瘢痕疙瘩microRNA表达谱的筛选及miR-199a-5p生物功能的初步研究

Identification of differently expressed microRNAs in keloid and pilot study on biological function of miR-199a-5p

目的探讨并筛选出瘢痕疙瘩相关微小RNAs（miRNAs），检测其对瘢痕疙瘩成纤维细胞（keloid fibroblasts，KF）增殖的影响。方法收集手术切除瘢痕疙瘩及正常皮肤组织标本各8例，采用基因芯片检测瘢痕疙瘩与正常皮肤组织差异表达的miRNA，并采用qRT．PCR验证，在瘢痕疙瘩成纤维细胞株中转染miRNA模拟物，模拟细胞中成熟miRNA的高表达，用EdU检测方法检测其对瘢痕疙瘩成纤维细胞增殖的影响。结果①通过芯片检测发现包括miR-199a．5p在内的17个差异表达的miRNAs。②qRT．PCR验证结果显示miR．199a．5p表达下调，与芯片检测结果相一致。③miR-199a-5p模拟物转染组与阴性对照组EdU的阳性率分别为（20．72±2．50）％和（27．68±4．92）％，miR-199a-5p模拟物转染组瘢痕疙瘩成纤维细胞的增殖率下降（t=2．183，P=0．047）。同时，细胞的生长周期分布也发生改变，MiR．199a-5p模拟物转染组S期与G2／M期细胞百分比分别为（33．93±1．30）％和（10．87±0．80）％，阴性对照组分别为（31．39±0．79）％和（9．27±0．46）％，差异有统计学意义（P〈0．05）。结论①瘢痕疙瘩中miRNA的差异表达有组织特异性；②miR．199a．5p在瘢痕疙瘩中的显著低表达，可影响瘢痕疙瘩成纤维细胞周期分布，抑制瘢痕疙瘩成纤维细胞增殖，提示miR-199a-5p可能参与了瘢痕疙瘩成纤维细胞增殖的调控。

[ Abstract] Objective To screen out related microRNAs in keloid tissue, and identify their effect on the proliferation of keloid fibroblasts. Methods 8 cases of keloid tissue and 8 cases of normal skin tissue were collected as specimens. The differently expressed miRNA in keloid tissue from normal skin tissue were screened out with gene chip（ Exiqon company） , which was validated with quantitative real-time PCR. Then miRNA mimics was transfected into keloid fibroblasts line to stimulate high expression of mature miRNA in cells. The effect on the proliferation of fibroblasts in keloid was tested by Edu. Results ①A total of 17 differently expressed microRNAs were found, including miR-199a-5p. ②The expression of miR-199a-5p had been verified by qRT-PCR to be down-regulated in keloid, which was consistent with the result of array. ③The positive rate of EdU in miR-199a-5p mimics transfected group and negative control group was （20.72±2.50）% and（27.68±4. 92）% , respectively. The proliferative rate of keloid fibroblasts turned down in miR-199a-5p-transfected group（ t = 2. 183, P =0. 047）. Besides that, the cell cycle changed after transfection. The percentage of S and G2/M phase in miR-199a-5p mimics transfected group was 33.93 ±1.50 and 10.87±0.80, respectively, while it was 51.39±0.79 and 9.27±0.46 in negative control group, and the difference was statistically significant. Conclusions ①The miRNA expression profile is different between keloid and normal skin; ②The expression of miR-199a-5p is down-regulated in keloid and miR-199a-5p can affect the cell cycle and suppress proliferation of keloid fibroblasts. It indicateds that miR-199a-5p may be involved in regulating fibroblastic proliferation.