摘要
背景:研究表明骨肉瘤的发生与多种miRNAs的异常表达有关,外泌体miRNA在细胞内通讯中受到越来越多的关注。目的:探讨骨肉瘤细胞来源外泌体miR-1307对骨肉瘤细胞增殖和凋亡的影响及其作用机制。方法:购买人骨肉瘤细胞系(143B、MG63、U2OS、Saos-2和SW1353)和人正常成骨细胞系(hFOB 1.19)。通过qRT-PCR实验检测miR-1307在5种骨肉瘤细胞系和正常成骨细胞系中的表达水平,最终选择SW1353作为此次骨肉瘤细胞功能验证的细胞系。培养SW1353骨肉瘤细胞和正常成骨细胞,通过差速离心法提取骨肉瘤细胞外泌体和正常成骨细胞外泌体,然后将miR-1307 inhibitor、miR-NC mimic转染至SW1353骨肉瘤细胞和正常成骨细胞,再提取SW1353骨肉瘤细胞外泌体和正常成骨细胞外泌体。取25 mg/L上述外泌体加入SW1353骨肉瘤细胞中干预24 h,采用CCK-8实验和流式细胞术观察骨肉瘤细胞的增殖和凋亡情况,使用Targetscan、MiRBase和miRDIP数据库在线预测miR-1307的靶基因,通过荧光素酶报告基因实验测定荧光素酶活性。使用qRT-PCR和Western blot检测骨肉瘤细胞中AGAP1的mRNA和蛋白表达水平。结果与结论:①与hFOB 1.19成骨细胞外泌体相比,SW1353骨肉瘤细胞外泌体明显促进骨肉瘤细胞增殖并抑制凋亡(P<0.01);与SW1353骨肉瘤细胞外泌体相比,SW1353骨肉瘤细胞外泌体中miR-1307下调后,对SW1353骨肉瘤细胞的增殖作用明显降低而凋亡率明显升高(P<0.01);②AGAP1被验证为miR-1307的直接靶基因,过表达miR-1307骨肉瘤细胞的AGAP1 mRNA和蛋白表达水平均明显低于miR-NC组(P<0.01)。与miR-NC组相比,miR-1307明显抑制野生型PGLO-AGAP1-WT 3'-UTR的荧光素酶活性(P<0.05);③结果表明,SW1353骨肉瘤细胞外泌体miR-1307通过靶向AGAP1促进SW1353骨肉瘤细胞的增殖并抑制凋亡。
BACKGROUND:Studies have shown that the occurrence of osteosarcoma is associated with abnormal expression of various microRNAs(miRNAs).Exosomes(Exos)containing miRNAs get much more attentions in intracellular communications.OBJECTIVE:To investigate the effects of exosomal miR-1307 from osteosarcoma on proliferation and apoptosis of osteosarcoma cells and its mechanism.METHODS:Human osteosarcoma cell lines(143B,MG63,U2OS,Saos-2 and SW1353)and human normal osteoblastic cell line(hFOB 1.19)were purchased.The expression level of miR-1307 in five kinds of osteosarcoma cell lines and normal osteoblastic cell line was detected by qRT-PCR.Finally,SW1353 cell line was selected for functional verification of osteosarcoma cells.Osteosarcoma cells and normal osteoblasts were cultured.Osteosarcoma exosomes and normal osteoblastic exosomes were extracted by differential centrifugation.Then miR-1307 inhibitor and miR-NC mimic were transfected into SW1353 osteosarcoma cells and normal osteoblasts,and then SW1353 osteosarcoma cell exosomes and normal osteoblast exosomes were extracted.The above exosomes(25 mg/L)were added to SW1353 osteosarcoma cells to intervene for 24 hours.Cell proliferation assay and flow cytometry were used to observe the proliferation and apoptosis of osteosarcoma cells.Targetscan,miRBase and miRDIP databases were used to predict the target genes of miR-1307.The activity of luciferin was assayed by luciferase reporter gene.mRNA and protein expression levels of AGAP1 were assayed by qRT-PCR and western blot assay in osteosarcoma cells.RESULTS AND CONCLUSION:(1)Compared with hFOB 1.19-exosomes of osteoblasts,SW1353 osteosarcoma cell exosomes significantly promoted the proliferation and inhibited the apoptosis of osteosarcoma cells(P<0.01).Compared with SW1353 osteosarcoma cell exosomes,the level of miR-1307 in SW1353 osteosarcoma cell exosomes was down-regulated;the proliferation of osteosarcoma cells was significantly decreased but the apoptosis rate was significantly increased(P<0.01).(2)AGAP1 was verified as a direct target gene of miR-1307.Compared with miR-NC,miR-1307 significantly inhibited the mRNA and protein levels of AGAP1(P<0.01).Compared with miR-NC,miR-1307 significantly inhibited the luciferase activity of wild-type PGLO-AGAP1-WT 3'-UTR(P<0.05).(3)The results show that exosomal miR-1307 of osteosarcoma promoted the proliferation and inhibited the apoptosis of osteosarcoma cells via targeting AGAP1.
作者
韩飞
蒲沛东
马青源
朱洲均
王梦雨
王超
石冲
史晨辉
王维山
Han Fei;Pu Peidong;Ma Qingyuan;Zhu Zhoujun;Wang Mengyu;Wang Chao;Shi Chong;Shi Chenhui;Wang Weishan(Department of Orthopedics,the First Affiliated Hospital of the Medical College,Shihezi University,Shihezi 832008,Xinjiang Uygur Autonomous Region,China;Medical College,Shihezi University,Shihezi 832008,Xinjiang Uygur Autonomous Region,China)
出处
《中国组织工程研究》
CAS
北大核心
2021年第13期2036-2042,共7页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金(81772407,81660374),项目负责人:史晨辉
兵团中青年科技创新领导人才计划(2016BC001),项目负责人:王维山。
