The Expression And Role Of SOX2 In Gastric Cancer Cell

Gastric cancer (GC) is one of the most common malignant tumors and severely endangers human health. At present the lack of effective methods on the early diagnosis,due to its etiology and pathogenesis is not clear. The effects of clinical treatment were not quite satisfied for the most patients with advanced stage, excepted early stages of gastric cancer. Th most of the patients has lost an opportunity of surgery when confirmation of the diagnosis,thus the majority of them died from the infiltrating, metastasis, recurrence or multidrug resistance(MDR) after cancer chemotherapy. The characteristics of tumor above-mentioned, such as tumors infiltration, metastasis, recurrence or MDR depends mainly on the biological characteristics of the malignant phenotype of gastric cancer cells and its performance characteristics Therefore, it is necessary to explore the molecular mechanism of malignant phenotype in gastric cancer cells for understanding the cancer cell proliferation, invasion, metastasis and MDR.SOX2 is one of the key regulatory genes during the early stages of development in embryos. Recently, the combining transcript factors SOX2, Oct3/4, KLF4 and c-MYC were sufficient to reprogram somatic cells to a pluripotent state, and have characteristics for rapid proliferation and self-renewal that is similar to ES cell phenotype, called induced pluripotent stem cells (iPS cells). This suggests that SOX2 plays an important role in cell differentiation growth and proliferation.Tumors often originate from complex Pathological phenomena such as obstacles of cell differentiation or out of control of proliferation. More recently, lots of researchers mainly focused on the mechanism of SOX2 in iPS cells, whether SOX2 could use to induce the differentiation and to reverse the malignant phenotype of GC cell?In our study, we firstly identified that the expression of SOX2 were significantly decreased in GC by using immunohistochemistry, PCR and WB. The impact of SOX2 in malignant phenotype of GC cell line were investigated by gain of function and loss of function methods.The high-throughput of ChIP-DSL were used for exploring the mechanisms of SOX2 involved in GC malignant transformation.Our study provide a new experimental and theoretical basis gene therapy for gastric cancer to guide further clinical application.Objectives:The aims of this study were to investigate the abnormal expression of SOX2 in GC and to clarify its roles and the underlying mechanisms in malignant transformation, to explore the new method to reversal the GC cells malignant phenotype and to provide a with some new theoretical and experimental evidences basis for treatment of GC.Methods:1. The expressions of SOX2 were investigated by immuohistochemistry and qRT-PCR and Western blot in gastric cancer tissues and adjacent non-tumor mucosa of GC tissues, respectively. And the relationship between the SOX2 expression and the clinicopathological characteristics was statistically analyzed.2. The lentivirus expression vector of SOX2 was constructed to up-regulate the expression of SOX2. The SOX2 expression vector and corresponding empty vector were respectively transfected into GC cell line MKN28. Stable clones were selected by Zeocin for 4 weeks, pooled cell colonies were obtained and to expand culture to establish the stable cell lines of MKN28-SOX2 and MKN28-FUW, Semi-quantitative RT-PCR and Western blot were used to detect the SOX2 expression in the transfected cells for confirmation of transfection.3. The effects of up-regulated SOX2 on the proliferation of gastric cancer cell lines MKN28-SOX2 were investigated; growth curve of gastric cancer cells were drawn by WST-1 assay, the ability of clone formation was studied by plate clone formation assay and subcutaneous tumorgenersis in nude mice using MKN28-SOX2 cells, respectively. The cell cycle distribution of MKN28-SOX2 was investigated by Flow cytometry. Annexin V /PI staining Flow cytometry assay was used to assess the Apoptosis of MKN28-SOX2.The MKN28-FUW was used as a negative control cells.4. Transwell migration and invasion assays and wound healing assay and tail vein injection metastasis assay were performed to observe the effect of SOX2 expression on metastasis and invasive of GC in vitro and in vivo.5. The promoter microarray using ChIP-DSL technology was employed to find the target genes which SOX2 directly regulated. The Whole Genome Gene Expression Microarray was applied to screen for differential expression genes in up-regulated SOX2 GC cell lines. The two microarray results were analyzed together to screen for SOX2 target genes, which can directly bind to SOX2 and also regulate directly by SOX2, and the functional as well as the pathways of those target genes involved in GC were analyzed by the Bioinformatics Software.Results1. SOX2 was significantly down-regulated in gastric cancer. Western blot and qRT-PCR demonstrated that SOX2 protein and mRNA were lower expressed in gastric carcinoma cell lines KATⅢ,AGS,MKN45,MKN28,and SGC7901 than that in the immortalized gastric mucosal cell line GES (p<0.05). In 8 out of 10 cases or 80% cancerous tissues SOX2 was down-expressed by qRT-PCR. Considering staining scores of Immuohistochemistry in gastric cancer tissue microarray, the average staining score in gastric cancer was significantly lower than the normal gastric mucosal .The expression of SOX2 were gradually decreased during the development of GC. The ratio of positive cases of SOX2 were 8% of normal mucosal, 7% of inflammation, 5% of atypical hyperplasia, 0% of adenocarcinoma. These results suggested that SOX2 expression level was significantly decreased in gastric carcinoma.2. Successfully established a stable up-regulated SOX2 in GC cell lines. To perform functional analysis of SOX2, we successfully constructed the SOX2 expression plasmids of lentiviral vector and then transfected into MKN28 cells, using empty vector as control. After selecting with Zeocin, western blot and qRT-PCR analysis confirmed that the up -regulated mix cell pooled stable cell clones of transfectants was compared to vector-transfected and parental MKN28 cells. 3. To determine the impact on proliferation after over expression of SOX2 in gastric cell lines, growth curves were generated by means of WST-1 assay over a 6-day period and cell-cycle analysis by flow cytometry. We found that MKN28-SOX2 showed significant growth inhibition compared with the control vector infected cells, the MKN28-SOX2 cells showed a higher proportion of cells in G1 phase (58.5%), compared with the control cells (33.1%), and concomitant decreases in the proportions of cells in S and G2/M phases . A significant increase in the percentage of apoptosis cells was observed in MKN28-SOX2(26.8%) compared with the control cells(5.1%)by flow cytometry analysis.4. Wound healing assay, transwell migration and invasion assays demonstrated that no statistically significant differences were observed on the invasion and metastasis of MKN28-SOX2 cells.5. We found 785 target genes that directly binding to the promoter region of SOX2 by ChIP-DSL technical. The Whole Genome Gene Expression Microarray demonstrated that 351 genes were up-regulated and 59 genes were down-regulated in MKN28-SOX2.Conclusions:1. SOX2 was significantly down-regulated in gastric cancer tissues and gastric cancer cell lines than in the adjacent nomor tissues and the immortalized gastric mucosal cell line, respectively, which suggested that SOX2 might play a certain role in the tumorgenesis or progression of gastric cancer.2. We successfully established the cell lines that over-expression of SOX2 in GC cell lines MKN28, which provided a cell model for studying the role of SOX2 in malignant phenotype of GC. 3. Up-regulation of SOX2 reduced the malignant phenotype of gastric cancer by both inhibiting the growth and inducing apoptosis of GC cells. There was no statistically significant difference in invasion and metastasis of GC cells.4. The SOX2 directly binding target genes and signaling pathways were screened out by gene microarrays, which provide new evidence for the further studying the role of SOX2 in gastric carcinoma.