TGFB1-induced extracellular expression of TGFBIp and inhibition of TGFBIp expression by RNA interference in a human corneal epithelial cell line.
TGFB1-induced extracellular expression of TGFBIp and inhibition of TGFBIp expression by RNA interference in a human corneal epithelial cell line.
复制标题
DOI：
10.1167/iovs.10-5362
复制
发表时间：
2011-02
影响因子：
4.4
通讯作者：
V. Yellore;S. Rayner;A. Aldave
中科院分区：
文献类型：
--
作者：
V. Yellore;S. Rayner;A. Aldave
目的观察TGF β 1诱导人角膜上皮细胞（HCECs）产生细胞外转化生长因子β诱导蛋白（TGF β 1 β）的情况，以及RNA干扰（RNAi）对诱导和未诱导HCECs产生TGF β 1 β的抑制作用。方法无血清培养HCECs，加入0或10 ng/mL TGF β 1处理72 h。将市售的靶向TGFB 1 mRNA的siRNA与转染试剂混合，并用于逆转转染TGFB 1诱导的和未诱导的HCEC。分别通过ELISA和Western印迹分析测定细胞外和细胞内TGF β 1 β浓度，并使用半定量RT-PCR测定TGF β 1 β RNA。结果HCECs组成型表达TGF β 1 β，TGF β 1处理导致细胞外TGF β 1 β的量增加4倍。四种市售的靶向TGFB 1 mRNA的siRNA在转染未诱导的HCEC后48小时内产生细胞外TGF β 1 β>70%的降低，但在转染TGF β 1诱导的HCEC后48小时细胞外TGF β 1 β降低<25%。细胞外TGF β 1 β产生的抑制与转染后细胞内TGF β 1 β产生和TGF β 1 mRNA表达的减少相关。结论：在暴露于TGF β 1后，HCECs的细胞外TGF β 1 β表达增加了数倍。HCEC-组成型和HCEC-诱导的TGF β 1 β产生都可以用RNA干扰抑制，尽管对于组成型的影响比诱导的TGF β 1 β产生更大且持续时间更长。鉴于TGFBI营养不良中的角膜沉积物由源自HCEC的TGF β 1 β组成，RNAi代表了抑制原发性营养不良存款形成和手术干预后复发的潜在手段。
PURPOSE To report the increased production of extracellular transforming growth factor β-induced protein (TGFBIp) by human corneal epithelial cells (HCECs) after induction by TGFB1 and the inhibition of TGFBIp production in induced and noninduced HCECs by RNA interference (RNAi). METHODS HCECs were cultured in serum-free medium and treated with 0 or 10 ng/mL TGFB1 over a period of 72 hours. Commercially available siRNAs targeting TGFBI mRNA were mixed with a transfection reagent and used to reverse transfect TGFB1-induced and noninduced HCECs. Extracellular and intracellular concentrations of TGFBIp were measured by ELISA and Western blot analysis, respectively, and TGFBI RNA was assayed using semiquantitative RT-PCR. RESULTS HCECs constitutively express TGFBIp, and treatment with TGFB1 results in up to a fourfold increase in the amount of extracellular TGFBIp. Four commercially available siRNAs targeting TGFBI mRNA produced a >70% decrease in extracellular TGFBIp within 48 hours after transfection of noninduced HCECs but a <25% decrease in extracellular TGFBIp by 48 hours after transfection of TGFB1-induced HCECs. The suppression of extracellular TGFBIp production correlated with a decrease in intracellular TGFBIp production and TGFBI mRNA expression after transfection. CONCLUSIONS Extracellular TGFBIp expression by HCECs is increased several fold after exposure to TGFB1. Both HCEC-constitutive and HCEC-induced TGFBIp production can be inhibited with RNA interference, though the effect was greater and lasted longer for constitutive than induced TGFBIp production. Given that the corneal deposits in the TGFBI dystrophies consist of TGFBIp derived from HCECs, RNAi represents a potential means to inhibit primary dystrophic deposit formation and recurrence after surgical intervention.