The Influence Of MiR-221 On The Proliferation And Migration Of Epithelial Ovarian Cancer Cells

Objective: Ovarian malignant carcinoma is the most common female gynecological malignancy.Due to its atypical symptoms and lacking of diagnostic methods in early stage,most of the ovarian cancer patients can't be diagnosed until advanced stage.The advanced cases lack of effective treatment and easy recurrence,therefore the lethality rate of ovarian malignant carcinoma take the first place in gynecological malignancy.The epithelial ovarian cancer(EOC)accounted for 85%-90% in ovarian malignant tumor to be the most common ovarian malignant tumor.MicroRNAs(miRNAs)are small noncoding RNAs that function as negative gene regulators at the post-transcriptional level by destabilizing mRNA or by directly repressing translation.People found that miR-221 are overexpression in variety of malignant tumors,including ovarian cancer.This indecaed that miR-221 expression associated with the occurrence of tumor.The levels of serum miR-221 is closely related to the grade and tumor stage of the international association of obstetrics and gynecology.High serum miR-221 expression is EOC independent poor prognostic factors.Studies have shown that hight level of miR-221 in human liver cancer,breast cancer,colon cancer and other tumors can inhibit cell apoptosis,promote cell proliferation and migration by negatively regulating target genes such as tumor suppressor genes CDKN1B(p27kip1),CDKN1C(p57kip2),MMAC1(PTEN)when they compelet transcription.It's reported that the direct target genes of miR-221 is diverse in different tumors.In ovarion cancer the direct target genes of mi R-221 has not been reported.However,Wurz K etc's reseach shows that p27 and p57 expressed in ovarian surface epithelium and down-regulated in ovarian carcinomas.Moreover,higher miR-222 and mi R-221 expression were significantly associated with decreased p57 expression.Related studies have shown that down-regulation of miR-221 can inhibit the proliferation of tumor cells and promote tumor cell apoptosis in colon cancer and bladder cancer.Most of ovarian cancer patients can't be diagnosed in early stage until advanced stage with extensive invasion,The estimated five survival rate for patients at early stage was 90%,but only 20%~30% at advanced stage.Nearly 80% of patients relapsed with postoperative chemotherapy.The leading cause of recurrence in patients with ovarian cancer is invasion and metastasis,Therefore the exploration the related genes about the invasion and metastasis has the significance of inhibition.MiR-221 as an oncogene,what kind of role play in ovarian tumor cell proliferation,migration? Studay about it is rare.Whether can we selective inhibition of miR-221 genes in ovarian cancer to repression the invasion and metastasis of ovarian cancer cells.Expecting to achieve the goal of diagnosis and treatment in early stage.In this study,The ovarian cancer cell SKOV3 was cultivated invitro,transfection the plasmid of miR-221 by transient to decreased the expression of miR-221 in ovarian cancer cell lines SKOV3.We observed the effect on the proliferation and migration of ovarian cancer cell,and aimed at studying the function of mi R-221 in the occurrence and development in choriocarcinoma,we explorde the molecular mechanisms,intending to provide new ideas that early diagnosis of oophoroma.Methods:1 Using TIANpure Midi Plasmid Kit extract the plasmid of CAG-EGFP-miR-221 sponge and CAG-EGFP,and using Agarose gel electrophoresis to test its quality.2 The SKOV3 cells were divided into three group and used LipofectamineTM2000 lipsome transfection reagent to transfect:(1)the group of experiments was transfect with CAG-EGFP-mi R-221 sponge plasmid;(2)negative control group was transfect with CAG-EGFP plasmid;(3)blank control group without anytreatment.We observe the transfection rate through fluorescence microscope at the time of 24 h after transfection.3 Real-time PCR: the expresstion level of miR-221 in different groups was determined by real-time quantitative PCR using SYBRGreen method at the time of 48 h after transfection.4 MTS assay tested the proliferation capabilities of SKOV3 cell in different groups at the time of 0h,24 h,48h,72 h.5 Wound Healing assay tested the migration capabilities of SKOV3 cell in different groups.6 Statistical method: Data were analysed by SPSS13.0 statistical software,and data were recorded as mean(10)standard differential((?)�S).Means of multiple groups were analyzed by single factor analysis of variance.P<0.05 was considered as significant difference.All experiments were repeated 3 times.Results: 1 The identification of CAG-EGFP-miR-221 sponge and CAG-EGFP.The A260/A280 of the plasmids were around 1.8,The concentration of the plasmid was around 700ng/uL.2 Transfection Efficiency were observed.At time of 24 h after transfected with CAG-EGFP-miR-221 sponge and CAG-EGFP,green fluorescence was observed by fluorescence microscopy on the cells rather than untranfected ones,and the result showed that tranfection reats were about 70%-80% and 60%-70% respectively,blank control group have not see green fluorescence.3 The expression level of miR-221 in groups were detected by Raal-time qPCR.RT-PCR analysis the expression level of miR-221 in each group at 48 h after transfection:SKOV3-mi R-221 spong group was 1.172�0.025;SKOV3-N group was 1.852�0.019,SKOV3 group was 1.921�0.023.The expression of miR-221 in SKOV3-mi R-221 sponge group decreased than SKOV3-N group and SKOV3 group,P<0.05,the difference has statistical significance.Compared SKOV3-N group with SKOV3 group,P>0.05,the difference has no statistical significance.4 The MTS experiment tested the proliferation of SKVO3 cells in groups at different times.0 hour: SKOV3-miR-221 sponge group 0.344�0.0318;SKOV3-N group 0.370�0.043;SKOV3 group 0.390�0.047.Compared experimental group with the control groups,P>0.05,the difference has no statistical significance;24 hour: SKOV3-miR-221 sponge group(0.421�0.068);SKOV3-N group(0.510�0.041);SKOV3 group(0.561�0.059);Compared SKOV3-miR-221 sponge group with SKOV3-N group,P<0.05,the difference has statistical significance;Compared SKOV3-miR-221 sponge group with SKOV3 group,P<0.05,the difference has statistical significance;Compared SKOV3-N group with SKOV3 group,P>0.05,the difference has no statistical significance;48 hour: SKOV3-miR-221 sponge group(0.518�0.0499);SKOV3-N group(0.614�0.035);SKOV3 group(0.709�0.038),Compared experimental group with the control groups,P<0.05,the difference has statistical significance;72 hour: SKOV3-miR-221 sponge group(0.5827�0.091);SKOV3-N group(0.719�0.026);SKOV3 group(0.816�0.038),Compared experimental group with the control groups,P<0.05,the difference has statistical significance.5 The scratch assay tested the migration of SKVO3 cells in groupsData were analysed by ImageJ software,the migration rate were computed after scratch 24 hours: SKVO3-miR-221 sponge group(17.39�5.28)%;SKOV3-N group(24.55�2.641)%;SKOV3 group(21.01�3.74)%;Compared experimental group with the control groups,P<0.05,the difference has statistical significance.Conclusion:1.The down-regulation of miR-221 can significantly suppress the migration of SKOV3 cells.2.The down-regulation of miR-221 can significantly suppress the proliferation of SKOV3 cells.