摘要
外泌体可参与肿瘤细胞侵袭、迁移和血管生成等生物学过程,而侵袭是胶质瘤患者死亡的主要原因。研究表明,肿瘤细胞分泌的外泌体能够携带miRNA到受体细胞中,调控受体细胞增殖、迁移和侵袭等生物学功能。miR-574-5P在多种肿瘤发生发展中发挥关键作用,但胶质瘤细胞来源的外泌体是否表达miR-574-5P,以及其在胶质瘤细胞生长和侵袭迁移中的作用未见报道。本研究将探讨胶质瘤细胞来源的外泌体miR-574-5P在细胞增殖、迁移和侵袭过程中的作用机制。运用电镜、纳米粒径跟踪和Western印迹表征外泌体。结果显示,所提取的圆形颗粒是外泌体,直径为30~100 nm;经免疫荧光检测外泌体的内化,结果显示,外泌体内化到LN229细胞中;运用生物信息学和网上数据找出胶质瘤细胞来源的外泌体差异miRNA,结果显示,外泌体差异miRNA为miR-574-5P,并预测大肿瘤抑制基因2(LATS2)可能是miR-574-5P的靶基因;双荧光素酶报告基因结果证实,miR-574-5P与LATS2的3′UTR区互补结合;转染实验、qRT-PCR和Western印迹检测miR-574-5P与LATS2的关系,结果显示,过表达miR-574-5P后,LATS2 mRNA的含量与对照组无显著差异(P>0.05),表明miR-574-5P对LATS2的调控作用通过抑制其翻译实现(P<0.05)。转染实验、CCK-8法、Transwell迁移和侵袭实验,检测miR-574-5P对LN229细胞增殖、迁移和侵袭能力的影响。结果显示,过表达miR-574-5P可显著促进LN229细胞的增殖、迁移和侵袭(P<0.05)。此外,Western印迹检测信号通路LATS2/YAP关键激酶蛋白质的表达情况,观察外泌体对此信号通路的影响。结果显示,外泌体下调LATS2表达,减少p-YAP磷酸化。综上所述,外泌体miR-574-5P通过靶向下调LATS2,激活LATS2/YAP信号通路,从而促进胶质瘤细胞的增殖、迁移和侵袭能力,这可为胶质瘤的诊断和治疗提供一个科学可靠的靶点。
Exosomes are involved in invasion,migration and angiogenesis of tumor cells,and invasion is the main cause of death in glioma patients.Studies have shown that the exosomes secreted by tumor cells can carry miRNA into the receptor cells and regulate the biological functions of the receptor cells,such as proliferation,migration and invasion.miR-574-5 p plays a key role in the occurrence and development of a variety of tumors.However,whether the exosomes derived from glioma cells express miR-574-5 p and its role in the growth,invasion and migration of glioma cells have not been reported.This study investigated the mechanism of the exosomal miR-574-5 p secreted from glioma cells in the process of cell proliferation,migration and invasion.The exosomes were characterized by electron microscopy,nanoparticle size tracking and Western blot.The results displayed that the extracted exosomes were round particles with a diameter of 30~100 nm.The internalization of exosomes was detected by immunofluorescence assay.The results showed that exosomes were internalized into LN229 cells;Bioinformatics and online data were used to screen the differentially secreted miRNA between LN229 and H4 glioma cells.The results showed that the differentially secreted miRNA was miR-574-5 p,and large tumor suppressor 2(LATS2)was predicted to be the target gene of miR-574-5 p;Duel luciferase reporter assay confirmed that miR-574-5 p was complementary to the 3′UTR region of LATS2;The transfection assay,qRT-PCR and Western blot was conducted to measure the relationship between miR-574-5 p and LATS2.The results demonstrated that there was no significant difference in LATS2 mRNA levels between the control group and the group with miR-574-5 P overexpression(P>0.05),suggesting the regulatory effect of miR-574-5 P on LATS2 was achieved by inhibiting its translation(P<0.05).CCK-8,Transwell migration and invasion assays were conducted to explore the effect of miR-574-5 p on proliferation,migration and invasion of LN229 cells.The results showed that overexpression of miR-574-5 p could significantly promote the ability of proliferation,migration and invasion of LN229 cells(P<0.05).In addition,Western blot was performed to measure the expression of kinase proteins involved in the LATS2/YAP signaling pathway,and the influence of the exosomes on this signaling pathway.The results revealed that the exosomes down-regulated the protein expression level of LATS2 and reduced p-YAP phosphorylation.In conclusion,the exosomal miR-574-5 p can promote the proliferation,migration and invasion of glioma cells by down-regulating LATS2 and activating LATS2/YAP signaling pathway,which may provide a potential biomarker for the diagnosis and target for the treatment of glioma.
作者
冯瑞军
郑远航
盛智梅
李可欣
董杰
肖钦沛
张宝刚
FENG Rui-Jun;ZHENG Yuan-Hang;SHENG Zhi-Mei;LI Ke-Xin;DONG Jie;XIAO Qin-Pei;ZHANG Bao-Gang(Department of Clinical Pathology,Department of Pathology,Weifang Medical University,Weifang 261042,Shandong,China)
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2021年第11期1520-1527,共8页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金(No.81872163,No.81672631)资助。
