Construction Of AQP1-shRNA Recombinant Adenovirus Expressing Vectors And Identification Of Its Function

Aquaporins (AQPs) are membrane water channels,which selectively transport water across cell plasma membranes. Previous studies have demonstrated that AQP1 is ubiquitously expressed in tumor vascular endothelium and is also expressed in many types of tumor cells. But it is still unclear from expression studies whether this is an aberrant phenomenon or whether it plays a role in tumor biology.In the paper, we constructed AQP1-shRNA recombinant adenovirus expressing vectors, and interfere the exogenous AQP1 and endogenious AQP1 by use of them, and then we study the the relationship between AQP1 and cell migration as well as the relations between AQP1 and water transporting function.Firstly we constructed the AQP1-shRNA recombinant adenovirus expressing vectors by use of Gatewaytm Colone System including entry vector pENTR2B and destination vector pAd/CMV/V5-DEST,DNA recombinant technology and LR recombinantion reaction. To identify the AQP1-shRNA recombinant adenovirus expressing vectors , we made use of two kinds of antibiotics selection,transformation selection with different competent cells and restriction endonucleases digestion.And then we transfected the AQP1-shRNA recombinant adenovirus expressing vectors which identified were correct at molecular level to CHO cells that express exogenous AQP1 and human liver tumor cells SMMC7221 that express endogenious AQP1, after that we identified its function via Western blot and immunofluorescence, the result demonstrated that AQP1-shRNA recombinant adenovirus expressing vectors could silence expression of exogenous AQP1 and endogenious AQP1, and the second recombinant plasmid——hAQP1-shRNA2 has the best gene silencing effection.Afterwards we did MTT experiments, Fluo-star water permeability experiments, scratch adhesion test and Western blot to study the relationship between AQP1 and cell migration as well as the relations between AQP1 and water transporting function by silencing AQP1 with AQP1-shRNA recombinant adenovirus expressing vectors. The result showed that AQP1-shRNA recombinant adenovirus expressing vectors could eminently inhibit exogenous AQP1 expressed in CHO cells and endogenious AQP1 expressed in liver tumor cells SMMC7221 mediated water transporting function, furthermore after silencing AQP1 with it, cell migration could be eminently inhibited, at the same time AQP1 could participate in cell movements by reconstruction and rehabilitation of cytoskeleton.