The Effect Of Deficiency Of Parathyroid Hormone Related Peptide Nuclear Localization Sequence And C-terminus On The Oxidative Stress Of Mice Spinal Cord

To explore the effect of deficiency of parathyroid hormone related peptide nuclear localization sequence and C-terminus on the oxidative stress of mice spinal cord,PTHrP KI mice(gifted from Dr.Karaplis)was used in this study.This animal model was generated by introducing an amino acid translation termination codon TGA into 84 amino of PTHrP gene and expressed only PTHrP1-84,and lacking the NLS and C-terminus.We use the techniques of immunohistochemistry,molecular biology,and RNA chips detection system to analyse the oxidative stress and DNA damage of spinal cord from PTHrP KI and their wild type(WT)littermates at postnatal day 3,7,and 14(P3,P7,and P14).Firstly,spinal cords were collected from P7 PTHrP KI and their WT littermates.Flow cytometry was used to detect the ROS level,and biochemical reagents detecting kits were selected to measure the SOD,CAT,and GSH-PX levels in spinal cord samples.Immunohistochemical staining was used to detect the expression of 8-OH-dG from P3,P7 and P14 PTHrP KI and their WT littermates.Western blot was used to measure the expression levels of p-chk2 and y-H2AX in spinal cord from P7 and P14 PTHrP KI and their WT littermates.Our results revealed that expression of ROS was increased,and SOD,CAT,and GSH-PX were decreased in spinal cord from PTHrP KI mice compared with their WT littermates.Expression of 8-OH-dG,p-chk2,and γ-H2AX were significantly increased in spinal cord from PTHrP KI mice.These results indicate that deficiency of PTHrP NLS and C-terminus can increase the ROS expression,and decrease the antioxidases’ expression,and active the DNA damage,thus resulting the delay of spinal cord development.To clarify whether the deficiency of PTHrP NLS and C-terminus lead to the delay of spinal cord development,and to explore the involving key regulators,we design the following experiments.Firstly,we prepared two group mice as PTHrP KI heterozygote(PTHrP KI+/-)pregnant mice fed with pyrroloquinoline quinone(PQQ,4mg/kg)added diet,and PTHrP KI+/-pregnant mice fed with normal diet.P7 PTHrP KI and their WT littermate controls were divided randomly into four groups,such as①group 1:P7 PTHrP KI mice fed with PQQ;②group 2:P7 PTHrP KI mice fed with normal diet;③group3:P7 WT mice fed with PQQ;④group 4:P7 WT mice fed with normal diet.4 cervical cords were collected from each above group and detected the expression of mRNA and lncRNA with CapitalBio Technology Mouse LncRNA Array v1.0 chips by Nanjing Subo Biotechnology Company.The data was collected and normalized by Feature Extraction,and GeneSpring software.Differential expressed genes were screened and analyzed for their protein-protein interaction network.Then,hub-genes were visually screened based on cytohubba,and KEGG pathway enrichment analysis was proceded based on GSEA.Finally,the mRNA expression profiles of total 12 spinal cord samples from group 1,2,and 4 were analyzed by Principal Component Analysis(PCA)to explore whether the delay of spinal cord development induced by deficiency of PTHrP NLS and C terminus can be rescued by PQQ.Based on the DEG screened from mRNA of PTHrP KI and WT mice,54 mRNA were up-regulated and 51 mRNA were down-regulated in PTHrP KI mice compared with their WT controls.Cellular proliferation related pathways such as cell cycle,DNA replication,nucleotide excision repair,and mismatch repair pathways were negative correlated with PTHrP KI phenotype related pathway,and 10 hub-genes from PTHrP KI mice as Ccna2,Ccnb1,Cdk1,Kif11,Pbk,Bub1,Prc1,Mki67,Rrm2,Shcbpl were all related to the cellular proliferation.Nine key lncRNAs related to the above top 3 genes as Ccna2,Ccnb1,Cdk1 were obtained based on lncRNA-mRNA co-expression net.Two of these lncRNAs were up-regulated,and other seven of them were down-regulated in spinal cord from PTHrP KI mice.At the same time,only one lncRNA as NONMMUT006126 was increased in PTHrP KI mcie and decreased after PQQ feeding,likewise,only one lncRNA as NONMMUT064530 was decreased in PTHrP KI mcie and increased after PQQ feeding.Considered of the backgroup expression of NONMMUT064530 was much higher than NONMMUT006126 in spinal cord,and NONMMUT064530 was highly expressed in brain tissue as hippocampus,we initially selected NONMMUT064530 as a target lncRNA,which will be verified by Q-PCR in the coming future.The results of PCA analysis revealed that PQQ could rescue the effect of deficiency of PTHrP NLS and C terminus on the spinal cord development.On a conclusion,our results reveal that deficiency of PTHrP NLS and C-terminus can increase the ROS expression,and decrease the antioxidases’ expression,and active the DNA damage,thus resulting the delay of spinal cord development,and RNA chip analysis data suggests that which may mediated by NONMMUT064530,and PQQ could at least partially rescue the effect of deficiency of PTHrP NLS and C terminus on the spinal cord development.