The Study Of Malignant Transformation Of NIH3T3 Cells Induced By Transfection Of HMGA1 Gene

Objective: Over expressed HMGA1 gene was found in many malignant cancer cells. The objective of this study was to establish a mouse fibroblast cell line NIH3T3 with extraneous HMGA1 gene stable-expression, and to observe the effect of HMGA1 gene expression on the biological characteristics of NIH3T3 cells. This will provide an experimental and theoretical basis for further study of the mechanism of HMGA1 and the relationship between HMGA1 gene expression and the occurrence, development and metastasis of tumor.Methods: 1. HMGA1 cDNA was subcloned into eukaryotic expressing vector pcDNA3.0, then transfected into the mouse fibroblast cell line NIH3T3 by lipofectamine-mediation. The positive cell clones were selected by G418 through 15 days. The insertion and expression of extraneous HMGA1 gene in NIH3T3 cells were analyzed by RT-PCR. 2. The cytomorphological alterations of NIH3T3 cells transfected with HMGA1 gene were observed under light microscope. The cell growth curve was measured through methods of MTT and flow cytometry. Soft agar colony formation was employed to analyze the ability of anchorage-independent growth of the NIH3T3 cells transfected with HMGA1 gene. Finally, we detected the expression of the immune inhibition factors of VEGF mRNA and FasL mRNA in these three kinds of cell lines.Results: 1. Establishment of mouse fibroblast cell line NIH3T3 with stable expression of extraneous HMGA1 gene.The plasmids pcDNA3.0-HMGA1 and pcDNA3.0 were transfected into the NIH3T3 cells by lipofectamine-mediation, which were incubated in selective medium containing G418. The survival cells formed the G418-resistant monoclone after about 15 days. The positive cell clones were isolated and expanded.Total RNA of the clones were extracted and analyzed by RT-PCR. The presence of HMGA1 was detected in cell clones stably inserted with HMGA1 gene, whereas not seen in the clones transfected with pcDNA3.0 and parental cells.2. Effects of extraneous HMGA1 gene expression on the biological characteristics of NIH3T3 cells.Morphology showed that cells transfected with HMGA1 gene arranged intensively, their size unequal, their nucleus size enlarging, mitoses and chromatin increasing. There were no diferences in morphology between cells transfected with empty vector and parental cells NIH3T3, whose arrangement showed sparsely distribution.The cell growth curve was measured by MTT methods. The result showed that the growth rate of cells tranfected with HMGA1 gene was accelerated, compared with the growth rates of the cells tranfected with empty vector and parental cells(p <0.05). No diference was showed in the growth rates between the cells tranfected with empty vector and parental cells (p>0.05).The analyzed result of cell cycle by FCM showed that the number of cells tranfected with HMGA1 gene at G0-G1 stage was reduced (p<0.05), which of S stage was increased (p<0.05) comparing to the normal parental cells group. No diference was showed between the cells tranfected with empty vector and parental cells (p>0.05).The proliferative capacity of three group cells in soft agar was assessed by soft agar colony formation. The result indicated that the HMGA1 gene transfectants acquired the ability to grow in soft agar, forming cell clones, cloning efficiency being 4.07%; whereas the parental cells and control cells could not.We detected the expression of the immune inhibition factors of VEGFmRNA and FasLmRNA in cells stably transfected with HMGA1, whereas no in parental cells and control cells.Conclusions: 1. The HMGA1 gene was successfully subcloned into eukaryotic expressing vector pcDNA3.0. The recombinant plasmid pcDNA3.0 - HMGA1 was successfully constructed.2. The recombinant HMGA1 expression vector was transfected into the mouse fibroblast cell line NIH3T3 by lipofectamine mediation. The positive cell lines were selected with G418.3. The results of RT-PCR confirmed that extraneous HMGA1 gene was inserted stably and expressed effectively in targeted cell NIH3T3.4. Extraneous HMGA1 gene stable expression in NIH3T3 cells caused their changes of cell morphology, promoted the proliferation of NIH3T3. The results of soft agar colony experiments verified that the HMGA1 gene transfectants acquired the ability of anchorage-independent growth to form cell colony in soft agar. And the expression of VEGF mRNA and FasL mRNA were detected in the transfectants.