SENP1 Has An Important Role In Lung Development And Influences The Differentiation Of AEC?

ObjectiveIn order to investigate the function of SENP1,the expressive levels of SENP1 and SUMO1 are assessed during lung development process of rats.At the same time,AEC? cells are transfected with SENP1?targeting small interfering RNA.And the proliferation,apoptosis and differentiated function of AEC? cells was subsequently evaluated.MethodsAccording to the histological stage of rats' lung development,neonatal rats were sacrificed at 1 d,4 d,7 d,14 d.HE staining was used to observe the lung morphologic appearance.RT-qPCR was used to detect the mRNA expressions of SUMO1 and SENP1 at each time?point.Western bolt was used to detect the protein expression.In this work,AEC? were cultured in vitro and Retinoic acid(RA)was used to promote differentiation.The differentiated efficiency of RA was examined by immunofluorescence and Western bolt.To investigate the suppressive impact of SENP1 on differentiation,cells were divided into two groups,one was transfected with a SENP1 siRNA as the experimental group and the other was transfected with NS siRNA with RA added as the control group.The suppressive influence of SENP1 with RA added was detected by RT?qPCR and western blot analysis.Protein and gene expressive levels of SP?C and AQP5 were measured to analyze the effects on differentiation.The protein expressive levels of the conjugated SUMO1 were also detected.At the same time,with SENP1 suppressed,CCK?8 was used to analyze cell proliferation and flow cytometry was used to analyze cell apoptosis.Results(1)The results of the RT-qPCR experiments indicated that SUMO1 mRNA was not significantly changed during lung development from P1 to P4(P>0.05).Free SUMO1 was increased at day 4 compared with day 1,was visibly decreased at day 7compared with day 4,and was expressed at a similar level until day 14(P<0.05).At western blot analysis,free SUMO1 and bands of higher molecular masscorresponding to SUMOylated proteins were detected.This test results indicate that naturally existing free SUMO1 at day 14 exhibited a sharp decrease compared with day 4(P<0.05),while no significant change was observed in total IV SUMO1.The results demonstrated an increase in SENP1 mRNA at P4 compared with P1(P<0.05),which was decreased at P7 and was maintained a steady level from P7 to P14.The protein levels of SENP1 exhibited a similar trend with gene expression during lung development.(2)The expressions of SP?C and AQP5 were first measured by immunofluorescence.These results demonstrated that the decrease in SP?C was accompanied by an increase in AQP5.The expression levels of SP?C,AQP5 and SENP1 were then detected by using western blot analysis.The expression of SP?C was decreased at 24 h and 72 h following treatment with RA.However,it was increased at 48 h,which may have been caused by cell proliferation.RA enhanced the expressions of AQP5 at 24 h,48 h and 72 h compared with untreated cells.The expression of SENP1 in the RA group was increased compared with the control group at 24 h,48 h and 72 h(P<0.05).(3)Compared to the control group,the inhibition of SENP1 decreased the protein level of AQP5 expression at 24,48 and 72 h.The protein expression of SP?C was increased at 24 and 48 h,but decreased at 72 h(P<0.05).The decrease of SP?C may have been caused by growth inhibition.The conjugated SUMO1 was increased by si?SENP1 and free SUMO1 was decreased(P<0.05).(4)Cell proliferation was reduced in the si?SENP1 groups as compared with si?NS(P<0.05).And the percentage of cells apoptosis in si?SENP1 groups were higher compared with the si?NS group(P<0.05).Conclusion(1)SENP1 shows a dynamic expression pattern in lung development,which may indicates it participates in the progress by influencing the post?translational modifications SUMOlation.(2)The experiments demonstrated that RA promoted the differentiation of AEC?.Taken together,these results suggest that SENP1 may participate in thedifferentiation of AEC?.To further prove this result,we find that inhibition of SENP1 expression interferes with deSUMOlation and hinders the differentiation of AEC?.SENP1 take part in the differentiation of AEC? by impairs the expression of conjugated SUMO1.(3)SENP1 is a key factor involved in normal lung development.It affects the proliferation and differentiation status of AEC? cells.