【摘要】 目的 探索骨髓间充质干细胞（bone mesenchymal stem cells,BMSCs）对子宫内膜癌Ishikawa细胞转录水平的影响。方法 用Lenti-EGFP慢病毒感染Ishikawa细胞，分别将表达增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）的Ishikawa细胞单独培养、与BMSCs接触共培养；通过流式细胞术分离Ishikawa细胞，并进行转录组测序分析筛选差异表达的mRNA和miRNA，通过miRWalk预测差异miRNA靶基因，对两者的交集基因进行GO、KEGG、蛋白网络互作分析；采用qRT-PCR和Western blot验证测序结果的准确性。结果 标记EGFP的Ishikawa细胞与BMSCs共培养后，分离出的Ishikawa-EGFP阳性细胞占33.6%，阴性细胞占10.5%。测序后共筛选出5 928个差异表达mRNA和111个差异表达miRNA。蛋白网络互作分析显示交集基因表达的蛋白之间相互作用，核心节点包括CDKN1A、JAK1、COL1A1、VCAN等。miRNA-mRNA网络图显示CDKN1A与hsa-let-7e-5p存在潜在靶向关系。GO分析和KEGG分析结果显示，其交集基因参与细胞外基质结构、细胞黏附分子结合等，并主要富集在PI3K-Akt、黏着斑、MAPK、EGFR酪氨酸激酶抑制剂耐药等信号通路。qRT-PCR和Western blot结果显示，共培养后Ishikawa-EGFP细胞中CDKN1A表达上调，hsa-let-7e-5p表达下调。结论 BMSCs可能通过细胞间的相互作用调节肿瘤局部微环境并促进子宫内膜癌的发生发展。更多还原

【Abstract】 ObjectiveTo investigate the effect of bone mesenchymal stem cells(BMSCs) on the transcription level of endometrial cancer Ishikawa cells.MethodsIshikawa cells were infected with Lenti-EGFP lentivirus. The Ishikawa cells expressing the enhanced green fluorescent protein(EGFP) were cultured separately and co-cultured by contacting with BMSCs; Ishikawa cells were isolated by flow cytometry, and the differentially expressed mRNA and miRNA were analyzed and screened by transcriptome sequencing. The differential miRNA target genes was predicted through miRWalk, and then the intersection genes of the two genomes were analyzed by GO, KEGG and protein-protein interaction network(PPI) analysis. The accuracy of the sequencing was verified by qRT-PCR and Western blot.ResultsAfter co-culture of EGFP-labeled Ishikawa cells with BMSCs, 33.6% Ishikawa-EGFP positive cells and 10.5%Ishikawa-EGFP negative cells were isolated. A total of 5 928 differentially expressed mRNA, 111 differentially expressed miRNA were screened after sequencing. PPI analysis showed that the interaction occurred among the proteins expressed by the intersection genes,and the core nodes included CDKN1A, JAK1, COL1A1, VCAN, etc. The miRNA-mRNA network diagram showed that a potential targeting relationship existed between CDKN1A and hsa-let-7e-5p. GO and KEGG analysis showed that the intersection genes were involved in extracellular matrix organization, cell adhesion molecule binding, and mainly enriched in PI3K-Akt, focal adhesion, MAPK,EGFR tyrosine kinase inhibitor resistance and other signaling pathways. qRT-PCR and Western blot showed that the expression of CDKN1A was up-regulated and the expression of hsa-let-7e-5p was down-regulated in Ishikawa-EGFP cells after co-culture.Conclusions The BMSCs may regulate the local tumor microenvironment through the interaction between cells and promote the occurrence and development of the endometrial cancer.更多还原