Radiological Intervention Center, Department of Radiology, Xiangya Hospital, Central South University, 87 Xiangya Road, Kai Fu District Changsha City, Hunan Province, 410008, China.
The Tumor Hospital of SUMC, Cancer Hospital of Shantou University Medical College, Shantou City, Guangdong Province, 515000, China.
Appl Biochem Biotechnol. 2023 May;195(5):3477-3490. doi: 10.1007/s12010-022-04279-8. Epub 2023 Jan 6.
In cell biological functions and viability, cyclin-dependent kinase 1 (CDK1) takes an essential part. miR-195-5p is pivotal in pathogenesis and development of hepatocellular carcinoma (HCC). But in HCC, whether there is a connection between CDK1 and miR-195-5p remains an unanswered question. In view of this, this study focuses on exploring the mechanism of miR-195-5p/CDK1 in the progression of HCC. The bioinformatics method was applied to predict target mRNA and upstream miRNAs, and further analyzes the signal enrichment pathway of target mRNA. We utilized qRT-PCR and Western blot for detecting expression of genes, as well as their corresponding protein levels. Cell cycle was assayed through flow cytometry. As for the examination of DNA replication, the EDU staining was employed. Cell proliferation was determined via plate colony formation assay. The combined application of bioinformatics analysis and dual-luciferase gene assay assisted in figuring out the binding relationship between miR-195-5p and CDK1. DNA damage was marked by immunofluorescence staining. CDK1 was overexpressed in HCC cells, and enriched in cell cycle and DNA replication pathway. Silencing CDK1 modulated cell cycle of HCC cells and inhibited DNA replication and proliferation. In HCC cells, miR-195-5p targeted and reduced CDK1 expression, inhibited the G1 phase-to-S phase transition, induced DNA damage response, and inhibited DNA replication and proliferation. miR-195-5p targeted CDK1 and repressed synthesis of new DNA in HCC cells, thus restraining HCC cell proliferation.
在细胞生物学功能和活力方面，细胞周期蛋白依赖性激酶 1（CDK1）起着至关重要的作用。miR-195-5p 在肝细胞癌（HCC）的发病机制和发展中起着关键作用。但是在 HCC 中，CDK1 和 miR-195-5p 之间是否存在联系仍然是一个悬而未决的问题。有鉴于此，本研究重点探讨了 miR-195-5p/CDK1 在 HCC 进展中的作用机制。应用生物信息学方法预测靶 mRNA 和上游 miRNA，并进一步分析靶 mRNA 的信号富集途径。我们利用 qRT-PCR 和 Western blot 检测基因的表达及其相应的蛋白水平，通过流式细胞术检测细胞周期，采用 EDU 染色检测 DNA 复制，通过平板集落形成实验测定细胞增殖。生物信息学分析和双荧光素酶基因检测联合应用有助于阐明 miR-195-5p 与 CDK1 之间的结合关系。通过免疫荧光染色标记 DNA 损伤。CDK1 在 HCC 细胞中过表达，并富集在细胞周期和 DNA 复制途径中。沉默 CDK1 调节 HCC 细胞的细胞周期，抑制 DNA 复制和增殖。在 HCC 细胞中，miR-195-5p 靶向并降低 CDK1 的表达，抑制 G1 期到 S 期的转变，诱导 DNA 损伤反应，抑制 DNA 复制和增殖。miR-195-5p 靶向 CDK1 并抑制 HCC 细胞中新 DNA 的合成，从而抑制 HCC 细胞的增殖。
