Expression And Clinical Significance Of Transcription Factor SOX7in Lung Cancer

BACKGROUNDLung cancer is a common malignant cancer. It is the first death of all cancers in China which threaten the health of the population. The development of lung cancer is a multistep process and associated with multiple factors including virus infection, abnormal gene expression and smoking. However, the precise molecular mechanisms of lung cancer remain unknown. The abnormal expression of SOX genes, which act as negative regulators in Wnt/β-catenin signaling pathway, are found to associate with the occurance and development of human tumors including glioma, colorectal and HCC.Part I Study on the SOX gene famliy protein expressionin lung cancerObjective To construct the protein expression profile of SOX genes and identify the possible relationship between SOX protein, and to figure out whether SOX protein expression profile is related to the clinical and pathological characteristics of lung cancer, and whether abnormal expression of SOX genes can induce the occurrence and development of lung cancer.Methods1. Lung-derived tumor cell lines and lung tissues from lung cancer patients. The cell lines were composed of A549, NCI-H520, SBC-5, HTB-56, Calu-6and WCQH-9801. We informed consent obtained from the subjects.40normal lung tissues,240pairs of HCC tissues and para-carcinoma tissues were collected from lung cancer patients.2. SOX protein profiles in candidate samples. West Blotting was performed to determine the SOX protein levels in lung cancer cell lines, normal lung tissues, lung cancer tissues and para-carcinoma tissues.β-actin was quantified as internal control.3. Data analysisThe SOX protein expression differences among lung cancer cell lines and normal tissue sample were analyzed by paired-Samples t Test, and that among these lung cancer tissues Wilcoxon Signed Ranks Test. Spearman Rank Correlation analytical method was used to study the interactions between SOX protein expression levels. P value less than0.05is considered significant. All data analyses were performed by using of SPSS18.0.Results1. SOX protein profiles in lung cancer cell linesThe protein expression levels of SOX1, SOX2, SOX7, SOX8and SOX17were down-regulated in6lung cancer cell lines, compared to40normal tissue samples, and their differences were statistically significant (t=-3.142, P=0.019; t=-2.864, P=0.031; t=-2.948, P=0.026; t=-3.285, P=0.015; t=-2.941, P=0.028).2. SOX protein profiles in lung cancer tissues and paracacinoma tissues. A statistically significant down-regulation of SOX2/SOX7, SOX1and SOX8was found in47.5%(114/240),52.5%(126/240),55.0%(132/240) of lung cancer tissues, respectively, when compared to corresponding paracarcinoma tissues (t=-2.886, P=0.005; t=-2.089, P=0.047; t=-2.094, P=0.041; t=-2.309, P=0.023). In contrast, SOX3showed its up-regulation with statistical significance in108/240(45%) of lung cancer tissues compared to paracarcinoma tissues (t=1.108, P=0.346).Conclusion1. SOX gene family express abnormally in lung cancer. They may stimulate the canonical Wnt/β-catenin signaling pathway and thereby contribute to carcinogenesis.2. It is presumed that SOX gene family may play a critical role in the development of lung cancer via their interaction with each other. Part II Expression and clinical significance of SOX7in lung cancerObjective To investigate the expression and clinical significance of SOX7in lung cancer tissues.Methods We evaluated SOX7expression by RT-PCR and western blot in lung cancer tissues. Bisulfite sequencing PCR(BSP) and combined bisulfite restriction analysis(COBRA) were used to test promoter methylation of SOX7gene in lung cancer tissues. Specimens from240participants were stained immunohistochemically by mouse anti-human SOX7monoclonal antibody. Staining was quantified using Image-Pro Plus software, and mean integrated optical density(mlOD) was measured on each section.Results The expression of SOX7in lung cancer tissues was significantly lower than those in corresponding paracarcinoma tissues either protein level or mRNA level. The grey level of SOX7expression in Lymph node positive groups and Lymph node negtive groups were17.3±4.1and26.3±5.6, there was a significant difference. The grey level of SOX7expression in methylation positive groups and methylation negtive groups were38.9±9.4and14.7±7.6, there was a significant difference.Conclusion Gene methylation induces the expression of SOX7increased. A negative correlation was found between SOX7protein and nodal metastatic, clinical staging in lung cancer. SOX7may inhibit the development of lung cancer. Part Ⅲ Impact of SOX-7expression on proliferation and cell cycles of human lung cell line A549Objective To investigate the effect of SOX-7protein1expression on the proliferative ability of human lung cancer cells in vitro and in vivo.Methods Eukaryotic expression vectors containing human SOX-7gene was stably transfected into panel cells with Lipofeetamine2000. The clones stably overexpressing SOX-7were identified by Rt-PCR and Western-blot. The cell growth activity was examined by MTT assay. Anchorage independent growth was detected by colony formation assay in soft agar. Flow cytometry was used to analyze the cell cycle and apoptosis. Tumor growth Was visualized by whole-body imaging system.Results pcDNA3.1-SOX7and pEGFP-N1-SOX7vectors were successfully constructed and transtected into the A549cells. Comparing with the pancl cells, the transfected cells exhibited a slower growth rate and formed fewer colonies in soft agar. The results of flow cytometry showed that over-expression of SOX7resulted in the cell cycle arrest at G1phase and increased the apoptotie cell fraction. Stable transfectants with GFP expression were inoculated subcutaneously into the nude mice. Tumorigenesis and proliferation ability of SOX7over-expressed transfectants were lower than those of the control cells.Conclusion SOX7gene expression inhibits proliferation of lung cancer cells and may serve as a useful marker for lung cancer proaression.