The Function And Regulation Mechanisms Of TIMP1in Colorectal Cancer

Background and objectiveThe tumor is one of the largest human health hazard factors. With the intensification of the world’s population growth and aging, the global burden of cancer continues to increase. GLOBOCAN estimated that, in2008, about12.7million new-onset cancer cases and7.6million deaths. Colorectal cancer is the third most common cancer in men and the second most common tumor in women. In the past few years, along with the development of surgery, radiotherapy and chemotherapy molecularly targeted biological treatment advances and early detection of the disease, the prognosis of colorectal cancer survival has been significantly improved. But the long-term prognosis of patients with colorectal cancer is still disappointing, especially advanced colorectal cancer5-year survival rate of only19%or less.It has been confirmed the occurrence of colorectal cancer is closely related to the abnormal expression of the multiple oncogenes and tumor suppressor genes. Hence, it is very important to find the gene level knot rectal cancer related genes, colorectal cancer diagnosis, treatment and prevention through in-depth study of colorectal cancer from cellular and molecular level. Therefore, we first use of gene chip technology to detect the four cases of colorectal adenocarcinoma tumor tissue and paired with four cases of peritoneal metastasis organizations genome-wide set of mRNA expression of differences. Compared with the primary tumor, we consider genes with significant expression differences as candidate genes. In view of the results of the literature study and preliminary experiments, we ultimately focus on the tissue inhibitor of matrix metalloproteinase-1(Tissue inhibitor of metalloproteases1, TIMP1) gene. Next, we applied immunohistochemical methods to detect the expression of TIMP1protein in112cases of colorectal cancer tissues and found that high expression of TIMP1protein in colorectal cancer tissues. Then we discussed the relation between pathological data of TIMP1expression levels and colorectal cancer specimens of patient survival. To further verify the role of TIMP1in colorectal cancer occurred in the development process, cell growth curve detected by MTT, clone formation, cell transfer and other methods were used to detect the TIMP1gene in colorectal cancer cell proliferation and tumor sphere forming ability and cells transferred invasion ability and colorectal cancer cells to5-FU drug sensitivity. Tumor metastasis and recurrence is the most common cause of cancer patients’death. We discussed TIMP1in colorectal cancer cells epithelial mesenchymal transition and EMT-associated transcription factor expression levels. In addition, through Targetscan, miRanda and PicTar4three miRNA analysis of biological information website, we forecast potential miRNAs targeting regulation of TIMP1expression, three prediction results showed the presence of miR-618and TIMP13’UTR complementary base the base sequence binding sites. We applied the luciferase reporter gene and Western blot molecular biological methods to further study the regulation and mechanism of TIMP1expression levels of miR-618. In situ hybridization and immunohistochemical methods detect miR-618and TIMP1expression levels in colorectal cancer and its interconnectedness. Through the study, we enlarged the understanding of the carcinogenic effects of TIMP1in the development of colorectal cancer and its molecular mechanism involved. We provide a new strategy and theory basis for the early diagnosis, treatment and prevention of colorectal cancer.MethodsChapterlFour cases of patients with colorectcal cancer were collected and the RNA from the four samples were extracted. Gene expression was detected by high-throughtput ChIP techolony. The expression of TIMP1in colorectal cancer tissue was detected by Immunohistochemical methods. The relationship between TIMP1expression and the samples from clinical parhology were also evaluated. We also analyzed the relationship between the expression of TIMP1and the survival prognosis in patents with colorectal cancer.Chapter2Because of the significance relations of TIMP1and the size of tumor, the transformation of tumor, the tumor clinical stage and the survival prognosis of patients, we further researched the effects of biological behavior which is related to the expression of TIMP1. First, we designed an siRNA which is specific to the TIMP1ORF (opening reading frame) and the siRNA was transfected to the cell line, SW-620. Then we detected the efficiency of siRNA through Western blot and Immunofluorescence technology. MTT growth curve, colony formation assay and cell transfer experiments, reconstituted basement membrane invasion assay were used to detect the expression of TIMP1level which changed the proliferation of colorectal cancer cells, tumor sphere formation and cell transfer invasive ability. MTT assay was also employed to detect the change of5-FU resistance in SW-620cells after transfection with si-TIMP1.Chapter3 For TIMP1can promote metastatic invasion of colorectal cancer cells, we characterized si-RNA expression levels of MT1-MMP by RT-PCR and Western blot. Meanwhile, in order to verify the TIMP1in colorectal cancer cell epithelial mesenchymal transition, we determined TIMP1downregulation of epithelial marker E-cadherin and mesenchymal markers Vimentin expression level by Western blot. To further clarify the cause of the epithelial-mesenchymal transformation mechanism, markers associated with EMT ZEB1, ZEB2and SANI1were detected by Western blot.Chapter4We predicted potential miRNAs sites which are possible to regulate human TIMP1expression by applying three biological information Targetscan, miRanda and PicTar4. Three sites predicted results show that miR-618has TIMP13’UTR binding sites. MiR-618transfected to SW-620cells. RT-PCR was used to detect the expression level of miR-618in the cells of miR-618/SW-620, and NC/SW-620two treatments. TIMP1protein expression of miR-618SW-620cells is detected by Western blot and immunofluorescence in miR-618/SW-620, and NC/SW-620. Build dual fluorescent reporter gene vector containing the full-length sequence of the coding region of TIMP1(pTIMPl-CDS), and sequenced to verify the sequence of the inserted fragment is correct or not. PTIMP1-CDS plasmid was transfected into SW-620and293T cells with miR-618mimetics or inhibitors processing. The application of the double fluorescent reporter gene assay was used to detect the fluorescein changes after different treatment of cells. In situ hybridization, immunohistochemical and QRT-PCR methods were used to detect the expression of TIMP1and miR-618in38cases of colorectal cancer tissues and the relationship between TIMP1and miR-618expression levels in colorectal cancer tissues was analyzed. Statistical AnalysisExperimental results used SPSS13.0statistical software for statistical processing and analysis of measurement data were presented as mean±standard deviation (x±s). TIMP1expression and the relationship of the clinical and pathological data used the chi-square test analysis; survival analysis used the Kaplan-Meier method, log-rank test comparing survival differences. Tumor Ball number was used to compare two independent samples T-test. The measurement data were presented as mean±standard deviation (x±s). MiR-618and TIMP1relationship in clinical samples used the chi-square test analysis. P<0.05was considered as statistically significant.ResultsChapter1Gene expression profile analysis was detected by using high-throughput gene chip technology in primary tumor tissue and peritoneal metastasis tissue samples. We found that24genes increased more than four times of expression in the peritoneal metastasis tissue. TIMPl gene in peritoneal metastasistissue was significantly higher than the primary tumor tissue,4.49times higher. TIMP1protein expression in112cases of colorectal cancer tissues detected by immunohistochemical methods and the results show that TIMP1expression, negative expression in10cases (8.9%) in the cancer cells pulp, low expression in26cases (23.2%), the degree of expression in42cases (37.5%), strongly expressed in34cases (30.4%). Combined with clinical data, through the results of immunohistochemistry analysis of tumor size, lymph node metastasis, clinical stage and TIMP1expression, respectively, and we found that TIMP1expression correlated with tumor size (P=0.000), lymph node metastasis (P=0.001significant correlation), clinical stage (P=0.004). A co-relation between TIMP1expression and survival prognosis in patients with colorectal cancer was analyzed by the Kaplan-Meier method.112cases of colorectal cancer patients all received a follow-up and results showed that the higher TIMP1expression, the shorter the time of the median survival. In contrast, expression of TIMP1was lower, the longer median survival time.Chapter2SiRNA-TIMP1was transfected in SW-620cells. The Interferance efficiency of TIMP1siRNA was detected by Western blot and immunofluorescenceexpression. Results showed that the SW-620cells were transfected with of TIMP1siRNA expressed significant lower protein expression compared to the control group. MTT assay was used continuously4d the observation si-TIMP1/SW-620cell proliferation speed and result showed low expression of TIMP1SW-620cell proliferation slowed from the second day. This result showed that si-TIMP1/SW-620had slower cell proliferation than NC/SW-620. Different concentrations of5-FU treated on NC/SW-620and si-TIMP1/SW-620for72h. The result showed that the IC50of the two groups of cells is significantly reduced in si-TIMP1/SW-620than in NC/SW-620resistance. Further NC/SW-620, and si-TIMP1/SW-620cells were cultured in serum-free medium. The growth of tumors diameter was significantly different after5d culture in two independent samples by T-test (t=13.929, P=0.000).Chapter3RT-PCR and Western blot were used to detect MT1-MMP expression levels in NC/SW-620, and si-TIMP1/SW-620cells. The results showed si-TIMP1/SW-620cell had significantly lower expression of MT1-MMP than NC/SW-620cells. Western blot analysis detected the epithelial marker E-cadherin in NC/SW-620and mesenchymal markers Vimentin in si-TIMP1/SW-620cell. The results showed the expression of E-cadherin increased in si-TIMP1/SW-620cells compared to NC/SW-620cells. While Vimentin expression reduced. The Western blot detected EMT markers of ZEB1, ZEB2and SANI1in NC/SW-620and si-TIMP1/SW-620cell. Results showed expressions of ZEB1, ZEB2and SANI1reduced in si-TIMP1/SW-620cells compared NC/SW-620cells.Chapter4We used the conventional miRNA target gene prediction methods, Targetscan miRanda and PicTar4, three of miRNA biological information site to predict the possible regulation of TIMP1potential miRNAs. Three prediction results showed that miR-618had TIMP13’UTR binding site. MiR-618may regulate the expression of TIMP1through a combination of TIMP13’URT. The Targetscan online analysis suggests that miR-618and TIMP1mRNA zone exists between82-88complementary binding sequences. MiR-618transfected SW-620cells and RT-PCR was used to detect the expression of miR-618. The results showed significantly higher expression of the miR-618/SW-620of cells than NC/SW-620cell miR-618. MiR-618transfected into SW-620cells and TIMP1expression is significantly lower by Western blot and immunofluorescence, compared to the control group. The SW-620cells transfected with miR-618expressed TIMP1protein, suggesting that miR-618can inhibit the expression of TIMP1. So we constructed pTIMP1-CDS, pTIMPl-CDS SW-620and transfected into293T cells to inhibit expression of miR-618for48hours of treatment. Double fluorescent reporter gene detection technology detected total cellular luciferase protein, and found that miR-618mimics the role of these two cell types after the Renilla luciferase activity was inhibited, only about50%of the control group. MiR-618inhibitor showed the different degrees of increase. These results indicated that miR-618affected the expression of the Renilla luciferase by acting on pTIMP1-CDS fragment. TIMP1and miR-618expression levels in38cases of colorectal cancer tissues were detected by In situ hybridization, immunohistochemical and QRT-PCR methods. Fisher exact test results showed that difference of the number of miR-618+/TIMP1-and miR-618+ /TIMP1+cases is statistically significant (P=0.000).Conclusion1. The gene expression of24genes including TIMP1increased more than four times in colorectal cancer peritoneal metastasis tissue. The expression level of TIMP1protein is also upregulated in colorectal cancer, and it was significantly associated with tumor size, lymph node metastasis, and clinical stage. TIMP1expression was closely related to survival prognosis of patients with colorectal cancer. It may become the ideal marker of progress and prognosis.2. Silencing TIMP1expression significantly inhibited the SW-620colorectal cancer cell proliferation, metastasis, invasion and tumor sphere forming ability, but enhanced the sensitivity of5-FU for SW-620cell. Further molecular biology experiments found the down-regulation expression of TIMP1can inhibit the protein expression of MT1-MMP, SW-620cells, mesenchymal markers Vimentin and EMT markers of ZEB1, ZEB2and Snail expression levels, and promote the epithelial marker E-cadherin.3. MiR-618could regulate protein expression of TIMP1by binding to3’UTR of TIMP1. Furthermore, MiR-618and TIMP1both expressed in the cytoplasm of cancer cells. In colon cancer specimens, there was a significant difference in the number of cases between miR-618+/TIMP1-and miR-618+/TIMP1+. This study helps to reveal the molecular mechanisms of colon cancer development and provide a theoretical basis and strategies for the early diagnosis, individual prevention and treatment.of colon cancer.