基于NOD2介导的AMPK/mTOR信号通路探讨宫颈癌细胞恶性行为的机制

Mechanism of malignant behavior in cervical cancer cells based on NOD2-mediated AMPK/mTOR signaling pathway

目的 基于核苷酸结合寡聚化结构域受体2(NOD2)介导的AMP活化蛋白激酶(AMPK)/雷帕霉素靶蛋白(mTOR)信号通路探讨宫颈癌(CC)细胞恶性行为的机制。方法 生物信息学分析确定NOD2在CC组织中的表达。将靶向NOD2(shNOD2)、shRNAs阴性对照(shNC)以及NOD2过表达(NOD2)质粒和载体(Vec)转染CC细胞。通过CCK-8测定、集落形成和Transwell细胞侵袭测定来确定NOD2对CC细胞生长的影响。通过高通量RNA测序(RNA-Seq)进行转录组分析。Western blot试验检测细胞系中NOD2、AMPK/mTOR信号通路和自噬蛋白的表达。24只雌性BALB/c裸鼠随机分为4组,每组6只:载体组(Vec组)、NOD2过表达组(NOD2组)、shNC组和shNOD2组。构建小鼠远处转移模型,监测肺转移的荧光强度,计数肺转移结节的数量。结果 在线数据库分析显示,NOD2在CC组织中表达明显高于正常组织,并且不同分期的CC中NOD2的mRNA表达差异有统计学意义(P<0.05)。此外,NOD2的高表达与较差的总生存期和无病生存期相关(P<0.05)。NOD2过表达对CC细胞增殖、集落形成、迁移和侵袭具有促进作用,而NOD2敲低则相反。与体外结果一致,在转移的小鼠尾静脉注射模型中,NOD2组CC细胞的肺定殖、肺转移灶较Vec组增加(P<0.05),而shNOD2组CC细胞的肺定殖、肺转移灶较shNC组减少(P<0.05)。RNA-Seq结果显示NOD2表达与AMPK信号激活、mTOR信号抑制、自噬调节途径激活和自噬体形成显著相关。与shNC组相比,shNOD2组磷酸化AMPK、LC3蛋白表达水平减少(P<0.05),磷酸化mTOR、p62蛋白表达水平增加(P<0.05);与Vec组相比,NOD2组LC3、AMPK蛋白表达水平增加(P<0.05),磷酸化mTOR、p62蛋白表达水平减少(P<0.05)。与shNC组相比,shNOD2组GFP-mRFP-LC3的点积累减少(P<0.05);与Vec组相比,GFP-mRFP-LC3的点积累增加(P<0.05)。结论 NOD2可能通过AMPK/mTOR信号促进CC增殖、迁移和侵袭,其作用机制部分涉及自噬激活。

Objective To explore the mechanism of malignant behavior of cervical cancer(CC) cells based on AMP-activated protein kinase(AMPK)/rapamycin target protein(mTOR) signaling pathway mediated by nucleotide-binding oligomerization domain receptor 2(NOD2).Methods Bioinformatics analysis was performed to determine the expression of NOD2 in CC tissue.Plasmids targeting NOD2(shNOD2) and shRNAs negative control(shNC),NOD2 overexpression(NOD2) and vectors(Vec) were transfected into CC cells.The effect of NOD2 on the growth of CC cells was determined by cell counting kit-8 assay,colony formation and Transwell cell invasion assay.Transcriptome analysis was performed by high throughput RNA sequencing(RNA-Seq).Western blot was used to detect the expression of NOD2,AMPK/mTOR signaling pathway and autophagy protein in the cell line.24 female BALB/c nude mice were randomly divided into four groups,with 6 mice in each group:vector group(Vec group),NOD2 overexpression group(NOD2 group),shNC group and shNOD2 group.The distant metastasis model was established in mice,and the fluorescence intensity of lung metastasis was monitored and the number of lung metastasis nodules was counted.Results On-line database analysis showed that the expression of NOD2 in CC tissues was significantly higher than that in normal tissues,and there were significant differences in the mRNA expression of NOD2 in different stages of CC(P<0.05).In addition,the high expression of NOD2 was associated with poor overall survival and disease-free survival(P<0.05).NOD2 overexpression promoted the proliferation,colony formation,migration and invasion of CC cells,while NOD2 knock-down was the opposite.Consistent with the results in vitro,the lung colonization and lung metastasis of CC cells in NOD2 group were significantly higher than those in Vec group(P<0.05),while those in shNOD2 group were significantly lower than those in shNC group(P<0.05).RNA-Seq results showed that the expression of NOD2 was significantly related to AMPK signal activation,mTOR signal inhibition,autophagy regulation pathway activation and autophagy formation.Compared with shNC group,the expression of phosphorylated AMPK and LC3 protein decreased significantly in shNOD2 group(P<0.05),and the expression levels of phosphorylated mTOR and p62 protein increased significantly(P<0.05).Compared with Vec group,the expression levels of LC3 and AMPK protein in NOD2 group increased significantly(P<0.05),and the expression levels of phosphorylated mTOR and p62 protein decreased significantly(P<0.05).Compared with shNC group,the point accumulation of GFP-mRFP-LC3 in shNOD2 group decreased significantly(P<0.05).Compared with Vec group,the point accumulation of GFP-mRFP-LC3 increased significantly(P<0.05).Conclusion NOD2 may promote the proliferation,migration and invasion of CC through AMPK/mTOR signal,and its mechanism partly involves autophagy activation.