PKD2 Lentivirus Restore Polycystin-2 Expression And Wnt/β-catenin Signaling Pathways In Mice ADPKD Cell Lines

Background:Autosomal dominant polycystic kidney disease（ADPKD）is a common hereditary kidney disease,which occurs frequently in adults,with a prevalence of about0.1%and a global population of 400-600 million.In our country,there are about 1.5million patients suffer from this disease.The main clinical symptom of ADPKD is the formation of numerous enlargeable cysts in both kidneys.Progressive cysts can destroy the structure and function of the kidney,and about 50%of patients develop to end-stage renal disease（ESRD）at the age of 60.ADPKD is mainly responsible for two causative genes,PKD1 and PKD2,while about 15%of ADPKD is caused by PKD2 gene mutations.At present,there are no validated methods for ADPKD therapy,except kidney transplantation.Gene therapy is considered as a new and promising method in treatment of hereditary disease,therefore,with the help of lentivirus,delivery PKD2 gene into mutant cells might become a new method for the treatment of ADPKD.Objective:To establish PKD2 gene recombinant lentivirus and to investigate its restorative effects on polycystin-2 and Wnt/β-catenin signaling pathways in Pkd2-null cell lines.Methods:PKD2 gene was cleaved from the pcDNA3.1-TM-PKD2 plasmid and was inserted into the pLV-sfGFP₂A_Puro by restriction enzymes XbaI and XhoI.Sequenced the recombinant pLV-sfGFP-PKD2 plasmid to verify a correct construction.Obtained recombinant lentiviruses by co-transfecting HEK293T cells with recombinant plasmid and packaging plasmids.B3/D3(Pkd2^+/-)and B2/E8(Pkd2^-/-)cell lines were used to evaluate the effectiveness of lentivirus,they were divided into experimental group,control group,blank group and treated with pLV-sfGFP-PKD2 virus,pLV-sfGFP virus or culture media,respectively.The expression of PC2 was detected by Western blotting and immunofluorescence staining.Cell proliferation was evaluated by detecting of proliferating cell nuclear antigen.The changes of Wnt/β-catenin signaling pathway were evaluated by real-time quantitative PCR.Results:Enzyme digestion analysis and DNA sequencing showed that the recombinant plasmid pLV-sfGFP-PKD2 was constructed successfully.After infected with pLV-sfGFP-PKD2 virus,the expression of PC2 in the experimental group B2 and E8 cells（0.668±0.013,0.763±0.021）was restored to the normal level,compared with control group B3and D3 cells,respectively（0.687±0.015,P=0.164,0.776±0.008,P=0.409）.The proliferative activity in experimental group B2 cells（0.573±0.010）was significantly lower than that in control group B2 cells（0.848±0.031,P<0.01）,and was returned to the level of blank B3 cells（0.585±0.017,P=0.369）.Re expression of PKD2 in experimental group B2 cells also reduced the expression of Wnt7a,β-catenin,Myc and Cyclin-D1 back to normal levels（P>0.05 compared to blank B3 cells）.Conclusion:The recombinant pLV-sfGFP-PKD2 lentivirus has been constructed successfully.The lentivirus could rectify the absence of PC2 in PKD2-null cell lines,by which the hyper activated Wnt/β-catenin signaling pathway and the abnormal cell proliferation caused by PC2 deficiency could be also restored to normal levels.