| Gastric cancer is a common malignant tumor worldwide which is threating people’s life, it is the second cause of cancer deaths after lung cancer. To date, more than 70% of gastric cancer cases occur in developing countries and its mortality is the highest in China. Although treatment of eraly stage gastric cancer has achieved great progress and has greatly improved the survival rate of patients, the five-year survival rate of advanced gastric cancer patients is still low. It’s not only due to the low diagnosis rate of early stage gastric cancer, but also due to the proliferation, invasion and migration abilities of tumor cell. Therefore, to further investigation of molecular mechanisms of invasion and metastasis in gastric cancer and to select the key intervention targets has important significance for the prevention and treatment of gastric cancer.Transcription factor is a kind of regulatory factor which plays a decisive role in the process of transcription by specifically binding on gene-specific sequences. There are so many transcription factors affect the occurrence and development of gastric cancer. micro RNA(mi RNA)is small non-coding RNA with single chain which can inhibit the transcription or lead to degradation of target gene by combining with target m RNA. mi RNA and transcription factor were involved in the regulation of gene transcription level as well as posttranscriptional level.Meanwhile, there is also a regulated relationship between mi RNA and transcription factors.Their mutual cooperation and regulatory interactions forms a complex regulatory network. To date, to establish a TF-mi RNA co-regulatory network has become an effective method to provide importans clues for cancer pathogenesis.Aberrant mi RNA expression abnormally modulates gene expression in cells and can contribute to tumorigenesis in humans. In the first chapter of this study, we screened differentially expressed genes by using Affymetrix Exon array and found 2540 differentially expressed genes(fold change>1.5) in gastric cancer, 715 of them were significantly expressed(fold change>2), most of these genes were up-regulated, only one-fifth were down-regulated. We screened differentially expressed mi RNA by mi RNA chips and found 93 significant differentially expressed mi RNA, 27 of them were up-regulated while 66 of them were down-regulated. Gene function analysis and pathway analysis were made to these 2540 differentially expressed genes, these data showed how these gastric cancer-related genes and differentially expressed genes involves in the process of gastric cancer. We predicted target genes of TF and mi RNAs by combining the data of gene chips and information obtained from databases such as TRED and Targetscan, then TF-mi RNA co-regulated network of gastric cancer was established. Analysis and annotation of genes in regulatory network were made, the results showed that these genes belong to two signaling pathways “extracellular matrix-receptor interaction” and “focal adhesion”, suggesting that these genes closly related to tumor invasion and might be key genes in invasion and metastasis procedure of gastric cancer. Node analysis of these genes were made and found two of these genes COL1A1 and NCAM1 have the most regulatory interactions, so emphatically studies of these two genes were made. q RT- PCR and Western blot data showed an increase in COL1A1 and decrease in NCAM1 m RNA and protein levels in gastric cancer tissues which is consistant with prediction result of regulatory network. From TF-mi RNA co-regulated network, we found that COL1A1 was regulated by let-7i directly. We detected the low expression of let-7i in gastric cancer tissues and cells by q PCR and western blot in order to find out its biological function in gastric cancer, and found its expression is related to tumor invasion and metastasis.This study identified functionally relevant differentially expressed genes using the transcription factors and mi RNA-co-regulated network analysis for gastric cancer. The TF-mi RNA co-regulatory network was constructed based on data obtained from c DNA microarray and mi RNA expression profiling of gastric cancer tissues. The network along with their co-regulated genes was analyzed using Database for Annotation, Visualization and Integrated Discovery(DAVID) and Transcriptional Regulatory Element Database(TRED).We found eighteen(17 up-regulated and 1 down-regulated) differentially expressed genes that were co-regulated by transcription factors and mi RNAs. KEGG pathway analysis revealed that these genes were part of the extracellular matrix-receptor interaction and focal adhesion signaling pathways. In addition, q RT- PCR and Western blot data showed an increase in COL1A1 and decrease in NCAM1 m RNA and protein levels in gastric cancer tissues. Thus, these data provided the first evidence to illustrate that altered gene network was associated with gastric cancer invasion. Further study with a large sample size and more functional experiments is needed to confirm these data and contribute to diagnostic and treatment strategies for gastric cancer. Gastric cancer cell lines SGC-7901 and MGC-803 were chose to further follow-up experiments, let-7i mimic was transfected into cancer cells to up-regulated the expression of let-7i artificially, the results showed that the proliferation, invasion and migration abilities were significantly decreased; Meanwhile, transplanted tumor model further verified that let-7i can inhibit tumor growth. In order to verify that let-7i participate in the process of gastric cancer by directly regulating the expression of COL1A1, we detected COL1A1 expression after transfecting let-7i mimic to cancer cells, and found its decrease expression in both m RNA level and protein level. Luciferase activity experiment was made to further prove that let-7i regulated the expression of COL1A1 by regulating 3’UTR region of COL1A1. Furthermore, the proliferation, migration and invasion abilities of cancer cell were significantly decrease after transfecting si RNA-COL1A1 in cancer cells to knockout its expression artificially, which showed consistent result with transfecting let-7i mimic. All these above results showed that COL1A1 is the direct target gene of let-7i.In conclusion, we predicted the regulatory interactions of let-7i and COL1A1 by establishing a TF-mi RNA co-regulatory network in gastric cancer, and verified that let-7i can decrease the proliferartion, migration and invasion abilities of cancer cells and inhibit tumor growth in vivo. The decreasing COL1A1 expression can lead to the drop of proliferartion, migration and invasion abilities of cancer cells. Let-7i can promote the process of gastric cancer by up-regulateing COL1A1 expression, whichindicate that let-7i might be a potential diagnostic target or a treatment target of gastric cancer. |
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