外泌体miR-20a-5p调控Sp1/CD147信号通路促进非小细胞肺癌的骨转移

Exosome miR-20a-5p regulates Sp1/CD147 signaling pathway to promote bone metastasis in non-small cell lung cancer

目的探究外泌体miR-20a-5p对非小细胞肺癌(NSCLC)骨转移的调控作用及机制。方法提取并鉴定16HBE、A549、PC9、H1299细胞以及转染miR-20a-5p mimic和miR-NC的A549细胞所分泌的外泌体。qRT-PCR检测各种细胞及外泌体中miR-20a-5p的表达。将A549和RAW264.7细胞分为PBS组、16HBE exo组、A549 exo组、miR-NC exo组和miR-20a-5p exo组。Transwell小室检测A549细胞迁移和侵袭能力,TRAP染色检测破骨细胞分化。Western blot检测各组RAW264.7细胞Sp1和CD147的表达。6周龄的雌性Balb/c裸鼠随机分为5组,分别注射PBS(A组)、16HBE exo(B组)、A549 exo(C组)、miR-NC exo(D组)和miR-20a-5p exo(E组)外泌体4周,构建NSCLC骨转移的小鼠模型;HE染色观察各组小鼠骨转移情况。结果透射电镜下观察到A549 exo、PC9 exo、H1299 exo、16HBE exo为具有双层膜的囊泡样结构,粒径在30~100 nm,且均表达CD9、CD81、HSP70标志蛋白。miR-20a-5p高表达于NSCLC细胞及其外泌体。与PBS组相比,A549 exo组肿瘤细胞迁移和侵袭能力显著增加(P<0.001),RAW264.7细胞中miR-20a-5p表达上调(P<0.001),破骨细胞分化显著增加(P<0.001),Sp1和CD147表达显著增加(P<0.001)。与miR-NC exo组相比,miR-20a-5p exo组A549细胞迁移和侵袭能力增加(P<0.001),RAW264.7细胞中miR-20a-5p表达上调(P<0.001),破骨细胞分化显著增加(P<0.001),Sp1和CD147表达显著增加(P<0.001)。在小鼠体内实验中,A组、B组骨组织骨小梁结构完整,组织中有少量肿瘤细胞,骨髓腔相对完整,病变较少;C组小鼠骨组织出现大量肿瘤细胞且细胞紧密排列,骨小梁显著破坏,核浆比例严重失调,病变显著增加;E组小鼠骨组织中出现大量肿瘤细胞,骨小梁破坏严重,病变较D组显著。结论外泌体miR-20a-5p可显著促进NSCLC的体内外转移,其机制可能为激活破骨细胞Sp1/CD147信号通路从而促进破骨细胞的分化。

Objective To investigate the regulatory effect and mechanism of exosome miR-20a-5p on bone metastasis in non-small cell lung cancer(NSCLC).Methods The exosomes secreted by 16HBE,A549,PC9,H1299 cells and A549 cells transfected with miR-20a-5p mimic and miR-NC were extracted and identified.The expression of miR-20a-5p in various cells and exosomes was detected by qRT-PCR.A549 and RAW264.7 cells were divided into the PBS group,the 16HBE exo group,the A549 exo group,the miR-NC exo group and the miR-20a-5p exo group.Transwell chamber was used to detect the migration and invasion abilities of A549 cells,and TRAP staining was used to detect osteoclast differentiation.Western blot was used to detect the expression of Sp1 and CD147 in RAW264.7 cells of each group.The 6-week-old female Balb/c nude mice were randomly divided into 5 groups and given PBS(group A),16HBE exo(group B),A549 exo(group C),miR-NC exo(group D)and miR-20a-5p exo(group E)exosomes for 4 weeks respectively,to establish a mouse model of NSCLC bone metastasis;HE staining was used to observe bone metastasis of mice in each group.Results Under transmission electron microscopy,A549 exo,PC9 exo,H1299 exo and 16HBE exo were bilayer vesicle-like structures with particle sizes ranging from 30 to 100 nm,and all of them expressed CD9,CD81 and HSP70 marker proteins.miR-20a-5p was highly expressed in NSCLC cells and their exosomes.Compared with the PBS group,the migration and invasion abilities of tumor cells in the A549 exo group were significantly increased(P<0.001),the expression of miR-20a-5p was up-regulated in RAW264.7 cells(P<0.001),the osteoclast differentiation was significantly increased(P<0.001),and the expressions of Sp1 and CD147 were significantly increased(P<0.001).Compared with the miR-NC exo group,the migration and invasion abilities of A549 cells in the miR-20a-5p exo group were increased(P<0.001),the expres⁃sion of miR-20a-5p was up-regulated in RAW264.7 cells(P<0.001),the osteoclast differentiation was significantly increased(P<0.001),and the expressions of Sp1 and CD147 were significantly increased(P<0.001).In vivo experiments of mice showed that the bone trabecular structures of the bone tissue in group A and group B were complete,there were a few tumor cells in the tissue,the bone marrow cavity was relatively complete with few lesions.In group C,a large number of tumor cells were found in the bone tissue of mice,and the cells were closely arranged,the bone trabeculae was seriously damaged,the ratio of nucleus to plasma was seriously disordered,and the lesions were significantly increased.A large number of tumor cells appeared in the bone tissue of mice in group E,and the bone trabeculae was seriously damaged,and the lesions were more serious than those in group D.Conclusion Exosome miR-20a-5p can significantly promote the metastasis of NSCLC in vivo and in vitro,and its mechanism may be that it can activate the Sp1/CD147 signaling pathway of osteoclasts to promote osteoclast differentiation.