沉默WNT4基因对稳定转染PAX2基因大鼠肾小管上皮细胞迁移和侵袭能力的影响

Effects of WNT4 gene silencing on migration and invasion of renal tubular epithelial cells of rats stably transfected with PAX2 gene

目的:探讨沉默WNT4基因对稳定转染配对盒基因2 (PAX2)大鼠肾小管上皮细胞迁移和侵袭能力的影响,阐明PAX2基因是否通过WNT4基因调控细胞生物学活性。方法:选取对数生长期的PAX2基因稳转细胞系,分为稳转组和对照组。稳转组细胞分为稳转对照组(不应用WNT siRNA沉默)和沉默72h组(应用WNT siRNA沉默72h)。Western blotting法检测稳转组和对照组大鼠肾小管上皮细胞中PAX2和WNT4蛋白的相对表达量,Real time PCR法检测稳转对照组和沉默72h组大鼠肾小管上皮细胞中WNT4mRNA相对表达量,细胞划痕实验检测稳转对照组和沉默72h组大鼠肾小管上皮细胞的细胞迁移率,Transwell实验检测稳转对照组和沉默72h组大鼠肾小管上皮细胞的穿膜细胞数。结果:Western blotting法检测,稳转组大鼠肾小管上皮细胞中PAX2和WNT4蛋白相对表达量高于对照组(P<0.05)。Real-time PCR法检测,沉默72h组大鼠肾小管上皮细胞中WNT4mRNA相对表达量低于稳转对照组(P<0.05)。细胞划痕实验10h后,沉默72h组大鼠肾小管上皮细胞的细胞迁移率低于稳转对照组,但组间比较差异无统计学意义(P>0.05);细胞划痕实验18h后,沉默72h组大鼠肾小管上皮细胞的细胞迁移率低于稳转对照组(P>0.05)。Transwwell实验,沉默72h组大鼠肾小管上皮细胞的穿膜细胞数低于稳转对照组(P<0.05)。结论:PAX2基因可能通过WNT4基因调控细胞的生物学活性。

Objective:To investigate the effects of WNT4 gene silecing on the migration and invasion of renal tubular epithelial cells of the rats stably transfected with paired box gene 2(PAX2)gene,and to clarify whether the PAX2 gene regulates the biological activities of renal tubular epithelial cells through WNT4 gene or not.Methods:The renal tubular epithelial cells transfected with PAX2 gene in the logarithmic phase were divided into stably transfected group and control group.The cells in stably transfected group were divided into stably transfected control group(without WNT4 siRNA silencing)and silencing 72 h group(with WNT4 siRNA silencing for 72 h).The relative expression amounts of PAX2 and WNT4 proteins in renal tubular epithelial cells of the rats in stably transfected group and control group were detected by Western blotting method.The relative expression amounts of WNT4 mRNA in renal tubular epithelial cells of the rats in stably transfected control group and silencing 72 h group were detected by Real time PCR method.The migration rates of renal tubular epithelial cells of the rats in stably transfected control group and silencing 72 h group were detected by cell wound scratch assay.The number of transmembrane renal tubular epithelial cells of the rats in stably transfected control group and silencing 72 h group was detected by Transwell experiment.Results:The Western blotting results showed that the relative expression amounts of PAX2 and WNT4 proteins in renal tubular epithelial cells of the rats in stably transfected group were higher than those in control group(P<0.05).The Real time PCR results showed that the relative expression amount of WNT4 mRNA in renal tubular epithelial cells of the rats in silencing 72 h group was lower than that in stably transfected control group(P<0.05).The cell wound scratch assay results showed that the migration rate of renal tubular epithelial cells in silencing 72 h group after 10 h was lower than that in stably transfected control group,but there was no significant difference(P>0.05);the migration rate of renal tubular epithelial cells in silencing 72 h group after 18 h was lower than that in stably transfected control group(P<0.05).The Transwell experiment results showed that the number of transmembrane renal tubular epithelial cells in silencing 72 h group was lower than that in stably transfected control group(P<0.05).Conclusion:PAX2 gene can regulate the biological activities of renal tubular epithelial cells through WNT4 gene.