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

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5×10^3/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(hsamiR-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。

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 realtime 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.5×10^3 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 downregulated 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.