细胞因子诱导的凋亡抑制因子1靶向miRNA预测及其在乳腺癌耐药细胞株中的表达

Prediction of miRNAs targeting cytokine induced apoptosis inhibitor 1 and their expression in multidrug.resistant breast cancer ceil lines

目的：预测并筛选与细胞因子诱导的凋亡抑制因子1（CIAPIN1）基因3′非翻译区（3′UTR）高度匹配的目标miRNA，并初步探讨乳腺癌亲本细胞株及耐药细胞株中CIAPIN1蛋白及目标miRNA表达间的靶向关系。方法应用TargetScan、PicTarmi、miRanda数据库预测CIAPIN1基因3′UTR可能存在的种子结合位点miRNA，并根据文献筛选出目的miRNA。 Western blot法检测易成瘤的乳腺癌细胞株MCF-7、三阴性乳腺癌细胞株MDA-MB-231、人表皮生长因子2（HER2）高表达的乳腺癌细胞株MDA-MB-453及相对应的3种耐紫杉醇的乳腺癌细胞株MCF-7／Tax、MDA-MB-231／Tax、MDA-MB-453／Tax中CIAPIN1蛋白的表达情况。实时定量聚合酶链反应（PCR）法检测乳腺癌亲本细胞及其耐药细胞中目的miRNA的表达情况。结果预测并筛选出目标miRNA，分别为miRNA143、miRNA571、miRNA647。Western blot法检测示，与乳腺癌亲本细胞相比，其耐药细胞中CIAPIN1蛋白相对高表达，差异有统计学意义[MCF-7／Tax比MCF-7、MDA-MB-231／Tax比MDA-MB-231、MDA-MB-453／Tax比MDA-MB-453的表达量（相对于内参GAPDH）分别为：0.80比0.21、0.70比0.31、1.00比0.35，均P＜0.05]。 PCR法检测示，与乳腺癌亲本细胞相比，耐药细胞中miRNA143、miRNA571、miRNA647相对低表达，差异有统计学意义（P＜0.05）。结论预测并筛选出的与 CIAPIN1基因3′UTR 区存在种子结合位点的 miRNA 有miRNA143、miRNA571、miRNA647，各miRNA在耐药细胞株中低表达，而CIAPIN1蛋白相对高表达，提示这些miRNA可能通过靶向调控CIAPIN1基因参与了乳腺癌的多药耐药过程。

Objective To predict and select the targeting miRNA highly matched the 3＇untranslated region （3＇UTR） of cytokine induced apoptosis inhibitorl （CIAPIN1） gene, and to primarily investigate the targeting correlation between CIAPINI and the target miRNA in parental cell lines and drug-resistant cell lines of breast cancer. Methods The miRNA potentially binding to seed side of CIAPIN1 3＂-UTR was predicted by the bioinformatics software as well as screening out the target miRNA according to the literatures TargetScan, PicTarmi and miRanda. The expression of CIAPIN1 protein in parental cell lines and resistant cell lines of breast cancer was detected by using Western blot. The expression of the target miRNA in parental cell lines and resistant cell lines of breast cancer was detected by real-time quantitative PCR （qRT-PCR）. Results The miRNAs binding to seed side of targeting CIAPIN1 3＇-UTR were predicted including miRNA143, miRNA571, miRNA647. Compared with parental cell lines, the CIAPIN1 protein in resistant cell lines of breast cancer was high expression detected by Western blot with significant difference （MCF-7/Tax vs. MCF-7：0.80 vs. 0.21; MDA-MB-231/Tax vs. MDA-MB-231：0.70 vs. 0.31; MDA-MB-453/Tax vs. MDA-MB-453：1.00 vs. 0.35, all P〈 0.035）. Compared with parental cell lines, miRNA143, miRNA571 and miRNA647 in resistant cell lines was low expression detected by qRT-PCR, and there were statistical differences. Conclusions The miRNAs binding to seed side of CIAPIN1 3＇-UTR are predicted and selected including miRNA143, miRNA571, miRNA647. Compared with in parental cell lines, these miRNAs are low expression, and the CIAP1N1 protein is high expression in resistant cell lines of breast cancer, which suggests these miRNAs may be involved in multidrug resistance in breast cancer by regulating C1APIN1 gene.