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| Study on the relationship between microRNA-142-5p and E2F7 gene and mechanism of its influence on the proliferation, invasion, metastasis of pancreatic cancer |
| Amutijiang·Mahemuti ZHENG Jianjiang Dixiati·Alimu |
Department of Pancreatic Surgery, People’s Hospital of Xinjiang Uygur Autonomous Region, Xinjiang Uygur Autonomous Region, Urumqi 830001, China
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Abstract Objective To investigate the relationship between microRNA-142-5P (miRNA-142-5p) and E2F7 gene and the mechanism of its influence on the proliferation, invasion, metastasis of pancreatic cancer (PC). Methods The cancer and adjacent tissues of 35 patients with PC who received surgical treatment in the People’s Hospital of Xinjiang Uygur Autonomous Region from December 2016 to January 2019 were selected. Real-time quantitative PCR was used to detect and compare the expression of miR-142-5p in cancer and adjacent tissues of PC patients. The expression of miR-142-5p in human PC cell lines (CFPAC-1, SW1990, Capan-1, PANC-1) and normal pancreatic duct epithelial cell lines (HPDE) was compared. Real-time quantitative PCR was used to detect and compare the differences of E2F7 mRNA in cancer and adjacent tissues of PC patients, the differences of E2F7 mRNA in Capan-1 and PANC-1 cells were compared, and the differences of E2F7 protein in Capan-1 and PANC-1 cells were compared by Western blot. The binding sites of miR-142-5p and E2F7 gene were predicted by Starbase database, and the miR-142-5p mimics group transfected with miR-142-5p mimics and the NC group transfected with meaningless sequences were set up, and the two groups were co-transfect E2F7 gene wild type (wt) and mutant (mut), respectively. Dual luciferase assay was used to verify the targeting relationship between miR-142-5p and E2F7 gene. The Capan-1 cells were divided into NC group, si-E2F7 group, E2F7 group, and miR-142-5p mimics+E2F7 group. CCK-8, flow cytometry, scratch assay, and Transwell assay were used to observe cell viability, apoptosis, migration, and invasion in each group. Twenty male BALB/c nude mice (4-6 weeks old) were selected to establish xeno-transplantation model of nude mice, and were divided into NC nude mice group, si-E2F7 nude mice group, E2F7 nude mice group, and miR-142-5p mimics+E2F7 nude mice group by random number table method, with five mice in each group. A total of 5×106 treated PC cells were mixed with 100 μl matrigel and injected subcutaneously into the skin below the front leg of nude mice. The four groups were killed at the 30th day after injection. The tumor volume and weight of the four groups were compared, and the tumor metastasis ability was observed by hematoxylin-eosin staining. Results The expression of miR-142-5p in cancer tissues of PC patients was lower than that in adjacent tissues, the expression of E2F7 mRNA was higher than that in adjacent tissues, and the expression of miR-142-5p in CFPAC-1, SW1990, Capan-1, and PANC-1 cells were lower than those in HPDE cells (P＜0.05). The expressions of E2F7 mRNA and protein in Capan-1 and PANC-1 cells were higher than those in HPDE cells (P＜0.05). Starbase database analysis showed that miR-142-5p was complementary to the 3’untranslated region of E2F7 gene. The results of dual luciferase reporter gene showed that after co-transfection with E2F7-wt, the luciferase activity of miR-142-5p mimics group was lower than that of NC group (P＜0.05). After co-transfection with E2F7-mut, there was no significant difference in luciferase activity between miR-142-5p mimics group and NC group (P＞0.05). The expression of E2F7 mRNA and protein, cell viability, scratch healing area, and relative number of invasive cells in E2F7 group were higher than those in NC group, and the apoptosis rate was lower than that in NC group (P＜0.05). The expression of E2F7 mRNA and protein, cell viability, scratch healing area, and relative number of invasive cells in miR-142-5p mimics+E2F7 group were lower than those in E2F7 group, and the apoptosis rate was higher than that in E2F7 group (P＜0.05). The tumor volume and weight of E2F7 nude mice group were higher than those of NC nude mice group, and the tumor volume and weight of miR-142-5p mimics+E2F7 nude mice group were lower than those of E2F7 nude mice group (P＜0.05). Hematoxylin-eosin staining showed that the tumor metastasis of E2F7 nude mice group was strong, while that of miR-142-5p mimics+E2F7 nude mice group was poor. Conclusion MiR-142-5p may affect the proliferation, migration, invasion, and apoptosis of PC cells by targeting E2F7gene.
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