背景:宫颈癌是最常见的妇科癌症之一。越来越多的证据表明,长链非编码RNA(lncRNAs)在宫颈癌的发生和发展中起着至关重要的作用,但是它们的具体功能和机制还有待进一步探索。
方法:采用RT-qPCR法检测NEAT1在宫颈癌组织和细胞株中的表达。CCK-8,集落形成,流式细胞术,西方印迹,和Transwell分析用于评估NEAT1对宫颈癌细胞恶性行为的影响。葡萄糖消耗,乳酸生产,ATP水平,ROS水平,MMP水平,检测糖酵解相关基因和三羧酸循环相关基因的mRNA表达,分析NEAT1对宫颈癌细胞代谢重编程的影响。免疫印迹法检测PDK1、β-catenin及WNT/β-catenin信号通路下游分子在宫颈癌细胞和组织中的表达,RT-qPCR,免疫荧光和免疫组织化学测定。
结果:本研究调查了lncRNA核旁斑点组装转录物1(NEAT1)在宫颈癌中的作用和可能的分子机制。我们的结果表明,NEAT1在宫颈癌组织和细胞系中高表达。下调NEAT1抑制增殖,迁移,宫颈癌细胞的侵袭和糖酵解,而NEAT1的过度表达导致相反的效果。机械上,NEAT1通过WNT/β-catenin信号通路上调丙酮酸脱氢酶激酶(PDK1),这增强了糖酵解,然后促进了宫颈癌的转移。此外,NEAT1维持了β-catenin的蛋白质稳定性,但不影响其mRNA水平。我们还通过RNA下拉测定排除了NEAT1与β-catenin蛋白的直接结合。NEAT1敲低对细胞增殖的抑制作用,入侵,β-catenin过表达拯救了迁移。WNT抑制剂iCRT3减弱了NEAT1过表达诱导的致癌作用。
结论:总之,这些结果表明,NEAT1可能通过激活WNT/β-catenin/PDK1信号轴参与宫颈癌的进展.
BACKGROUND: Cervical cancer is one of the most common gynecological cancers. Accumulated evidence shows that long non-coding RNAs (lncRNAs) play essential roles in cervical cancer occurrence and progression, but their specific functions and mechanisms remain to be further explored.
METHODS: The RT-qPCR assay was used to detect the expression of NEAT1 in cervical cancer tissues and cell lines. CCK-8, colony formation, flow cytometry, western blotting, and Transwell assays were used to evaluate the impact of NEAT1 on the malignant behavior of cervical cancer cells. Glucose consumption, lactate production, ATP levels, ROS levels, MMP levels, and the mRNA expressions of glycolysis-related genes and tricarboxylic acid cycle-related genes were detected to analyze the effect of NEAT1 on metabolism reprograming in cervical cancer cells. The expressions of
PDK1, β-catenin and downstream molecules of the WNT/β-catenin signaling pathway in cervical cancer cells and tissues were detected by western blotting, RT-qPCR, immunofluorescence and immunohistochemistry assays.
RESULTS: This study investigated the role and possible molecular mechanism of lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in cervical cancer. Our results showed that NEAT1 was highly expressed in cervical cancer tissues and cell lines. Downregulation of NEAT1 inhibited the proliferation, migration, invasion and glycolysis of cervical cancer cells, while overexpression of NEAT1 led to the opposite effects. Mechanistically, NEAT1 upregulated pyruvate dehydrogenase kinase (
PDK1) through the WNT/β-catenin signaling pathway, which enhanced glycolysis and then facilitated cervical cancer metastasis. Furthermore, NEAT1 maintained the protein stability of β-catenin but did not affect its mRNA level. We also excluded the direct binding of NEAT1 to the β-catenin protein via RNA pull-down assay. The suppressive impact of NEAT1 knockdown on cell proliferation, invasion, and migration was rescued by β-catenin overexpression. The WNT inhibitor iCRT3 attenuated the carcinogenic effect induced by NEAT1 overexpression.
CONCLUSIONS: In summary, these findings indicated that NEAT1 may contribute to the progression of cervical cancer by activating the WNT/β-catenin/
PDK1 signaling axis.