Construction Of Racl Mutant Gene Plasmids By Overlap Extension And Role Of Racl In Hepatocyte EMT

Background and aims:Hepatic cirrhosis is a major clinical problem at present, and oxygen and nutrition shortage which is caused by many reasons result in hepatocyte regenerative dysfunction plays a key role in hepatic cirrhosis development. Rac1belongs to a superfamily of Rho GTPases, many researches elucidate that Rac1can induce cell to undergo Epithelial-mesenchymal transition(EMT) in different kinds of cancer cells. EMT endows cells with migratory and invasive properties, induces stem cell properties and prevents apoptosis and senescence. So we speculate that Racl can increase the regeneration of hepatocyte by promoting hepatocyte undergoing EMT which induces stem cell properties. There are two mutant Rac1gene, constitutively active mutation Rac1V12as Gly at codon12of Rac1CDS is mutated into Val, and dominant negative mutation Rac1N17as Thr at codon17is mutated into Asn. Overlap extension is a widely used methodology throughout the biological sciences to make hybrid genes, its principle is to incorporation a new sequence which does not exist in original gene into DNA, required primers only need to combine with template effectively instead of completely match. Therefore our research is aimed to construct plasmids contains Rac1mutant gene by Overlap extension, and further investigate the role of Rac1plays in hepatocytes’ EMT.Methods:①Total RNA was extracted from hepatocyte LO2using Trizol Reagent, Rac1cDNA and CDS were cloned by Reverse Transcription-Polymerase Chain Reaction, The CDS of Racl and pEGFP-C1were ligated after purification and double digestion.②Rac1gene site-directed mutagenesis which was applied to construct two mutant plasmids was carried out by Overlap extension through designing mutant primer and three steps PCR, and cDNA was used as a template.③Target genes were proved by double digestion of the recombinant plasmids, Rac1gene sequence and mutant site were verified by gene sequencing.④All kinds of plasmids were transfected into cell by cationic liposome.⑤Expression of EGFP was examined by fluorescence microscope, and exogenous EGFP-Rac1fusion protein was determined by Western blotting.⑥Twenty-four hours after transfection, Western Blotting was employed to detect the expression of E-cadherin.Results:cDNA and about580bp CDS of Racl were successfully cloned. CDS of Racl was inserted into the pEGFP-C1vector. upstream sequence and downstream sequence of Rac1genes with mutant sites were successfully cloned, Gly-to-Val mutagenesis at codon12and Thr-to-Asn mutagenesis at codon17of the Racl CDS was achieved by overlap extension of upstream and downstream sequence, mutant Rac1also inserted into pEGFP-Cl vector. There were about580bp DNA fragments after double digestion of all three recombinant plasmids, and both Rac1gene sequence and mutant sites were correct. The recombinant plasmids were named pEGFP-C1-Rac1, pEGFP-C1-Rac1V12and pEGFP-C1-Rac1N17respectively. Empty vector and three recombinant plasmids were successfully transfected into human hepatocyte LO2, transfection efficiency was almost15-22%. Expression of exogenous EGFP-Rac1fusion protein was confirmed in cells transfected by three recombinant plasmids. Constitutively active mutant pEGFP-C1-Rac1V12and dominant negative mutant pEGFP-C1-Rac1N17were used to up-regulation and down-regulation of Rac1activity, Rac1V12can significantly down-regulate the expression of E-cadherin, however Rac1N17is opposite.Conclusions:Overlap extension is a highly effective tool to reconstruct gene, and Gly-to-Val mutagenesis at codon12and Thr-to-Asn mutagenesis at codon17of the Racl CDS were successfully achieved by it. Three recombinant plasmids pEGFP-Cl-Racl, pEGFP-C1-Rac1V12and pEGFP-C1-Rac1N17were successfully constructed. Rac1may induce hepatocytes’EMT through inhibiting the expression of E-cadherin.