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OBJECTIVE To analyze the role of miR-214-5p in proliferation and metastasis of pancreatic cancer (PC) cells, as well as its underlying mechanism. PATIENTS AND METHODS 30 pairs of PC tissues and adjacent normal tissues were collected in our Department. The expression level of miR-214-5p was detected by quantitative Real-time-polymerase chain reaction (qRT-PCR). Biological information analysis and luciferase report gene assay were used to verify potential target genes of miR-214-5p. Cell counting kit-8 (CCK-8) and transwell methods were applied to observe the interference of miR-214-5p on invasion and migration of PC cells. Western blot (WB) assay was applied to determine the expression changes of Jagged 1 (JAG1) and epithelial-mesenchymal transition (EMT)-related genes in PC cells. RESULTS QRT-PCR results showed that the expression level of miR-214-5p is significantly down-regulated in PC tissues and cells. Bioinformatics software and luciferase report gene assay identified that JAG1 is a target gene of miR-214-5p. The negative correlation between protein expressions of miR-214-5p and JAG1 was assessed by Western Blot assay. Furthermore, miR-214-5p could suppress cell proliferation, invasion and migration, and it also blocked the EMT in PC cells in vitro. Meanwhile, JAG1 overexpression reversed the inhibitory effects of miR-214-5p on proliferation, invasion and migration of PC cells. CONCLUSIONS Overexpressing miR-214-5p could significantly inhibit malignant behavior of PC cells through targeted regulation of JAG1. Thus, miR-214-5p might be a potential therapeutic target for treatment of PC.