胃癌细胞株MKN-45肿瘤干细胞中microRNA差异表达研究

目的筛选胃癌干性相关的mircoRNA(miRNA),探讨其差异表达的意义。方法体外成球实验筛选胃癌干细胞,应用miRNA芯片技术筛选spheroid及attachment培养胃癌细胞株MKN-45差异表达的miRNA。同时,选取10例新鲜胃癌组织及其癌旁组织运用qRT-PCR对差异表达的miRNA进行验证。运用靶基因预测软件预测差异表达miRNA可能的调控靶基因。结果筛选得到胃癌干细胞较胃癌细胞表达明显上调的miRNA共有7个,分别为miR-29a-3p、miR-21-5p、miR-22-3p、miR-4270、miR-483-5p、let-7b-3p、miR-34a-5p。7个miRNA在球体细胞与亲代贴壁细胞间的相对表达量存在明显差异。其中,miR-29a-3p、miR-21-5p、miR-22-3p、miR-4270、miR-483-5p的表达水平与芯片检测结果一致,符合率71%;let-7b-3p、miR-34a-5p的表达水平与芯片检测结果不一致。MiRanda、Target Scan及Pictar在线数据库的交集中,miR-27a-3p、miR-34a-5p、miR-21-5p、miR-4270、let-7b-3p以及miR-29a-3p可预测到靶基因,而miR-483-5p与miR-16-5p未能预测到靶基因。结论miRNA在胃癌细胞株MKN-45肿瘤干细胞中存在特异性的表达谱,其差异表达的miRNA可能与胃癌耐药相关,可作为诊断胃癌和评判预后的重要标准,并为抵胃癌耐药提供可能靶点。

Differential expression of microRNA in gastric cancer MKN-45 cancer stem cell

Objective To investigate the significance of differential expression of microRNA( miRNA) by screening associated gastric cancer stem cells. Methods Gastric cancer stem cells was cultured by spheroid and the attachment,the different miRNA expressions in gastric cancer cell MKN-45 were detected by miRNA array. At the same time,10 fresh gastric cancer samples and paired adjacent normal tissue samples were collected,and the discovered miRNA were confirmed by real time RT-PCR. Using the forecast of the target gene prediction software may discover differentially expressed miRNA regulating target genes. Results A total of 7 miRNA which ex-pression were obviously up-regulated,such as miR-29a-3p,miR-21-5p,miR-22-3p,miR-4270,miR-483-5p,let-7b-3p,miR-34a-5p, which had statistically significant difference in ball somatic cells and parental relative expression between the adherent cells detected by miRNA microarray in MKN-45. Among them,5 miRNA detected from real time RT-PCR such as miR-29a-3p,miR-21-5p,miR-22-3p,miR-4270,miR-483-5,the expression level and results detected by microarray were in agreement,the coincidence rate was 71%;the expression level and results detected by microarray between let-7b-3p and miR-34a-5p were in disagreement. In MiRanda,Tar-getScan and Pictar online database of intersection,miR-27a-3p,miR-34a-5p,miR-21-5p,miR-4270,let-7b-3p and miR-29a-3p can be detected predicted target genes;miR-483-5p and miR-16-5p were failed to detect predicted target genes. Conclusion miRNA in gas-tric cancer cell exists specific expression spectrum in MKN-45 cancer stem cells,the differential expression of miRNA may be associat-ed with gastric cancer drug resistance,which can be used as the important standard in the diagnosis of gastric cancer and judging prog-nosis,and provide possible targets for gastric cancer drug resistance.