Construction Vector Of Tumor Suppressor Gene PTEN And Its Identification

Background and ObjectivePTEN is a multifunctional enzyme with dual protein and lipid phosphatase. PTEN inhibits tumor cell growth, invasion and migration, promotes apoptosis through dephosphorylating its substrates such as PIP3 and FAK.The loss of PTEN tumor suppressor function is observed in various tumors, which is caused by gene mutation, promoter methylation, protein ubiquitination, oxidation and phosphorylation. This study is intended to construct pLenti6/V5-PTEN expression vector and to verify PTEN expression in LOVO and HepG2 cells. This may provide the base for further study of PTEN in the mechanism of tumorigenesis.Method1. Construction of PTEN expression vector RNA was obtained with TRIZOL method from healthy human leukocyte; PTEN gene was amplified by RT-PCR, and inserted into pLenti6/V5 expression vector by digestion ligation. The recombinant vector pLenti6/V5-PTEN was transformed into E. coli Stb13. Positive clone was analyzed by PCR, and sent to DNA sequencing subsequently.2.Culture of LOVO and HepG2 cells.3. The expression and verification of PLenti6/V5-PTEN vector in LOVO and HepG2 cellsThe recombinate vector pLenti6/V5-PTEN were transfected into mammalian cell line LOVO and HepG2 with Lipofectamine 2000.24h and 48h after transfection, RNA and protein were extracted from cells, and then RT-PCR and western bolt were performed to detect the mRNA and protein expression of PTEN. Every experiment was divided into three groups:including blank, negative vector and pLenti6/V5-PTEN vector.ResultThe human gene PTEN was amplified and cloned into pLenti6/V5. RT-PCR and western blotting confirmed that elevated expression of PTEN mRNA and protein LOVO/HepG2 cells 48 h after transfection. Compared to untransfected cells, the transfected cells grew much more slowly.ConclusionThese results demonstrated that the pLenti6/V5-PTEN expression vector was successfully constructed. The vector upregulated the expression of PTEN and reduced the proliferation of mamanlian cell lines at the meanwhile.