18-25nucleotides longand involved in post-transcriptional gene repression via degradation of target mRNA or inhibition of translation. They are found in both plants and animals and confirmedto be involved in diverse biological response in human being due to one miRNAcould regulate more than hundreds of gene. Recent studies indicate that miRNAmutations or mis-expression correlate with various human cancers and suggest thatmiRNAs can function as tumour suppressors or oncogene via activate or inactivatethe expression of TSCs-related genes. Further investigation into the expression andfunction of miRNAs in TSCs play an important role for the study of mechanism oftumor generation and recurrence as well as prove useful approaches for thediagnosis.the treatment and the prognosis of cancer. Wang ZX et al. reported thatmiR-21was frequently high-expressed in advanced breast cancer. Patients with highexpression of miR-21were found to have a significantly poor survival. Themechanisms of miR-21predict the poor prognosis of breast cancer patients have notbeen clarified. Human miR-21located in Chromosome17q23.2, which had beendemonstrated could regulate the gene of Ki-67in breast cancer, target the tumorsuppressor RhoB in colorectal cancer cells, downregulate the gene of Pdcd4inglioblastoma, and downregulate the expression of programmed cell death4(PDCD4)in HeLa cervical carcinoma cells.result in the acceleration of tumor cellsproliferation.whatmore, miR-21could restrain caspase9and3in glioma cell,upregulate the gene of Bcl-2in MIA PaCa-2pancreatic cancer cells and target thetumor suppressor RhoB in colorectal cancer cells. result in the inhibition of tumorcells apoptosis. But the pathway in ovarian tumor stem cell has not been verified.ObjectivemiRNAs mis-expression in CD133/1+and CD133/1-subpopulation wereidentified using microarray in our previous study. The results showed that miR-21was over-expressed in CD133/1-cells compared with CD133/1+cells. To furtherdemonstrate the expression of mature miR-21in CD133/1+cells, and study theeffect of miR-21on proliferation and apoptosis of CD133/1+cells, so as to provideexperimental evidence for the development of TSCs targeting therapeutic strategiesfor ovarian cancer. Methods1) CD133/1+cells sorting: Human ovarian cancer cell line OVCAR3was grown inDMEM, cells in logarithmic phase were trypsinized and resuspended in PBS, thenincubated with Mouse anti-human CD133/1-PE monoclonal antibodies following themanufacturer’s instruction. After finishing staining, BD FACSAria was use to sortCD133/1+cells and CD133/1-cells.2) The expression level of mature miR-21in CD133/1+and CD133/1-cells: TotalRNA from CD133/1+and CD133/1-cells was extracted with TriZol Reagent, Firststrand cDNA synthesis and amplification was performed using a stem-loop primers,SYBR Green quantitative PCR amplifications were performed in a7500SequenceDetection System, U6snRNA as an endogenous control.3) MTT assay: MTT kit was used to investigate the proliferation of CD133/1+cellstransfected with miR-21mimic or negative control.4) EdU assay: EdU kit was used to investigate the proliferation of CD133/1+cellstransfected with miR-21mimic or negative control.5) Mitochondrial membrane potential experimental determination: Rhodamine123was used to investigate the mitochondrial membrane potential of CD133/1+cellstransfected with miR-21mimic or negative control，Through further analyze theapoptosis of CD133/1+cells.6) The cell apoptosis assay:Annexin V-FITC Apoptosis Detection Kit was directlyused to research the level of the apoptosis in different groups transfected withmiR-21mimic or negative control.7) Statistic analysis:Results were calculated as mean±SD.SPSS16.0was used for allanalysis,and P<0.05was considered statistically significant.Results1. The proportion of CD133/1positive in OVCAR3cell line was0.3%.2. The expression level of mature miR-21in CD133/1+cells was much less thanCD133/1-cells（1.00±0.00vs2.16±0.25，P=0.015）. 3. MTT result showed that the value of OD in miR-21group was distinctly reduced,Compared with negative control and blank group, the difference was significant（P<0.05）. the value of OD was approximate between negative control and blankgroup, the difference was meaningless（P>0.05）.4. Effects of miR-21on proliferation of CD133/1+cells: In EdU experiment,the cellnumber that is proliferating in miR-21group(48.0±5.3) was significantly less thanthat in negative control group (117±6.4) and blank group(122±4.9)．So eachrespectively，the rate of increase is30.4%,47.5%,48.6%，The proliferation ofCD133/1+cells treated with miR-21was weakened than the other twogroups(P<0.05).5. The outcome of Mitochondrial membrane potential experimental determinationshowed that the relative fluorescence intensity in miR-21group that was (85137±276) was significantly less than that in negative control group (98365±362) andblank group(98887±259).the mitochondrial membrane potential of CD133/1+cellstreated with miR-21was declined than the other two groups(P<0.05).6. The outcome of Annexin V-FITC Apoptosis Detection Kit showed that the rate ofapoptosis in miR-21group (16.7%) was higher than that in negative controlgroup(9.7%) and blank group (9.3%), the difference was significant（P<0.05）.Conclusions1. There was a small subset of CD133/1+TSCs in ovarian cancer OVCAR3cell line,mature miR-21was low-expressed in CD133/1+TSCs.2. Over-expression of miR-21in CD133/1+TSCs inhibited its proliferation.3. Over-expression of miR-21in CD133/1+TSCs induced its apoptosis. |
