外泌体为载体的微小RNA-1基因运送抑制人口腔鳞状细胞癌CAL-27细胞增殖的研究

microRNA-1 gene delivery mediated by exosomes suppresses CAL-27 cell proliferation

目的用供体细胞基因过表达的方法构建微小RNA-1(miR-1)高表达的细胞内源性外泌体,探讨以外泌体为载体,运送miR-1对口腔鳞状细胞癌CAL-27细胞增殖的作用。方法采用超速离心法提取过表达miR-1的HEK293细胞的外泌体,并用透射电子显微镜、纳米颗粒分析仪、Western blot及定量聚合酶链反应(qPCR)进行外泌体鉴定。过表达miR-1的HEK293细胞外泌体(miR1-EXO)、HEK293细胞的外泌体(CON-EXO)及等体积磷酸盐缓冲液(PBS)分别与CAL-27细胞共培养,用免疫荧光检测外泌体向细胞的转运,用qPCR检测CAL-27细胞miR-1及下游靶基因MET的表达变化,用噻唑蓝比色法(MTT)检测CAL-27细胞增殖情况,用流式细胞术进行细胞周期检测。同时,miR1-EXO、CON-EXO及等体积PBS分别与人正常口腔黏膜上皮角化细胞(NOK)共培养,用MTT法检测NOK细胞增殖情况。结果miR1-EXO、CON-EXO均呈典型的球形或杯状结构,直径约110 nm,并高表达外泌体标志性蛋白CD9、Alix及Tsg101。miR1-EXO中miR-1的表达量为285.80±14.33,CON-EXO的表达量为1.00±0.06,二者间有统计学差异(P<0.0001)。免疫荧光及qPCR结果显示,与CAL-27细胞共培养后,miR1-EXO被CAL-27细胞摄取,miR1-EXO CAL-27细胞的miR-1表达水平高于CONEXO及PBS,miR-1下游靶基因MET表达水平下调。MTT及细胞周期结果显示,与CAL-27细胞共培养后,miR1-EXO G0/G1期细胞比例高于CON-EXO和PBS,抑制CAL-27细胞增殖,而miR1-EXO对NOK细胞增殖的影响很小。结论供体细胞基因过表达的方法可使过表达miR-1的HEK293细胞分泌高表达miR-1的外泌体,并且高表达miR-1的外泌体可以将miR-1运送到CAL-27细胞,下调MET基因表达,抑制CAL-27细胞增殖。

Objective This study aims to construct endogenous exosomes abundantly loaded with miR-1 and investigate the role of exosome-mediated microRNA-1(miR-1)delivery on CAL-27 cell proliferation.Methods Exosomes secreted by miR-1-overexpressing HEK293 cells(miR1-EXO)were purified via ultracentrifugation and subjected to transmission electron microscopy,nanoparticle analysis,Western blot analysis,and quantitative polymerase chain reaction(qPCR).CAL-27 cells were cocultured with exosomes secreted by HEK293 cells(CON-EXO)and miR1-EXO and equivalent phosphate buffer saline.The intracellular transport of exosomes was measured by using immunofluorescence,the expression of miR-1 and its target gene MET were investigated via qPCR,CAL-27 cell proliferation was measured through MTT assay,and cell cycle state was determined by applying flow cytometry.Results Electron microscopy revealed that miR1-EXO and CON-EXO were spherical or cup-shaped with an average diameter of approximately 110 nm.The well-known exosome markers CD9,Tsg101,and Alix were enriched.The expression of miR-1 in miR1-EXO was higher than that in CON-EXO(285.80±14.33 vs 1.00±0.06,P<0.0001).After coculture with CAL-27 cells,miR1-EXO was internalized and unloaded miR-1 into CAL-27 cells.After coculture with miR1-EXO,the expression of miR-1 in CAL-27 cells was upregulated,whereas that of MET,the target gene of miR-1,was suppressed and the proliferation of CAL-27 cells was inhibited significantly.Normal oral keratinocyte cell proliferation was negligibly affected after coculture with miR1-EXO.Conclusion Exosomes secreted from miR1-EXO cells could load abundant miR-1.Exosomal miR-1 delivered into CAL-27 cells by using miR1-EXO suppressed the expression of MET mRNA and inhibited cell proliferation.