MTA Gene Expression And Its Impact On Biologic Features In Human Non-Small Cell Lung Cancer

Lung cancer, especially non-small cell lung cancer (NSCLC), has become the most common malignant tumor in the world. In recent years, although the diagnosis especially the treatments such as surgical treatment, chemotherapy radiation have developed sharply, the long-term survival rates (especially the 5-year survival rate) have no significant improvements. The main reason for difficulties on treatment is associated with the malignant biological phenotype of lung cancer, which including excessive proliferation, antiapoptosis, angiogenesis, invasion and metastasis. So it has important significance and clinical application value to identify the key genes and mechanisms involved in the malignant biological behavior of lung cancer for overcoming it. MTA is a gene family closely related with carcinogenesis and cancer progression whose member overexpressed in a wide range of human cancers. According to many studies recently, MTA, especially MTA1, may be one of main regulatory molecules in the procession of carcinogenesis and progression.At present, research on the relationship between MTA and human non-small cell lung cancer is little. For this reason, the current study was performed from four aspects as follows:Part1:Expression of mRNA of MTA family in human non-small cell lung cancer and its significanceHuman NSCLC tissue and human normal lung tissue of 40 cases were investigated by Real-time PCR for expression of mRNA of MTA family and 2 human lung cancer cells (A549 and SK-MES-1) were investigated for expression of mRNA of MTA1 gene.The results showed that, mRNA of MTA1 and MTA2 were expressed in human NSCLC tissue and human normal lung tissue and mRNA of MTA3 were expressed in 60%human NSCLC tissue and 40%human normal lung tissue. The expression levels of MTA family were significantly higher in NSCLC tissue than normal lung tissue and have close relationship with TNM stage and lymph nodes metastasis. Expressions of mRNA of MTA1 gene were high enough for RNAi in the 2 human lung cancer cells. The results suggested that, MTA family associated closely with initiation and progression of human NSCLC.Part2:MTA1 protein expression and its relationship with clinicopathological features, proliferation and angiogenesis in human non-small cell lung cancer.80 cases of human NSCLC tissue (40squamous carcinoma and 40adenocarcinoma) and 10 cases of human normal lung tissue were investigated by immunohistochemistry for protein expression of MTA1. The results showed that, MTA1 protein expressed in 43 cases of NSCLC tissue (53.75%), and in 21 cases of squamous carcinoma (52.5%) and in 21 cases of adenocarcinoma (55%) respectively. MTA1 protein expression has close relationship with TNM stage and lymph nodes metastasis.No expression was detected in 10 cases of human normal lung tissue.Further, expression of proliferating cell nuclear antigen(PCNA), CD34 and vascular endothelial growth factor (VEGF) were investigated by immunohistochemistry. Proliferative index (PI) and microvessel density (MVD) in lung cancer were measured respectively. The results showed that PI, MVD and VEGF expression were extremely higher in MTA1 positive group than MTA1 negative group, the difference is significant(P<0.05). The results suggested that, MTA1 may closely associate with initiation, progression, malignant proliferation and angiogenesis of NSCLC,Part3:Construction of hMTAl-siRNA lentiviral vector.According to known sequence of hMTAl gene mRNA in the genebank to determine the appropriate target site, synthesize DNA templates encoding siRNA, connect MTAlsiRNA annealed template oligonucleotide to linearized pGCSIL-GFP expression vector, and to construct hMTAl-siRNA lentivirus expression vector by sequencing and enzyme digestion. Construction of target gene over-expression vector:primer design, the use of PCR to amplify MTA1 functional gene, to connect with eukaryotic over-expression vector after digestion or linearization, to construct pEGFP-Nl-3FLAG-MTAl over-expression vector by sequencing and enzyme digestion. Selection of exogenous target by Western blot:cotransfection of 293T cells with hMTAl-siRNA lentivirus expression vector and pEGFP-Nl-3FLAG-MTAl over-expression vector, exogenous target selection with Western blot. Packaging and titer determination of hMTA1-siRNA lentivirus vector:to pack three plasmid vector with the best target hMTA1-siRNA lentivirus expression vector and pHelper 1.0 vector and vector pHelper 2.0 in 293T cells, and to demarcate virus titer. The sequencing and enzyme digestion results showed that we successful constructed hMTA1-siRNA lentivirus expression vector and target gene over-expression vector pEGFP-N1-3FLAG-MTA1. Co-transfection of 293T cells was performed with greater than 90% fluorescence expression of the rate. Exogenous selection by Western blot showed that target 2# is the best target. High-titer lentivirus liquid was prepared after packaging and enrichment of hMTA1-siRNA lentivirus vector. The virus titer was 6E+8 TU/ml, which was measured and calibrated in 293T cells.Part4:Influence of transfecting human lung cancer cells with hMTA1-siRNA lentiviral vector on its biological features in vitro.Transfect human lung cancer A549 and SK-MES-1 cell line with hMTA1-siRNA lentivirus expression vector; apply Real-time PCR to determine transcription levels of MTA1 gene expression after transfection; the changes of biological features of lung cancer A549 cell, such as cell proliferation and invasion ability were detected by MTT and the basement membrane invasion assay (TRANSWELL). Real-time PCR results showed that expression level of MTA1 mRNA was significantly reduced after transfecting A549 cells with hMTA1-siRNA lentivirus expression vector. MTT and basement membrane invasion assay showed that cell proliferation and invasion ability of A549 cells was reduced significantly after tranfecting.The results showed that, hMTA1-siRNA lentivirus expression vector can inhibit proliferation, invasion ability of human lung cancer A549 cells in vitro. The results suggested that, MTA1 may play a very important role in malignant proliferation, invasion and metastasis of NSCLC; MTA1 is a potential target point for lung cancer therapy in the future.