Effects And Its Mechanism Of Targeting Regulation Of Pim-1 In Repairing Injuried Neuron-like Cells

Purpose:Insufficient regenerative capacity of central nervous system neurons is the main reason leading to the failure of damaged neuron regeneration and functional recovery.Target genes that are coordinately regulated between multiple intracellular signal pathways after neuronal injury can significantly promote neuron survival,enhance the intrinsic regeneration capacity of neurons,and enable long-distance axon regeneration,neural circuit reconstruction and functional recovery of damaged neurons become possible.Therefore,pinpointing the target gene to improve the intrinsic regeneration capacity of damaged central neurons is the key to treating central nervous injury.The provirus integration site for Moloney murine leukemia virus(Pim-1)is a proto-oncogene that encodes a serine/threonine protein kinase.Pim-1 is a common downstream effector molecule of many intracellular cytokines and growth factor signaling pathways.It plays an important role in cell proliferation,survival and apoptosis by phosphorylating downstream protein serine/threonine sites.Based on the fact that Pim-1 is a common downstream effector of multiple signaling pathways,and has an important role in cell proliferation and survival,this study first observed the expression changes of Pim-1 in injured central neuron-like cells in vitro,the effects of ciliary neurotrophic factors(CNTF)and Neuritin(Nrn1)regulating downstream signaling pathways on Pim-1 expression,as well as the such effects on injured neurons and related mechanisms.Using Pim-1 gene as a target,Pim-1 was overexpresed with a recombinant lentivirus vector,or specifically interfered with Pim-1 silencer,and exploring the direct regulation of Pim-1 expression on damaged neurons activity and neurite regeneration,providing a new method for clinical treatment of central neuron injury and its experimental and theoretical mechanism.This study is also based on the research about long non-coding RNA(Lnc RNA)regulating neural development and injury and the close relationship between Lnc-Pim1 and Pim-1 gene loci and sequences.The sequence length,expression localization,expression changes after injury of Lnc-Pim1 in vitro neurons was observed,as well as its interaction with Pim-1.Using the Lnc-Pim1 gene as the target,Lnc-Pim1 was overexpressed with a recombinant lentiviral vector,or specifically interfered with Lnc-Pim1 siliencer,and exploring effect of regulating Lnc-Pim1 expression to repair damaged neuron-like cells in vitro.At the same time,CHIRP-MS and RNA-seq techniques were used to explore the preliminary molecular mechanism of Lnc-Pim1's function.Methods:(1)Neuro-2a cells were induced into neuron-like cells(N-2a-N)with 20 ?mol/ml trans retinoic acid(RA)in vitro,and Neuro-2a cells proliferation were inhibited with 50 ?mol/L deferoxamine sulfinate(DFO).N-2a-N cell neurite was injured with a concentration of 1 mmol/L acrylamide(ACR),and N-2a-N cells were divided into normal control group,PBS group,300 ?g/L CNTF group,and 500 ?g/L Nrn1 group.The length of cell neurite was measured with Image J 6.0,and cell viability was measured by CCK-8 method.Immunofluorescence cytochemistry was used to detect the phenotype of N-2a-N cells and Pim-1 protein expression.Real-time PCR and Western blotting were used to detect the changes of Pim-1 expression in each group.Western blotting were used to detect related signal transduction molecules(Stat-3,p-Stat-3,Erk1/2,p-Erk1/2)that regulate Pim-1 activity,cell survival and apoptosis related molecules(Caspase-3,Cleaved Caspase-3,Bax,Bcl-2)and axon regeneration related molecules GAP-43 expression changes.(2)The full-length of Lnc-Pim1 sequence in N-2a-N cells was obtained by the RACE method.Prediction of Lnc-Pim1 encoding capacity was performed using Phylo CSF,CPC and CNCI software.The subcellular localization of Lnc-Pim1 in Neuro-2a cells,normal N-2a-N cells,and damaged N-2a-N cells was detected by FISH and nuclear/cytoplasm separation techniques.The expression of Lnc-Pim1 in damaged N-2a-N cells at different time points was detected by Real-time PCR.(3)We constructed Pim-1 and Lnc-Pim1 recombinant lentiviral vectors,and used Real-time PCR to detect virus titers.(4)We designed Pim-1 and Lnc-Pim1 specific interference reagents Pim-1 silencer and Lnc-Pim1 silencer,and using Real-time PCR to detect effects of different concentrations of interference reagents on Pim-1 and Lnc-Pim1 expression in N-2a-N cells.(5)Neuro-2a cells were infected with a Pim-1 recombinant lentiviral vector carrying a blasticidin resistance at an MOI value of 40,and to purify recombinant lentivirus-infected Neuro-2a cells with 80 ?g/ml blasticidin.The N-2a-N cells overexpressed by Pim-1 were damaged with 1mmol/L ACR.The experiments were divided into Ctrl group,ACR group,LV-Blank/ACR group and LV-Pim-1/ACR group,and the detections were performed 24 hours after injury caused by ACR;N-2a-N cells were transfected with 200 nmol/ml negative control(NC)silencer or Pim-1 silencer.The experiment was divided into NC silencer group and Pim-1 silencer group,and the detections were performed 48 hours after transfection.The N-2a-N cells of each group were photographed under an inverted phase contrast microscope,and the length of the cell neurite was measured and counted with Image J 6.0.Cell activity of each group was detected by CCK-8,Pim-1 gene and protein expression of each group was detected by Real-time PCR and Western blotting,and GAP-43 protein expression of each group was detected by Western blotting.(6)Neuro-2a cells were infected with the Lnc-Pim1 recombinant lentivirus vector carrying a puromycin resistance at an MOI value of 40,and 20 ?g/ml of puromycin was used to purify recombinant lentivirus-infected Neuro-2a cells.The N-2a-N cells overexpressed by Lnc-Pim1 were damaged with 1mmol/L ACR.The experiment was divided into Ctrl group,ACR group,LV-Blank/ACR group and LV-Lnc-Pim1/ACR group,and the detections were performed 24 hours after injury;N-2a-N cells were transfected with 100 nmol/ml negative control(NC)silencer or Lnc-Pim1 silencer.The experiment was divided into NC silencer group and Lnc-Pim1 silencer group,and the detections were performed 48 hours after transfection.The cell viability of each group was detected by CCK-8 method.N-2a-N cells of each group were photographed under an inverted phase-contrast microscope.Image J was used to measure and count the cell neurite length.The expression of Lnc-Pim1 was detected by Real-time PCR,and the expressions of Erk1/2 and p-Erk1/2 were detected by Western blotting.(7)Pim-1 and Lnc-Pim1 were overexpressed with recombinant lentiviral vectors in N-2a-N cells,respectively.Pim-1 and Lnc-Pim1 in N-2a-N cells were knocked down with 200 nmol/ml Pim-1 silencer and 100 nmol/ml Lnc-Pim1 silencer.Real-time PCR was used to detect the effects of overexpression or knockdown of Pim-1 and Lnc-Pim1 on each other's gene expression.(8)Lnc-Pim1 interacting proteins in normal N-2a-N cells were detected using CHIRP-MS method,and the detection of differential genes expression caused by overexpression or knockdown of Lnc-Pim1 in normal N-2a-N cells was performed using RNA-seq technology.GO and KEGG enrichment analysis were performed on interacting proteins and differentially expressed genes,respectively.Results:(1)Cell immunofluorescence showed that N-2a-N cells expressed neuronal marker molecules ?-? Tubulin and Neurofilament-200,and Pim-1 was mainly expressed in the cytoplasm of N-2a-N cells.50 ?mol/L DFO effectively inhibited the proliferation of Neuro-2a cells.1 mmol/L ACR successfully established a model of N-2a-N cell neurite injury.After N-2a-N cell injury,the expression of Pim-1 gene showed a trend of first decrease,then increase,and then decrease.Compared with the PBS group,the proportion of the longest neurites in the CNTF group and the Nrn1 group increased significantly.Apoptosis-related molecules Cleaved Caspase-3 and Bax were down-regulated,anti-apoptosis related molecules Bcl-2 were up-regulated,and neurite growth-related molecules GAP-43 were up-regulated.(2)The total length of the Lnc-Pim1 sequence in N-2a-N cells obtained by the RACE method was 2262 bases;Phylo CSF,CPC,and CNCI software confirmed that Lnc-Pim1 had a lower potential for encoding proteins;FISH and nuclear/cytoplasm separation technology had confirmed that Lnc-Pim1 was mainly expressed in the cytoplasm in Neuro-2a cells,normal N-2a-N cells and damaged N-2a-N cells,and a small part was expressed in the nucleus.After N-2a-N cells were injured,Lnc-Pim1 gene expression showed a trend of first decrease,then increase,and then decrease,and the phase of change was earlier than that of Pim-1.(3)The LV-Pim-1 and LV-Lnc-Pim1 overexpression vectors were successfully constructed.The LV-Pim-1 titer was 2.64×108 TU/ml,and the LV-EGFP titer was 6.82×108 TU/ml;The LV-Lnc-Pim1 titer was 3.65×108 TU/ml,and the LV-mcherry titer was 1.18×109 TU/ml.(4)200 nmol/ml Pim-1 silencer and 100 nmol/ml Lnc-Pim1 silencer could specifically interfere with the expression of Pim-1 and Lnc-Pim1 in N-2a-N cells,respectively.(5)Pim-1 recombinant lentiviral vector with MOI value of 40 had a high infection rate and didn't affect cell viability.80 ?g/ml blasticidin could effectively purify virus-infected Neuro-2a cells and after virus infection purifing for 5 generations,the infection rate of Neuro-2a cells could reach more than 85%.After purification and subculture,Neuro-2a cells had higher Pim-1 expression levels.Overexpression of Pim-1 could enhance the cell viability of damaged N-2a-N cells,up-regulated the expression of GAP-43 protein,and then promoted the neurite regeneration of damaged N-2a-N cells.200 nmol/ml Pim-1 silencer could lead to down-regulation of Pim-1 m RNA and protein expression in N-2a-N cells,decreased cell viability,and down-regulated of GAP-43 protein expression,and significantly inhibitd the growth of N-2a-N cell neurite.(6)Lnc-Pim1 recombinant lentivirus with a MOI value of 40 had a high infection rate and didn't affect cell viability.20 ?g/ml puromycin could effectively purify virus-infected Neuro-2a cells and after virus infection purifing for 5 generations,the infection rate of Neuro-2a cells could reach more than 90%.After purification and subculture,Neuro-2a cells had higher Lnc-Pim1 expression levels.Lnc-Pim1 overexpression could activate the Erk1/2 signaling pathway in Neuro-2a cells,had the potential to promote cell differentiation,and could promote the neurite regeneration of damaged N-2a-N cells.Lnc-Pim1 silencer knocked down Lnc-Pim1 expression in N-2a-N cells,inhibited the growth of N-2a-N cell processes,and didn't affect cell viability.(7)Overexpression of Pim-1 in N-2a-N cells could lead to down-regulation of Lnc-Pim1,and down-regulation of Pim-1 expression had no significant effect on Lnc-Pim1 expression.Overexpression of Lnc-Pim1 in N-2a-N cells could promote up-regulation of Pim-1 expression,and down-regulation of Lnc-Pim1 had no significant effect on Pim-1 expression.(8)CHIRP-MS had detected 183 Lnc-Pim1-specific interacting proteins in N-2a-N cells.GO and KEGG enrichment analysis showed that Lnc-Pim1-specific interacting proteins were mainly distributed in N-2a-N cell cytoplasm,a small part distributed in the nucleus.These interacting proteins were related to various biological processes in N-2a-N cells such as the regulation of genes and proteins expression,cell-to-cell adhesion,exosome secretion,proteasome activity,and substance metabolism.Among these interacting proteins,Tubulin and Histone H2 had the highest confidence value.RNA-seq results showed that overexpression of Lnc-Pim1 in N-2a-N cells resulted in 140 differentially expressed genes,of which 93 were up-regulated and 47 down-regulated;Lnc-Pim1 knockdown in N-2a-N cells resulted in 982 differentially expressed genes,of which 732 were up-regulated and 250 were down-regulated.GO and KEGG enrichment analysis showed that the MAPK and PI3K-Akt signaling pathways might play an important role in the effect of Lnc-Pim1 on the growth of N-2a-N cell neurites.After overexpression or knockdown of Lnc-Pim1,Ccl2,Ccl5,Ghr,Hgf,and Il1 b genes co-expressed in some pathways might be target genes for Lnc-Pim1 action.Conclusion:(1)Pim-1 and Lnc-Pim1 are mainly expressed in the cytoplasm of N-2a-N cells.Repairing damaged N-2a-N cells requires overexpression of Pim-1 and Lnc-Pim1.(2)Neurotrophic factors CNTF and Nrn1 can activate the Erk1/2 and Stat3 signaling pathways of damaged N-2a-N cells,up-regulate Pim-1,thereby enhancing cell activity,reducing apoptosis,and up-regulating GAP-43 expression,thereby promoting damaged N-2a-N cells to regenerate neurites.(3)Pim-1 overexpression can enhance the activity of damaged N-2a-N cells,up-regulate the expression of GAP-43,and then promote neurite regeneration.By knocking down the expression of Pim-1 in N-2a-N cells,cell activity,GAP-43 expression,and neurite growth are significantly inhibited.(4)Lnc-Pim1 overexpression can promote the regeneration of damaged N-2a-N cells and may also promote the differentiation of Neuro-2a cells.Down-regulation of Lnc-Pim1 expression inhibits neurite out growth of N-2a-N cells.(5)Lnc-Pim1 overexpression may enhance Pim-1 expression in cis,and overexpression of Pim-1 may inhibit Lnc-Pim1 expression.(6)CHIRP-MS and RNA-seq detects some Lnc-Pim1 interacting proteins in N-2a-N cells and differentially expressed genes regulated by Lnc-Pim1,which provides preliminary experimental basis for the next study of the mechanism of Lnc-Pim1 effects.In short,Pim-1 and Lnc-Pim1 of N-2a-N cells are mainly expressed in the cytoplasm.Repairing injured N-2a-N cells requires overexpression of Pim-1 and Lnc-Pim1;Up-regulation of Pim-1 expression with exogenous CNTF or Nrn1 and over-expression of Pim-1 and Lnc-Pim1 with recombinant LV vectors can promote the neurite's regeneration of damaged N-2a-N cells,and some related mechanisms are clarified.