| Background:Epithelial ovarian cancer (EOC) is the most lethal gynaecologic malignancy, the5-year survival rate is only about30%in patients with advanced stage. Currently, cytoreductive surgery combined with platinum-based chemotherapy were efficient in almost70%of advanced cases at initial treatment, but most of these patients will eventually develop chemo-resistance and recurrence. Therefore, an in-depth understanding of EOC and effectively improve its poor prognosis is needed.Recent studies have identified the close relationship between chemo-resistance and epithelial mesenchymal transition (EMT) in EOC. EMT consists of a reversible change of cell phenotype during which epithelial cells loosen cell-cell adhesion structures including adherens junctions and desmosomes, modulate their polarity and rearrange their cytoskeleton, transformed into mesenchymal cell phenotype. Previous studies have demonstrated EMT played a central role in regulating tumor invasion, metastasis and chemoresistance. Most importantly, recent studies have found that EOC cancer stem cells (CSCs) harboured post-EMT characteristics. CSCs theory believes:tumor is composed of a heterogeneous population of cells; in which very small amount of subpopulation with stem cell properties were the root of tumor progression, metastasis and chemoresistance. Therefore, inhibition of EMT may be a promising strategy to improve the prognosis of EOC.MicroRNA (miRNA, miR) is a large class of small noncoding RNAs; approximately18-24nucleotides (nt) in length, as critical post-transcriptional regulators of gene expression through targeting mRNA3’untranslated region (3’-untranslated region,3’-UTR) base pair recognition. A growing body of evidence indicated that miRNAs played an important role in the regulation of EMT, and aberrant miRNAs profile were the defining feature in many types of tumours. Integrated genomic analyses confirmed that mesenchymal phenotype was associated with poor prognosis of EOC, and miR-200a, miR-141and miR-506were included in the eight key miRNAs predicted to target the mesenchymal subtype in459cases of serous ovarian cancer.MiR-200a and miR-141are belong to the miR-200family (miR-200s, including miR-200a/b/c, and miR-141/429of5miRNA). MiR-200s, which are significantly involved in inhibition of EMT, repression of CSCs self-renewal, differentiation, modulation of cell division, apoptosis and reversal of chemoresistance in many types of human cancer. Accumulating evidences showed that miR-200s were associated with progression-free survival (PFS), overall survival(OS) and chemo-sensitivity in EOC. These foundings showed that investigation miR-200s as potential therapeutic strategy for EOC, has theoretical feasibility and positive clinical significance.Studies have reported that overexpression of miR-200c may increase EOC cell line Hey’s. sensitivity to paclitaxel by targeting TUBB3. Previously, we verified CD133+cells have characteristics of CSCs in EOC cell lines-OVCAR-3; further demonstrated that the profile of miRNAs was different between CD133+and CD133-cells; in which miR-200a was down-regulated about2.16times in CD133+cells compared with CD133-cells, additionally, restoration of miR-200a expression by synthetic miR-200a mimics could reduce the ZEB2-mediated migration and invasion of CD133+ovarian CSCs, which initially confirmed miR-200a has a positive effect in inhibiting metastasis in EOC. However, miR-200a regulation of chemotoxicity, CSCs phenotype and its clinical significance in EOC remains unclear.We found transient transfection of miR-200a mimics was only suitable to observe the effects of miR-200a on ovarian cancer cells in a short term; additionally, the percentage of CD133+cells by sorting is very low (about0.2%～1%); furthermore, CD133+cells will gradually differentiate into CD133-cells in vitro, these limitations will impact our in-depth explore the biological function of miR-200a in the chemosensitivity of EOC. Recently, Wang et al. found that SDCs (spheriod derived cells) formed from OVCAR-3cells growth in serum-free culture medium, maintained CSCs characteristics and were ideal models in vitro for CSCs-targeted investigations in EOC.Considering the above factors, herein we first constructed EOC cells model, in which miR-200a was stably over-expressed via a lentiviral expression vector, then verified the role of miR-200a on chemosensitivity in both normal2D and3D culture; then further explored the role of miR-200a in regulation proliferation, CSCs and its mechanisms, in order to bring more powerful treatment strategies resulting in improving the prognosis of EOC.Chapter1Establishment miR-200a over-expression EOC cells model Objective:Establishment of miR-200a over-expression EOC cells via a lentiviral expression vector in both2D and3D culture, for further exploring the biological role and mechanism of miR-200a in EOC.Methods:1. Identification of miR-200a over-expression lentiviral vector and control empty vector.Over-expression of miR-200a lentivirus vector pLV-miR-200a and control empty vector pLV-control were purchased from Shenzhen, China Anping Kang Biotechnology Co., Ltd., through plasmid transformation, plasmid amplification, plasmid extraction, PCR amplification, DNA electrophoresis and DNA sequencing, identification miR-200a expression lentiviral vector and control empty vector.2. Packaging miR-200a over-expression and control lentivirus.Lentiviral packaging components psPAX2and pMD2.G were kept by the Southern Medical University Cancer Institute. Lentiviral packaging systems was composed by three plasmids pLV-miR-200a or pLV-control, psPAX2and pMD2.G.293T cells transfected with lentiviral packaging system by Lipofectamine2000, were utilized to produce lentivirus carrying the vector over-expression of miR-200a and control empty vector.3. Infection EOC cells by miR-200a over-expression and control lentivirus.Infection EOC cell line OVCAR-3by miR-200a over-expression and control lentivirus, then monitor the infection efficiency by green fluorescent protein (GFP) expression under inverted fluorescence microscope, and sorting GFP+cells by flow cytometry.4. Verify miR-200a expression level in EOC cells after infection.Extraction total RNA from infected EOC cells, then detection the level of miR-200a by qRT-PCR. Frozen and expanded miR-200a over-expression ovarian cancer cells and control cells for subsequent experiments.5. Establish suspended growth model of EOC cells over-expression of miR-200a.Collecting miR-200a over-expression EOC cells and control cells, washing twice with PBS, resuspending in tumor sphere medium (containing EGF20ng/ml, bFGF lOng/ml,0.4%BSA, insulin5μg/ml,100U/ml penicillin and100U/ml streptomycin DMEM/F12), inoculated at ultra-low petri dishes with conventional conditions, adding appropriate amount of tumor sphere medium72h later, collecting spheroid cells7days later. The collected cells were digested with0.02%EDTA-2Na and a1:1mixture of0.25%trypsin at37℃at about lmin to form single cells, re-suspend in tumor sphere medium and continue culture with the above method, then obtaining suspended growth of EOC cells. Detected the level of miR-200a in EOC cells grown in suspension by qRT-PCR.6. Statistical Analysis. Statistical analyses were performed using SPSS version16.0, one-sample t-test was utilized to analyze the differential expression of miR-200a in adherent and suspension growth of ovarian cancer cells, the definition of significant level α=0.05, P<0.05was considered statistically significant, all data are presented as "mean values±SD". Results:1. DNA sequencing confirmed that the sequence of miR-200a over-expression lentiviral vector and control empty vector were correct.2. Successfully produced miR-200a over-expression and control lentivirus for infection EOC cell line OVCAR-3. The expression of GFP was approximately20%-30%under inverted fluorescence microscope, after sorting GFP+cells by FACS, the infection efficiency was approximately95%.3. Obtained miR-200a over-expression adhesive cells model of EOC. qRT-PCR validation the expression level of miR-200a increased about (3.67±0.45) times compared with the control, and the difference between the two group was statistically significant(t=10.187, P=0.009).4. Obtained miR-200a over-expression suspended spheroid cells model of EOC. qRT-PCR validation the expression level of miR-200a increased about (2.05±0.39) times compared with the control, and the difference between the two group was statistically significant (t=4.666, P=0.043).Conclusion:1. Successfully integrated miR-200a into EOC cells, and achieved its high expression. Establishing a stable miR-200a over-expression EOC cells in the presence of adherent and suspended growth.Chapter2The roles and mechanisms of miR-200a regulation on chemosensitivity, proliferation and CSCs in EOCSection1The role of miR-200a regulation on chemosensitivity in EOC Objective:Verifying the relationship between miR-200a and the chemo-sensitivity of paclitaxel and cisplatin in adherent and suspension growth of EOC cells model.Method:1. MTT detection of miR-200a regulation on chemosensitivity in adherent growth of EOC cells.MiR-200a over-expressing or control adherent growth of EOC cells were collected and resuspended in RPMI1640containing10%FBS medium.5×103miR-200a over-expressing or control adherent growth of EOC cells were seeded in normal96-well plates, respectively, routine cultured12-16h later, paclitaxel (final concentration:2nM,4nM,8nM,16nM,32nM) or cisplatin (final concentration:1ug/mL,2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mL), were added in the cells, each concentration was repeated four holes,48h after drug treatment, added20νL of cell viability assay MTT (5mg/mL) to each well, continue regular culture4h, the supernatant was carefully discarded and added100uL DMSO in each well, after mixing in dark, absorbance values (OD) of each well was measured by enzyme-linked detector, detection wavelength was490nm. Cell viability (SR) according to the following formula:(the average OD of dosing wells/the average OD of control wells) x100%. Draw viability curves with SR value in each group. This experiment was repeated three times.2. MTT detection of miR-200a regulation on chemosensitivity in suspension growth of EOC cells.MiR-200a over-expressing or control suspension growth of EOC cells were collected and resuspended in tumour spheroid medium,1×104miR-200a over-expressing or control suspension growth of EOC cells were seeded in ultra-low attach96-well plates, respectively, routine cultured24h later, paclitaxel (final concentration:2nM,8nM,32nM,128nM,512nM) or cisplatin (final concentration:2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mL), were added in the cells, each concentration was repeated four holes.72h after drug treatment, added20μL of cell viability assay MTT (5mg/mL) to each well, continue regular culture4h, box-type centrifuge3000rpm,15min, then visible tumor cells aggregates in the side plates, using1mL syringe suction supernatant carefully, added100uL DMSO in each well, after mixing in dark, absorbance values (OD) of each well was measured by enzyme-linked detector, detection wavelength was490nm. Cell viability (SR) according to the following formula:(the average OD of dosing wells/the average OD of control wells) x100%. Draw viability curves with SR value in each group. This experiment was repeated three times. 3. Statistical Analysis. Statistical analyses were performed using SPSS version16.0, factorial design analysis of variance and two independent samples t-test were utilized to analyze the differences of cell viability in cytotoxicity tests. P<0.05was considered statistically significant, data are presented as "mean values±SD".Results:1. The cell survival rate (%) of miR-200a over-expressing adherent growth of EOC cells and control cells, after treated with a final concentration of paclitaxel:2nM,4nM,8nM,16nM,32nM, were (85.86±4.16vs.87.80±2.22),(74.66±3.19vs.80.62±2.60),(65.85±4.24vs.76.15±4.46),(54.91±4.43vs.69.53±3.31),(54.62±1.76vs.66.91±3.41), respectively. These results showed that, the difference of paclitaxel cytotoxicity between miR-200a over-expressing adherent growth of EOC cells and control cells, was statistically significant (F=63.464, P<0.001).The cell survival rate (%) of miR-200a over-expressing adherent growth of EOC cells and control cells, after treated with a final concentration of cisplatin (final concentration:1ug/mL,2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mL), were (84.26±5.50vs.90.73±5.19),(71.95±6.68vs.66.96±3.57),(66.46±2.89vs.60.52±4.93),(57.48±3.70vs.54.45±2.63),(48.91±2.40vs.51.48±1.52),(43.54±4.34vs.45.87±4.19), respectively. These results showed that, the difference of cisplatin cytotoxicity between miR-200a over-expressing adherent growth of EOC cells and control cells, was not statistically significant (F=0.247, P=0.622).2. The cell survival rate (%) of miR-200a over-expressing suspension growth of EOC cells and control cells, after treated with a final concentration of paclitaxel:2nM,8nM,32nM,128nM,512nM, were(69.27±3.63vs.83.43±4.01),(57.03±2.88vs.78.91±0.86),(57.47±1.31vs.80.89±1.29),(57.69±1.35vs.80.1±1.80),(55.17±0.60vs.77.96±3.75), respectively. These results showed that, the difference of paclitaxel cytotoxicity between miR-200a over-expressing suspension growth of EOC cells and control cells, was statistically significant (F=721.813, P<0.001).The cell survival rate (%) of miR-200a over-expressing suspension growth of EOC cells and control cells, after treated with a final concentration of cisplatin (final concentration:2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mL), were (50.80±2.35vs.54.37±1.86),(42.64±2.80vs.41.71±11.62),(49.66±2.03vs.46.92±6.37),(46.84±7.72vs.51.57±5.25),(42.66±2.98vs.45.54±5.36), respectively. These results showed that, the difference of cisplatin cytotoxicity between miR-200a over-expressing suspension growth of EOC cells and control cells, was not statistically significant (F=1.191, P=0.284).Conclusion:Over-expression of miR-200a increased the sensitivity of adherent and suspension growth of EOC cells to paclitaxel, without affecting the sensitivity of adherent and suspension growth of EOC cells to cisplatin.Section2The role of miR-200a regulation on proliferation in EOCObjective:Given miR-200a enhanced paclitaxel sensitivity in adherent and suspension growth of EOC cells, but no such effect to cisplatin. Considering paclitaxel is a drug targeting cell cycle, while cisplatin is not a cell cycle dependent drug, we intended to further explore the effects of miR-200a on proliferation in EOC through a series in vitro and in vivo assays.Methods:1. Colony forming assay was utilized to detect the impact of miR-200a on the growth state of adherent EOC cells.MiR-200a over-expressing or control adherent growth of EOC cells were collected and resuspended in RPMI1640containing10%FBS medium.1.5×102over-expression miR-200a or control ovarian cancer cells, were seeded in normal6-well plates, respectively, three wells in each group, routine cultured10d, observed the morphology of cell clones, calculating the number of clone more than50cells, cloning efficiency=(clone number/seeded cells)×100%. Discard cells medium, carefully rinsed cells with PBS three times, fixed with methanol lOmin, hematoxylin5-10min, carefully rinsed cells with water, photographed cloning appearance.2. Growth curve (CCK8) assay was utilized to detect the impact of miR-200a on the growth state of adherent and suspended EOC cells.MiR-200a over-expressing or control adherent growth of EOC cells were collected and resuspended in RPMI1640containing10%FBS medium.2.0×103over-expression miR-200a or control adherent growth of EOC cells were seeded in normal96-well culture plates, respectively, six wells in each group, when cultured6h,1,2,3,4,5,6d, adding CCK8with culture medium mixture110μL (ratio of1:10),37℃cultured1.5h, absorbance values (OD) of each well was measured by enzyme-linked detector, detection wavelength was450nm, blank control wells as zero, draw growth curves with the average OD value from6wells in each group.MiR-200a over-expressing or control suspension growth of EOC cells were collected and resuspended in tumour spheroid medium.4.0×103over-expression miR-200a or control suspended growth of ovarian cancer cells were seeded in ultra-low attach96-well culture plates, respectively, six wells in each group, when cultured1,2,3,4,5,6,7d, adding10μL CCK8,37℃cultured4h, absorbance values (OD) of each well was measured by enzyme-linked detector, detection wavelength was450nm, blank control wells as zero, draw growth curves with the average OD value from6wells in each group.3. Flow cytometry was utilized to detect the impact of miR-200a on the cell cycle progression of adherent and suspended EOC cells.Collecting over-expression miR-200a and control EOC cells growth in adherent and suspension, respectively, rinsed twice with cold PBS, adding cold70%ethanol,4℃fixed overnight or-20℃long-term fixed, cells were rinsed once with PBS before detection, incubated with a solution containing50μg/mL ethidium bromide (PI),100μg/mL RNase A,0.2%Triton X-100in500μL PBS for30min at room temperature in the dark, using a standard procedure by flow cytometry, counting from10,000to20,000cells, the results were intended by the cell cycle software ModFit analysis.4. In vivo xenograft experiment was utilized to detect the impact of miR-200a on the proliferation of EOC cells. Selected4-5week-old female BALB/c-nu/nu mice as transplant objects, subcutaneously transplanted nude mice back at right and left side with over-expression miR-200a and control EOC cells growth in adherent, respectively. Each point transplantation1.5x106cells, the total volume of the cell suspension was1100μL. The number of nude mice used in this experiment was eight. Observed the general status, measured the growth of mass in vaccination site, and record the weight of nude mice every three days. Timely drawn and correlation analysis based on tumor growth.5. Immunohistochemistry assay was utilized to detect the expression of Ki67in transplanted tumor tissue, photographed under an optical microscope, randomly selected three high-power fields, calculate the proportion of positive cells per field. The percentage of positive cells per field=the number of positive cells/total cells per field×100%.6. Statistical Analysis. Statistical analyses were performed using SPSS version16.0, two independent samples t-test was utilized to analyze the differences of colony formation rate, Ki67-positive rate and cell cycle distribution, and factorial design analysis of variance was utilized to analyze the differences of growth curves (CCK8) and xenograft in nude mice. P<0.05was considered statistically significant, all data are presented as "mean values±SD".Results:1. Colony forming assay shown, over-expression miR-200a adherent growth of EOC cells became smaller in size, arranged closer, more inclined epithelial phenotype compared with control group. The colony formation rate(%) of over-expression miR-200a adherent growth of ovarian cancer cells and control cells was (92.52±5.60vs.79.12±4.41). Statistical analysis showed that the difference of colony formation rate between miR-200a over-expressing adherent growth of EOC cells and control cells, was statistically significant (t=-4.461, P=0.001).2. Growth curves (CCK8) experiment shown, the OD value of miR-200a over-expressing adherent growth of EOC cells and control cells, cultured in8h,1,2,3,4,5,6d, were (0.278±0.008vs.0.275±0.002),(0.500±0.096vs.0.433±0.035), (0.624±0.058vs.0.528±0.046),(1.028±0.115vs.0.710±0.048),(1.530±0.160vs.1.000±0.171),(1.993±0.206vs.1.239±0.159),(2.450±0.233vs.1.428±0.161), respectively. Statistical analysis showed that, the difference of cell growth between miR-200a over-expressing adherent growth of EOC cells and control cells, was statistically significant (F=199.507, P<0.001).The OD value of miR-200a over-expressing suspended growth of EOC cells and control cells, cultured in1,2,3,4,5,6,7d, were (0.218±0.015vs.0.216±0.013),(0.333±0.013vs.0.323±0.014),(0.462±0.028vs.0.437±0.021),(0.720±0.078vs.0.565±0.050),(0.952±0.081vs.0.688±0.062),(1.149±0.116vs.0.803±0.054),(1.434±0.135vs.0.957±0.080), respectively. Statistical analysis showed that, the difference of cell growth between miR-200a over-expressing suspended growth of EOC cells and control cells, was statistically significant (F=159.036, P<0.001).3. Flow cytometry cell cycle analysis shown, G0/G1, S, G2/M proportion (%) in over-expression miR-200a adherent growth of EOC cells and control cells, were (55.86±2.55vs.72.41±3.22),(26.15±2.30vs.21.76±2.11),(13.52±1.99vs.6.82±0.76), respectively. Statistical analysis showed that, the difference of G0/G1,S, G2/M ratio between miR-200a over-expressing adherent growth of EOC cells and control cells (t=9.861, P<0.001),(t=-3.445, P=0.006),(t=-7.686, P<0.001) were statistically significant.The G0/G1, S, G2/M proportion (%) in over-expression miR-200a suspended growth of EOC cells and control cells, were (69.52±3.24vs.90.48±2.36),(21.35±2.10vs.5.49±0.52),(11.31±1.43vs.3.32±0.28), respectively. Statistical analysis showed that, the difference of G0/G1, S, G2/M ratio between over-expression miR-200a suspended growth of EOC cells and control cells (t=11.470, P<0.001),(t=-17.926, P=0.006),(t=-13.386, P<0.001) were statistically significant.4. In vivo xenograft experiment shown, both miR-200a overexpression EOC cells and control cells could form xenograft in nude mice. The tumor volume (mm3) of over-expression miR-200a and control group after subcutaneous inoculation transplantation50,53,56,59,62d, were (110.27±57.84vs.58.19±19.55),(146.88±73.64vs.67.44±32.58),(167.03±75.68vs.82.25±48.15),(234.01±80.85vs.108.74±42.98),(366.36±99.87vs.137.44±43.13), respectively. Statistical analysis showed that the difference of tumor volume between over-expression miR-200a and control group, was statistically significant (F=63.44, P<0.001).5. Immunohistochemistry assay shown, the positive expression rate of Ki67(%) between over-expression miR-200a xenograft and control was (70.91±5.18vs.15.21±3.52). Statistical analysis showed that the difference positive expression rate of Ki67between over-expression miR-200a and control group, was statistically significant (t=-21.156, P<0.001).Conclusion:Over-expression miR-200a increased EOC cells cloning efficiency, promoted cells growth, induced cells enter into cell cycle, and promoted the subcutaneous xenograft growth in nud mice, suggesting miR-200a faciliated the proliferation of EOC cells, and this effect of promoting proliferation may be one of the mechanisms involved in enhancing the chemosensivitity of paclitaxel regulated by miR-200a.Section3The roles and mechanisms of miR-200a regulation on CSCs in EOCObjective:The above findings showed miR-200a has a certain influence on paclitaxel sensitivity and proliferation in EOC cells. Then we intended to further explore the effects of miR-200a regulation on CSCs in EOC through tumor sphere formation assay, detection the side population (SP) by FACS, and validation stem-related genes expression regulated by miR-200a.Methods:1. Tumor sphere formation assay was utilized to detect the impact of miR-200a on the self-renewal capacity in EOC cells.MiR-200a over-expressing or control suspension growth of EOC cells were collected and resuspended in tumour spheroid medium,1×103over-expression miR-200a or control EOC cells were seeded in ultra-low attach24-well culture plates, respectively, three wells in each group, routine cultured7d, observed the morphology of tumor spheroids, calculating the number of tumor spheroids diameter≥70μm. The experiment was repeated three times.2. Investigation the proportion of side population (SP) by FACS was utilized to detect the impact of miR-200a on CSCs ratio.Collecting miR-200a over-expression EOC cells and control cells, resuspended in RPMI1640medium containing2%FBS preheated to37℃, cells’density was adjusted to1×106/mL. Prepare two bottles of cells in each group, Hoeehst33342was added with final concentration of5ug/mL in all four bottles of cells, randomly selected one bottle of cells in each group and verapamil was added with final concentration of50μmol/L, incubated at37℃shaker for90min in the dark, and terminated the reaction on ice.4℃1000rpm centrifuge3min, supernatant was removed, washed with pre-cooling PBS once.40μm filter filtering cells before detection, propionate iodide (PI) was added with final concentration1μg/mL to label dead cells. SP ratio was detected by FACS, Hoeehst33342excitation wavelength350nm,405/30bandpass collected blue,570/20bandpass collect red, PI was excitated by488nm blue,630/30bandpass collected red. Dead cells were directly removed by PI staining, Hoechst Red X-axis, Hoechst Blue Y-axis for the two-dimensional scatter plot. Low Hoechst Red and low Hoechst Blue and verapamil missing region was SP. Comparison the proportion of SP between miR-200a over-expression EOC cells and control cells.3. qRT-PCR and Western Blot were utilized to detect the impact of miR-200a on regulation CSCs related genes expression.qRT-PCR detection method is as follows, total RNA in miR-200a over-expression EOC cells and control cells was extracted, then reversed to cDNA, and qRT-PCR detected (SYBR method) SOX2and OCT4mRNA levels, GAPDH used as an endogenous control.Western blot methods is as follows:the proteins in over-expression miR-200a or control EOC cells were extracted with RIPA lysis buffer, BCA protein assay kit was employed to assess protein concentrations, protein samples (30μg per lane) were resolved using SDS-PAGE, and then transferred to PVDF membranes, after blocking the nonspecific binding sites with3%BSA, PVDF membranes were incubated with primary antibodies at4℃overnight, specifically, rabbit monoclonal antibody to SOX2and OCT4, followed by incubation with the corresponding secondary antibodies at room temperature for1h, immunoblots were visualized using an enhanced chemiluminescence (ECL) Western Blot Detection System, P-actin used as an endogenous control.4. Statistical Analysis. Statistical analyses were performed using SPSS version16.0, two independent samples t-test was utilized to analyze the differences of tumor sphere formation and the ratio of SP, one-sample t-test was utilized to analyze the different expression of SOX2and OCT4by qRT-PCR and Western Blot. P<0.05was considered statistically significant, all data are presented as "mean values±SD".Results:1. Tumor sphere formation assay showed, over-expression of miR-200a induced the attachment of suspended tumor spheroid cells, even in ultralow attachment dishes, and showed a gradual proliferation state, which could not be seen in control groups. The number of tumor spheroids(/1,000cells) in over-expression miR-200a and control EOC cells was (7.17±1.17vs.17.50±1.87). Statistical analysis showed that, the difference number of tumor spheroids between over-expression miR-200a and control EOC cells was statistically significant (t=11.474, P<0.001).2. SP ratio by FACS experiments show that two groups of cells, the lower left corner show low fluorescence or negative cell populations, after adding verapamil these cells disappear. SP ratio (%) of over-expression miR-200a and control EOC cells was (0.233±0.076vs.0.850±0.100). Statistical analysis showed that, the differences of SP ratio between over-expression miR-200a and control EOC cells was statistically significant (t=8.488, P=0.001).3. qRT-PCR showed that SOX2and OCT4mRNA levels in over-expression miR-200a EOC cells, decreased to (0.467±0.085) and (0.361±0.064) times, respectively, compared with control. Statistical analysis showed that, the difference mRNA levels of SOX2and OCT4between over-expression miR-200a EOC cells and control cells (t=10.932, P<0.001),(t=17.416, P=0.003) were statistically significant.Western Blot results Show that SOX2and OCT4levels in over-expression miR-200a EOC cells, decreased to (0.546±0.077) and (0.518±0.086) times, respectively, compared with control. Statistical analysis showed that, the difference levels of SOX2and OCT4between over-expression miR-200a EOC cells and control cells (t=10.191, P=0.001),(t=9.659, P=0.011) were statistically significant.Conclusion:miR-200a weakened the stem characteristics of CSCs in EOC by blocking the formation of tumour spheroids, inducing the differentiation process in3D culture, reducing the proportion of CSCs and down-regulating the CSCs related genes SOX2and OCT4. |
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