水通道蛋白3通过调控hnRNPQ/p53延缓皮肤成纤维细胞光老化的作用及机制研究

Role and action mechanism of aquaporin 3 in alleviating photoaging of skin fibroblasts by regulating hnRNPQ/p53

目的探讨水通道蛋白3(AQP3)在皮肤光老化过程中的作用及机制。方法将正常人皮肤成纤维细胞(NHDF)分为NHDF组(未转染)、AQP3 cDNA组(转染AQP3 cDNA)、AQP3 siRNA组(转染AQP3 siRNA)、核内不均一核糖核蛋白Q(hnRNPQ)cDNA组(转染hnRNPQ cDNA)、hnRNPQ siRNA组(转染hnRNPQ siRNA)、AQP3-hnRNPQ cDNA组(转染AQP3 cDNA和hnRNPQ cDNA)、AQP3-hnRNPQ siRNA组(转染AQP3和hnRNPQ siRNA)、cDNA空载体组(转染cDNA空载体)、siRNA空载体组(转染siRNA空载体)。用长波紫外线(UVA,10 J·cm-^(2)·d^(-1))连续照射已经转染或未转染的NHDF 3 d来建立细胞衰老模型,同时以未接受UVA照射的NHDF作为对照。用细胞计数法评估各实验组细胞增殖活性,衰老相关-β半乳糖苷酶染色试剂盒对各实验组进行染色来评估HDF的衰老水平,用荧光素酶报告基因技术检测p53转录调控活性,用Western印迹分析NHDF中AQP3、hnRNPQ及衰老相关蛋白p53、p21的表达水平。两组间比较采用独立样本t检验,多组间比较采用ANOVA检验。结果用UVA连续照射各组NHDF 3 d后,NHDF中p53、p21的表达水平和β半乳糖苷酶阳性细胞百分率明显高于未照射的对照组(P<0.05),但是AQP3的表达水平和照射后第5、6、7天细胞增殖活性明显低于对照组(P<0.05)。在接受UVA连续照射3 d后,AQP3 siRNA组中UVA诱导的NHDF的衰老相关表型较siRNA空载体组明显加重,两组间p53、p21、hnRNPQ的表达水平和β半乳糖苷酶阳性细胞百分率、p53转录调控活性、细胞增殖活性差异均有统计学意义(均P<0.05),进一步沉默hnRNPQ能够逆转上述反应。与siRNA空载体组相比,hnRNPQ siRNA组中UVA诱导的NHDF的衰老相关表型明显减轻,两组间p53、p21表达水平、β半乳糖苷酶阳性细胞百分率、p53转录调控活性和细胞增殖活性差异均有统计学意义(均P<0.05)。在接受UVA连续照射3 d后,cDNA空载体组中p53、p21、hnRNPQ的表达水平、β半乳糖苷酶阳性细胞百分率、p53转录调控活性和细胞增殖活性分别为0.56±0.08、0.70±0.07、0.92±0.03、(81.53±7.62)%、7.16±0.25、(2.15±0.23)×10^(6)/ml,而AQP3 cDNA组中UVA诱导的NHDF的衰老相关表型明显轻于cDNA空载体组,其上述指标分别为0.25±0.06、0.23±0.06、0.82±0.09、(31.23±6.54)%、2.52±0.36、(2.93±0.33)×10^(6)/ml,两组比较,差异均有统计学意义(均P<0.05),进一步过表达hnRNPQ能够逆转上述效应。在接受UVA连续照射3 d后,hnRNPQ cDNA组中p53、p21的表达水平、β半乳糖苷酶阳性细胞百分率、p53转录调控活性和细胞增殖活性分别为1.41±0.09、1.42±0.06、(91.06±4.24)%、12.35±0.88、(1.23±0.41)×10^(6)/ml,与cDNA空载体组相比,hnRNPQ cDNA组中UVA诱导的NHDF的衰老相关表型明显加重,两组比较,上述指标差异均有统计学意义(均P<0.05)。结论AQP3可能通过调控hnRNPQ下调p53的表达来减缓UVA诱导的NHDF衰老。

Objective To investigate the role and action mechanism of aquaporin 3(AQP3)in skin photoaging.Methods Normal human skin fibroblasts(NHDF)were divided into several groups:NHDF group receiving normal culture without transfection,AQP3 cDNA group transfected with AQP3 cDNA,AQP3 siRNA group transfected with AQP3 siRNA,heterogeneous nuclear ribonucleoprotein Q(hnRNPQ)cDNA group transfected with hnRNPQ cDNA,hnRNPQ siRNA group transfected with hnRNPQ siRNA,AQP3-hnRNPQ cDNA group transfected with AQP3 and hnRNPQ cDNAs,AQP3-hnRNPQ siRNA group transfected with AQP3 and hRNPQ siRNAs,cDNA empty vector group transfected with a cDNA empty vector,and siRNA empty vector group transfected with a siRNA empty vector.Transfected or untransfected NHDFs were irradiated with ultraviolet A(UVA)at a dose of 10 J·cm-^(2)·d^(-1)for 3 consecutive days to establish a model of cellular senescence,and NHDF receiving no UVA irradiation served as a control.A cell counting method was used to evaluate the cellular proliferative activity,a senescence-relatedβ-galactosidase staining kit to evaluate the senescence level of NHDFs in each experimental group,and luciferase reporter gene technology to assess the transcriptional regulation activity of p53.Western blot analysis was performed to determine the expression of AQP3,hnRNPQ and senescence-related proteins p53 and p21 in NHDFs.Two-independent-sample t test was used for comparisons between two groups,and one-way analysis of variance for comparisons among multiple groups.Results After 3-day consecutive irradiation with UVA,the expression of p53 and p21 in NHDFs and the percentage ofβ-galactosidase-positive cells significantly increased compared with the unirradiated control group(all P<0.05),but the expression of AQP3 and cellular proliferative activity on days 5,6 and 7 significantly decreased in the UVA group compared with the unirradiated control group(all P<0.05).After 3-day consecutive irradiation with UVA,aggravated senescence-related phenotypes of UVA-induced NHDFs were observed in the AQP3 siRNA group compared with the siRNA empty vector group,and there were significant differences in the expression of p53,p21 and hnRNPQ,percentage ofβ-galactosidase-positive cells,p53 transcriptional regulation activity and cellular proliferative activity between the 2 groups(all P<0.05).Further silencing of the hnRNPQ gene could reverse the above effects.Compared with the siRNA empty vector group,the senescence-related phenotypes of UVA-induced NHDFs were attenuated in the hnRNPQ siRNA group,and significant differences were observed between the 2 groups in terms of the expression of p53,p21 and hnRNPQ,percentage ofβ-galactosidase-positive cells,p53 transcriptional regulation activity and cellular proliferative activity(all P<0.05).After 3-day consecutive irradiation with UVA,the senescence-related phenotypes of UVA-induced NHDFs were significantly attenuated in the AQP3 cDNA group compared with the cDNA empty vector group(all P<0.05),manifesting as significantly decreased expression of p53(0.25±0.06 vs.0.56±0.08),p21(0.23±0.06 vs.0.70±0.07)and hnRNPQ(0.82±0.09 vs.0.92±0.03),percentage ofβ-galactosidase-positive cells(31.23%±6.54%vs.81.53%±7.62%)and p53 transcriptional regulation activity(2.52±0.36 vs.7.16±0.25),but increased cellular proliferative activity([2.93±0.33]×10^(6)/ml vs.[2.15±0.23]×10^(6)/ml),and further overexpression of hnRNPQ could reverse the above effects.After 3-day consecutive irradiation with UVA,the expression of p53,p21,percentage ofβ-galactosidase-positive cells,p53 transcriptional regulation activity and cellular proliferative activity in the hnRNPQ cDNA group were 1.41±0.09,1.42±0.06,91.06%±4.24%,12.35±0.88 and(1.23±0.41)×10^(6)/ml respectively,and the senescence-related phenotypes of UVA-induced NHDFs were significantly aggravated in the hnRNPQ cDNA group compared with the cDNA empty vector group(all P<0.05).Conclusion AQP3 may alleviate the UVA-induced senescence of NHDFs by regulating hnRNPQ and downregulating p53 expression.