丹参活性成分调控人肺腺癌A549细胞外泌体miRNA的变化及外泌体对HUVEC的作用

Active components of Salvia miltiorrhizae to regulate the changes of exosomal miRNA in human lung adenocarcinoma A549 cells and the effects of exosomes on HUVEC

目的:检测隐丹参酮、丹参酮IIA对人肺腺癌A549细胞分泌的外泌体中内源性非编码小RNA(microRNA,miRNA)的差异表达情况。观察外泌体对HUVEC细胞的作用,并初步探索其作用机制。方法:取对数生长期的A549细胞,分别以隐丹参酮6μg/mL及丹参酮IIA 4μg/mL给药,用无血清的培养基培养24小时后,细胞上清液按照说明书提取外泌体。采用高通量测序技术,检测各组A549细胞中miRNA的表达情况,对外泌体中hsa-miR-1246进行验证,对获得的差异基因进行GO和KEGG分析。以各组外泌体加入HUVEC细胞中,并采用CCK-8实验和划痕实验,分别检测各组外泌体对HUVEC细胞的抑制作用和迁移作用,检测HUVEC细胞中CD31的基因和蛋白表达。结果:高通筛选结果发现隐丹参酮能够上调109个miRNA,下调78个miRNA;丹参酮IIA能够上调35个miRNA,下调40个miRNA。将有差异的miRNA进行功能与通路的富集分析,显示隐丹参酮、丹参酮IIA差异表达基因主要与内吞作用、癌症通路、MAPK信号通路、hippo信号通路、Wnt信号通路、癌症中的microRNAs、Rap1信号通路、肌动蛋白细胞骨架的调节等相关。细胞增殖实验发现,空白外泌体和隐丹参酮外泌体对HUVEC细胞的增殖均具有显著的抑制作用,且隐丹参酮外泌体的抑制作用明显高于空白外泌体。细胞划痕实验发现,外泌体给药后,迁移率均有显著下降,外泌体作用48 h后,隐丹参酮外泌体和丹参酮IIA外泌体作用后HUVEC的迁移率均显著低于空白外泌体。隐丹参酮处理后的A549细胞外泌体中hsa-miR-1246显著升高,而隐丹参酮外泌体作用后的HUVEC细胞中CD31的基因和蛋白的表达显著降低。结论:隐丹参酮、丹参酮IIA对A549细胞外泌体中miRNA有一定的调控作用,且富集分析与多个癌症有关信号通路相关。隐丹参酮、丹参酮IIA处理下的A549细胞外泌体对HUVEC有抑制作用,进一步从外泌体角度探析隐丹参酮及丹参酮IIA抑制血管内皮细胞增殖可能的作用机制,旨在为肺癌的治疗策略提供新的思路。

Objective:To investigate the differential expression of cryptotanshinone and Tanshinone IIA on endogenous small non-coding RNA(miRNA)in exosomes secreted by human lung adenocarcinoma A549 cells.To observe the effect of exosomes on HUVEC cells and explore its mechanism.Methods:A549 cells at logarithmic growth stage were treated with 6μg/mL cryptotanshinone and 4μg/mL tanshinone IIA,respectively,and cultured in serum-free medium for 24 hours.Exosomes were extracted from the cell supernatant according to the instructions.High-throughput sequencing technology was used to detect miRNA expression in A549 cells of each group,and the obtained differential genes were analyzed by GO and KEGG.Exosomes of each group were added to HUVEC cells,and the inhibition and migration of exosomes of each group on HUVEC cells were detected by CCK-8 test and scratch test,CD31 gene and protein expression was detected in HUVEC cells.Results:Cryptotanshinone could up-regulate 109 miRNAs and down-regulate 78 miRNAs.Tanshinone IIA could up-regulate 35 miRNAs and down-regulate 40 miRNAs.The function and pathway enrichment analysis of the different miRNAs showed that the differential expression genes of cryptotanshinone and tanshinone IIA were mainly related to endocytosis,cancer pathway,MAPK signaling pathway,hippo signaling pathway,Wnt signaling pathway,microRNAs in cancer,Rap1 signaling pathway,and actin cytoskeleton regulation.The cell proliferation experiment showed that both the exosomes and cryptotanshinone treated-exosomes had significant inhibitory effects on the proliferation of HUVEC cells,and the inhibitory effect of cryptotanshinone treated-exosomes was significantly higher than that of exosomes.The cell scratch test showed that the mobility of all exosomes decreased significantly after exosomes were administered.After 48 h of exosomes,HUVEC mobility of both cryptotanshinone treated-exosomes and tanshinone IIA treated-exosomes was significantly lower than that of empty exosomes.Hsa-miR-1246 in exosomes of A549 cells treated with cryptotanshinone was significantly increased,while the gene and protein expression of CD31 in HUVEC cells treated with cryptotanshinone exosomes was significantly decreased.Conclusion:Cryptotanshinone and Tanshinone IIA have certain regulatory effects on miRNA in A549 exosomes,and the enrichment analysis is related to several cancer-related signaling pathways.Exosomes of A549 cells treated with cryptotanshinone and tanshinone IIA have inhibitory effects on HUVEC.The possible mechanism of inhibition of vascular endothelial cell proliferation by cryptotanshinone and tanshinone IIA from the perspective of exosomes is further explored,aiming to provide new ideas for the treatment of lung cancer.