| Ovarian cancer is a gynecological malignancy in women that leads to high mortality rate due to metastatic progression and recurrence. Mi RNAs are small endogenous noncoding RNAs as tumor suppressors or oncogenes in various human cancers. We investigated the role of mi R-203 in ovarian cancer and found that mi R-203 was downregulated while its target gene snai2(slug) was upregulated in human ovarian serous carcinoma compared to normal ovary controls. In addition,mi R-203 expression was associated with long-term survival rate of ovarian cancer patients. Lentiviral mediated mi R-203 expression inhibits cell proliferation, migration and invasion in SKOV3 and OVCAR3 cells. Furthermore, mi R-203 inhibits epithelial to mesenchymal transition(EMT) and counteracts the transforming growth factor β(TGFβ)-induced EMT in ovarian cancer cells. Silencing Snai2 using lentiviral sh RNA mimics mi R-203 mediated EMT inhibition and tumor cell invasion.Xenografting mi R-203 expressing ovarian cancer cells in immunodeficient mice leads to significantly reduced tumor initiation and growth. Our data demonstrate that mi R-203 functions as a tumor suppressor by downregulating Snai2 in ovarian cancer.We analyzed the leptin expression in ovarian cancer tissues from patients by using western blot, PCR and ELISA assay. We found that leptin expressed higher in ovarian carcinoma than normal ovaries.ObjectiveWe evaluated the important role of mi R-203 and leptin in ovarian cancer patients,and in SKOV3 and OVCAR3 cell lines, the result demonstrated that mi R-203 is a tumor suppressor and inhibits cell proliferation, migration, and invasion as well as inhibits tumor growth in xenograft mouse model by directly targeting mesenchymal marker Snai2. Both leptin and mi R-203 play important roles and have obviously correlated to the prognosis of ovarian cancer patients.MaterialsThe ovarian cancer cell lines SKOV3 and OVCAR3 were obtained from ATCC and cultured in Dulbecco’s Modified Eagle Medium(DMEM) supplemented with10% FBS(Hyclone; Logan, UT), 100 U/ml penicillin, and 100 μg/ml streptomycin(Invitrogen; Carlsbad, CA).mi R-203 and EGFP lentiviral vectors were packaged in HEK293 FT cells and produced as described previously(31).1x106 mi R-203 expressing SKOV3 and OVCAR3 cells were labeled using lentiviral vector expressing luciferase(p Lenti-UBC-Luc2-T2A-m Kate, VVC at UTHSC, Memphis)and then subcutaneously injected into two months old immunodeficient NSG female mice. All the patients from the third affiliated hospital of Zhengzhou University and the hospital of University of Tennessee health science center, respectively.MethodsCell culture- The ovarian cancer cell lines SKOV3 and OVCAR3 were obtained from ATCC and cultured in Dulbecco’s Modified Eagle Medium(DMEM)supplemented with 10% FBS(Hyclone; Logan, UT), 100 U/ml penicillin, and 100μg/ml streptomycin(Invitrogen; Carlsbad, CA). HEK293 FT cells were cultured in DMEM media with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 1%glutamine, 1% nonessential amino acid, and geneticin with a final concentration of 1μg/ml.Lentiviral vector production- mi R-203 and EGFP lentiviral vectors were packaged in HEK293 FT cells and produced as described previously(31). Mi R-203 stable and control SKOV3 and OVCAR cell lines were established by transducing the cells with the purified viruses and selected with 5 μg/ml puromycin.Cell colony formation assay- SKOV3 and OVCAR3 cells transduced with mi R-203 or EGFP overexpression viruses(200 cells/well each) were plated in triplicate into 6-well plates and then stained with 0.1% crystal violet following two week culture, and cell colonies were counted as described previously(32).MTT assay- Cells were plated 8000 per well in 96-well plates and cultured for 24 h. Thereafter, 10 μl of MTT reagent were added to each well and incubated for 4 h.The reaction was terminated by adding 100 μl detergent reagent and incubated at22°C in the dark for 2 h; and then the absorbance was measured at 570 nm wavelength.Cell migration assay –The transwell migration assay was performed using a modified chamber(BD Falcon?, San Jose, CA). These chambers were inserted into a24-well plate. Cells(3 × 104) in 300 μl serum-free DMEM were added to the upper chamber. 10% DMEM was added into the lower chamber of each well and cells were incubated for 24 h. The medium and non-migrated cells in the upper chamber were removed whereas, the migrated cells in the lower side of the membranes were fixed with methanol and stained with Crystal Violet. Pictures were taken at 10 X magnification. Cells in at least three different fields were counted.Cell invasion assay- SKOV3 and OVCAR3 cells(5 × 105) transduced with mi R-203 and EGFP lentiviral vectors were seeded in serum-free DMEM onto inserts precoated with Matrigel(BD Bio Coat TM, 24-well Tumor Invasion System(BD Bio Sciences, San Jose, CA). DMEM containing 10% FBS was added to the bottom chamber of the invasion system as the chemoattractant. After 24 h, the transwell inserts were stained using 4 μg/ml of Calcein AM(Life Technologies,Grand Island, NY) at 37°C for 1 h. The fluorescent intensity was measured using the Bio Tek Synergy TM(Winooski, VT) plate reader at excitation and emission wavelengths of 485 nm and 528 nm, respectivelyCell apoptosis assay- mi R-203 expressing stable cell lines SKOV3 and OVCAR3 were treated with different doses of cisplatin for 48 h. Cell apoptosis was examined by measuring caspase3 and 7 activity using luciferase assay kit(Promega,Madison, WI).Real-time RT-PCR- Total RNA was extracted from ovarian cancer cells using Trizol and from formalin-fixed, paraffin-embedded(FFPE) Blocks of human ovarian carcinoma and normal ovaries and FFPE RNA extraction kit according to manufacturer’s instruction(Life Technologies). Total RNA was used to perform RT-PCR or poly A tailing real time RT-PCR as published previously(31).Immunofluorescent staining- Ovarian cancer cells were fixed for 10 min using4% PFA, washed three times with 0.1% Tween20 in PBS(PBST), and sections from human and mouse tumors were antigen retrieved and then incubated with blocking buffer(5% normal goat serum, 3% bovine serum albumin, and 0.1% Triton-X 100 in PBS) for 1 h. The primary antibodies to E-cadherin, Snail2, Vimentin(1:200, Cell Signaling, Danvers, MA) and PCNA(1:200, Satan Cruz), were incubated with fixed cells overnight. After rinsing three times for 5 min with PBST, Alexa 488 or 594 conjugated goat anti-rabbit(1:200 dilution, Life Technologies) antibodies were added for 1 h at room temperature. Cell nuclei were counterstained with DAPI(Vector Laboratories, Inc.; Burlingame, CA). Images were taken using a Nikon inverted fluorescence microscope.Western blot- Ovarian and breast cancer cells were collected in RIPA buffer(Thermo Scientific; Rockford, IL) containing 1% Halt Proteinase Inhibitor Cocktail(Thermo Scientific; Rockford, IL). An equal amount of protein(40 μg/lane) was loaded onto 10% SDS-PAGE gels and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk for 1 h and incubated with primary antibodies against GAPDH(Sigma; St. Louis, MO), vimentin, E-cadherin, or snai2(Cell Signaling).Xenograft mouse model- 1x106 mi R-203 expressing SKOV3 and OVCAR3 cells were labeled using lentiviral vector expressing luciferase(p Lenti-UBC-Luc2-T2 Am Kate, VVC at UTHSC, Memphis) and then subcutaneously injected into two month old immunodeficient NSG female mice. Tumor initiation and progression were monitored twice a week using Xenogen image system. Tumor sizes were measured using a hand held caliper and mice were sacrificed at two months following cell injection and tumors were weighed and collected for H.E and immunostaining.ELISA ASSAY-Allow all reagents to reach room temperature before use. Gently mix all liquid reagents prior to use.Add 50-100 μL of prepared standard and sample to wells. Cover plate and incubate at room temperature for 2 hours.Thoroughly aspirate or decant solution from wells and discard the liquid.Wash wells 4 times using a squirt wash bottle or an automated 96-well plate washer.Add 100 μL of diluted detection antibody to wells. Cover plate and incubate at room temperature for 1hour.Thoroughly aspirate or decant solution from wells and discard the liquid.Wash wells 4 times.Add 100 μL of diluted HRP conjugate to each well. Cover plate and incubate at room temperature for 30 minutes.Thoroughly aspirate or decant solution from wells and discard the liquid.Wash wells 4 times.Add 100 μL of chromogenic substrate to each well.Develop plate at room temperature in the dark for 30 minutes.Add 100 μL of stop solution to each well. The solution in the wells should change from blue to yellow.The plate must be evaluated within 30 minutes of stopping the reaction. Read the absorbance of each well at 450 nm and 550 nm.Subtract 550 nm values from 450 nm values to correct for optical imperfections in the microplate.Use curve-fitting statistical software to plot a four-parameter logistic curve fit to the standards and then calculate results for the test samples.TCGA database query-To examine how mi R-203 and its target gene Snai2 expression are associated with clinical features of human ovarian cancer tissues, we queried the TCGA database. The data set was filtered for samples on mi R-203, Snai2 and clinical data. Statistical analyses were performed using Graphpad Prism.Patient samples: tumor tissues from the tissue bank an department of pathology of Zhengzhou University, routine tissue HE staining and specific staining.statistics approach: Significant differences were determined from two or three independent experiments performed in triplicate and presented as means ± S.D. using Student’s t-test. p < 0.05 was considered significant.Results1.Mi R-203 expression level is associated with long term survival of ovarian cancer patients, and downregulated m RNA and protein levels in ovarian cancertissues.2.Mi R-203 inhibits cancer cells proliferation, migration and invasion.3.Mi R-203 inhibits cancer cells EMT and counteracts TGFβ induced EMT in ovarian cancer cells4.Mi R-203 inhibits tumor growth in vivo5.Silencing Snai2 expression mimics mi R-203 mediated function in ovarian cancer cells6.Snai2 is associated with short term survival and upregulated in human ovarian cancer7.Leptin has higher expression in ovarian carcinoma.Conclusions1. Mi R-203 functions as a tumor suppressor by inhibiting cell proliferation,migration and invasion in ovarian cancer cells.2.Mi R-203 inhibits tumor growth in vivo through downregualting Snai2 and counteracting TGFβ-induced EMT.3.Mi R-203 and leptin have great potential to be useful biomarkers for ovarian cancer prognosis and even preclinical screening. |
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