| BACKGROUND and OBJECTIVE:Ovarian cancer is a common cause of death for women from malignant gynecological tumors.Its lethality is mainly due to the difficulty to detect the disease early and the difficulty of getting effective treatment for patients with end-stage or recurrent patients due to extensive tumor invasion and metastasis in the body.MicroRNA can be involved in the tumorigenesis,proliferation,apoptosis,metastasis,invasion,and drug resistance of tumor cells,and related literature has reported that it can play different roles of oncogenes or tumor suppressor genes.Our previous experiments showed that miR-135b-3p expression in benign ovarian tissue was significantly lower than that in ovarian tumor tissue.Therefore,we propose to speculate whether the miR-135b-3p gene also functions as an oncogene as reported in related literatures such as pancreatic cancer and colorectal cancer,which can promote the invasion and metastasis of ovarian cancer cells and regulate epithelial-mesenchymal transition(EMT)related protein expression.Whether it can be a valuable early marker for serological diagnosis of ovarian cancer.This experiment mainly explores the role and mechanism of miR-135b-3p in enhancing epithelial ovarian cancer proliferation,invasion and metastasis,and epithelial-mesenchymal transition.The main research content of this topic includes the following three aspects:1.Expression of miR-135b-3p in epithelial ovarian cancer and its clinical pathology correlation of features2.Expression of miR-135b-3p in OVCAR-3 and CAOV-3 cells and its effect on the proliferation,migration and invasion of ovarian cancer cells3.Related Mechanisms and Clinical Significance of miR-135b-3p in Promoting Invasion and Metastasis of Ovarian Cancer CellsMETHODS:1.qRT-PCR was used to detect the expression of miR-135b-3p in ovarian tumor tissue and benign ovarian tissue,and to analyze the correlation between miR-135b-3p expression and clinicopathological characteristics of ovarian cancer.2.Use qRT-PCR to detect the expression of miR-135b-3p in various ovarian cancer cells and screen for cancer cell lines;small molecule inhibitor miR-135b-3p inhibitor group and its control group inhibitor NC against OVCAR-3,CAOV-3 cells were transiently transfected to detect the expression of miR-135b-3p in cells before and after transfection;CCK-8 experiments were performed to observe the effect of low expression of miR-135b-3p on the proliferation rate of OVCAR-3 and CAOV-3 cells;Scratch healing experiment and Transwell migration experiment to observe the changes of OVCAR-3 and CAOV-3 cell migration ability after low expression of miR-135b-3p;Transwell invasion experiment to observe the change of cell invasion ability;Western blot experiment was used to detect The expression of marker molecules related to epithelium and mesenchyme was studied to study the effect of miR-135b-3p on epithelial-mesenchymal transition pathway.3.Predict the target genes of miR-135b-3p on the three online prediction softwares:TargetScan,miRDB and PicTar respectively.Through gene ID conversion,take the intersection of the above prediction results on venny2.1.0 software and determine 9 target genes.Query miRTarBase and the latest literature reports for experimentally verified target genes related to invasion and metastasis,and then take the intersection as the miR-135b-3p target gene set for subsequent analysis;Western blot experiments were performed to observe the changes of the expression levels of ROBO1,ERK1/2,and MMP-9 in OVCAR-3 and CAOV-3 cells by regulating the expression of miR-135b-3p.Whether the gene ROBO1 has a direct regulatory effect,and explore the mechanism of miR-135b-3p on promoting the invasion and metastasis of ovarian cancer.4.qRT-PCR was used to detect the expression level of miR-135b-3p in the serum of 26 ovarian cancer patients and 26 healthy people.The expression characteristics of miR-135b-3p were analyzed,and its value in clinical diagnosis of ovarian cancer patients was discussed.RESULTS:1.miR-135b-3p is highly expressed in ovarian cancer and may play an oncogene function.The high expression of miR-135b-3p is closely related to the degree of differentiation,clinical stage and lymph node metastasis of patients with ovarian cancer(all p<0.05).2.Exogenous low expression miR-135b-3p significantly inhibited the proliferation,migration and invasion of ovarian cancer cell lines(OVCAR-3,CAOV-3).Epithelial and mesenchymal marker molecules of ovarian cancer cells with low exogenous expression of miR-135b-3p compared with the control group.E-cadherin expression was down-regulated,expression of N-cadherin and vimentin is upregulated.3.Exogenously low-expressing miR-135b-3p can target negative feedback to regulate ROBO1 and reduce the relative expression of phosphorylated ERK and MMP-9.The double luciferase report shows that in HEK293T cells,exogenously overexpressing miR-135b-3p significantly inhibited 3’-UTR luciferase activity carrying wild-type ROBO1.The 3’-UTR luciferase activity of the mutant ROBO1 was not affected.4.Compared with the healthy group,the expression level of miR-135b-3p in the serum of ovarian cancer patients was significantly increased(p<0.01),which has certain clinical diagnostic value for ovarian cancer patients.CONCLUSION:1.The expression of miR-135b-3p was significantly up-regulated in ovarian cancer tissues and OVCAR-3 and CAOV-3 cancer cell lines.miR-135b-3p promotes ovarian cancer cell proliferation,migration and invasion,promotes epithelial-mesenchymal transition,and promotes distant metastasis of epithelial ovarian cancer.2.ROBO1 is one of the target genes directly bound by miR-135b-3p.MiR-135b-3p activates the ERK/MMP-9 signaling pathway by inhibiting the expression of ROBO1.Regulation of ROBO1/ERK/MMP-9 signaling axis plays an important role in the invasion and metastasis of ovarian cancer.3.miR-135b-3p shows abnormally high expression in the serum of patients with ovarian tumors,and is expected to become a biomarker for early diagnosis of ovarian cancer. |
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