微小 RNA-181a 通过靶向磷酸酶张力蛋白同源物促进肥厚性瘢痕成纤维细胞的增殖并抑制其凋亡。
microRNA-181a promotes the proliferation of hypertrophic scar fibroblasts and inhibits their apoptosis via targeting phosphatase and tensin homolog.
机构信息
Traumatic Surgery, Xinxiang Central Hospital, Xinxiang, China.
The Fourth Clinical College of Xinxiang Medical University, Xinxiang, China.
出版信息
Ann Palliat Med. 2021 Apr;10(4):4563-4571. doi: 10.21037/apm-21-604.
BACKGROUND
This study aimed to compare the expression of microRNA (miR)-181a and phosphatase and tensin homolog (PTEN) in hypertrophic scar tissue and cells and to explore the effects of miR-181a and PTEN on the proliferation and apoptosis of the human scar fibroblast cell line HSFb.
METHODS
HSFb cells were transfected with miR-negative control (miR-NC), miR-181a mimics, miR-181a inhibitor, pcDNA3.1-PTEN (pc-PTEN), or small interfence-PTEN (si-PTEN) plasmid using a Lipofectamine 2000 transfection kit. The effects of miR-181a and PTEN on the proliferation and apoptosis of HSFb were determined using a Cell Counting Kit (CCK)-8 experiment and flow cytometry, respectively. The effects of miR-181a and PTEN on the expression of apoptosis-related proteins in HSFb, including type I collagen (Col-1) and type III collagen (Col-3), were measured by western blot. Finally, the relationship between miR-181a and PTEN was explored by the dual-luciferase reporter gene experiment.
RESULTS
The miR-181a in hypertrophic scar tissues and HSFb were significantly up-regulated compared to embryo skin fibroblast (ESF-1) cells and normal tissues (P<0.05), whereas the opposite results were seen for PTEN expression (P<0.05). Inhibiting miR-181a or upregulating the expression of PTEN significantly suppressed the proliferation of HSFb (P<0.05) and induced their apoptosis (P<0.05). Western blot revealed that inhibiting and upregulating miR-181a and PTEN, respectively, decreased the expression of the B-cell lymphoma-2 (Bcl-2), Col-1, and Col-3 proteins in HSFb, but significantly up-regulated the expression of Bcl-2-associated X protein (Bax), cleaved caspase-3 (c-caspase-3), and cleaved caspase-9 (c-caspase-9) (P<0.05). The dual-luciferase reporter gene experiment results confirmed PTEN to be the downstream target gene of miR-181a. Simultaneous upregulation of miR-181a and PTEN expression had no significant effect on the proliferation and apoptosis of HSFb.
CONCLUSIONS
miR-181a promotes the up-regulation of Col-1 and Col-3, and regulates the proliferation and apoptosis of HSFb by targeting PTEN, thereby enhancing the formation of hypertrophic scarring (HS). Therefore, miR-181a and PTEN may be potential therapeutic targets for the treatment of HS.
背景
本研究旨在比较增生性瘢痕组织和细胞中微小 RNA（miR）-181a 和磷酸酶张力蛋白同源物（PTEN）的表达，并探讨 miR-181a 和 PTEN 对人瘢痕成纤维细胞系 HSFb 增殖和凋亡的影响。
方法
使用 Lipofectamine 2000 转染试剂盒将 miR-阴性对照（miR-NC）、miR-181a 模拟物、miR-181a 抑制剂、pcDNA3.1-PTEN（pc-PTEN）或小干扰 RNA-PTEN（si-PTEN）质粒转染至 HSFb 细胞。通过细胞计数试剂盒（CCK）-8 实验和流式细胞术分别测定 miR-181a 和 PTEN 对 HSFb 增殖和凋亡的影响。通过 Western blot 测定 miR-181a 和 PTEN 对 HSFb 中细胞凋亡相关蛋白，包括Ⅰ型胶原（Col-1）和Ⅲ型胶原（Col-3）的表达的影响。最后，通过双荧光素酶报告基因实验探讨 miR-181a 和 PTEN 之间的关系。
结果
与胚胎皮肤成纤维细胞（ESF-1）和正常组织相比，增生性瘢痕组织和 HSFb 中的 miR-181a 表达明显上调（P<0.05），而 PTEN 的表达则相反（P<0.05）。抑制 miR-181a 或上调 PTEN 的表达可显著抑制 HSFb 的增殖（P<0.05）并诱导其凋亡（P<0.05）。Western blot 显示，分别抑制和上调 miR-181a 和 PTEN 可降低 HSFb 中 B 细胞淋巴瘤-2（Bcl-2）、Col-1 和 Col-3 蛋白的表达，但显著上调 Bcl-2 相关 X 蛋白（Bax）、裂解的半胱天冬酶-3（c-caspase-3）和裂解的半胱天冬酶-9（c-caspase-9）的表达（P<0.05）。双荧光素酶报告基因实验结果证实 PTEN 是 miR-181a 的下游靶基因。同时上调 miR-181a 和 PTEN 的表达对 HSFb 的增殖和凋亡无显著影响。
结论
miR-181a 通过靶向 PTEN 促进 Col-1 和 Col-3 的上调，并调节 HSFb 的增殖和凋亡，从而增强增生性瘢痕的形成（HS）。因此，miR-181a 和 PTEN 可能是 HS 治疗的潜在治疗靶点。
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