磁性分选肺腺癌始动细胞的异常miRNAs验证

目的利用磁性活细胞分选法（magnetic activated cell sorting,MACS）从人A549肺腺癌细胞中分离得到CD133＋标记细胞,通过CD133/CD326双阳性检测探讨分离效果,初步分析差异表达miRNAs对该亚群细胞的调控功能。方法将对数生长期的A549细胞离心收集,重悬于无血清培养基中,培养至第二代后利用CD133磁珠标记后分选,流式细胞术及免疫荧光验证分选后细胞的CD133/CD326双阳性率;结合前期实验miRNA芯片结果,挑选兴趣分子进行定量PCR验证。结果利用CD133磁珠分选得到的阳性细胞亚群高表达CD133/CD326分子,结合前期miRNA芯片结果,选出在CD133＋/CD326＋细胞亚群中表达上调的miR-663,miR-183,miR-125a-5p,miR-127,miR-520h及表达下调的miR-18b,miR-29ab,miR-17和miR-155行定量PCR检测证实miR-29ab,miR-155,miR-183,miR-127-3p及miR-17的表达趋势与芯片结果相符。结论利用磁珠分选方式能获得CD133＋/CD326＋高表达肺腺癌始动细胞亚群且包括miR-183等在内的6条分子与芯片结果一致,可能在肺腺癌始动细胞生物学行为的调控中发挥重要作用。

Aberrant miRNA validation in lung adenocarcinoma initiating cells spared by magnetic bead from A549

Objective To validate magnetic activated cell sorting （MACS） is another means in enriching lung adenocarcinoma initiating cells from normal A549 cells and based on quantitative RT-PCR to analyze regulatory roles of this subpopulation. Methods After obtaining the lung adenocarcinoma initiating cells by MACS, we utilize flow cytometry analysis and imrnunofluoreseence to verify CD 133/CD326 expression of this subpopulation and choose 10 miRNAs to perform quantitative RT-PCR. Results We obtained CD133/CD326 high expression subpopulation by MACS. 10 miRNAs chose for quantitative RT-PCR and 6 miRNAs expression trend were consist with army data including miR-29ab, miR-155, miR-183, miR-127-3p, miR-17. Conclusion MACS can enrich CD133＋/CD326＋ subpopulation and 6 miRNAs expression trend were consist with array data, which may play important roles in regulating the biobehavior of lung adenocarcinoma initiating cells.