miR-9对甲状腺乳头状癌干细胞体外增殖及迁移能力的影响

Effects of microRNA-9 on proliferation and migration of thyroid papillary cancer stem cells in vitro

背景:miR-9在各类型肿瘤及肿瘤不同发生发展阶段的作用不一样,而对于其在甲状腺乳头状癌中的作用目前尚不清楚。目的:分析miR-9基因对甲状腺乳头状癌干细胞体外增殖及迁移能力的影响。方法:实验采用流式细胞仪从人乳头状甲状腺癌细胞中分选ALDH1阳性细胞,实时反转录PCR检测miR-9基因的表达。取对数生长期的ALDH1阳性细胞,分别转染mi R-9模拟物、mi R-9抑制物或mi R模拟物对照,48 h后采用q RT-PCR检测mi R-9转染效率,MTT实验、Tunel法、Transwell小室实验检测细胞增殖、凋亡、迁移能力,Western Blot检测凋亡相关蛋白Bcl-2、Bax及迁移相关蛋白基质金属蛋白酶2、基质金属蛋白酶9的表达。结果与结论:(1)miR-9模拟物组miR-9基因表达高于miR模拟物对照组(P<0.05),miR-9抑制物组miR-9基因表达低于miR模拟物对照组(P<0.05),说明实验转染成功;(2)与miR模拟物对照组比较,mi R-9模拟物组细胞增殖能力和迁移能力下降,细胞凋亡增多,而mi R-9抑制物组细胞增殖能力和迁移能力增强,细胞凋亡减少;(3)与miR模拟物对照组比较,mi R-9模拟物组Bcl-2蛋白减少、Bax蛋白增多(P均<0.05),mi R-9抑制物组Bcl-2蛋白增多、Bax蛋白减少(P均<0.05);(4)与mi R模拟物对照组比较,mi R-9模拟物组基质金属蛋白酶2、基质金属蛋白酶9表达减少(P<0.05),miR-9抑制物组基质金属蛋白酶2、基质金属蛋白酶9表达增多(P <0.05);(5)结果显示,mi R-9可抑制甲状腺乳头状癌干细胞的体外增殖及迁移,促进细胞凋亡。

BACKGROUND:MicroRNA-9(miR-9)exerts different effects in different types of tumors and at different stages of tumorigenesis,but its role in thyroid papillary carcinoma remains unclear.OBJECTIVE:To analyze the effects of miR-9 on the proliferation and migration of thyroid papillary cancer stem cells in vitro.METHODS:ALDH1 positive cells and ALDH1 negative cells were separated from human papillary thyroid cancer cells by flow cytometry.Expression of miR-9 mRNA was detected using reverse transcription PCR.ALDH1 positive cells in logarithmic growth phase were transfected with miR-9 mimetic,miR-9 inhibitor or miR mimetic control.After 48 hours,the transfection efficiency of miR-9 was detected by qRT-PCR;cell proliferation,apoptosis and migration were detected by MTT,TUNEL,and Transwell chamber assay,respectively.The expression of apoptosis-related proteins(Bcl-2 and Bax)and migration-related proteins(matrix metalloproteinase-2 and matrix metalloproteinase-9)was detected by western blot.RESULTS AND CONCLUSION:Successful transfections were confirmed by increased miR-9 gene expression in the miR-9 mimetic group and decreased miR-9 expression in the miR-9 inhibitor group relative to the miR mimetic control group(P<0.05).Compared with the miR mimetic control group,the miR-9 mimetic group showed decreased cell proliferation and migration ability,and increased apoptosis,while in the miR-9 inhibitor group,cell proliferation and migration abilities were increased,and cell apoptosis decreased.Compared with the miR mimetic control group,the expression of Bcl-2 protein was decreased and the expression of Bax protein increased in the miR-9 mimetic group(P<0.05),and the expression of Bcl-2 protein increased and expression of Bax protein decreased in the miR-9 inhibitor group(P<0.05).Compared with the miR mimetic control group,the expression of matrix metalloproteinase 2 and matrix metalloproteinase-9 was decreased in the miR-9 mimetic group(P<0.05),and the expression of matrix metalloproteinase 2 and matrix metalloproteinase-9 in the miR-9 inhibitor group was increased(P<0.05).To conclude,miR-9 can inhibit the proliferation and migration of thyroid papillary cancer stem cells in vitro,but increase cell apoptosis.