Prkaa2对肾小管上皮细胞纤维化的调控作用及Prkaa2和Rps6ka1间的关系研究

Regulatory effect of Prkaa2 on renal tubular epithelial fibrosis and relationship between Prakaa2 and Rps6ka1

目的探讨腺苷酸活化蛋白激酶亚基α2(protein kinase AMP-activated catalytic subunit alpha 2,Prkaa2)与核糖体蛋白S6激酶A1(ribosomal protein S6 kinase A1,Rps6ka1)的关系及Prkaa2对转化生长因子β1(transforming growth factor-β,TGF-β1)诱导的肾小管上皮细胞纤维化的影响。方法用重组蛋白TGF-β1诱导NRK-52e细胞制备纤维化的细胞模型。采用实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,qRT-PCR)、蛋白质印迹法检测大鼠肾小管上皮细胞系NRK-52e细胞(对照组)和10 ng/ml TGF-β1诱导12、24、36 h的NRK-52e细胞中Prkaa2的表达水平。于NRK-52e贴壁生长达30%时加入Prkaa2低表达慢病毒,转染72 h后收集细胞,并于Prkka2低表达慢病毒组及Prkaa2空病毒组中用qRT-PCR检测Prkaa2敲减效率。设立TGF-β1诱导、Prkaa2RNAi+TGF-β1诱导和Prkaa2空病毒+TGF-β1诱导3个实验组,同时以正常未作任何处理的NRK-52e细胞系为对照组。通过蛋白质印迹法检测Prkaa1、Prkaa2、α-平滑肌肌动蛋白(Alpha-smooth muscle actin,α-SMA)、E钙粘蛋白(E-cadherin)、波形蛋白(Vimentin)、Bcl-2相关X蛋白(Bax)、含半胱氨酸的天冬氨酸蛋白水解酶9(cysteinyl aspartate specific proteinase9,Caspase9)和p53基因(p53)的表达。从Prkaa2RNAi+TGF-β1诱导组和Prkaa2空病毒+TGF-β1诱导组中分别选取3个生物学重复样本,通过Affymetrix基因表达谱芯片测序结果筛选差异表达的基因。然后,在Prkaa2RNAi+TGF-β1、Prkaa2空病毒+TGF-β1诱导2个实验组中验证Prkaa2、Rps6ka1的表达。结果qRT-PCR及蛋白印迹检测结果显示,TGF-β1诱导12 h时NRK-52e细胞中Prkaa2的mRNA和蛋白的相对表达量分别为1.368±0.083和1.311±0.007,与对照组1.000±0.123和1.159±0.009相比,差异有统计学意义(P<0.05)。qRT-PCR实验结果显示Prkaa2空病毒组相对表达量为1.042±0.302,较Prkaa2低表达慢病毒组为0.171±0.165,Prkaa2 mRNA表达下调,差异具有统计学意义(P=0.023)。蛋白质印迹法检测结果显示,TGF-β1诱导组E-cadherin相对表达量为2.89±0.04,较对照组3.82±0.03下调,差异有统计学意义(P<0.05);TGF-β1诱导组Prkaa1相对表达量为1.04±0.04,较对照组1.06±0.05未见明显变化,差异无统计学意义(P>0.05);TGF-β1诱导组α-SMA、Vimentin和Bax、Caspase9、p53相对表达量分别为3.60±0.03、1.07±0.07、1.81±0.02、1.17±0.04、0.62±0.01,均较对照组2.95±0.04、0.74±0.03、1.00±0.04、0.77±0.05、0.37±0.01上调,差异均有统计学意义(P<0.05)。Prkaa2RNAi+TGF-β1诱导组E-cadherin相对表达量为3.57±0.03,较空病毒+TGF-β1诱导组3.04±0.15上调,差异有统计学意义(P<0.05);Prkaa2RNAi+TGF-β1诱导组Prkaa1相对表达量为1.03±0.05,较空病毒+TGF-β1诱导组1.05±0.06未见明显变化,差异无统计学意义(P>0.05);Prkaa2RNAi+TGF-β1诱导组Prkaa2、α-SMA、Vimentin和Bax、Caspase9、p53相对表达量分别为0.54±0.02、3.21±0.07、0.44±0.03、1.16±0.08、0.94±0.03、0.44±0.03,均较空病毒+TGF-β1诱导组0.89±0.01、3.49±0.06、0.98±0.07、1.87±0.02、1.21±0.04、0.65±0.03下调,差异均有统计学意义(P<0.05)。Affymetrix基因表达谱芯片测序结果显示核糖体蛋白S6激酶A1(ribosomal protein S6 kinase A1,Rps6ka1)下调。蛋白质印迹法和qRT-PCR验证结果提示,Prkaa2RNAi+TGF-β1诱导组Rps6ka1表达相对表达量为0.60±0.02、0.590±0.026,较Prkaa2空病毒+TGF-β1诱导组1.15±0.04、1.003±0.080下调,差异有统计学意义(P<0.05)。结论Prkaa2与Rps6ka1存在正向调控关系,抑制Prkaa2可以改善TGF-β1诱导的肾小管上皮细胞纤维化。

Objective To explore the relationship between protein kinase AMP-activated catalytic subunit alpha 2(Prkaa2)and ribosomal protein S6 kinase A1(Rps6ka1)and the effect of Prkaa2on TGF-β1 induced renal tubular epithelial fibrosis.Methods NRK-52e induced by recombinant TGF-β1 was utilized for establishing cell model of fibrosis.Quantitative real-time polymerase chain reaction(qRT PCR)and Western blot were used for detecting the expression of Prkaa2 after an induction of 10 ng/ml TGF-β1 for 12/24/36h respectively.Prkaa2 low-expression lentiviral vector was added when NRK-52e grew to 30%.Cells were collected after 72h transfection.Knockdown efficiency of Prkaa2 was detected by qRT PCR.Three experimental groups were established:TGF-β1 induction,Prkaa2RNAi+TGF-β1 and empty virus+TGF-β1.Normal NRK-52e group served as control.Western blot was employed for detecting the expressions of Prkaa1,Prkaa2,α-SMA,E-cadherin,vimentin,Bax,caspase-9 and p53.Low-expression lentivirus Prkaa2 was transfected under an induction of TGF-β1(empty virus in control group).The differentially expressed genes were sequenced and screened by Affymetrix gene expression profile chip.Then differential gene expression was verified in two experimental groups of Prkaa2RNAi+TGF-β1 and Prkaa2 empty virus+TGF-β1.Results The relative expression levels of Prkaa2 mRNA and protein were(1.368±0.083)and(1.311±0.007)in TGF-β1 group.Both were significantly higher than those in control group[(1.000±0.123)and(1.159±0.009)](P<0.05).Compared with transfected empty virus group(1.042±0.302)and transfected Prkaa2 low expression virus group(0.171±0.165),the expression of Prkaa2 mRNA was down-regulated(P=0.023).Western blot indicated that the relative expression of E-cadherin in induction group was 2.89±0.04 in TGF-β1 group,which was significantly lower than that in control group(3.82±0.03)(P<0.05);The relative expression of Prkaa1 in induction group was 1.04±0.04,which had no significant change compared with 1.06±0.05 in control group(P>0.05);The relative expressions ofα-SMA,vimentin,Bax,caspase 9 and p53 were 3.60±0.03,1.07±0.07,1.81±0.02,1.17±0.04 and 0.62±0.01 in TGF-β1 group,respectively,which were significantly higher than those in control group(2.95±0.04,0.74±0.03,1.00±0.04,0.77±0.05 and 0.37±0.01,P<0.05).The relative expression of E-cadherin in Prkaa2RNAi+TGF-β1 induction group was 3.57±0.03,compared with empty virus+TGF-β1 induction group up-regulated by 3.04±0.15,and the difference was statistically significant(P<0.05);The relative expression of Prkaa1 in Prkaa2RNAi+TGF-β1 induction group was 1.03±0.05,compared with empty virus+TGF-β1 there was no significant change in induction group(1.05±0.06)(P>0.05);Compared with induction group(0.89±0.01,3.49±0.06,0.98±0.07,1.87±0.02,1.21±0.04 and 0.65±0.03),the relative expressions of Prkaa2,α-SMA,vimentin,Bax,caspase 9 and p53 in Prkaa2RNAi+TGF-β1 induction group were(0.54±0.02,3.21±0.07,0.44±0.03,1.16±0.08,0.94±0.03 and 0.44±0.03).There was a down-regulation and the difference was statistically significant(P<0.05).Affymetrix gene expression profile chip sequencing hinted that ribosomal protein S6 kinase A1(Rps6ka1)was down-regulated.Western blot and qRT-PCR showed that the relative expression of Rps6ka1 in induction group was(0.60±0.02 and 0.590±0.026)in Prkaa2RNAi+TGF-β1 group.It was higher than that of Prkaa2 empty virus+TGF-β1 in induction group(1.15±0.04 and 1.003±0.080).There was a down-regulation and the difference was statistically significant(P<0.05).Conclusion A positive regulatory relationship exists between Prkaa2 and Rps6ka1.And an inhibition of Prkaa2 improves TGF-β1-induced renal tubular epithelial fibrosis.