[Construction of stable human umbilical vein endothelial cells line expressing short hairpin RNA (shRNA) targeting high mobility group box-1 gene].

Abstract

OBJECTIVE To construct the short hairpin RNA (shRNA) targeting high mobility group box-1 (HMGB1) and culture the stable human umbilical vein endothelial cell (HUVEC) line expressing this shRNA. METHODS Based on the HMGB1 gene sequence, shRNA was designed, synthesized and subcloned into the pRNA-u6.1/Neo vector, while negative controls were also established. Then the recombinant vector was transfected into HUVEC cell line and the cell was screened with G418 and assayed by using real time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. RESULTS Restriction endonuclease digestion test and sequencing verification showed that the recombinant pRNA-u6.1/Neo vector expressing this shRNA targeting HMGB1 was successfully constructed and the stable HUVEC cell line expressing this shRNA was developed. The real time RT-PCR and Western blotting was used to detect that recombinant plasmid in HUVEC cell effect on expression of HMGB1 was reduced. (mRNA: 0.4635 ± 0.0342 vs. 1.0340 ± 0.0352, protein: 0.4510 ± 0.0200 vs. 1.0210 ± 0.0110, both P<0.05). CONCLUSION The recombinant pRNA-u6.1/Neo vector expressing shRNA targeting HMGB1 was successfully constructed and the stable HUVEC cell line expressing this shRNA was developed, and therefore allowed further investigation regarding the function of HMGB1 gene in the HUVEC cell line.