| 胡新红,曹天宇,吕雅洁,刘 涛,周 芳.miR-20a靶向CCND1作用PI3K/AKT信号通路抑制皮肤鳞状细胞癌的发展[J].,2023,(18):3416-3424 |
| miR-20a靶向CCND1作用PI3K/AKT信号通路抑制皮肤鳞状细胞癌的发展 |
| miR-20a Inhibits the Development of Cutaneous Squamous Cell Carcinoma through Targeted by CCND1 in the PI3K/AKT Signaling Pathway |
| 投稿时间：2023-03-21 修订日期：2023-04-16 |
| DOI：10.13241/j.cnki.pmb.2023.18.003 |
| 中文关键词: 皮肤鳞状细胞癌 miR-20a CCND1 PI3K/AKT信号通路 |
| 英文关键词: Cutaneous squamous cell carcinoma miR-20a CCND1 PI3K/AKT signaling pathway |
| 基金项目:国家自然科学基金青年基金项目（81703119） |
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| 中文摘要: |
| 摘要 目的：探究miR-20a与CCND1蛋白在皮肤鳞状细胞癌（CSCC）中的作用关系，以及其可能涉及的信号通路分子机制。方法：分别收集皮肤鳞状细胞癌患者的皮肤癌组织及其邻近正常皮肤组织，采用qRT-PCR分析组织中miR-20a和CCND1基因表达水平。为探究miR-20a对CSCC细胞的影响，将SCL-1细胞分为对照组（不转染）、miR-NC组（转染miR-NC）和miR-20a mimics组（转染miR-20a mimics）；为探究CCND1与PI3K/AKT信号通路的关系，将SCL-1细胞分为对照组（不转染）、si-NC组（转染si-NC）和si-CCND1组（转染si-CCND1）；为探究miR-20a与CCND1间的作用关系及对CSCC细胞的影响，将SCL-1细胞分为miR-NC组（转染miR-NC）、miR-20a mimics组（转染miR-20a mimics）、mimics+pcDNA组（共转染miR-20a mimics和pcDNA）和mimics+CCND1组（共转染miR-20a mimics和pcDNA-CCND1）。采用Western blot分析p-AKT、AKT、p-PI3K、PI3K和GSK-3β蛋白表达水平；采用MTT检测细胞增殖情况；采用流式细胞术检测细胞凋亡情况；采用Transwell分析细胞迁移和侵袭情况；采用双荧光素酶报告基因检测分析miR-20a与CCND1的靶向关系。结果：CSCC癌组织和SCL-1中miR-20a均低表达，CCND1高表达。与对照组和miR-NC组比较，miR-20a mimics组SCL-1细胞增殖水平以及侵袭和迁移数量均降低（P<0.05），SCL-1细胞凋亡水平升高（P<0.05），PI3K和AKT蛋白磷酸化水平降低（P<0.05）。TargetScanHuman数据库分析和双荧光素酶报告基因检测结果显示miR-20a与CCND1存在靶向作用关系。与对照组和si-NC组比较，si-CCND1组SCL-1细胞中CCND1和GSK-3β蛋白表达水平以及PI3K和AKT蛋白磷酸化水平均降低（P<0.01）。与miR-20a mimics组或mimics+pcDNA组比较，mimics+CCND1组SCL-1细胞增殖水平以及侵袭和迁移数量均升高（P<0.05），SCL-1细胞凋亡水平降低（P<0.05），PI3K和AKT蛋白磷酸化水平均升高（P<0.05）。结论：过表达miR-20a可能通过靶向抑制CCND1的表达而抑制PI3K/AKT信号通路的激活，从而抑制CSCC细胞的增殖、侵袭和迁移，并促进癌细胞凋亡。 |
| 英文摘要: |
| ABSTRACT Objective: To explore the relationship between miR-20a and CCND1 protein in cutaneous squamous cell carcinoma (CSCC), as well as the possible signaling pathway molecular mechanism involved. Methods: Skin cancer tissues and adjacent normal skin tissues of the CSCC patients were collected, respectively. And the expression levels of miR-20a and CCND1 genes in tissues were analyzed by qRT-PCR. In order to investigate the effect of miR-20a on SCL-1 cells of CSCC cell line, SCL-1 cells were divided into control group (non-transfection), miR-NC group (transfection of miR-NC) and miR-20a mimics group (transfection of miR-20a mimics). In order to investigate the relationship between CCND1 and PI3K/AKT signaling pathway, SCL-1 cells were divided into control group (non-transfection), si-NC group (transfection of si-NC) and si-CCND1 group (transfection of si-CCND1). In order to explore the interaction between miR-20a and CCND1 and its effect on CSCC cells, SCL-1 cells were divided into miR-NC group (transfection of miR-NC), miR-20a mimics group (transfection of miR-20a mimics), and mimics+pcDNA group (co-transfected miR-20a mimics and pcDNA) and mimics+CCND1 group (co-transfected miR-20a mimics and pcDNA-CCND1). The protein expression levels of p-AKT, AKT, p-PI3K, PI3K and GSK-3β were analyzed by Western blot. Cell proliferation was detected by MTT. The apoptosis was detected by flow cytometry. Cell migration and invasion were analyzed by Transwell. Dual luciferase reporter assay was used to analyze the targeting relationship between miR-20a and CCND1. Results: The expression level of miR-20a in CSCC cancer tissues and SCL-1 cell were low, and the expression level of CCND1 in CSCC cancer tissues and SCL-1 cell were high. Compared with control group and miR-NC group, the proliferation level and the number of invasion and migration of SCL-1 cells in the miR-20a mimics group were decreased (P<0.05), the apoptosis level of SCL-1 cells was increased (P<0.01), and the protein phosphorylation levels of PI3K and AKT were decreased (P<0.01). Target Scan Human database analysis result and dual luciferase reporter assay results showed that miR-20a had a targeting relationship with CCND1. Compared with control group and si-NC group, the protein expression levels of CCND1 and GSK-3β and the protein phosphorylation levels of PI3K and AKT in SCL-1 cells in the si-CCND1 group were decreased (P<0.01). Compared with miR-20a mimics group or mimics+pcDNA group, the proliferation level and the number of invasion and migration of SCL-1 cells in mimics+CCND1 group were increased (P<0.05), while the apoptosis level of SCL-1 cells was decreased (P<0.01), and the protein phosphorylation levels of PI3K and AKT were increased (P<0.01). Conclusion: Overexpression of miR-20a may inhibit the activation of PI3K/AKT signaling pathway by targeting the expression level of CCND1, thereby inhibiting the proliferation, invasion and migration of CSCC cells, and promoting the apoptosis of cancer cells. |
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