| Backgroud According to cancer statistics in 2014, ovarian cancer is the fifth cause of death in women in the United States. Its annual incidence is 21980, but the number of deaths is 14270. Early diagnosis of ovarian cancer is very difficult because of the lack of specificity of the abdominal-pelvic symptoms and sensitive marker, which causes the majority of patients have been diagnosed in advanced stage. So the mortality rate of ovarian cancer is very high and the prognosis is extremely poor. 5-year overall survival (OS) rate of ovarian cancer is less than 30%. Owing to the absence of specific clinical symptoms and lack of sensitive biomarker in ovarian cancer, it is very urgent to find a tumor marker for early diagnosis of ovarian cancer, and to select the best and personalized treatment plan.MicroRNAs (miRNAs), which are a group of short, non-coding and single-strand RNAs. are emerging as novel posttranscriptional regulator by directly base-pairing to 3’-UTR of target mRNA. MiRNAs have been proved to be involved in a multitude of physiological processs, such as cell cycle, metabolism, angiogenesis, differentiation, apoptosis and so on. The abnormal expression of miRNAs may be associated with several tumors by regulating the different genes and signal pathways to be involved in development, processing, transferring and drug resistance for tumors. Lucikly, there are evidences showed miRNA is produced in the cytoplasm, which not only can affect the function of the cell, but also can release into the blood in order to play the function of regulating the distant target gene. Compared with other nucleic acids in circulation, the miRNA level in the circulation is more stable. MiRNAs are successfully evaluated in a variety of solid tumors, and these can be used as new and noninvasive early diagnostic markers for disease.Studies have proved that the miR-15a, miR-16, miR-31, miR-200 family were upregulated in EOC, and let-7, miRNA-34a/b/c, miRNA-100, miR-199a/214 and miR-99a were downregulated in EOC, in which miR-99a is located at 21q21 which is related to many diseases. It had been found to be abnormally expressed in hepatocellular carcinoma, prostate cancer, head and neck squamous cell carcinoma and other cancers. So far, only one report showed that the miR-99a was differentially expressed between normal and cancerous ovarian tissues through miRNA microchip technology detection. However, the biological function of the abnormal expression of miR-99a in EOC is still unclear. Therefore, it is necessary to verify the role of miR-99a in ovarian cancer. In our study, the expression of miR-99a in clinical specimens of EOC and EOC cell lines were detected respectively. In addition, the mechanism of miR-99a in EOC has been speculated through the study of fibroblast growth factor receptor3 (FGFR3).Part One:The expression of miR-99a in tissues, serum of the ovarian cancer patients and in ovarian cancer cell linesObjective In order to explore the possibility of miR-99a as a new serum marker in patients with epithelial ovarian cancer, we need to detect the expression of miR-99a in serum of the ovarian cancer patients, ovarian tissues and ovarian cancer cells.Methods 1.Serum samples from 15 patients with ovarian cancer and 3 normal volunteers were collected, and the expression of miR-99a was detected by quantitative real-time PCR.2. Tissue samples of epithelial ovarian cancer were collected, and the expression of miR-99a in epithelial ovarian cancer tissues and normal ovarian tissues were detected by quantitative real-time PCR, too.3.In the end, we used the same method to detect the expression of miR-99a in ovarian cancer cell line SKOV3 and normal ovarian tissue cell line OSE.Results 1.The expression of miR-99a in serum of patients with epithelial ovarian cancer was down regulated.2. The expression of the miR-99a was also down regulated in epithelial ovarian cancer tissues.3. The expression of miR-99a in ovarian cancer cell line SKOV3 was decreased compared with normal ovarian tissue cells OSE.Conclusion 1.MiR-99a was down regulated in epithelial ovarian cancer, suggesting that miR-99a may be a tumor suppressor gene in ovarian cancer.2. The expression of miR-99a was down regulated in serum of ovarian cancer patients, which provided a new biomarker for early diagnosis of EOC.Part Two:Prediction and verification of miR-99a target gene FGFR3Objective Target genes of miR-99a need to be predicted by biological information method, and the role of miR-99a on target gene in ovarian cancer cells need to be investigated, which can provide some theoretical basis for further elucidation of the pathogenesis of ovarian cancer.Methods 1.MiRNA target prediction software database (TargetScan, PicTar, miRanda) was used to search for the miRNA target genes. And RNA hybridization was performed on the initial measurement (http://bibiserv.techfak.uni-bielefeld.de/ mahybrid/).2. RT-PCR and Western Blot were used to detect the expression of FGFR3 mRNA and protein in OSE cells and SKOV3 cells, thus to verify whether the FGFR3 gene was associated with EOC.3. We constructed the luciferase expression plasmid pGL3-FGFR3, which contains 3’-UTR of FGFR3, co-transfected with miR-99a in SKOV3 cells, the effected of miR-99a on the activity of pGL3-FGFR3 was observed. Compared with no treatmen group (No-treated) and mutation group (pGL3-mut), we can further estimate whether miR-99a can directly regulate the 3’-UTR of FGFR3.4. In order to further verify the relationship between miR-99a and FGFR3, we transiently transfected miR-99a into SKOV3 cells, and the expression level of miR-99a was detected by qRT-PCR. Then we detected the mRNA and protein of FGFR3 by RT-PCR, qRT-PCR and Blot Western when miR-99a was over expressioned.Results 1. FGFR3 was predicted the target genes of miR-99a by biological information analysis.2. The expression of FGFR3 in SKOV3 cells was significantly higher than that in OSE cells.3. The 3’-UTR of FGFR3 was regulated by miR-99a.4. The mRNA and protein expression of FGFR3 in SKOV3 cells were down regulated by miR-99a.Conclusion FGFR3 is a target gene of miR-99a, miR-99a can directly regulate the 3’-UTR of FGFR3, and down regulate the expression of mRNA and protein of FGFR3.Part Three:The effect of miR-99a on the proliferation of ovarian cancer cells by targeting FGFR3Objective In order to explore the effect of miR-99a on the ovarian cancer cell line SKOV3 proliferation, the study of the proliferation of ovarian cancer cell line SKOV3 by knocking down FGFR3 need to be done.Methods 1.CCK-8 assay was used to detect the effect of miR-99a overexpression on the proliferation of SKOV3 cells.2. The expression of FGFR3 in ovarian cancer cells was decreased by siRNA technology.3. The effect of FGFR3 low expression on the proliferation of SKOV3 cells was detected by CCK-8 assay.Results 1. Over expression of miR-99a significantly inhibited the proliferation of ovarian cancer cells.2. After silence of FGFR3 gene by siRNA, we found FGFR3 in ovarian cancer cell SKOV3 in both the mRNA level and protein level were significantly decreased, and the growth of SKOV3 cells obviously decreased.Conclusion The vitro experiments confirmed that miR-99a might inhibit proliferation of ovarian cancer cells by regulating the expression of FGFR3. |
PDF Full Text Request