胶质瘤细胞外泌体通过miR-125b对细胞增殖凋亡的影响

Glioma cell exosomes effect cell proliferation and apoptosis through miR-125b

目的探索胶质瘤细胞来源的外泌体对细胞增殖凋亡的影响,并探讨其作用机制。方法高速离心法提取胶质瘤细胞U251外泌体,通过电镜及纳米粒径分析外泌体形态,Western blot检测外泌体特征性蛋白表达。在U251细胞培养基中分别加入浓度为100μg/ml的外泌体悬液30μl(外泌体组)或等量不含外泌体培养基(对照组),通过双荧光染色法验证外泌体能否进入U251细胞;CCK-8及流式细胞法检测细胞增殖凋亡的变化。通过RT-PCR法筛选U251外泌体与人星型胶质细胞外泌体中差异性表达的miRNAs。最后,将miR-NC、miR-125b mimics或miR-125b inhibitor转染U251细胞,重新提取外泌体后加入细胞培养基,采用CCK-8及流式细胞法检测U251细胞增殖凋亡变化。结果U251细胞分离的外泌体为圆形或卵圆形囊泡状小体,直径范围约100 nm,富含CD63和CD9蛋白。在U251细胞培养基中加入外泌体悬液后,外泌体能够被U251细胞吞噬。CCK-8及流式细胞法显示:外泌体组细胞增殖活性高于对照组,凋亡比例低于对照组。RT-PCR显示:U251细胞外泌体miRNA-125b含量高于人星型胶质细胞外泌体。将miR-NC、miR-125b mimics或miR-125b inhibitor转染U251细胞后,分别提取三组细胞外泌体并加入U251细胞培养基,miR-125b inhibitor组细胞增殖活性低于miR-NC组,凋亡比例高于miR-NC组;miR-125b mimics组细胞增殖活性高于miR-NC组,凋亡比例低于miR-NC组。差异均有统计学意义(P<0.05)。结论胶质瘤细胞来源的外泌体可通过miR-125b,促进胶质瘤细胞增殖,抑制细胞凋亡。

Objective To explore the mechanism of exosomes derived from glioma cells on cell proliferation and apoptosis.Methods The U251 exosomes were extracted by high-speed centrifugation.The morphology of exosomes was analyzed by electron microscopy and nano particle size,and the characteristic protein expression of exosomes was detected by Western blot.30μl of exosome suspension with concentration of 100μg/ml(exosome group)or equal amount of medium without exosomes(control group)was added to U251 cell culture medium,and the double fluorescence staining method was used to verify whether exosomes could enter U251 cells.The changes of cell proliferation and apoptosis were detected by CCK-8 and flow cytometry.The miRNAs differentially expressed in U251 exosomes and human astroglial cells were screened by RT-PCR.Finally,the U251 cells were transfected with miR-NC,miR-125b mimicsor miR-125b inhibitor,exosomes were extracted and added into cell culture medium,and the proliferation and apoptosis of U251 cells were detected by CCK-8 and flow cytometry.Results The exosomes isolated from U251 cells were round or oval vesicular bodies,with the diameter around 100 nm and rich in CD63 and CD9 proteins.After adding exosomes suspension in U251 cell culture medium,exosomes could bepackaged by U251 cells.CCK-8 and flow cytometry showed that the proliferation activity of exosomes was higher than that of the control group,and the apoptosis rate was lower.RT-PCR showed that the content of miRNA-125b in U251 cells exosomes was higher than that in human astrocytes exosomes.After transfection of U251 cells with miR-NC,miR-125b mimicsor miR-125b inhibitor,the exosomes of the three groups were extracted and added into U251 cell culture medium.The proliferative activity of miR-125b inhibitor group was lower than that of miR-NC group,and the apoptosis rate was higher than that of miR-NC group.The cell proliferation activity of miR-125b mimics group was higher than that of miR-NC group,and the apoptosis rate was lower than that of miR-NC group.The differences were statistically significant(P<0.05).Conclusion By enriching miR-125b,exosomes derived from glioma cellscan promote the proliferation of glioma cells and inhibit cell apoptosis.