| Hepatocellular carcinoma(HCC) has become the second leading cause of cancer-related deaths, and the incidence continues to rise worldwide. Globally, approximately 750,000 new cases of liver cancer are reported each year. Population-based studies show that the incidence rate continues to parallel the death rate, which indicates that most individuals with HCC die from this disease. Despite the advances in the surveillance of high-risk patients, surgical intervention for patients with early-stage disease and chemotherapy for advanced patients, the overall outcome of patients with HCC remains poor. It is therefore necessary to find more effective treatment strategies and to further investigate the detailed mechanisms of this deadly disease.Insulin-like growth factor binding protein-1(IGFBP-1) is one of the six soluble binding proteins which can affect the bioactivities of insulin-like growth factors(IGFs) through binding IGFs with high affinity. IGFBP-1, as well as IGFBP-3,-4 and-6 usually impair the access of IGFs to the IGF-receptor(IGF-R),and therefore, they diminish the effects if IGFs on target cells, including cancer cells. Other IGFBPs such as IGFBP-2 and-5 seem to promote the bioavailability of IGF ligands. IGFBP-1, which is produced primarily by hepatocytes and is secreted into the serum, is also synthesized by the kidney and ovarian granulosa cells and, in pregnant women, by decidualized uterine endometrium. Serum IGFBP-1 modulates cell growth, differentiation and metabolism in an IGF-dependent manner. In addition, IGFBP-1 functions in IGF-independent effects on proliferation, migration and apoptosis of different cell types through its interaction with cell surface molecules. It is therefore conceivable that the biological activity of IGFBP-1 is more complicated than we previously presumed. ObjectTo further study the function of Notch in the mechanism on its invasiveness in liver cancer; investigate the expression of the IGFBP-1 in human HCC tissues and elucidate the relationship between IGFBP-1 and the clinical pathologic features and prognostic significance, then to explore the biological functions and underlying mechanisms of IGFBP-1 in progression of HCC in vitro, to clarify the mechanism on its invasiveness in liver cancer from a new stage, and provide theoretical therapy molecular target for the treatment of liver cancer in the future. Methods1. Blocking Notch signaling pathway via DAPT, then using Cytokines chiptechnology to detect the related molecular changes.2. To verify the exactness of the cytokines chip by using RT-PCR and ELISA.3. To evaluated the expression of IGFBP-1 in 90 paired HCC tissues and adjacentnon-cancerous liver tissues and analyzed its clinical and prognostic significance.4. In vitro, the apoptosis and invasion of the HCC cell lines Hep G2 and MHCC97-H,which were treated with recombinant IGFBP-1 was analyzed via Flow cytometryand Transwell assays.5. To discuss the possible molecular mechanisms by detecting ERK、MMP-2、MMP-9via Western-blot and ELISA Result1. High expression of IGFBP-1 was observed in 96.7%(87/90) of the adjacentnon-cancerous liver tissues, while 36.7%(33/90) of the HCC samples demonstratedlow IGFBP-1 expression. The difference in IGFBP-1 expression that was observedbetween the HCC samples and the adjacent non-cancerous liver tissue wasstatistically significant(P < 0.05). We found that decreased expression of IGFBP-1was correlated with tumor differentiation, liver cirrhosis, microvascular invasion ormetastasis, and TNM stage, but there was no significance with respect to gender,age, tumor number, tumor size, HBV infection and serum AFP level. Furthermore,to determine the potential role of IGFBP-1 expression as an independent prognosticindicator in the prediction of the outcomes of patients with HCC, multivariate Coxregression analyses were performed. In this analysis, microvascular invasion, TNMstage and IGFBP-1 expression were recognized as independent prognosticindicators of the overall survival of the patients.2. We evaluated the expression of IGFBP1 in a human liver non-tumor cell line(HL-7702) and in HCC cell lines(Hep G2, SMMC-7721, MHCC97-H). This resultshowed that the expression of IGFBP-1 decreases gradually in the above cell lines,which suggests that IGFBP-1 may participate in the cellular invasion process ofHCC cells.3. The apoptosis of the HCC cell lines Hep G2 and MHCC97-H, which were treatedwith different dose recombinant IGFBP-1 was analyzed via Flow cytometry, thisresult showed that IGFBP- 1 has no effect on apoptosis.4. The invasion of the HCC cell lines Hep G2 and MHCC97-H, which were treatedwith different dose recombinant IGFBP-1 was analyzed via Transwell assays, thisresult showed that the invasion capability of Hep G2 and MHCC97-H cells wassignificantly impaired after IGFBP-1 treatment in a dose-dependent manner.5. We found that the protein expression levels and proteolytic acticity of MMP-2 、MMP-9、ERK1/2 were decreased in Hep G2 and MHCC97-H cell lines by usingWestern-blot and ELISA. Conclusion1. IGFBP-1 expression is uniformly and significantly down-regulated in patients withHCC compared to adjacent non-cancerous liver tissues, it is associated with theclinicopathological features, and low expression of IGFBP-1 is an independentfactor that predicts poor prognosis in patients with HCC. 2. By adding recombinant IGFBP-1 to the cultured HCC cell lines Hep G2 andMHCC97-H, IGFBP-1 could inhibit the invasion of Hep G2 and MHCC97-H, but ithas no effect on apoptosis. 3. IGFBP-1 inhibits the invasion and metastasis of HCC cells via down-regulateERK1/2、MMP-2 、MMP-9. |
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