基于miRNA调控网络探究miR-653在肺癌干细胞干性维持中的作用

Probing the Role of miR-653 in Stemness Maintenance of Lung Cancer Stem Cells Based on miRNA Regulatory Network

目的:基于miRNA调控网络探究miR-653在肺癌干细胞干性维持中的作用及其相关机制。方法:miRNA-seq检测肺癌患者肿瘤组织和癌旁组织,根据差异miRNA绘制调控网络,并对靶基因进行KEGG分析。通过脂质体转染法将miR-NC、miR-653、oe-NC、oe-MYC转染至H460肺癌细胞系,分组为:miR-NC组、miR-653组、miR-653+oe-NC组、miR-653+oe-MYC组。qRT-PCR检测各组miR-653、MYC表达;流式细胞术检测CD133、CD24蛋白表达;CCK8检测细胞增殖水平;有限稀释法检测细胞成球比例;通过荧光素酶报告基因检测试剂盒检测miR-653和MYC靶向性。结果:与癌旁组织比较,肿瘤组织中有326个miRNA表达升高,363个miRNA表达降低,其中miR-653表达显著降低(P<0.05)。调控网络预测miR-653靶向22个mRNA,参与细胞干性维持等信号通路。在野生型MYC中,miR-653组荧光强度低于miR-NC组(P<0.05)。与miR-NC组比较,miR-653组miR-653表达升高,MYC表达降低;CD133、CD24蛋白降低,细胞增殖和成球比例降低(P<0.05);与miR-653+oe-NC组比较,miR-653+oe-MYC组MYC表达升高;CD133、CD24蛋白升高,细胞增殖和成球比例增加(P<0.05)。结论:miRNA调控网络参与肺癌细胞干性维持,miR-653靶向调控MYC抑制肺癌细胞干细胞维持。

Objective:To explore the role and related mechanism of miR-653 in the maintenance of stemness of lung cancer stem cells based on the miRNA regulatory network.Methods:miRNA-seq was used to detect tumor tissues and adjacent tissues of lung cancer patients,and the regulatory network was drawn according to the difference of miRNA,and the target genes were analyzed by KEGG analysis.miR-NC,miR-653,oe-NC,and oe-MYC were transfected into the H460 lung cancer cell line by liposome transfection method.The cells were divided into the miR-NC group,miR-653 group,miR-653+oe-NC group,and miR-653+oe-MYC group.The expression of miR-653 and MYC were detected by qRT-PCR.The protein expression of CD133 and CD24 was detected by flow cytometry.CCK8 was used to detect cell proliferation.The proportion of pellet formation was detected by the limited dilution method.The targeting of miR-653 and MYC was detected by a luciferase reporter assay kit.Results:Compared with adjacent tissues,245miRNAs were up-regulated and 363 miRNAs were down-regulated in tumor tissues,among which miR-653 was significantly down-regulated(P<0.05).The regulatory network predicted that miR-653 targeted 22 miRNAs and was involved in signaling pathways such as cell stemness maintenance.The fluorescence intensity of the miR-653 group was lower than that of the miR-NC group in wild-type MYC(P<0.05).Compared with the miR-NC group,the expression of miR-653 was increased and the expression of MYC was decreased in the miR-653 group.The protein expression of CD133 and CD24 was decreased,and the proportion of cell proliferation and pellet formation ratio was decreased(P<0.05).Compared with the miR-653+oe-NC group,the expression of MYC in the miR-653+oe-MYC group was increased.The protein expression of CD133,and CD24 was increased,and cell proliferation and pellet formation ratio was increased(P<0.05).Conclusion:The miRNA regulatory network is involved in the stemness maintenance of lung cancer cells,and miR-653 regulates MYC to inhibit the maintenance of lung cancer cell stem cells.