miR-21与舌鳞癌SCC9细胞干细胞样特性及顺铂敏感性的实验研究

The effect of miR-21 on the stem cell characteristics of human tongue squamous cell carcinoma and cisplatin sensitivity

目的观察舌鳞癌SCC9细胞干细胞样特性、顺铂敏感性以及miR-21表达对其影响。方法选取人舌鳞癌SCC9细胞分别通过贴壁、悬浮培养法培养获得SCC9a和SCC9f细胞;体外转染法将miR-21 mimics、miR-21 inhibitor分别转染至SCC9f细胞,设为SCC9m和SCC9i细胞。实时荧光定量PCR(QPCR)法检测干细胞相关转录因子(Oct3/4、Sox2、Nanog)及miR-21表达水平,ALDEFLOR kit检测ALDH阳性细胞的比例。MTT法、Annexin V/PI双染法分别检测顺铂对舌鳞癌细胞增殖及凋亡的影响。结果 SCC9f细胞中Oct3/4、Sox2和Nanog表达以及ALDH阳性比例高于SCC9a细胞(P<0.05)。顺铂对SCC9f细胞的增殖抑制率、凋亡率均低于SCC9a细胞。miR-21表达在SCC9f细胞中高于SCC9a细胞(P<0.05),而在SCC9m、SCC9i细胞中表达分别较SCC9f细胞明显升高与降低(均P<0.05)。Oct3/4、Sox2和Nanog在SCC9m细胞中表达高于SCC9f细胞,其中Oct3/4上调差异具有统计学意义(P<0.001);而Oct3/4、Sox2和Nanog在SCC9i细胞中表达均较SCC9f细胞呈显著下调(P<0.05)。SCC9a、SCC9m和SCC9i细胞中ALDH阳性率分别与SCC9f细胞中ALDH阳性率比较,差异均有统计学意义(P<0.05)。SCC9m、SCC9i细胞的凋亡率分别较SCC9f减低与增高(均P<0.05)。结论舌鳞癌SCC9细胞采用悬浮培养法培养获得的SCC9f细胞具有干细胞特性,对顺铂更为耐受;抑制miR-21表达可增强SCC9细胞对顺铂的敏感性,可能与miR-21调节其肿瘤干细胞相关转录因子表达有关。

Objective To investigate the effect of expression of miR-21 on the stem cell characteristics of human tongue squa- mous cell carcinoma and cisplatin sensitivity. Methods Human tongue squamous cell carcinoma SCC9 ceils were cultured by adherent and suspension cultures to obtain SCC9a and SCC9f cells. The miR-21 mimics and mir-21 inhibitor were transfected to the SCC9f cells respectively. Stem cell related transcription factors （Oct3/4, Sox2, Nanog） and miR-21 expression was detected by quantitative real- time PCR（QPCR）. The proportion of ALDH positive cells was detected by ALDEFLOR kit. The cell proliferation was determined by MTT assay, and AnnexinV/PI method was used to measure cell apoptosis. Results The expression of Oct3/4, Sox2 and Nanog and the positive rate of ALDH in SCC9f ceils was significantly higher than that of SCC9a cells. The inhibition rate and apoptosis ratio of SCC9f cells treated with cisplatin was lower than those of SCC9a cells. The expression of miR-21 in SCC9f was higher than that of SCC9a （ P〈 0. 05）. The expression of miR-21 in SCC9m was higher than that of SCC9f （P〈0. 05）, while that of SCC9i was lower than that of SCC9f （P〈O. 05）. The expression of Oct3/4 in SCC9m cells was higher than that in SCC9f ceils （P〈O. 05）. The expression of Oct3/ 4, Sox2 and Nanog in SCC9i cells was significantly lower than that in SCC9f cells （P〈0. 05）. The positive ratio of ALDH in SCC9a, SCC9m and SCC9i cells was significantly different from that in SCC9f cells （P〈0. 05）. The apoptosis rate of SCC9f ceils was lower than that of SCC9i cells, and higher than that of SCC9m cells （ all P〈0. 05）. Conclusion The SCC9f cultured by suspension culture meth- od have stem cell characteristics, and more tolerable to cisplatin. Inhibition of miR-21 expression could enhance the sensitivity of SCC9 cells to cisplatin. It may be related to tumor stem cells which are regulated by the expression of miR-21.