SIRT6通过TGF-β1/Smad信号通路调控瘢痕疙瘩成纤维细胞增殖、侵袭和胶原合成

SIRT6 regulates proliferation, invasion and collagen synthesis in keloid fibroblasts by regulating TGF-β1/Smad signaling pathway

目的 探讨沉默信息调节因子6(silent information regulator 6, SIRT6)对瘢痕疙瘩成纤维细胞增殖、侵袭和胶原合成的调控作用和分子机制。方法 收集瘢痕疙瘩和正常皮肤样本,采用实时定量PCR(real-time quantitative PCR,RT-qPCR)检测SIRT6 mRNA表达水平,采用Western blot检测SIRT6蛋白表达水平。采取组织块贴壁法从瘢痕疙瘩组织中分离养成纤维细胞。构建SIRT6重组腺病毒(adenovirus, Ad),感染瘢痕疙瘩成纤维细胞实现SIRT6过表达。将瘢痕疙瘩成纤维细胞分为空白对照组、Ad-NC组和Ad-SIRT6组。采用RT-qPCR检测SIRT6 mRNA表达水平。采用Western blot检测SIRT6、Ⅰ型胶原(collagen typeⅠ,ColⅠ)、转化生长因子-β1(transforming growth factor-β1,TGF-β1)和SMAD家族成员2/3(SMAD family member 2/3,Smad2/3)蛋白的表达水平。采用5-乙炔基-2′-脱氧尿苷(5-ethynyl-2′-deoxyuridine, EdU)方法检测细胞增殖。采用Transwell实验检测细胞侵袭能力。结果 与正常皮肤组织比较,SIRT6在瘢痕疙瘩组织中表达水平显著降低(P<0.01)。与空白组和Ad-NC组比较,Ad-SIRT6组中SIRT6表达量显著升高,细胞增殖显著减少,细胞侵袭水平显著降低,ColⅠ、TGF-β1、p-Smad2/3蛋白表达量显著下降(均P<0.01)。结论 过表达SIRT6通过下调TGF-β1/Smad信号通路抑制瘢痕疙瘩成纤维细胞增殖、侵袭和胶原合成。

Objective To investigate the regulatory effect of silent information regulator 6(SIRT6)on the cell proliferation and invasion and the collagen synthesis in keloid fibroblasts,and explore the underlying molecular mechanism.Methods The mRNA and protein expression levels of SIRT6 in keloid tissues and normal skin tissues were detected by real-time quantitative PCR and Western blot.The fibroblasts were isolated from keloid tissues using tissue explants adherent method.Recombinant adenovirus(Ad)expressing SIRT6 was constructed and transduced into keloid fibroblasts to achieve SIRT6 overexpression.Keloid fibroblasts were divided into blank control group,Ad-NC group and Ad-SIRT6 group.SIRT6 mRNA expression was detected by RT-qPCR.Protein levels of SIRT6,collagen typeⅠ(ColⅠ),transforming growth factor-β1(TGF-β1),and SMAD family member 2/3(Smad2/3)were examined by Western blot.Cell proliferation was assessed by 5-ethynyl-2′-deoxyuridine(EdU)assays.Cell invasion was evaluated by Transwell assay.Results Compared with normal skin tissues,SIRT6 expression was significantly decreased in keloid tissues(P<0.01).Compared with blank control group and Ad-NC group,the expression of SIRT6 was significantly upregulated in Ad-SIRT6 group,the proliferation was significantly decreased,the invasive ability was significantly downregulated,and the protein levels of ColⅠ,TGF-β1 and p-Smad2/3 were significantly reduced(P<0.01).Conclusion Overexpression of SIRT6 could inhibit the cell proliferation and invasion and the collagen synthesis in keloid fibroblasts by inhibiting TGF-β1/Smad signaling pathway.