| Colorectal cancer (CRC) is the third most common cancer in the world. In male tumor patients, its incidence is third, however, in female, the incidence is second. Each year, there are about1,000,000new diagnosed cases, more than600,000people died of it. Comprehensive treatment based on surgical radical operation is the main method for treatment of colorectal cancer. Although we have made much progress in operation method in recent years, there are still about half of the patients showed disease progress in5years after radical resection. Metastasis is the main causes responsible for failure of surgical operation and lead to die in treating colorectal carcinoma. The detection of metastasis in the early phase can improve the prognosis of the patients, which is essential to develop the treatment strategy. So finding the specific biomarkers, which prompt the early identification of tumor or hint of metastasis, play a decisive role in tumor treatment.AGR2is a gene, which show highly expressed in many human adenocarcinomas, was proved to be associated with formation of adenocarcinoma in recent years. Moreover, the expression level of AGR2protein is proved to responsible for the formation of tumor and the metastasis expressed in tumor tissue. In many malignant tumors, such as breast cancer, prostate cancer, lung cancer, ovarian cancer, pancreatic cancer and so on, AGR2was expressed in high level. And that the expression level of correlated with poor prognosis.It was found that AGR2was expressed in the mucus secreting cells of the intestinal tract, it located in the endoplasmic reticulum of these cells, involved in the process of the secretion of the MUC2protein, play an important role in maintain the integrity of the intestinal mucosal barrier.It was found that AGR2is a marker of circulating tumor cells in colorectal cancer. This is consistent with the cases mentioned above. They also found that high AGR2mRNA level in the serum of the patient with colon cancer show tumor infiltrating pT3/T4or higher level stage; while AGR2expressed high level of the patients, the disease free survival is reduced, even the patients was in early stage. However, AGR2was one of the differentially expressed secretory proteins between the colon cancer cell lines, with the same genetic background but different metastatic potential SW480and SW620.Present investigation of AGR2on biological behavior of tumor only involved in breast cancer, lung cancer, Prostate cancer, bladder cancer and so on. In order to investigate the expression levels of AGR2in human colon cancer tissue and normal colon tissue, and explore the relationship between the clinicopathologic characters and the levels of AGR2, we designed this research. Firstly, we detected the AGR2expression level between colon cancer tissue and normal colon mucous tissue, and then explore the relationship between the clinicopathologic characters and the levels of AGR2. AGR2was envisaged as a promotive factor of colon cancer, high-expressed in colon cancer tissue and to associate with proliferation and migration of colon cancer cell. Furthermore, it was also assumed that gene silence of AGR2-targeting could effectively inhibit malignant progression of colon cancer. Accordingly, the design of this experiment was as follows:1. To investigate the expression levels of AGR2in human colon cancer, normal colon tissue and to explore the relationship between the clinicopathologic characters and the levels of AGR2, lymph node metastasis and tumor dlflbrefltiation.2. Designing and screening the si-RNA sequence which can effectively inhibit the expression of AGR2in human colon cancer cell line SW620for constructing the recombinant lenti-viral vector of AGR2si-RNA (lentivirus-AGR2si-RNA, LV-AGR2si-RNA). Build the vector of lentivirus:LV-AGR2siRNA. and transfected it into the colon cancer cell lines SW620. Explored the effects of AGR2on biological behaviors of colon cancer after colon cancer cell SW620was transfected with LV-AGR2si-RNA. Explored the inhibitory effect of AGR2mRNA on tumor invasion and angiogenesis, detected the mRNA and protein expression levels of metastasis related factors S100A4and VEGF-A, explored the mechanisms of AGR2on the biological behavior of colorectal cancer in molecular level.3. Detect the expression of VEGF-A and S100A4in cancer tissue in irnmunohistochemistry assay, to investigate the relationships among the level of AGR2and VEGF-A, S100A4expression in clinical specimens.First, we studied the expression level and significance of AGR2in human colon cancer tissues, normal colon tissues, and the relationship between the AGR2levels and the pathological characteristics. We tested the determination of AGR2protein expression in human colon cancer tissue and normal colon tissue by immunohistochemical (IHC) assay. Combined with clinical data, analysed the relationships between the AGR2expression levels and the clinical pathological characteristics of colorectal cancer.We found that1. Expressions of AGR2were found both in cancer tissue and normal colon tissue. There is higher expression in colon cancer tissues. The difference was statistically significant (p<0.05). There is a higher AGR2expression in the patients with lymph node metastasis in colorectal cancer. The difference was statistically significant (p<0.05).2. In different depth of invasion in colon cancer lesions, the expression of AGR2protein increased with the depth of invasion, with a significant difference (p<0.05).Then, for further research, we screened effective sequence of AGR2si-RNA and constructing the AGR2si-RNA. Three si-RNA interference sequences of AGR2-targeting gene were designed for constructing recombinant plasmid si-RNA, respectively. A small amount of si-RNA lenti-viral vectors with3kinds of plasmid si-RNA were transfected into human colon cancer cells SW620. AGR2expressions of mRNA and protein were detected by Q-PCR and WB. Then the effects of gene silence were determined for screening the effective sequences. We selected the most effective sequence AGR2si-RNA-1to construct LV-AGR2si-RNA. Then constructed a large number of LV-AGR2si-RNA, and then detect the titer of virus. The titer of virus detected was4.71×107V.P./mlAfter that we studied the proliferative activities of human colon cancer cell lines SW620after the AGR2gene was inhibited. To study the Changes of the metastasis related gene S100A4, VEGF-A mRNA and protein in colon cancer cells after inhibited AGR2gene. The process was as follows:The human colon cancer cells SW620were divided into3groups:The Experimental group, the negative control group and the blank control group. The experimental group was administrated with LV-AGR2si-RNA, negative control group with LV si-RNA and blank control group with nothing. AGR2mRNA expressions of every group were detected using Q-PCR. The proliferative activities of every group were detected using cell active assay (MTT). Detected the expressions of S100A4and VEGF-A in the three groups by Q-PCR and WB assay.We found that:The expression of AGR2mRNA was lower in experimental group than the negative control group and blank control group (p<0.05), but no significant difference (p<0.05) was found between the two control groups. It is prompted that LV-AGR2si-RNA can effectively silence the gene expression of AGR2in SW620cell line. And the cell growth curve of experimental group was flatter than the negative control group and blank control group, and the level of cell growth was significantly lower (p<0.05). The cell growth curve of negative control group was lower than blank control group. These results suggest that the gene silencing of AGR2-targeting significantly inhibit the proliferative activity of colon cancer cell. In Q-PCR and WB assay:The mRNA and protein expression of S100A4and VEGF-A were tested in the three groups. It is found that the mRNA and protein of both S100A4and VEGF-A lower than the other two groups, the difference was significant (p<0.05). The mRNA and protein of S100A4and VEGF-A in the other two groups show no significant difference. AGR2-targeting silence can inhibited the proliferation of the colon cell line S W620. When the expression of AGR2was reduced, the mRNA and protein of S100A4and VEGF-a was reduced, too. So it indicated that the gene AGR2may be involved in the infiltration and metastasis of colon cancer.Lastly, we investigated the expression of S100A4and VEGF-A in clinical specimens and the relationship between expression of AGR2and VEGF-A, S100A4in human colon cancer tissues. We tested the determination of S100A4and VEGF-A protein expression in human colon cancer tissue and normal colon tissue in immunohistochemical (IHC) assay. Combined with clinical data, analyzed the relationship among the AGR2expression levels and the S100A4and VEGF-A expression of colorectal cancer.We found that S100A4expressed both in the colon cancer tissue and normal colon tissue. The expression of S100A4was higher in the patient with lymph node metastasis, the difference was significant (P<0.05). VEGF-A was not expressed in all colon cancer tissue and normal colon tissue. The expression of VEGF-A was higher in the patient with pT3/T4. |
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