The Effect Of Overexpression Of Retinoic Acid Receptor ? On Epithelial Mesenchymal Transition In Renal Tubular Epithelial Cells Induced By Hypoxia

Objective To explore the effect of overexpression of retinoic acid receptor ?(RAR?)on epithelial to mesenchymal transition(EMT)in renal tubular epithelial cells induced by hypoxia.Methods The lentiviral vectors overexpressing RAR? and negative control lentiviral vectors were constructed and generated.Then the lentiviral vectors over-expressing RAR? or negative control lentiviral vectors were used to infect the NRK-52 E cells cultured in vitro respectively.At 72 h post-transfection,puromycin(2.0 mg/L)was added to the culture medium for screening to establish the stable clone cell line.The transfection efficiency of the NRK-52 E cells was evaluated using fluorescent microscopy and flow cytometry.The NRK-52 E cells were divided into four groups: normal group,hypoxia model group,transfection group,negative control group.Then the cells were subjected to hypoxia for 48 h,and the normal group without any treatment.The change of cellular morphology was observed by light microscope;The mRNA and protein expressions of RAR? ? E-cadherin ? ?-smooth muscle actin(?-SMA)and vimentin in NRK-52 E cells were detected by real time fluorescent quantitative reverse transcription polymerase chainreaction(RT-qPCR)and Western blotting(WB).Results 1.The NRK-52 E cells in transfection group and negative control group were observed to be green fluorescence positively and the cells were in good condition.And the transfection efficiency of the NRK-52 E cells was no less than 97% measured by flow cytometry.Moreover,both of the mRNA and protein expressions of RAR? in the transfected NRK-52 E cells were markedly upregulated,compared with normal group and negative control group.2.The NRK-52 E cells were exposed to hypoxia for 48 h,then the light microscope revealed that both hypoxia model group and negative control group cells became atrophic and elongated,which consistent with the morphology of myofibroblasts.But the cells in transfection gr oup were cubic,formed epithelial monolayers.3.Compared with normal group,the mRNA and protein expressions of RAR? ?E-cadherin in hypoxia model group were dramatically reduced(all P<0.05),but the mRNA and protein expressions of ?-SMA?vimentin were higher(all P<0.05).4.Compared with hypoxia model group and negative control group,the mRNA and protein expressions of RAR? and E-cadherin in transfection group were significantly increased(all P<0.05).Conversely,the mRNA and protein expressions of ?-SMA?vimentin were obviously lower in transfection group(all P<0.05).The differences in the mRNA and protein expressions of RAR??E-cadherin??-SMA and vimentin between hypoxia group and negative control group were no significant(all P>0.05).Conclusion Lower expression of RAR? might involve in EMT in renal tubular epithelial cells induced by hypoxia.Overexpression of RAR? alleviated EMT in renal tubular epithelial cells induced by hypoxia.