微小RNA-29c表达水平与胃癌生物学行为的关系

Relationship between the expression level of miR-29c and biological behavior of gastric cancer

目的探讨微小RNA-29c（miR-29c）在胃癌发生、发展过程中的作用。方法采用微小RNA（miRNAs）微阵列芯片技术检测胃癌组织中miRNAs表达谱，并筛选差异表达的miRNAs；采用实时定量PCR检测miR-29c在胃癌组织和相应正常胃上皮组织、正常胃上皮细胞系GES-1、胃癌细胞系BGC-823和SGC-7901中的表达水平；采用四甲基偶氮唑蓝（MTF）法和流式细胞术检测miR-29c对胃癌细胞增殖、细胞周期及药物敏感性的作用；应用实时定量PCR、Westernblot和荧光素酶报告基因技术研究髓样细胞白血病1（Mcl-1）与miR-29c之间的关系。结果与正常胃上皮组织比较，胃癌组织中表达上升超过2倍的miRNAs有7个，分别为miR-374b、miRPlus-E1212、miR-338-5p、miR-297、miR-21、miR-135b、miR-18a；表达下降的miRNAs有9个，分别为miR-29b-2、miR-1260、miRPlUS-E1241、miR-S1-5p、miR-48a、miR-29c、miR-647、miR-196b和ebv-miR-BART5。miR-29c在胃癌组织和正常胃上皮组织中的相对表达量分别为0．70±0．34和1．00±0．06（P〈0．05），分化越差的细胞系miR-29c表达水平越低（P〈0．05）。miR-29c表达与肿瘤直径、淋巴结转移、临床分期、Laur6n分型、Bon＇mann分型和Ming分型有关（均P〈0．05）。在BGC-823胃癌细胞中，miR-29c过表达可以抑制细胞增殖，促进细胞凋亡，使细胞周期受阻于S期，并增加肿瘤细胞对化疗药物多西紫杉醇的敏感性（均P〈0．05）。Mcl-1mRNA在胃癌组织和正常胃上皮组织中的相对表达量分别为3．47±1．34和1．00±0．20（P〈0．01），miR-29c和Mcl-1mRNA在胃癌组织中的表达呈负相关（r=一0．633，P〈0．05）。miR-29e直接靶向调控Mcl-1在胃癌细胞中的表达。结论胃癌组织具有自己独特的miRNAs表达谱。miR-29c的表达水平与胃癌的生物学行为相关。miR-29c参与对Mcl-1的靶向调控，可能是胃癌的发病机制之一。

Objective To study the function and clinicopathological significance of RNA-29c （miR- 29c） in the carcinogenesis and development of gastric cancer. Methods MicroRNA microarray was applied to assess the miRNAs expression profile of gastric cancer. Quantitative real-time PCR was used to detect the expression of miR-29c in 64 cases of gastric cancer tissues and corresponding normal gastric epithelium, as well as cell lines GES-1, BGC-823 and SGC-7901 ceils. MTT assay and flow cytometry were applied to detect the effects of forced expression of miR-29c in gastric cancer BGC-823 cells including cell proliferation, apoptosis, cell cycle and drug sensitivity. Quantitative real-time PCR, Western blot and luciferase reporter assay were used to explore the targeted relationship between miR-29c and myeloid cell leukemia-1 （ Mcl-1 ）. Results Compared with normal gastric epithelium, seven microRNAs （ miR-374b , miRPlus-E1212, miR-338-Sp, miR-297, miR-21, miR-135b, miR-18a） were significantly up-regulated more than 2-folds, and nine microRNAs （miR-29b-2 , miR-1260, miRPlus-E1241, miR-S1-Sp, miR- 148a, miR-29c, miR-647, miR-196b , ebv-miR-BARTS） were significantly down-regnated in gastric cancer tissues. The average expression level of miR-29c in gastric cancer tissues was 0.70 ± 0.34 and in corresponding normal epithelium was 1.00 ± 0.06 （P 〈 0.05 ）. miR-29c expression was related to tumorsize, lymph node metastasis, clinical stage, Laur6n classification, Borrmann classification and Ming classification （ P 〈 O. 05 ）. The poorer differentiation degree of gastric cell lines, the lower was miR-29c expression level （P 〈 0.05 ）. Overexpression of miR-29c in gastric cancer BGC-823 cells suppressed cell proliferation, stimulated cell apoptosis, induced cell cycle arrest in S phase and increased the chemotherapy sensitivity to drug docetaxel （ all were P 〈 0.05 ）. The average expression level of Mcl-1 mRNA in gastric cancer tissues was 3.47 ± 1. 34 and corresponding epithelialium was 1.00± 0.20 （ P 〈 0.05 ）. The expression level of miR-29c was negatively related with that of McM mRNA in gastric cancer tissues, miR- 29c directly targeted to regulation of Mcl-1 expression. Conclusions There are special miRNA expression profile in gastric cancer. The expression of miR-29c is closely related to biological behavior of human gastric cancer, miR-29c is involved in targeted regulation of Mcl-1, and may be one of mechanisms of the carcinogenesis of gastric cancer.