| 罗 好,刘怡波,颜崇兵,翁博雯,蔡 成.长链非编码RNA 1010001N08Rik和GATA6在高氧暴露小鼠肺癌细胞中表达和意义[J].,2024,(15):2805-2816 |
| 长链非编码RNA 1010001N08Rik和GATA6在高氧暴露小鼠肺癌细胞中表达和意义 |
| Expression and Significance of Long non-coding RNA 1010001N08Rik and GATA 6 in Hyperoxia-exposed LLC Cells |
| 投稿时间：2024-02-10 修订日期：2024-03-12 |
| DOI：10.13241/j.cnki.pmb.2024.15.002 |
| 中文关键词: 高氧暴露 lncRNA1010001N08Rik GATA6 高氧肺损伤 Wnt/β-catenin信号通路 |
| 英文关键词: Hyperoxygen exposure lncRNA 1010001N08Rik GATA6 Hyperoxygen injury Wnt/β-catenin signaling pathway |
| 基金项目:2023年"科技兴蒙"上海交通大学行动计划专项项目；2023年上海市儿童医院院级临床研究培育专项（2023YLY02） |
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| 中文摘要: |
| 摘要 目的：探讨长链非编码RNA（long noncoding RNA，lncRNA）1010001N08Rik和GATA6在高氧暴露小鼠肺癌细胞（Lewis lung carcinoma cell，LLC细胞）中表达量的变化与相关性，及其介导Wnt/β-catenin信号通路参与高氧肺损伤的机制。方法：（1）将LLC细胞分为空气组、高氧暴露24 h组和高氧暴露48h组，应用RT-PCR和Western blot法分别检测lncRNA1010001N08Rik、GATA6、Wnt及β-catenin的mRNA及相关蛋白表达水平。（2）将LLC细胞分为无干扰空气组、无干扰高氧组、NC-siRNA干扰高氧组、siRNA干扰高氧组。应用RT-PCR和Western blot法分别检测lncRNA1010001N08Rik、GATA6、Wnt及β-catenin的mRNA及相关蛋白表达水平，流式细胞术检测各组细胞凋亡。（3）利用GraphPad Prism 9.4.1进行数据可视化。（4）采用SPSS 26.0统计软件进行数据处理，两组间数据比较采用两独立样本t检验，多组间比较采用单因素方差分析。（5）将Wnt、Fzd2、GATA6等相关基因输入string11.0数据库构建PPI网络并导出PPI网络图。结果：（1）与空气组相比较，高氧暴露24 h组和48h组GATA6的mRNA表达量下降，lncRNA1010001N08Rik、Wnt、β-catenin的mRNA表达量增加，差异具有统计学意义（P＜0.05）。（2）与空气组相比，高氧暴露24 h组和48 h组GATA6蛋白表达量下降，Wnt、β-catenin蛋白表达量增加，差异具有统计学意义（P＜0.05）。（3）与无干扰空气组相比，无干扰高氧组、NC-siRNA干扰高氧组的GATA6的mRNA表达量和蛋白表达量下降，1010001N08Rik的mRNA表达量、Wnt、β-catenin的mRNA表达量和蛋白表达量增加；无干扰高氧组与NC-siRNA干扰高氧组的比较差异无统计学意义；与干扰高氧组与NC-siRNA干扰高氧组相比，siRNA干扰高氧组GATA6的mRNA表达量和蛋白表达量增加，1010001N08Rik的mRNA表达量、Wnt、β-catenin的mRNA表达量和蛋白表达量下降，差异均具有统计学意义（P<0.05）。（4）与无干扰空气组相比，无干扰高氧组、NC-siRNA干扰高氧组细胞凋亡率增加；无干扰高氧组与NC-siRNA干扰高氧组的比较差异无统计学意义；与无干扰高氧组与NC-siRNA干扰高氧组相比，siRNA干扰高氧组的细胞凋亡率下降，差异均具有统计学意义（P<0.05）。（5）根据string11.0数据库导出Wnt、Fzd2、GATA6等相关基因的PPI网络图显示，GATA6与Wnt有密切相关。结论：高氧暴露下，LCC细胞中lncRNA1010001N08Rik可能通过下调GATA6的表达激活Wnt/β-catenin信号通路的传导，从而促进高氧肺损伤的形成过程。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the expression changes and correlation of long non-coding RNA (lncRNA) 1010001N08Rik and GATA6 in hyperoxic LLC cells, and the mechanism of Wnt/β-catenin signaling pathway involved in hyperoxic lung injury. Methods: (1)LLC cells were divided into air group. The expression levels of GATA6, Wnt, β-catenin, 1010001N08Rik in each group were detected by RT-PCR and the expression levels of related proteins in each group were detected by Western blot. (2) LLC cells were divided into non-interference air group, non-interference hyperoxia group, NC-siRNA interference hyperoxia group and siRNA interference hyperoxia group. The expression levels of GATA6, Wnt, β-catenin, 1010001N08Rik in each group were detected by RT-PCR and the expression levels of related proteins in each group were detected by Western blot.The apoptosis was detected by flow cytometry. (3) Data visualization using GraphPad Prism 9.4.1. (4) SPSS 26.0 statistical software was used for data processing. The data of two groups were compared by t test. One-way analysis of variance was used for comparison among multiple groups. (5) Wnt, Fzd2, GATA6 and other related genes were input into string11.0 database to construct the PPI network and derive the PPI network diagram. Results: (1) Compared with the air group, the expression of GATA6 mRNA decreased in the hyperoxia exposure group for 24 h and hyperoxia exposure group for 48h, the expression of lncRNA1010001N08Rik mRNA, Wnt mRNA, β-catenin mRNA expression increased. The difference was statistically significant (P＜0.05). (2) Compared with the air group, the expression of GATA6 protein decreased in the hyperoxia exposure group for 24 h and hyperoxia exposure group for 48 h, whlie the expression of Wnt protein, β-catenin protein increased. The difference was statistically significant (P＜0.05). (3) Compared with the non-interference air group, the expression of GATA6 mRNA and protein in the non-interference hyperoxia group and the NC-siRNA interference hyperoxia group decreased, and the expression of 1010001N08Rik mRNA, Wnt mRNA and protein, β-catenin mRNA and protein increased. There was no significant difference between non-interference hyperoxia group and NC-siRNA interference hyperoxia group. Compared with the non-interference hyperoxia group and the NC-siRNA interference hyperoxia group, the expression of GATA6 mRNA and protein in the siRNA interference hyperoxia group increased, and the expression of 1010001N08Rik mRNA, Wnt mRNA and protein, β-catenin mRNA and protein in the siRNA interference hyperoxia group decreased. The difference was statistically significant (P＜0.05). (4) Compared with the non-interference air group, the apoptosis rate of the non-interference hyperoxia group and the NC-siRNA interfered hyperoxia group increased. There was no significant difference between non-interference hyperoxia group and NC-siRNA interference hyperoxia group. Compared with the non-interference hyperoxia group and the NC-siRNA interference hyperoxia group, the apoptosis rate of siRNA interference hyperoxia group decreased. The differences were statistically significant (P<0.05). (5) According to the protein-protein interaction(PPI) network diagram of Wnt, Fzd2, GATA6 and other related genes derived from the string11.0 database, it can be seen that GATA6 is closely related to Wnt. Conclusion: In LCC cells exposed to hyperoxia, 1010001N08Rik may activate Wnt/β-catenin signaling pathway by down-regulating the expression of GATA6, thus promoting the formation process of hyperoxic lung injury. |
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