Construction Of Recombinant Lentiviral Vector Expressing Cdc42-shRNA And Its Biologic Effects On Bladder Cancer Cells

Background Bladder cancer is the second most common urological malignancy inthe Western society, but it is the most common urological malignancy in China with arising incidence. However, the bladder cancer at late-stage has poor long-termsurvival rates due to the absence of effective treatment methods. In order to developeffective anti-cancer therapies, one of the key issues is to identify molecular markersthat can effectively detect and distinguish the progression and malignancy of bladdertumors. Cdc42 (Cell division cycle 42), a member of Rho GTPases, acts as aGTP-binding protein switch to regulate multiple signal transduction pathways thatcontrol key cellular processes such as cell proliferation, survival, cytoarchitecture,adhesion, migration, and transcriptional regulation. Some data suggest that RhoGTPases including Cdc42 are more abundant in bladder tumor tissues than innon-tumor bladder tissues. Furthermore, the overexpression of Cdc42 is associatedwith carcinogenesis and progression of many tumors. RNAi (RNA interference), aspecific and efficient method for gene silencing, is widely used to analyze thefunctional roles of genes associated with cancer initiation and progression. And thetherapeutic potential of the RNAi technique has been demonstrated in many cancercells.Objectives To construct a lentivirus-vector plasmid expressing shRNAs againstCdc42 and evaluate its effects on the expression of Cdc42 mRNA and protein,morphological changes, cell growth, cell apoptosis, cell adhesion and STAT3expression after transfected into two human bladder cancer cell lines, EJ and T24cells.Methods Chemically synthesized oligonucleotides were composed of two 19 ntsequences placed in opposition to each other with the insertion of a 7 nt spacer sequence and the attachment of five Ts at the 3'-end of the sequence. The templateoligonucleotides were annealed and ligated into the linearized pSIH-H1 vector togenerate the recombinant lentiviral vector plasmid pSIH-H1-Cdc42-shRNA. Thisvector was evaluated by using enzyme digestion, PCR and DNA sequencing. EJ andT24 cells were transfected with pSIH-H1-Cdc42-shRNA and pSIH-H1-Luciferase-shRNA, respectively. The morphological changes were observed by phase-contrastmicroscopy and fluorescence microscope. Moreover, both the proliferation and thecell cycle of bladder cancer cells were detected by MTT assay and flow cytometry,respectively. Cell adhesion to the extracellular matrices was measured by an adhesionassay. Hoechst 33258 staining and DNA ladder electrophoresis were used as cellapoptosis analysis. In addition, the levels of Cdc42 mRNA and STAT3 mRNAexpression were evaluated by semi-quantitative RT-PCR assay, whereas theexpression of Cdc42 and STAT3 proteins was determined by Western blotting.Resultsâ‘ The recombinant lentiviral vector plasmid pSIH-H1-Cdc42-shRNA wassuccessfully constructed.â‘¡Its transfection efficiency in EJ and T24 cells was morethan 50% 72 h after transfection. The ablation of Cdc42 led to the morphologicalchanges of tumor cells, which are mainly to become round shape.â‘¢Cdc42expression was gradually decreased at its mRNA and protein levels after transfectionwith pSIH-H1-Cdc42-shRNA, and at 72 h after transfection the levels of its mRNAand protein were decreased by 89% and 81% in EJ cells, and by 90% and 85% in T24cells, respectively, whereas no changes were observed in EJ and T24 cells transfectedwith pSIH-H1-Luciferase-shRNA.â‘£Cdc42 silencing can significantly decrease thelevels of p-STAT3 protein in two bladder cancer cells. However, no difference wasobserved at the levels of STAT3 mRNA and protein compared with two controlgroups.⑤After Cdc42 silencing, the proliferation ability of T24 or EJ cells wasmarkedly decreased compared with that of two control groups. Furthermore, thesilencing of Cdc42 exhibited a significantly decreased percentage of S-phase cells(decreased by 71.2% in EJ cells and by 48.0% in T24 cells) and a compensatoryincrease in the population of G₁-phase cells.â‘¥After Cdc42 deletion, an increasedlevel of nuclear fragmentation and apoptotic bodies was detected by Hoechst 33258 staining, and ladders of DNA fragmentation were observed by agarose-gelelectrophoresis of DNA in EJ cells. In addition, Cdc42 silencing can markedly reducethe adhesive ability of tumor cells to the extracellular matrices (by 65.3% in EJ cellsand by 74.5% in T24 cells).Conclusion The constructed lentiviral vector plasmid targeting Cdc42 caneffectively knock-down the expression of Cdc42 mRNA and protein, result in themorphological changes, inhibit cell proliferation, induce cell apoptosis, decrease celladhesion and down-regulate the levels of p-STAT3 in human bladder cancer EJ andT24 cells. These data suggest that RNAi-mediated Cdc42 silencing may be a novelapproach for gene therapy of bladder cancer.