瞬时受体电位类香草醛5和骨桥蛋白在泌尿系统结石患者中的表达及其与微小RNA-22-3p的关系

Expression of transient receptor potential vanilla 5 and osteopontin in patients with urinary calculi and its relationship with microRNA-22-3p

目的检测瞬时受体电位类香草醛5(TRPV5)和骨桥蛋白(OPN)在泌尿系统结石患者中的表达及其与微小核糖核酸-22-3p(miR-22-3p)的关系。方法荧光定量反转录-聚合酶链式反应(RT-qPCR)检测健康体检者、非泌尿系统结石患者和泌尿系统结石血清TRPV5、OPN和miR-22-3p表达并分析其相关性。将模型+miR-con组(转染miR-con)、模型+miR-22-3p组(转染miR-22-3p)转染至HK-2细胞并草酸钙结石晶体溶液(100 mg/L)处理。流式细胞仪检测细胞凋亡;蛋白质印迹法(Western blot)检测抗裂解的半胱氨酸天冬氨酸蛋白酶-3(cleaved Caspase-3)、B淋巴细胞瘤-2(bcl-2)和bcl-2相关X蛋白(bax)表达。两组间比较采用t检验,多组间比较采用单因素方差分析联合SNK-q检验。采用Pearson相关性分析法分析相关性。结果与健康体检者或非泌尿系统结石患者比较,泌尿系统结石患者血清TRPV5和miR-22-3p表达显著降低(TRPV5分别为0.66±0.15、1.06±0.26、0.77±0.21,miR-22-3p分别为0.62±0.18、1.00±0.23、0.83±0.19),OPN表达显著升高(2.78±0.49、1.07±0.21、1.60±0.20),差异有统计学意义(F=43.908、300.788、41.410,P<0.05)。泌尿系统结石患者血清中miR-22-3p的表达量与TRPV5的表达呈正相关(r=0.52,P<0.05),与OPN的表达呈负相关(r=-0.69,P<0.05)。与对照组比较,模型组HK-2细胞中TRPV5和miR-22-3p表达显著降低(TRPV5为0.43±0.07比0.98±0.10,miR-22-3p为0.72±0.16比1.01±0.13),OPN表达显著升高(3.24±0.36比1.02±0.12),差异有统计学意义(t=13.517、75.794和17.551,P<0.05),细胞凋亡率[(17.37±2.09)%比(3.78±0.49)%]、cleaved Caspase-3(0.42±0.06比0.19±0.05)和bax(0.68±0.07比0.28±0.06)蛋白质表达均显著升高,bcl-2蛋白表达显著降低(0.14±0.03比0.31±0.05),差异有统计学意义(t=21.431、17.233、13.655、31.782,P<0.05)。与模型+miR-con组比较,模型+miR-22-3p组细胞上述检测指标均发生显著的负向调控。结论TRPV5和OPN在泌尿系统结石患者中的表达与miR-22-3p存在明显的相关性。

Objective To detect the expression of transient receptor potential vanilla 5(TRPV5)and osteopontin(OPN)in patients with urinary calculi and its relationship with microRNA-22-3p(miR-22-3p).Methods Quantitatie reverse transcriptional polymerase chain reaction(RT-qPCR)was used to detect the expression of TRPV5,OPN and miR-22-3p in patients with urinary calculi,and the correlation was analyzed with pearson correlation analysis.In HK-2 cells,the model+miR-con group(transfected with miR-con),and the model+miR-22-3p group(transfected with miR-22-3p)were treated with a calcium oxalate stone crystal solution(100 g/ml).Apoptosis was detected by flow cytometry.The expression of cleaved Caspase-3,B-cell lymphoma-2(bcl-2)and bcl-2-associated X(bax)was detected by Western blotting.A t-test was used for two-group comparisons and a one-way ANOVA combined SNK-q test for multiple groups.Results Compared with healthy persons or patients without urinary calculi,the expression of TRPV5 and miR-22-3p significantly decreased(TRPV5:0.66±0.15 vs.1.06±0.26 or 0.77±0.21;miR-22-3p:0.62±0.18 vs.1.00±0.23 or 0.83±0.19,respectively);the expression of OPN significantly increased(2.78±0.49 vs.1.07±0.21 or 1.60±0.20,respectively)(F=43.908,300.788,41.410,P<0.05)in patients with urinary calculi.The expression of serum miR-22-3p in patients with urinary calculi was positively correlated with the expression of TRPV5(correlation coefficient r=0.52,P<0.05),and negatively correlated with the expression of OPN(r=-0.69,P<0.05).Compared with the control group,the expression of TRPV5 and miR-22-3p significantly decreased(TRPV5:0.43±0.07 vs.0.98±0.10;miR-22-3p:0.72±0.16 vs.1.01±0.13),OPN significantly increased(3.24±0.36 vs.1.02±0.12)with the differences being statistically significant(t=13.517,75.794,17.551,respectively,P<0.05),the apoptosis rate[(17.37±2.09)%vs.(3.78±0.49)%],cleaved caspase-3(0.42±0.06 vs.0.19±0.05)and bax(0.68±0.07 vs.0.28±0.06)significantly increased in the model group(t=21.431,17.233,13.655,31.782,P<0.05).Compared with model+miR-con group,the above indexes were significantly negatively regulated in model+miR-22-3p group.Conclusion The expression of TRPV5 and OPN in patients with urinary calculi were significantly correlated with miR-22-3p.