Effect Of 4.1N Gene Transfection On Biological Characteristics Of Human Lung Cancer Cell A549

BackgroundLung cancer is one of the most common malignant tumor which is hazarding the people’s health and life in the world. In recent years, the incidence and mortality of lung cancer showed a continued rising trend, Tumor invasion and metastasis is not only the malignant marker and characteristics of lung cancer but also the main factors affecting survival and mortality. Statistics show that nearly 90% of patients with lung cancer died of tumor metastasis and its complications, tumor metastasis are still an important factor to affect the 5-year survival of patients with lung cancer even if the primary tumor has been completely removed by surgery. Although the means of radiotherapy and chemotherapy has been widely used in inoperable metstatic lung cancer, but its efficacy is not satisfactory. Therefore, looking for a new lung cancer metastasis-related genes and protein markers to clear the molecular mechanisms of lung cancer invasion and metastasis and targeted control the metastasis of lung cancer is the hope to improve the efficacy of lung cancer.Protein4.1 is encoded by the EPB41L1 gene, located in human chromosome 20q11.2-q12, which was originally discovered in the nervous system. Belong to the member of the protein 4.1 gene superfamily the same as 4.1B,4.1G and 4.1R. These family members have three conserved domains:SABD (spectrin-actin-binding domain), FERM domain (4.1 protein-Ezrin-Radixin-Moesin) or membrane binding domain, and CTD domain (C-terminal domain).Recent studies found that the protein 4.1 decrease or loss was occurred in breast cancer, meningioma, lung cancer, colorectal cancer and other tumors, and more and more experimental data shows that the 4.1 gene family can be used as a negative regulatory factors involved in tumor development. In addition, as cytoskeletal component 4.1 proteins play an important role in the process of tumor metastasis because it is very likely involved in the maintaining of cell-cell and cell-matrix interactions. It has been confirmed that 4.1B was related to metastasis of prostate cancer and sarcoma cell lines, and Protein 4.1N with breast cancer metastasis are also closely related. The preliminary study found that protein 4.1N expression was decreased in lung cancer, and closely related with the degree of differentiation, but whether it has the function of inhibiting the growth of tumor suppressor genes has not been reported. In this study,We preliminary explore the expression of 4.1N in before and after transfect ion A549 cells, and the changes of some indicators which are closely related to tumor metastasis, such as proliferation, invasion, migration,etc.ObjectTo detect the expression of protein4.1N in human lung cancer A549 cells, and the effects of proliferation, invasion and metastasis abilities which has transfected 4.1N genes A549 cells that growth in vitro, and to investigate the affect of 4.1N to cell cycle, laying the foundation for the further study of its mechanism.MethodsVitro cultured human lung adencarcinoma cell line A549 as the research object, transfected the eukaryotic expression vector pEGFP-4.1N into A549 via lipofectamine 2000 mediation, at equal pace keno-carrier control, blank control were set up, the transfection efficiency and cell morphology was observed by fluorescence microscope at 24 hposttransfection; The mRNA and protein expression difference of 4.1N was examined by semi-quantitative RT-PCR and Western blotting in every group cell after 48h; The proliferation capability was determined by MTT assay. Invasion capability of every group A549 cell in vitro was evaluated by using Scratches, adhesion experiments and Tran swell chamber model; Then we detected the impact on lung cancer cell cycleby using flow cytometry after Transfected plasmid 24h and 48h.Results1, The pEGFP-4.1N plasmid was successfully transfected into human lung adencarcinoma cell line A549, and transfection efficiency was 50%, the EGFP-4.1N fusion protein locates in the cytoplasm, nucleus did not express.2, At 48h posttransfection, the expression of 4.1N mRNA and protein in pEGFP-4.1N plasmid transfection group was significantly enhanced (p<0.05), (p<0.05); the proliferation capability of A549 cells descended extremely (p<0.05); the adhesion capacity, Migration and invasion capability of A549 cells in vitro decreased substantially (p<0.05).3, Flow cytometry showed that the proportion of G1 phase increased and the S phase、G2 phase decreased when the 4.1N plasmid transfected into A549 cell.Conclusion1, Transfected with 4.1N gene can significantly increase the expression levels of 4.1N mRNA and protein in lung adenocarcinoma A549 cells which are highly metstatic in human.2, Cell behavior in vitro studies found that 4.1N can inhibit the proliferation, adhesion, invasion and migration of A549 cells, play an important role in the metastasis of lung cancer, and can arrest cells in G1 phase, may become a molecular marker for metastatic lung cancer.