目的：探讨miR-502-3p通过靶向GTP结合蛋白2（GTPBP2）调控结直肠癌干细胞（CCSC）增殖、细胞周期和凋亡的分子机制。方法：利用免疫磁珠分选技术从结直肠癌细胞HCT116中分选CCSC（CD133+CD44+双阳性细胞和CD133-CD44-双阴性细胞），用qPCR法检测细胞中miR-502-3p表达水平。利用脂质体转染法分别将miR-NC、miR-502-3p、si-miR-NC、si-miR-502-3p、miR-502-3p+vector和 miR-502-3p+GTPBP2 转染至 CD133+CD44+细胞中，记作 miR-NC、miR-502-3p、si-miR-NC、si-miR-502-3p、miR-502-3p+vector和miR-502-3p+GTPBP2组。用qPCR法检测细胞中miR-502-3p、GTPBP2 mRNA表达水平，MTT法、流式细胞术分别检测细胞增殖率、细胞周期和凋亡率，WB法检测细胞中 Ki67、CDK1、Bcl2、BAX和GTPBP2蛋白 的 表 达 水 平 。 双 荧光 素 酶 报 告基因实验验证miR-502-3p与GTPBP2基因的靶向关系。结果：CD133+CD44+细胞中miR-502-3p表达水平显著低于CD133-CD44-细胞（P<0.01）。与miR-NC组比较，miR-502-3p 组细胞增殖率、S 期细胞比例显著降低（均 P<0.01），凋亡率、G0/G1期细胞比例显著升高（均 P<0.01），细胞中 Ki67、CDK1、Bcl2 蛋白表达均显著下调（均P<0.01）、BAX蛋白表达显著上调（P<0.01）。miR-502-3p 靶向调控 GTPBP2 的表达 ，过表达 GTPBP2可逆转上调miR-502-3p对CCSC增殖、周期和凋亡的作用。结论：上调miR-502-3p表达抑制CCSC增殖和阻滞细胞周期、诱导凋亡，其作用机制可能与过表达GTPBP2有关。

Objective: To investigate the molecular mechanism by which miR-502-3p regulates the proliferation, cell cycle and apoptosis of colorectal cancer stem cells (CCSCs) by targeting GTP binding protein 2 (GTPBP2) gene. Methods: The immunomagnetic bead sorting technique was used to sort CCSCs (CD133+CD44+ cells and CD133-CD44- cells) from colorectal cancer HCT116 cells, and the expression level of miR-502-3p in the sorted cells was detected by qPCR. CD133+CD44+ cells were divided into different groups according to different transfections using liposome method, namely miR-NC group, miR-502-3p group,si-miR-NC group, si-miR-502-3p group, miR-502-3p+vector group and miR-502-3p+GTPBP2 group. The mRNA expression levels of miR-502-3p and GTPBP2 in cells were detected using qPCR method. The proliferation rate, cell cycle and apoptosis rate were detected by MTT assay and flow cytometry, and the protein expression levels of Ki67, CDK1, Bcl2, BAX and GTPBP2 were detected by WB. Dual-luciferase reporter gene assay adopted to verify the targeting relationship between miR-502-3p and GTPBP2 gene. Results: The expression level of miR-502-3p in CD133+CD44+ cells was significantly lower than that in CD133-CD44- cells (P<0.01). Compared with the miR-NC group, the cell proliferation rate and the proportion of S phase cells were significantly reduced (all P<0.01), the apoptosis rate and proportion of cells in G0/G1 phase were significantly increased (all P<0.01), the protein expression of Ki67, CDK1 and Bcl2 was significantly down-regulated (all P<0.01), and BAX protein expression was significantly up-regulated (P<0.01) in the miR-502-3p group. miR-502-3p targetedly regulated the expression of GTPBP2. Overexpression of GTPBP2 could reverse the effects of up-regulation of miR-502-3p on the proliferation, cell cycle and apoptosis of CCSCs. Conclusion: Up-regulating the expression of miR-502-3p can inhibits the proliferation, arrests the cell cycle, and induces the apoptosis of CCSCs. The mechanism may be related to the overexpression of GTPBP2 gene.

武汉市卫生健康委员会科研项目（No.WX20A07）