| Background:Epithelial ovarian carcinoma is the most lethal gynecological cancer, accounting for75%~90% of all ovarian cancer, and serous ovarian cancer accounts for 60%~70% of all epithelial ovarian carcinoma. Because there is no obvious early symptoms and no effective screenings and no early diagnosis methods, more than 70% of patients have been advanced stages when they were diagnosed. The 5- year survival rate is below 30%,mortality rate is the first one of all female genital tract malignant tumour. Micro RNAs(mi RNAs) are endogenous non-coding small RNA, regulating the expression of genes in the post-transcriptional level. They have an important role on cell proliferation,differentiation, metabolism, apoptosis and other biological processes. They closely related to the occurrence of a wide variety of tumor development. According to different target genes, mi RNAs can be used as tumor suppressor genes or oncogenes, involving in tumorigenesis, metastasis, chemoresistance and other pathological processes. At present,the studies about mi RNAs and ovarian cancer have been reported, but the mechanism is still not very clear. In recent years, scholars have found that serous ovarian cancer originated from the normal fimbria of fallopian tube tissues. We assessed the mi RNAexpression profiles in serous ovarian cancer compared with normal fimbria of fallopian tube tissues using mi RNA microarray in our previous studies. We found that mi R-450a-5p was signi?cantly down-regulated in serous ovarian cancer tissues, and its function and mechanism have not been reported in ovarian cancer research. Compared with normal fimbria of fallopian tube tissues, mi R-450a-5p was chose as our study gene and we hope that mi R-450a-5p could became an important biological molecular for early diagnosis,treatment and prevention of serous ovarian cancer. Basing on the results of our previous mi RNA microarray,(1) Real-time fluorescent quantitative PCR technology was used to detect the expression differences of mi R-450a-5p in large samples, and to analyze the relationship between mi R-450a-5p expression level and the clinical pathological features of ovarian cancer;(2) Mi R-450a-5p was over-expressed in SKOV3 cells and tumor biological behavior of the SKOV3 cells was analyzed by cell function experiments;(3)western blot and dual-luciferase reporter gene assay were used to explore the molecular signaling pathways of mi R-450a-5p in carcinogenesis and development of serous ovarian cancer, which can provide new ideas and theoretical basis for clinical treatment of serous ovarian cancer.Objective:1. To investigate the expression of mi R-450a-5p in serous ovarian cancer and analyze the correlation of its expression with clinicpathological features.2. To investigate the effect of mi R-450a-5p on the biological behavior of serous ovarian cancer SKOV3 cells by cell function experiments; To validate the target gene of mi R-450a-5p and explore the role of mi R-450a-5p on the carcinogenesis and development of serous ovarian cancer.Methods:1. Paraffin-embedded tissue blocks of normal fallopian tube tissues(50 samples) and serous ovarian cancer(101 samples) were obtained from the department of pathology. All samples were confirmed by two pathologists.2. Quantitative real-time PCR was used to assess the expression of mi R-450a-5p between serous ovarian cancer tissues and normal fimbria of fallopian tube tissues in large samples.The statistical method was used to analyze the relationship between mi R-450a-5p expression level and the clinical pathological features of ovarian cancer.3. Serous ovarian cancer SKOV3 cells were transfected with mi R-450a-5p mimics by hiperfect transfection reagents as experimental group compared to negative control group(NC group) and blank group. Cell proliferation ability was evaluated by MTT assay. Cell migration ability was detected by transwell migration assay and wound healing assay. Cell invasion ability was detected by transwell invasion assay. Cell cycle and apoptosis were detected by flow cytometry.4. We used several biological softwares to predict possible target genes of mi R-450a-5p and reviewed literatures in order to select the possible target gene.5. The application of western blot and dual-luciferase reporter gene assay were used to validate the potential target gene of mi R-450a-5p.6. The SPSS19.0 software was adopted to analyse the statistical results, using independent sample t-test, P < 0.05 was statistically significant.Results:1. The expression of mi R-450a-5p was significantly down-regulated in serous ovarian cancer tissues than that in normal fimbria of fallopian tube tissues.2. There was no significant correlation between the expression of mi R-450a-5p and clinicopathological features in serous ovarian cancer.3. No significant difference was found in proliferation ability between mimics group and control groups by MTT assay.Over-expression of mi R-450a-5p could lead significant inhibition effect on cell migration and invasion ability in SKOV3 cells detected by Transwell assay.The migration ability of mimics group was significantly inhibited by wound healing assay.Flow cytometry analysis displayed that mi R-450a-5p significantly induced SKOV3 cell G1 arrest, but no significant difference was found in cell apoptosis.4. The biological software was used to predict the possible target gene of mi R-450a-5p, combining with related literature, DUSP10 may be the target gene of mi R-450a-5p.5. The expression of DUSP10 protein was significantly down-regulated in mi R-450a-5p mimics group confirmed by western blot. DUSP10 may be the target gene of mi R-450a-5p verified by dual-luciferase reporter gene assay.Conclusions:1. Mi R-450a-5p was significantly down-regulated in serous ovarian cancer.Mi R-450a-5p may be involved in the occurrence and development of serous ovarian cancer.2. Over-expression of mi R-450a-5p may inhibit the invasion and migration ability of SKOV3 cells and may arrest SKOV3 cells in G1 phase.3. The target gene of mi R-450a-5p may be DUSP10. Mi R-450a-5p may exert functions through mi R-450a-5p /DUSP10 / MAPKs signal pathway.4. Mi R-450a-5p may play an important role in carcinogenesis and development of serous ovarian cancer as a tumor-suppressor and may be a new candidate biomarker for earlier diagnosis and target treatment of serous ovarian cancer. |
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