The Evaluation Of Serum Exosomal MicroRNAs In The Diagnosis Of Ovarian Cancer And The Effects Of MiR-1246 On Biological Behavior Of Ovarian Cancer Cells In Vitro

Part Ⅰ The evaluation of serum exosomal microRNAs in the diagnosis of ovarian cancerPurpose: miRNAs packaged in exosomes might play functional role in the development of tumor.The aims of our study were to evaluate the serum exosomal miRNAs as a potential biomarkers in patients diagnosed with ovarian cancer.Methods:1.Preoperative serum samples from 40 patients with ovarian cancer,15 patients with benign ovarian tumor and 30 healthy participants were collected from The Forth Hospital Hebei Medical University.2.Serum exosomes were extracted by Minute efficient exosome precipitation method and the expression of exosome proteins CD9 and CD63 was detected by Western blot.miRNAs were extracted from exosomes using miRNA purification kit.3.The quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression levels of serum exosome miRNAs.4.All the statistical analysis were performed using SPSS21.0 and the difference was statistically significant at P < 0.05.Results:1.We extracted exosomes from serum by exosome precipitation method.Western blot results showed that the extracted exosomes highly expressed CD9 and CD63.2.Four tumor-associated microRNAs were screened for miR-101-3P,miR-320 a,miR-3184 and miR-1246.We collected 15 ovarian cancers and 15 healthy controls in the primary screening group.Real-time quantitative PCR results showed that miR-101-3P,miR-320 a and miR-1246 were stably expressed in ovarian cancer group and healthy control group.However,further analysis revealed that There was no significant difference in the expression of 101-3P and miR-320 a between the ovarian cancer group and the healthy control group in the primary screening group(P>0.05).Only miR-1246 was differentially expressed between the two groups in the primary screening group(P<0.05).3.Validation group followed by 25 ovarian cancer patients,15 benign ovarian tumors and 15 healthy controls.Real-time quantitative PCR results showed that the expression of miR-1246 in the ovarian cancer group was significantly higher than that in the benign ovarian tumor group and the healthy control group,the difference was significant(P<0.05).The expression of miR-1246 in benign ovarian tumors was higher than that in healthy controls,but the difference was not significant(P>0.05).4.ROC curve analysis was performed on 25 patients with ovarian cancer.The area under the ROC curve of miR-1246,CA125 and the combination of the two were 0.819,0.898,and 0.909,respectively.The cutoff value of miR-1246 was 0.947 by ROC curve.The sensitivity of miR-1246 in serum exosomes and CA125 were 0.680 and 0.800,the specificities were 0.897 and 0.735,respectively.Combined diagnosis could improve sensitivity and diagnostic performance of miR-1246.5.Bivariate correlation analyses showed that there was a significant positive correlation between the serum levels of exosomal miR-1246 and CA125(r=0.644,P=0.001)、HE4(r=0.581,P=0.002).6.Bivariate correlation analysis showed that the relative expression level of miR-1246 was not related to age(r=-0.291,P=0.703)and histological type of ovarian cancer(r=0.111,P=0.597).miR-1246 was associated with disease stage(r=0.607,P=0.005),abdominal pelvic and distant metastasis(r=0.485,P=0.014),and CA125 positive expression level(r=0.430,P=0.032).Conclusions: This study found that miR-1246 in serum exosomes is differentially expressed in ovarian cancer and has potential as a new generation of tumor markers for the diagnosis of ovarian cancer.Part Ⅱ The effect of mi R-1246 on the biological behavior of ovarian cancer cells in vitroObjective: The first part of the results showed that mi R-1246 has significant differences in serum exosomal expression in ovarian cancer patients.This study investigated the effects of mi R-1246 on migration,invasion,proliferation and apoptosis of ovarian cancer cells.Methods: 1.The experiment was divided into two groups,the mi R-1246 low expression group included mi R-1246 inhibitor group and mi R-1246 inhibitor NC group,and mi R-1246 high expression group mi R-1246 mimic group and mi R-1246 mimic NC group,respectively.SKOV3 cells were transfected with mi R-1246 inhibitor,mi R-1246 inhibitor NC,mi R-1246 mimic and mi R-1246 mimic NC,respectively.2.The quantitative real-time polymerase chain reaction(q RT-PCR)was used to detect the expression of mi R-1246 in SKOV3 cell lines.3.The migration and invasion abilities of SKOV3 cells after transfected was evaluated by wound-healing assays and transwell invasion assays.4.The proliferation of cell was detected by CCK-8 assay and Flow cytometry was used to detect the apoptotic rate of cells after transfection.Results: 1.The results of real-time PCR showed that the expression level of mi R-1246 in SKOV3 cells of mi R-1246 mimic group was significantly higher than that of mi R-1246 mimic NC group(P<0.05),and the expression of mi R-1246 in mi R-1246 inhibitor group was significantly lower than that of the mi R-1246 inhibitor NC group(P<0.05).2.The result of wound-healing assays indicates that the migratory rate was decreased in the mi R-1246 inhibitor group and increased in the mi R-1246 mimic group compared with the mi R-1246 inhibitor NC group and mi R-1246 mimic NC group(P<0.05),respectively.3.Compared with each other nagitive control groups,the number of invasive cells significantly increased in the mi R-1246 mimic group and decreased in the mi R-1246 inhibitor group(P<0.05),It indicated that mi R-1246 inhibitor significantly inhibited the migration and invasion ability of SKOV3 cells(P<0.05).4.Cell proliferation was assessed using an CCK-8 assay,the rate of cell proliferation decreased in the mi R-1246 inhibitor group(P<0.05)and increased in the mi R-1246 mimic group(P<0.05),when compared with the mi R-1246 inhibitor NC group and mi R-1246 mimic NC group.This experiment show that mi R-1246 can significantly promote the proliferation of SKOV3 ovarian cancer cells(P<0.05).5.Apoptosis experiments showed that the apoptosis rate of mi R-1246 mimic group was significantly lower than that of mi R-1246 mimic NC group(P<0.05),and the apoptosis rate of mi R-1246 inhibitor group was significantly higher than that of mi R-1246 inhibitor NC group(P<0.05).This indicates that mi R-1246 can inhibit the apoptosis of SKOV3 ovarian cancer cells.Conclusions: Through in vitro experiments of ovarian cancer cells,mi R-1246 plays an important role in the development of ovarian cancer,which can promote the migration、invasion and proliferation of SKOV3 ovarian cancer cells and inhibit the apoptosis of SKOV3 cells.