摘要
目的 探讨结直肠癌(CRC)细胞来源外泌体miR-21对CRC细胞转移和侵袭的作用及机制。方法 采用实时荧光定量反转录聚合酶链反应(qRT-PCR)检测miR-21在人CRC细胞系HCT116和人正常结肠上皮细胞系NCM460中的表达差异,以及在2种细胞来源外泌体内的表达差异。通过小干扰RNA转染的方法构建miR-21敲低的HCT116细胞株。采用共培养试验检测HCT116细胞对荧光标记外泌体的摄取。采用伤口愈合试验检测摄取外泌体后HCT116细胞迁移能力变化。采用Transwell试验检测摄取外泌体后HCT116细胞侵袭能力变化。采用Western blot试验检测摄取外泌体后HCT116细胞内缺口同源物1(Notch1)、磷脂酰肌醇3激酶(PI3K)、磷酸化PI3K(p-PI3K)、蛋白激酶B(Akt)、磷酸化Akt(p-Akt)、哺乳动物雷帕霉素靶蛋白(mTOR)和磷酸化mTOR(p-mTOR)蛋白水平。采用qRT-PCR检测Notch1转录信使水平。结果 HCT116细胞及其外泌体内miR-21水平均明显高于NCM460细胞及其外泌体,差异均有统计学意义(P<0.05)。共聚焦显微镜观察结果显示,HCT116细胞成功摄取了其他HCT116细胞来源外泌体。伤口愈合试验结果显示,摄取miR-21敲低的外泌体后HCT116细胞迁移能力受到抑制(P<0.05)。Transwell试验结果显示,摄取miR-21敲低的外泌体后HCT116细胞侵袭能力明显减弱(P<0.05)。Western blot试验结果显示,摄取了miR-21敲低外泌体的HCT116细胞内Notch1 mRNA、PI3K、Akt、mTOR蛋白水平与摄取了miR-21敲低对照外泌体的细胞比较,差异均无统计学意义(P>0.05);但摄取了miR-21敲低外泌体的HCT116细胞内Notch1、p-PI3K、p-Akt、p-mTOR蛋白水平与摄取了miR-21敲低对照外泌体细胞比较明显降低,差异均有统计学意义(P<0.05)。结论 CRC细胞来源外泌体miR-21通过靶向Notch1/PI3K/Akt/mTOR通路可促进肿瘤转移和侵袭。
Objective To investigate the effect and mechanism of colorectal cancer(CRC)cell-derived exosomal miR-21 on the metastasis and invasion of CRC cells.Methods Real-time quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR)was used to detect the expression of miR-21 in human CRC cell line HCT116 and human normal colonic epithelial cell line NCM460,as well as the expression of Mir-21 in exosomes derived from the two cell lines.HCT116 cell line with knockdown of miR-21 was constructed by small interfering RNA transfection.Co-culture assay was used to detect the uptake of fluorescently labeled exosomes by HCT116 cells.Wound healing assay was used to detect the migration ability of HCT116 cells after exosome ingestion.Transwell assay was used to detect the changes in the invasion ability of HCT116 cells after exosome uptake.Western blot was used to detect the protein levels of nick homolog 1(Notch1),phosphatidylinositol 3 kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B(Akt),phosphorylated Akt(p-Akt),mammals rapamycin target protein(mTOR)and phosphorylated mTOR(p-mTOR)in HCT116 cells after exosome uptake,and qRT-PCR was used to detect the mrna level of Notch1.Results The level of miR-21 in HCT116 cells and their exosomes was significantly higher than that in NCM460 cells and their exosomes,and the differences were statistically significant(P<0.05).Confocal microscopy showed that HCT116 cells successfully ingested other HCT116 cell-derived exosomes.The results of wound healing assay showed that the migration ability of HCT116 cells was inhibited after the uptake of exosomes with knockdown of miR-21(P<0.05).Transwell assay showed that the invasion ability of HCT116 cells was significantly decreased after the uptake of exosomes with knockdown of miR-21(P<0.05).Western blot results showed that there was no significant difference in Notch1 mRNA,PI3K,AKT and mTOR protein levels between HCT116 cells that ingested exosomes from miR-21 knockdown and those that ingested exosomes from control miR-21 knockdown(P>0.05).However,the levels of Notch1,p-PI3K,p-Akt and p-mtor proteins in HCT116 cells that ingested exosomes from miR-21 knockdown were significantly lower than those in HCT116 cells that ingested exosomes from control miR-21 knockdown(P<0.05).Conclusion CRC cell-derived exosomal miR-21 promotes tumor metastasis and invasion by targeting the Notch1/PI3K/AKT/mTOR pathway.
作者
马利辉
魏芳芳
王楠
霍浩然
魏琦
张蕾
MA Lihui;WEI Fangfang;WANG Nan;HUO Haoran;WEI Qi;ZHANG Lei(Department of Emergency Surgery,Handan Central Hospital,Handan,Hebei 056001,China;Department of General Surgery,Handan Central Hospital,Handan,Hebei 056001,China;Department of General Surgery,Handan People′s Hospital,Handan,Hebei 056000,China)
出处
《检验医学与临床》
2025年第12期1702-1706,共5页
Laboratory Medicine and Clinic
基金
河北省医学科学研究课题计划(20220035)
河北省邯郸市科学技术研究与发展计划(23422083207)。
