微RNA-142-5p与E2F7基因的关系及其影响胰腺癌增殖、侵袭、转移的机制研究

doi:10.20047/j.issn1673-7210.2023.26.03

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摘要目的探讨微RNA-142-5p（miRNA-142-5p）与E2F7基因的关系及其影响胰腺癌（PC）增殖、侵袭、转移的机制。方法选择2016年12月至2019年1月于新疆维吾尔自治区人民医院接受手术治疗的35例PC患者的癌及癌旁组织，采用实时定量PCR检测并比较PC患者癌及癌旁组织中miR-142-5p的表达情况，比较人PC细胞系（CFPAC-1、SW1990、Capan-1、PANC-1）和正常胰管上皮细胞系（HPDE）中miR-142-5p的表达情况。采用实时定量PCR检测并比较PC患者癌及癌旁组织中E2F7 mRNA的差异，比较Capan-1、PANC-1细胞E2F7 mRNA的差异，采用蛋白质印迹法比较Capan-1、PANC-1细胞中E2F7蛋白的差异。通过Starbase数据库预测miR-142-5p与E2F7基因的结合位点，同时设置转染miR-142-5p模拟物的miR-142-5p mimics组和转染无意义序列的NC组，两组分别共转染E2F7基因野生型（wt）及突变体（mut），采用双萤光素酶实验验证miR-142-5p与E2F7基因的靶向关系。将Capan-1细胞设为NC组、si-E2F7组、E2F7组及miR-142-5p mimics +E2F7组，采用CCK-8、流式细胞仪、划痕实验、Transwell实验观察各组细胞活力、凋亡、迁移、侵袭情况。选取20只雄性BALB/c裸鼠（4～6周龄）用于建立裸鼠异种移植模型，采用随机数字表法将其分为NC裸鼠组、si-E2F7裸鼠组、E2F7裸鼠组及miR-142-5p mimics+E2F7裸鼠组，每组5只。将5×106个相应处理的PC细胞与100 μl的人工基膜混合后皮下注射于裸鼠前腿下方的皮肤，于注射后第30天处死四组动物，比较四组肿瘤体积及重量，采用苏木精-伊红染色观察肿瘤转移能力。结果 PC患者癌组织miR-142-5p表达低于癌旁组织，E2F7 mRNA表达高于癌旁组织，CFPAC-1、SW1990、Capan-1、PANC-1细胞miR-142-5p表达低于HPDE细胞（P＜0.05）。Capan-1、PANC-1细胞E2F7 mRNA及其蛋白表达高于HPDE细胞（P＜0.05）。Starbase数据库分析结果显示，miR-142-5p与E2F7基因的3’非翻译区存在互补。双萤光素酶报告基因结果显示，共转染E2F7-wt后，miR-142-5p mimics组萤光素酶活性低于NC组（P＜0.05）；共转染E2F7-mut后，miR-142-5p mimics组萤光素酶活性与NC组比较，差异无统计学意义（P＞0.05）。E2F7组E2F7 mRNA及其蛋白表达、细胞活力、划痕愈合面积、相对侵袭细胞数均高于NC组，细胞凋亡率低于NC组（P＜0.05）。miR-142-5p mimics+E2F7组E2F7mRNA及其蛋白表达、细胞活力、划痕愈合面积、相对侵袭细胞数均低于E2F7组，细胞凋亡率高于E2F7组（P＜0.05）。E2F7裸鼠组肿瘤体积、重量高于NC裸鼠组，miR-142-5p mimics+E2F7裸鼠组肿瘤体积、重量低于E2F7裸鼠组（P＜0.05）。苏木精-伊红染色显示，E2F7裸鼠组肿瘤具有较强的转移能力，而miR-142-5p mimics+E2F7裸鼠组肿瘤的转移能力较差。结论 miR-142-5p可能通过靶向E2F7基因影响PC细胞增殖、迁移、侵袭和凋亡。

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	阿木提江·马合木提　　郑坚江　　迪里夏提·阿力木

关键词 ：微RNA-142-5p, E2F7基因, 胰腺癌, 细胞增殖, 细胞侵袭

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.

Key words： MicroRNA-142-5p E2F7 gene Pancreatic cancer Cell proliferation Cell invasion

基金资助:新疆维吾尔自治区科学技术厅科研项目（2021D03019）。

引用本文:

阿木提江·马合木提　　郑坚江　　迪里夏提·阿力木. 微RNA-142-5p与E2F7基因的关系及其影响胰腺癌增殖、侵袭、转移的机制研究[J]. 中国医药导报, 2023, 20(26): 16-21,42.
Amutijiang·Mahemuti ZHENG Jianjiang Dixiati·Alimu. Study on the relationship between microRNA-142-5p and E2F7 gene and mechanism of its influence on the proliferation, invasion, metastasis of pancreatic cancer. 中国医药导报, 2023, 20(26): 16-21,42.

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