The Effect Of NPC1 Gene Mutation On Adhesion,Proliferation,and Apoptosis Of Renal Cells

BackgroundNiemann-pick disease type C1（NPC1）is a lysosomal lipid storage disease caused by NPC1 gene mutation.It is a rare and progressive autosomal recessive neurovisceral disease.At present,the cellular and molecular mechanism of NPC1 pathogenesis is still not well understood.Most of the existing studies focused on the effects of NPC1 gene mutation on the nervous system,liver and spleen,while there were few reports on kidney.In this study,NPC1 gene knockout human embryonic kidney（HEK）293T cell lines were constructed using CRISPR-Cas9 technology to further study the effects of NPC1 gene mutation on cell morphological,adhesion,proliferation,apoptosis and canonical Wnt signaling pathway,and to explore the basic structure and pathological changes of kidney tissue in mice with Npc1 gene mutation.Objective1.To study the effects of NPC1 gene mutation on the morphology,adhesion,proliferation,apoptosis and canonical Wnt signaling pathway of HEK 293T cells.2.To study the basic structure and pathologic changes of kidney tissue in Npc1 gene mutant mice aged 4 and 8 weeks.Methods1.The recombinant knockout vector（PX459-NPC1-sg RNA）was constructed and transfected into HEK 293T cells by liposome transfection technique.Stable cell lines were obtained by puromycin and limited dilution method screening.DNA sequencing,western blotting and immunofluorescence were used to verify the effect of NPC1 gene knockout,and filipin staining was used to detect the change of unesterified cholesterol content in cells.2.The effects of NPC1 gene mutation on HEK 293T cell morphology,cell adhesion,cell proliferation,cell apoptosis and Wnt signal pathway were detected by cell adhesion test,CCK-8 test,colony formation test,flow cytometry,q RT-PCR and western blotting.3.HE staining and oil red O staining were used to detect the kidney structure,pathological changes and lipid metabolism of Npc1 gene mutant mice at 4 and 8 weeks.Western blotting and immunofluorescence were used to detect apoptosis,renal fibrosis and canonical Wnt signaling pathway related proteins expression in kidney tissues of Npc1gene mutant mice at 4 and 8 weeks.Results1.The results of DNA sequencing,western blotting and immunofluorescence showed that the NPC1 gene knockout HEK 293T cell line(NPC1^KO HEK 293T)had a mutation in the exon 6 of Npc1 gene,resulting in no expression of NPC1 protein.Filipin staining showed abnormal accumulation of unesterified cholesterol in NPC1^KO HEK 293T cells.2.Phalloidin staining results showed that NPC1 gene mutation led to cell aggregation,synaptic shortening,nuclear enlargement and cytoskeleton protein filamentous actin（F-actin）thinning of HEK 293T cells.3.Cell adhesion assay showed that NPC1 gene mutation significantly enhanced cell adhesion ability.Western blotting results showed that,compared with NPC1^WT HEK 293T cells,the expression levels of E-cadherin andγ-catenin in NPC1^KO HEK 293T cells were significantly increased,whileα-catenin was significantly decreased.4.Colony formation assay,CCK-8 assay and cell cycle assay showed that NPC1 gene mutation significantly inhibited cell proliferation.q RT-PCR and western blotting showed that the expression levels of Wnt3a,c-Myc and cyclin D1 were significantly decreased in NPC1^KO HEK 293T cells.These results indicated that the NPC1 gene mutation inhibited cell proliferation through the canonical Wnt signaling pathway.5.q RT-PCR and western blotting results showed that,compared with NPC1^WT HEK293T cells,the expression levels of BCL-2 and BCL-XL in NPC1^KO HEK 293T cells were significantly decreased,while the expression levels of BAX was significantly increased.The flow cytometry with Annexin V-FITC and PI staining showed that NPC1 gene mutation significantly increased apoptosis.6.HE staining showed pathological changes in the kidney tissue structure of Npc1^-/-mice,with obvious vacuoles in the cytoplasm of kidney tubular epithelial cells and loose foam-like cell bodies.Oil red O staining showed no obvious lipid in Npc1^-/-mice kidney tissue.Western blotting and immunofluorescence assay showed that apoptosis and fibrosis occurred in the kidney tissues of Npc1^-/-mice,and the canonical Wnt signaling pathway was abnormally activated.Conclusion1.NPC1 gene mutation resulted in unesterified cholesterol deposition,cell morphological changes,cell adhesion enhancement,cell proliferation inhibition and cell apoptosis increase of HEK 293T cells.2.Npc1 gene mutation can cause obvious vacuoles in mice kidney tissue,abnormal lipid metabolism,increased apoptosis,abnormal activation of canonical Wnt signaling pathway and accelerated renal fibrosis.