结肠癌患者血清中miR-186-5p表达的研究

doi:10.12092/j.issn.1009-2501.2020.10.003

中国临床药理学与治疗学 ›› 2020, Vol. 25 ›› Issue (10): 1097-1104.doi: 10.12092/j.issn.1009-2501.2020.10.003

结肠癌患者血清中miR-186-5p表达的研究

程建平¹，李珍¹，赵晓琳¹，杨梦媛¹，冀希炜²，于久飞¹

¹民航总医院消化肿瘤科，北京100123；²北京大学第一医院临床药理研究所，北京100191

收稿日期:2020-05-08 修回日期:2020-08-09 出版日期:2020-10-26 发布日期:2020-11-03
通讯作者: 于久飞，男，硕士研究生，主任医师，研究方向：消化病学及消化道肿瘤。 E-mail: yujiufei1962@163.com 冀希炜，男，博士研究生，助理研究员，研究方向：抗肿瘤药物药理学。 E-mail: jxezz@sina.com
作者简介:程建平，男，硕士研究生，主治医师，研究方向：消化道肿瘤。 E-mail: chenjianping1209@163.com
基金资助:
国家自然科学基金资助项目（81803614）

Research on the expression of miR-186-5p in serum of patients with colon cancer#br#

CHENG Jianping ¹, LI Zhen ¹, ZHAO Xiaolin ¹, YANG Mengyuan ¹, JI Xiwei ², YU Jiufei ¹

¹Department of Gastroenterology and Oncology, Civil Aviation General Hospital, Beijing 100123, China; ²Institute of Clinical Pharmacology, Peking University First Hospital, Beijing 100191, China

Received:2020-05-08 Revised:2020-08-09 Online:2020-10-26 Published:2020-11-03

摘要/Abstract

摘要： 目的：探讨miR-186-5p在结肠癌中的表达及其对于结肠癌的影响，为结肠癌的治疗和预后治疗提供新的治疗靶点。方法：收集本院符合条件的病人血清样本，分为健康组、临床I、II、III、IV期5个组别（n=20），利用miRVana microRNA分离试剂盒提取病人血清中的miRNA，利用荧光实时定量PCR检测miR-186-5p在不同阶段结肠癌病人血清中表达的差异。将miR-186-5p的全长基因克隆到pENTR/D-Topo载体，构建miR-186-5p高表达的细胞系，利用miR-186-5p抑制剂，降低miR-186-5p表达。利用细胞增殖ELISA试剂盒，将5×103/孔的细胞接种到96孔板中，孵育24 h后加入BrdU标记24 h，在370和492 nm测定吸光度，测定miR-186-5p对肿瘤细胞增殖的影响。用MTT法检测miR-186-5p对结肠癌细胞系对化疗药物敏感性的影响。结果：结肠癌病人血清中miR-186-5p呈低表达状态，与正常组相比，临床I、II、III、IV期病人血清中miR-186-5p表达明显减少（P<0.01）。利用质粒构建miR-186-5p高表达的细胞系，利用miR-186-5p抑制剂（hsa-miR-186-5p inhibitor）降低SW480、HCT116两种细胞内miR-186-5p的表达。与对照组相比，过表达miR-186-5p的HCT116细胞和SW480细胞增殖能力减弱，说明miR-186-5p表达增多抑制了HCT116细胞和SW480细胞的增殖。miR-186-5p抑制剂处理后，与对照组相比，HCT116细胞和SW480细胞的增殖能力均升高，说明miR-186-5p表达降低可以增加HCT116细胞和SW480细胞的增殖。不同浓度顺铂处理24 h后，高表达miR-186-5p的HCT116和SW480细胞死亡率均高于对照组（P<0.01），miR-186-5p表达降低后，死亡率低于对照组（P<0.01）。在miR-186-5p高表达的结肠癌细胞系中，PLK1蛋白表达降低，而抑制miR-186-5p表达后，PLK1蛋白表达升高。结论：结肠癌患者中miR-186-5p表达降低，过表达miR-186-5p可以抑制结肠癌细胞增殖生长，并降低结肠癌细胞的耐药性，抑制miR-186-5p表达可以增加结肠癌细胞增殖，并增强细胞的耐药性。

关键词: 结肠癌, miR-186-5p, 质粒, 细胞增殖, 顺铂

Abstract: AIM: To investigate the expression of miR-186-5p in colon cancer and the effect on colon cancer, and provide a new therapeutic target for the treatment and prognosis of colon cancer. METHODS: Serum from eligible patients were collected and divided into five groups (n=20) including the healthy group and clinical stages I, II, III and IV. The miRNAs in patients' serum were extracted by miRVana microRNA isolation kit. The differences of miR-186-5p expression in patients' serum of colon cancer at different stages were detected with real-time quantitative PCR. The full-length gene of miR-186-5p was cloned into pENTR/D-Topo vector to construct cell lines with high expression of miRNA. 5×103 cells were seeded in 96-well plates. The expression of mir-186-5p was decreased by using miR-186-5p inhibitor. The proliferation of cells was detected with cell proliferation ELISA kit after 24 hours of incubation with BrdU and absorbance was measured at 370 and 492 nm. The effect of miR-186-5p on the sensitivity of colon cancer cell lines to chemotherapeutic drugs was detected by MTT assay. RESULTS: The expression of miR-186-5p was down-regulated in the serum of patients with colon cancer compared to normal group. Expression of miR-186-5p in serum of patients with stage I, II, III and IV was significantly decreased (P<0.01) compared with the normal group. Colon cells were transfected with a plasmid integrating the miR-186-5p gene, and miR-186-5p was highly expressed in the HCT116 and SW480 cell lines (P<0.01). The expression of mir-186-5p was decreased by mir-186-5p inhibitor (hsa-miR-186-5p inhibitor) in HCT116 and SW480 cells. The proliferation rate of HCT116 and SW480 cells with high expression of miR-186-5p was lower than control group (P<0.01). After treating with mir-186-5p inhibitor, the cell proliferation rate was higher than that of the control group (P<0.01). After treatment with different concentrations cisplatin for 24 hours, the mortality of HCT116 and SW480 cells with high expression of mir-186-5p was higher than that of the control group (P<0.01). The mortality of the cells with low expression of mir-186-5p was lower than that of the control group (P<0.01). The protein level of PLK1 was decreased after transfecting with miR-186-5p plasmid and increased after stimulating with miR-186-5p inhibitor in HCT116 and SW480 cells. CONCLUSION: The expression of miR-186-5p is decreased in colon cancer patients. Overexpression of miR-186-5p can inhibit the proliferation of colon cancer cells and reduce the drug resistance of colon cancer cells; inhibition of miR-186-5p expression can increase the proliferation and drug resistance of colon cancer cells.

Key words: colon cancer, miR-186-5p, plasmid, cell proliferation, cisplatin

中图分类号:

程建平, 李珍, 赵晓琳, 杨梦媛, 冀希炜, 于久飞. 结肠癌患者血清中miR-186-5p表达的研究[J]. 中国临床药理学与治疗学, 2020, 25(10): 1097-1104.

CHENG Jianping, LI Zhen, ZHAO Xiaolin, YANG Mengyuan, JI Xiwei, YU Jiufei. Research on the expression of miR-186-5p in serum of patients with colon cancer#br#[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2020, 25(10): 1097-1104.

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结肠癌患者血清中miR-186-5p表达的研究

Research on the expression of miR-186-5p in serum of patients with colon cancer#br#

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