重楼皂苷Ⅰ通过调控LncRNA CEBPA-AS1/miR-195-5p通路抑制瘢痕疙瘩成纤维细胞增殖及促进细胞凋亡的初步研究

张翠　王珺　王凯波　林伟　孙田

[摘要]目的：探討重楼皂苷Ⅰ（PPⅠ）是否通过调控长链非编码RNA CEBPA-AS1（LncRNA CEBPA-AS1）/微小RNA-195-5p（miR-195-5p）通路影响瘢痕疙瘩成纤维细胞增殖及凋亡。方法：原代培养人瘢痕疙瘩成纤维细胞，使用不同浓度的PPⅠ处理细胞，si-CEBPA-AS1、pcDNA-CEBPA-AS1分别转染至细胞;采用MTT检测细胞增殖;应用流式细胞仪检测细胞周期;采用流式细胞术检测细胞凋亡率;采用qRT-PCR法检测LncRNA CEBPA-AS1、miR-195-5p的表达量;双荧光素酶报告实验检测LncRNA CEBPA-AS1与miR-195-5p的靶向关系。结果：与Control组比较，PPⅠ可明显降低细胞存活率、S期细胞比例、Bcl-2蛋白水平、LncRNA CEBPA-AS1的表达水平，提高G0/G1期细胞比例、凋亡率、miR-195-5p的表达水平，差异均有统计学意义（P<0.05）。与si-NC组比较，si-CEBPA-AS1组细胞存活率、S期细胞比例降低，G0/G1期细胞比例、凋亡率升高，差异均有统计学意义（P<0.05）。双荧光素酶报告实验证实LncRNA CEBPA-AS1可靶向调控miR-195-5p;转染pcDNA-CEBPA-AS1可明显逆转PPⅠ对细胞增殖、细胞周期及凋亡的作用。结论：重楼皂苷Ⅰ可能通过下调LncRNA CEBPA-AS1的表达及上调miR-195-5p的表达从而抑制瘢痕疙瘩成纤维细胞增殖及促进细胞凋亡。

[关键词]重楼皂苷Ⅰ;LncRNA CEBPA-AS1;miR-195-5p;瘢痕疙瘩;成纤维细胞;增殖;凋亡

[中图分类号]R619+.6    [文献标志码]A    [文章编号]1008-6455（2021）09-0012-05

Polyphylin Ⅰ Inhibits Keloid Fibroblast Proliferation and Promotes Apoptosis by Regulating the LncRNA CEBPA-AS1/miR-195-5p Pathway

ZHANG Cui，WANG Jun，WANG Kai-bo，LIN Wei，SUN Tian

（Department of Dermatology，Shenyang Seventh People's Hospital，Shenyang 110000，Liaoning，China）

Abstract： Objective  To investigate whether PPⅠ affects keloid fibroblast proliferation and apoptosis by regulating the LncRNA CEBPA-AS1/miR-195-5p pathway. Methods  Primary culture of human keloid fibroblasts， and treatment of cells with different concentrations of PPⅠ. si-CEBPA-AS1 and pcDNA-CEBPA-AS1 were transfected into cells respectively. MTT was used to detect cell proliferation. Application of flow cytometry to detect cell cycle. Flow cytometry was used to detect the apoptosis rate. The expression of LncRNA CEBPA-AS1， miR-195-5p was detected by qRT-PCR method. The dual luciferase report experiment detected the targeting relationship between LncRNA CEBPA-AS1 and miR-195-5p. Results  Compared with the Control group， PPⅠ could significantly reduce the cell survival rate， S phase cell ratio， the protein level of Bcl-2， the expression level of LncRNA CEBPA-AS1， the differences were statistically significant （P<0.05）， and increase the G0/G1 phase cell ratio， apoptosis rate， the expression level of miR- 195-5p （P<0.05）. Compared with the si-NC group， the cell survival rate and the proportion of S-phase cells in the si-CEBPA-AS1 group was decreased （P<0.05）， and the proportion of cells in the G0/G1 phase and the apoptosis rate were increased （P<0.05）. The dual luciferase report experiment confirmed that LncRNA CEBPA-AS1 could target miR-195-5p. Transfection of pcDNA-CEBPA-AS1 could obviously reverse the effects of PPⅠ on cell proliferation， cell cycle and apoptosis. Conclusion  PolyphylinⅠmay inhibit the proliferation of keloid fibroblasts and promote apoptosis by down-regulating the expression of LncRNA CEBPA-AS1 and up-regulating the expression of miR-195-5p.

Key words： Polyphylin Ⅰ; LncRNA CEBPA-AS1; miR-195-5p; keloid; fibroblast; proliferation; apoptosis

瘢痕疙瘩是皮肤纤维组织增生异常引起的，天然植物提取物对瘢痕具有明显的抑制作用，但关于其作用机制尚未完全阐明[1-3]。重楼是我国中医药治疗中的重要药物之一，其主要成分重楼皂苷Ⅰ（Polyphylin Ⅰ，PPⅠ）具有抑制肿瘤细胞增殖的作用[4]。但重楼皂苷Ⅰ对瘢痕疙瘩成纤维细胞增殖及凋亡的影响尚不明确。长链非编码RNA CEBPA-AS1（LncRNA CEBPA-AS1）在肝癌中呈高表达，并可能促进肝癌发生及发展[5]。通过生物信息学分析显示微小RNA-195-5p（miR-195-5p）可能是LncRNA CEBPA-AS1的靶基因，miR-195-5p在肺癌细胞中表达下调，上调其表达可抑制肺癌细胞增殖、迁移及侵袭[6]。但重楼皂苷Ⅰ是否可通过调控LncRNA CEBPA-AS1/miR-195-5p通路影响瘢痕疙瘩成纤维细胞增殖及凋亡尚未可知。因此，本研究主要探讨重楼皂苷Ⅰ对瘢痕疙瘩成纤维细胞增殖及凋亡的影响，探究其对LncRNA CEBPA-AS1/miR-195-5p通路的调控作用。……