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目的分析miR-146a、miR-206、miR-223、let-7c-1等细胞分化相关miRNAs,在小鼠Lewis肺癌细胞株(theLewis lung cancer cell lines,LLC)CXCR4+与CXCR4-亚群间的差异表达。方法免疫磁珠分选CXCR4+和CXCR4-LLC细胞,Trizol法抽提细胞总RNA,实时荧光定量PCR(TaqMan探针)检测miRNAs表达,对表达差异最为显著的miRNAs进行潜在靶基因预测,应用免疫组化方法初步分析具有研究价值的关键分子在两个不同亚群小鼠种植瘤组织中的表达情况,并通过BLAST对其3′非翻译端(untranslated region,UTR)进行直向同源基因序列比对分析。结果CXCR4+与CXCR4-LLC比较,各检测mirna表达均较低,其中以miR-223差异最为显著(Fold Change=8.26),通过软件分析预测,miR-223与IGF1R、IGFBP5、Pik3cb、ELK-1和E2F1等基因均具有可能靶位点,免疫组化检测发现CXCR4+亚群种植瘤组织IGF1R表达显著高于CXCR4-亚群,IGF1R3′UTR的238～244nt和688～695nt两个结合位点具有高度的进化保守性。结论miR-223在CXCR4+LLC中显著低表达,可能与肺腺癌细胞IGF1R信号通路调控有关。
Objective To analyze the differences between CXCR4 + and CXCR4 subsets in mouse Lewis lung cancer cell lines (LLC) induced by miR-146a, miR-206, miR-223 and let-7c- expression. Methods The CXCR4 + and CXCR4-LLC cells were sorted by immunomagnetic beads, the total RNA was extracted by Trizol method, the miRNAs were detected by real-time fluorescent quantitative PCR (TaqMan probe), and the potential target genes were predicted by miRNAs with the most significant difference. Methods Preliminary analysis of the expression of key molecules with research value in the implanted tumor tissues of two different subgroups in mice and analysis of their orthologous gene sequences by 3 ’untranslated region (UTR) by BLAST Analysis. Results Compared with CXCR4-LLC, the expression of miR-223 was significantly lower than that of CXCR4-LLC. The most significant difference was found in miR-223 (Fold Change = 8.26) E2F1 and other genes have the possible target sites. Immunohistochemistry showed that the expression of IGF1R in CXCR4 + subsets was significantly higher than that in CXCR4- subsets. The two binding sites of 238 ~ 244nt and 688 ~ 695nt of IGF1R3’UTR had high Evolutionary conservativeness. Conclusions miR-223 is significantly down-regulated in CXCR4 + LLC cells, which may be related to the regulation of IGF1R signaling pathway in lung adenocarcinoma cells.