The Expression And Mechanism Of MiR-1247-5p In Human Breast Cancer

Objetive:1 Based on the expression information of miRNA of breast cancer(BC)in TCGA database,a miRNA-based prognosis model was constructed by Cox proportional hazard regression model.2 To detect the expression levels of miR-1247-5p in human BC tissues,and analyze the clinical significance and function of miR-1247-5p.3 To explore the potential mechanism of miR-1247-5p.Methods:1 The differentially expressed miRNA in BC and adjacent normal tissues in TCGA database were analyzed and screened out using Morpheus software,and the best prognostic model based on miRNA was established by Cox proportional hazard regression model.We tested the miRNA-based prognosis model using Kaplan-Meier method,Log-rank test,and ROC curve analysis.Chi-square test or Fisher exact test was used to analyze the correlation between prognostic model and clinicopathological characteristics in BC patients.Univariate and multivariate Cox regression analysis was carried out to determine whether the miRNA-based prognosticmodel was an independent prognostic factor for BC.2 To analyze the expression levels of miR-1247-5p in BC patients with complete clinicopathological features in TCGA database,and RT-qPCR was used to validate in 24 pairs of BC and adjacent normal tissues.Kaplan-Meier method,Log-rank test,univariate and multivariate Cox regression analysis were carried out to analyze the correlation between miR-1247-5p and overall survival(OS)and recurrence-free survival(RFS).CCK8 and flow cytometry asssys were used to detect the effects of miR-1247-5p on proliferation and apoptosis of BC cells.3 Online database and literature review were used for screening the potential target of miR-1247-5p.Dual-luciferase reporter assays,RT-qPCR,and western blot experiments were conducted to validated the target of miR-1247-5p.RT-qPCR and IHC were carried out to detect the expression levels of miR-1247-5p and DVL1 respectively,and Pearson correlation analysis was used to analyze the correlation between them.WB assays were used to detect the effect of miR-1247-5p on downstream signaling pathway.Results:1 By analyzing the differently expressed miRNA in 103 pairs of BC and adjacent normal tissues in TCGA database,we screened out 100 differentially expressed miRNA,including 50 up-regulated miRNA and 50down-regulated miRNA.Subsequently,839 BC patients with completeclinicopathological features were randomly divided into training set and testing set groups,a three miRNA-based prognostic model,incluiding miR-1247-5p,miR-29a-3p and miR-148b-3p,was constructed by Cox proportional hazard regression model in training set,and validated in testing set and entire set,the results showed that the prognosis of low-risk group was better than that of high-risk group,and the miRNA-based model had high diagnositic sensitivity and specificity.Multivariate Cox regression analysis showed that the miRNA-based prognostic model was an independent prognostic factor for BC patients.2 Compared with adjacent normal tissues,the expression of miR-1247-5p in human BC tissues was significantly lower,and it had high diagnostic sensitivity and specificity.The correlation analysis between miR-1247-5p and clinicopathological features suggests that miR-1247-5p was associated with patient age and tumor size.The prognostic analysis indicated that the expression levels of miR-1247-5p were positively correlated with OS and RFS in BC patients,and it was an independent prognostic factor for OS and RFS.CCK8 experiment and flow cytometry analysis indicated that miR-1247-5p inhibited proliferation and induced apoptosis in BC cells,respectively.3 We identified DVL1 was the potential target of miR-1247-5p through online database analysis and literature review.Dual-luciferase reporter assay results showed that miR-1247-5p notably suppressed theluciferase activity of the wide-type DVL1 3'-UTR,but not the mutant DVL1 3'-UTR.RT-qPCR assays showed that miR-1247-5p slightly decreased DVL1 mRNA levels,while WB assays indicated that miR-1247-5p significantly decreased DVL1 protein levels,indicating the inhibitive effect of miR-1247-5p on DVL1 was largely through posttranscriptional regulation.Moreover,an inverse correlation of miR-1247-5p and DVL1 expression was observed in BC tissue specimens by Pearson correlation analysis.And then,WB assays demonstrated that miR-1247-5p inhibited the expression levels of DVL1,and subsequently inhibited the downstream factors of the Wnt/?-catenin signaling pathway,including active ?-catenin,CyclinD1,c-Myc,and MMP7.Conclusion:1 The three-miRNA prognostic model,including miR-1247-5p,miR-29a-3p and miR-148b-3p,is an independent prognostic factor for BC patients.2 MiR-1247-5p,acts as a tumor suppressor gene in BC,is an independent prognostic factor for OS and RFS in BC patients.Functional analysis suggests that miR-1247-5p can inhibit proliferation and prompt apoptosis in BC cells.3 Mechanistic investigations showes that miR-1247-5p directly targeted DVL1,and subsequently inhibits the canonical Wnt/?-catenin signaling pathway.