目的:研究全反式维甲酸(ATRA)对视黄醇脱氢酶5(RDH5)的影响,基质金属蛋白酶-2(MMP-2)和转化生长因子-β2(TGF-β2)转录水平,以及RDH5对视网膜色素上皮(RPE)细胞MMP-2和TGF-β2的影响。
方法:用梯度浓度的ATRA(0-20μmol/L)干预成年RPE细胞系-19(ARPE-19细胞)24h后,流式细胞术检测各组细胞的增殖和凋亡,定量实时聚合酶链反应(qRT-PCR)检测RDH5、MMP-2和TGF-β2mRNA的表达。然后,用三种不同的siRNA靶标转染ARPE-19细胞48h后,qRT-PCR检测各组的RDH5敲除效率以及其中MMP-2和TGF-β2mRNA的表达。
结果:流式细胞术结果显示,ATRA能抑制RPE细胞的增殖,促进RPE细胞的凋亡,当ATRA浓度超过5µmol/L时,与正常对照组相比,凋亡差异有统计学意义(分别为P=0.027和P=0.031)。qRT-PCR结果显示,ATRA能显著抑制RDH5mRNA的表达(P<0.001),促进MMP-2和TGF-β2mRNA的表达(P=0.03,P<0.001),呈剂量依赖性,特别是用5μmol/LATRA处理时。RDH5siRNA的敲低效率随靶标的不同而不同,其中RDH5siRNA-435的敲低效率最高,即,低于阴性对照组50%以上(P=0.02)。当RDH5被击倒48h时,qRT-PCR结果显示MMP-2和TGF-β2mRNA表达明显上调(P<0.001)。
结论:ATRA抑制RDH5的表达,促进MMP-2和TGF-β2的表达,进一步RDH5敲低显著上调MMP-2和TGF-β2。这些发现表明RDH5可能参与了ATRA介导的RPE细胞的上皮-间质转化。
OBJECTIVE: To investigate the effect of all-trans retinoic acid (ATRA) on retinol dehydrogenase 5 (RDH5), matrix metalloproteinase-2 (MMP-2) and transforming growth factor-β2 (TGF-β2) transcription levels, and the effect of RDH5 on MMP-2 and TGF-β2 in retinal pigment epithelium (RPE) cells.
METHODS: After adult RPE cell line-19 (ARPE-19 cells) intervened with gradient concentrations of ATRA (0-20 µmol/L) for 24h, flow cytometry was used to detect the proliferation and apoptosis of cells in each group, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect RDH5, MMP-2 and TGF-β2 mRNA expression. Then, after ARPE-19 cells transfected with three different siRNA targets for 48h, the RDH5 knockdown efficiency of each group and expression of MMP-2 and TGF-β2 mRNA within them was detected by qRT-PCR.
RESULTS: Flow cytometry results showed that ATRA could inhibit the proliferation of RPE cells and promote the apoptosis of RPE cells, and the difference of apoptosis was statistically significant when the ATRA concentration exceeded 5 µmol/L and compared with the normal control group (P=0.027 and P=0.031, respectively). qRT-PCR results showed that ATRA could significantly inhibit the expression level of RDH5 mRNA (P<0.001) and promote the expression of MMP-2 and TGF-β2 mRNA (P=0.03 and P<0.001, respectively) in a dose-dependent manner, especially when treated with 5 µmol/L ATRA. The knockdown efficiency of RDH5 siRNA varies with different targets, among which RDH5 siRNA-435 had the highest knockdown efficiency, i.e., more than 50% lower than that of the negative control group (P=0.02). When RDH5 was knocked down for 48h, the results of qRT-PCR showed that the expressions of MMP-2 and TGF-β2 mRNA were significantly up-regulated (P<0.001).
CONCLUSIONS: ATRA inhibits the expression of RDH5 and promotes MMP-2 and TGF-β2, and further RDH5 knockdown significantly upregulates MMP-2 and TGF-β2. These findings suggest that RDH5 may be involved in an epithelial-mesenchymal transition of RPE cells mediated by ATRA.