METHODS: In the present study, we presented the expression of RNH1 in LUAD based on the database and confirmed it by western blot detection of RNH1 in human LUAD tissues. Lentiviral infection was constructed to silence or overexpress RNH1 in NCI-H1395 and NCI-H1437 cells. We assess the role of RNH1 on proliferation in LUAD cells by MTT assay, colony formation assays, and cell cycle detection. Hoechst staining and flow cytometry were used to evaluate the effects of RNH1 on apoptosis of LUAD cells. The function of RNH1 in invasion and migration was investigated by Transwell assay. Dual luciferase assay, ChIP detection, and pull-down assay were conducted to explore the association of E2F1 in the maintenance of RNH1 expression and function. The regulation of E2F1 on the functions of RNH1 in LUAD cells was explored. Mouse experiments were performed to confirm the in-vivo role of RNH1 in LUAD. mRNA sequencing indicated that RNH1 overexpression altered the expression profile of LUAD cells.
RESULTS: RNH1 expression in LUAD tissues of patients was presented in this work. Importantly, RNH1 knockdown improved the proliferation, migration and invasion abilities of cells and RNH1 overexpression produced the opposite effects. Dual luciferase assay proved that E2F1 bound to the RNH1 promoter (-1064 ∼ -1054, -1514 ∼ -1504) to reduce the transcriptional activity of RNH1. ChIP assay indicated that E2F1 DNA was enriched at the RNH1 promoter (-1148 ∼ -943, -1628 ∼ -1423). Pull-down assays also showed the association between E2F1 and RNH1 promoter (-1148 ∼ -943). E2F1 overexpression contributed to the malignant behavior of LUAD cells, while RNH1 overexpression reversed it. High-throughput sequencing showed that RNH1 overexpression induced multiple genes expression changes, thereby modulating LUAD-related processes.
CONCLUSIONS: Our study demonstrates that binding of E2F1 to the RNH1 promoter may lead to inhibition of RNH1 expression and thus promoting the development of LUAD.
方法:在本研究中,我们基于数据库提供了RNH1在LUAD中的表达,并通过蛋白质印迹检测人LUAD组织中的RNH1来证实。构建慢病毒感染以沉默或过表达NCI-H1395和NCI-H1437细胞中的RNH1。我们通过MTT法评估RNH1对LUAD细胞增殖的作用,集落形成试验,和细胞周期检测。用Hoechst染色和流式细胞术评价RNH1对LUAD细胞凋亡的影响。通过Transwell实验研究了RNH1在侵袭和迁移中的功能。双荧光素酶测定,ChIP检测,并进行下拉实验以探讨E2F1在维持RNH1表达和功能中的相关性。探讨了E2F1对LUAD细胞RNH1功能的调控。进行小鼠实验以证实RNHl在LUAD中的体内作用。mRNA测序表明RNH1过表达改变了LUAD细胞的表达谱。
结果:这项工作介绍了患者LUAD组织中RNH1的表达。重要的是,RNH1敲除增进了增殖,细胞的迁移和侵袭能力以及RNH1过表达产生了相反的作用。双荧光素酶实验证明,E2F1与RNH1启动子(-1064~-1054,-1514~-1504)结合,降低了RNH1的转录活性。ChIP分析表明,E2F1DNA富集在RNH1启动子(-1148~-943,-1628~-1423)。下拉法也显示了E2F1和RNH1启动子之间的关联(-1148~-943)。E2F1过表达有助于LUAD细胞的恶性行为,而RNH1过表达则逆转了它。高通量测序显示,RNH1过表达可诱导多基因表达改变,从而调节LUAD相关过程。
结论:我们的研究表明,E2F1与RNH1启动子的结合可能导致RNH1表达的抑制,从而促进LUAD的发展。