hMLH1和miR-148a表达与散发性结直肠癌微卫星不稳定性的关系

The relationship between the expression of hMLH1 and miR-148a and microsatellite instability in sporadic colorectal cancer

目的检测散发性结直肠癌(sCRC)组织中人类错配修复基因1(hMLH1)蛋白和miR-148a表达及其与微卫星不稳定性(MSI)的关系。方法收集黄冈市中心医院病理科保存的sCRC组织标本90例及其对应癌旁正常组织。收集患者临床病历资料,采用多重荧光PCR法检测组织DNA微卫星不稳定性,免疫组化染色法检测组织hMLH1蛋白表达,实时荧光定量PCR(qPCR)检测组织miR-148a表达量,双荧光素酶报告实验分析miR-148a和hMLH1 mRNA靶向关系。结果90例sCRC组织中,检出MSI 29例(32.22%),hMLH1蛋白表达缺失24例(26.67%)。经荧光素酶报告基因分析,miR-148a mimics可抑制含有hMLH1 mRNA 3′UTR-WT序列的报告基因质粒的荧光素酶活性;而且hMLH1蛋白表达缺失组织中miR-148a相对表达量高于未缺失组织(P<0.05);此外MSI组织中hMLH1蛋白表达缺失率和miR-148a相对表达量均高于微卫星稳定性(MSS)组织。右半结肠和直肠部位、pN_(0/1)期、pM _(0)分期hMLH1蛋白表达缺失率和MSI比例高于左半结肠、pN_(2)期、pM_(0)期(P<0.05);另外肿瘤直径>5.0 cm、右半结肠和直肠、pT_( 3/4)期、pN_(2)期、pM_(1)分组织miR-148a相对表达量低于肿瘤直径≤5.0 cm、左半结肠、pT_(1/2)期、pN_(0/1)期、pM_(0)期组织(P<0.05)。结论约30%的sCRC患者存在hMLH1蛋白表达缺失和MSI状态;sCRC肿瘤组织miR-148a相对表达量降低,导致对hMLH1基因表达的抑制作用减弱,可能是影响sCRC肿瘤细胞MSI状态的重要原因之一。

Objective To detect the expression of human mismatch repair gene 1(hMLH1)protein and miR-148a in sporadic colorectal cancer(sCRC)tissue and their relationship with microsatellite instability(MSI).Methods Ninety sCRC tissue specimens and their corresponding normal adjacent tissues were collected from the pathology department of our hospital.Collect clinical medical records of patients,detect tissue DNA microsatellite instability using multiplex fluorescence PCR,detect tissue hMLH1 protein expression using immunohistochemistry staining,detect tissue miR-148a expression using real-time fluorescence quantitative PCR(qPCR),and analyze the targeting relationship between miR-148a and hMLH1 mRNA using dual luciferase reporter assay.Results Among the 90 sCRC tissues,MSI was detected in 29 cases(32.22%)and hMLH1 protein expression was missing in 24 cases(26.67%).According to luciferase reporter gene analysis,miR-148a mimics can inhibit the luciferase activity of reporter gene plasmids containing the 3′UTR-WT sequence of hMLH1 mRNA;Moreover,the relative expression level of miR-148a in hMLH1 protein expression deficient tissues was higher than that in non deficient tissues(P<0.05);In addition,the loss rate of hMLH1 protein expression and the relative expression level of miR-148a in MSI tissues are higher than those in microsatellite stable(MSS)tissues.The loss rate and MSI ratio of hMLH1 protein expression in the right colon and rectum,pN_(0/1) stage,and pM_(0) stage were higher than those in the left colon,pN_(2) stage,and pM 0 stage(P<0.05);In addition,the relative expression level of miR-148a in tissues with tumor diameter>5.0 cm,right colon and rectum,pT _(3/4) phase,pN_(2) phase,and pM_(1) phase was lower than that in tissues with tumor diameter≤5.0 cm,left colon,pT_(1/2) phase,pN _(0/1) phase,and pM_(0) phase(P<0.05).Conclusion About 30%of sCRC patients have hMLH1 protein expression deficiency and MSI status.The relative expression level of miR-148a in sCRC tumor tissue is reduced,leading to a weakened inhibitory effect on hMLH1 gene expression,which may be one of the important reasons affecting the MSI status of sCRC tumor cells.