Expression Of Exogenous HSP70 Gene In Human Lung Cancer A549 Cells And Its Effect On The A549 Cells Growth

Objectives:This study is to provided experimental evidence of the molecular mechanisms of HSP70 act on pulmonary carcinoma by observing expression of exogenous HSP70 gene in human lung cancer A549 cells and its effect on the A549 cells growth.Methods:(1)Alkaline lysis method was used to extract plasmid DNA, including recombinant eukaryotic expression plasmid pIRES2-EGFP-HSP70 and control plasmid pIRES2-EGFP. (2)A549 cells were cultured in LG-DMEM base medium which contained 10% FBS and saturated with 95% O2and 5% CO2 at 37℃. (3)Recombinant eukaryotic expression plasmid and control plasmid were transfected into A549 cells with LipofectamineTM2000, respectively. (4)A549 cells were divided into four groups:normal control group (NC), transfection reagent control group (TC), control plasmid group (EC), the experimental group (HSP70). (5)The green fluorescent were observed by fluorescence microscopy. After transfection 36h, transfection efficiency was examined by flow cytometry (FCM). (6)HSP70 mRNA expression level was detected by real-time fluorescence quantitative PCR. (7)MTT was used to assess the proliferative effect of HSP70 gene on A549 cells. (8)Flow cytometry was used to examined the effects of HSP70 gene on cell cycle.Results:(1)The transfected positive A549 cells showed green fluorescence under fluorescence microscopy. After transfection 36h, recombinant eukaryotic expression plasmid pIRES2-EGFP-HSP70 transfection efficiency was 38.57%; (2)HSP70 mRNA expression levels of experimental group were higher than control group, which demonstrated that the HSP70 mRNA level of exogenous HSP70 gene was highly efficient expression in transfected A549 cells; (3)Cell proliferation of the experimental group was inhibited significantly; (4)Compared to control group, the percentage of cell increased in Go/G1 phase dramatically and decreased in S phase after exogenous HSP70 gene transfection.Conclusions:The recombinant eukaryotic expression plasmid pIRES2-EGFP-HSP70 transfected successfully into human lung cancer A549 cells, and exogenous HSP70 gene is able to expression with high efficiency, exogenous HSP70 gene expression can inhibit the proliferation A549 cells significantly, and cause a cell cycle arrest in G0/G1 phase.