肝细胞癌侧群/非侧群细胞中miRNA-21表达差异的检测

Differential expression of miRNA-21 between SP and NSP hepatocellular carcinoma cells

目的采用流式细胞仪优化Hoechst33342染料浓度,检测肝细胞癌(HCC)MHCC-97H细胞中侧群(SP)细胞与非侧群(NSP)细胞中微小RNA-21(miRNA-21)的表达情况。方法常规制备MHCC-97H单细胞悬液,采用不同浓度(5、6、7、8、9、10、11、12 mg/L)的荧光染料Hoechst33342染色,以维拉帕米拮抗组为对照,优化最适合的Hoechst33342染料浓度,通过流式细胞仪分离SP、NSP细胞;采用RT-PCR法检测SP、NSP细胞中miRNA-21的表达情况。结果优化Hoechst33342浓度为10 mg/L,活细胞比例较高,是最适染色的浓度,此时MHCC-97H中SP细胞比例为(8.4±0.7)%;从SP、NSP细胞中提取的总RNA纯度为1.8~2.0,其中没有DNA和蛋白质混杂;SP细胞的miRNA-21表达水平是NSP细胞的(7.30±0.78)倍,差异有统计学意义(P<0.05)。结论优化的Hoechst33342浓度可以提高SP细胞的纯度,同时miRNA-21表达可能与HCC干细胞分化有关。

Objective To optimize the dye concentration of Hoechst33342 in flow cytometry and detect the relative expression of microRNA-21（miRNA-21） between side population（SP） and non-side population（NSP） cells in MHCC-97H cell line. Method MHCC-97H single cells suspension was prepared and stained with different final concentration（5, 6, 7, 8, 9, 10, 11, 12 mg/L） of fluorescent dye Hoechst33342, verapamil antagonist was used as control, the flow cytometry was used to separate SP and NSP cells with the most suitable Hoechst33342 dye concentration. The relative expression of miRNA-21 was determined by RT-PCR between SP and NSP in the MHCC-97H cell. Result The optimized dye concentration was 10 mg/L for Hoechst33342, with the highest proportion of living cells, the proportion of SP cells was（8.4± 0.7）% in the MHCC-97H at the optimal concentration of dyeing; the overall purity of RNA extracted from SP and NSP cells was 1.8-2.0, with no DNA and residual proteins; the level of miRNA-21 in SP cells was（7.30±0.78） times of that in NSP cells, showing statistically significant difference（P〈0.05）. Conclusion Optimized concentration of dye Hoechst33342 could improve the purity of SP cells, and the expression of miRNA-21 may be related to the differentiation of HCC cells.