This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA （shRNA） targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside （PAN）-induced ...This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA （shRNA） targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside （PAN）-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein （GFP）-contained plasmids （pGC） to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein ex-pression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups： control group, PAN treatment group, PAN＋TRPC6 shRNA transfected group and PAN＋scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.展开更多
基金supported by grants from the National Natural Science Foundation of China(No.31050110433,No.81170600 and No.81170662)
文摘This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA （shRNA） targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside （PAN）-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein （GFP）-contained plasmids （pGC） to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein ex-pression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups： control group, PAN treatment group, PAN＋TRPC6 shRNA transfected group and PAN＋scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.
