Ma Xiaogen, Xiang Fei, Pei Zhuo, Miao Jiafeng, Wu Pan, Song Xiaofeng, Li Yuhong, Zhang Yiming
Department of Plastic and Cosmetic Surgery, Xinqiao Hospital, Army Medical University, Chongqing, 400037, PR China.
Department of Cell Biology, Army Medical University, Chongqing, 400038, PR China.
Genes Dis. 2020 Jan 9;8(3):364-372. doi: 10.1016/j.gendis.2020.01.001. eCollection 2021 May.
Circular RNAs are a large class of noncoding RNAs. Smad5 functions in cell differentiation, cell proliferation and metastasis. It has been reported that lnc-Smad5 can inhibit the proliferation of diffuse large B cell lymphoma. However, the function of circ-Smad5 has not yet been reported. Lentivirus vectors were constructed to establish circ-Smad5 upregulated and circ-Smad5 downregulated cell models. A CCK-8 assay was used to detect the proliferation of JB6 cells. FACS was used to analyze the cell cycle in the cell models. Western blot, immunofluorescence staining and TOP/FOP flash dual luciferase activity assays were used to determine the activity of the Wnt signaling pathway. The results revealed that the expression level of circ-Smad5 in JB6 cells was significantly lower than the expression level of linearized-Smad5. Compared with the control group, the percentage of S phase cells and the expression level of cyclin D1 protein were significantly higher in the sh-circ-Smad5 group. In the sh-circ-Smad5 group, β-catenin and LEF-1 were significantly increased, p-β-catenin was significantly decreased, and the relative activity of the TOP/FOP reporter gene was higher compared to the control group levels. These phenomena could be reversed by treating with Wnt signaling inhibitor PNU-74654. We conclude that the circ-Smad5 retards the proliferation and the cell cycle progression of JB6 cells. Thus, circ-Smad5 may function by inhibiting the activation of Wnt/β-catenin/Lef 1 signaling, which inhibits the expression of cyclin D1. To the best of our knowledge, we are the first to report the function of circ-Smad5.
环状RNA是一大类非编码RNA。Smad5在细胞分化、细胞增殖和转移过程中发挥作用。据报道，lnc-Smad5可抑制弥漫性大B细胞淋巴瘤的增殖。然而，circ-Smad5的功能尚未见报道。构建慢病毒载体以建立circ-Smad5上调和circ-Smad5下调的细胞模型。采用CCK-8法检测JB6细胞的增殖情况。运用流式细胞术分析细胞模型中的细胞周期。采用蛋白质免疫印迹法、免疫荧光染色法和TOP/FOP闪光双荧光素酶活性测定法来确定Wnt信号通路的活性。结果显示，JB6细胞中circ-Smad5的表达水平显著低于线性化Smad5的表达水平。与对照组相比，sh-circ-Smad5组中S期细胞的百分比和细胞周期蛋白D1蛋白的表达水平显著更高。在sh-circ-Smad5组中，β-连环蛋白和淋巴样增强因子1显著增加，磷酸化β-连环蛋白显著降低，与对照组水平相比，TOP/FOP报告基因的相对活性更高。用Wnt信号抑制剂PNU-74654处理可逆转这些现象。我们得出结论，circ-Smad5可延缓JB6细胞的增殖及细胞周期进程。因此，circ-Smad5可能通过抑制Wnt/β-连环蛋白/Lef 1信号的激活来发挥作用，从而抑制细胞周期蛋白D1的表达。据我们所知，我们是首个报道circ-Smad5功能的研究。
