Research On The KiSS-1 Gene Suppress The Metastasis Of Endometrial Carcinoma

ObjectivesMetastasis is the most lethal attribute of cancer, which severely affects the effectiveness and prognosis of cancer patients. The development of distant metastasis is the most important predictor of death from endometrial carcinoma. nm23, KAI-1, KiSS-1, MKK4, and BRMS1, reported to be implicated in tumor invasion and metastasis. The discovery of metastasis suppressor genes will provide important clues for the predictive diagnosis and interferential therapies of metastasis. However, there have been few metastasis suppressor genes discovered till now. Recently, the KiSS-1 gene has been reported to be a novel metastasis suppressor gene in human melanoma and breast carcinoma cells . KiSS-1 was identified as a metastasis suppressor gene encoding an array of structurally related peptides, namely kisspeptins, which acting through the G protein-coupled receptor GPR54 are able to inhibit tumor progression. KiSS-1 is a human metastasis suppressor gene whose product, metastin, was recently identified as the endogenous agonist for a novel Gq/11 coupled receptor (metastin receptor). The KiSS-1 gene encodes a 54-amino acid peptide that is the endogenous ligand of an orphan G-protein-coupled receptor (GPR54). The metastasis suppressor function of the KiSS-1 gene product is reported to act after binding with GPR54. After binding with GPR54, the KiSS-1 gene product inhibits the chemotaxis, invasion, and metastasis of cells. Active MMP-2, MMP-9, MT1-MMP, MT3-MMP and MT5-MMP cleaved the Glyll8-Leu119 peptide bond of not only full-length KiSS-1 protein but also metastin decapeptide. The 92-kDa type IV collagenase (MMP-9) plays a critical role in tissue remodeling. In the present study, we analyzed the expression levels ofKiSS-lmRNA in endometrial carcinoma. The expression profile and the role of KiSS-1 in cancer progression are largely unknown in most of the cancers, including endometrial carcinoma. We undertook a study to determine whether the KiSS-1 gene, previously shown to suppress cancer spread (metastases), negatively regulates MMP-9 expression. And this kind of research has not been reported domestically yet. It includes three parts in our report . The first part, Title: The expression and significance of KiSS-1 and MMP-9 in endometrial carcinoma tissues. Objective: The aim of this study is to detect the expression and correlation of KiSS-1 and MMP-9 mRNA in endometrial carcinoma tissues to probe the clinical significance and correlation of them. The second part, Title: Research on transfection KiSS-1 gene into the HEC-1B cell lines. Objective: To transfect KiSS-1 into the HEC-1B cell lines in a safe, high-efficiency and low toxicity way, and then to find out whether KiSS-1 affects the metastasis ability of cell lines. The third part, Title: Research on the effect that the KiSS-1 gene suppress the metastasis of a xenografted tumour model of endometrial carcinoma in nude mice. Objective: We establish a xenografted tumour model of endometrial carcinoma in nude mice and research the effect and way of gene therapy that KiSS-1 suppress the metastasis of endometrial carcinoma.1. The expression, correlation and their clinical significance of KiSS-1 and MMP-9mRNA in 32 patients with endometrial carcinoma, 10 patients with EIN and 12 patients with normal endometrium were detected by reverse transcriptase polymerase chain reaction (RT-PCR) method.2. Lipofect was used to transfect KiSS-1 into HEC-1B cell lines. The difference in expression of KiSS-lmRNA, MMP-9mRNA and metastin between the transfected and non-transfected cell lines was detected by RT-PCR and western blot. In the present study, we used real-time RT-PCR to evaluate the quantitative expression level of the target gene in transfected and non-transfected cell lines. Then by use of trans well-room and millicell-room , the difference in invasion and metastasis ability between the transfected and non-transfected cell lines was tested.3. To establish a tumour model of endometrial carcinoma in nude mice: the nude mice were randomly divided into the following three groups: the control group ( HEC-IB cells of endometrial carcinoma were directly injected into subcutaneously of nude mice, formed the tumours after 5-10 days); no-KiSS-1 plasmid transfection group (HEC-IB cells and pcDNA3.0 plasmid were directly injected into subcutaneously of nude mice, formed the tumours after 5-10 days. pcDNA3.0 plasmid were injected three times one week for tow weeks); KiSS-1 plasmid transfection group(HEC-lB cells and pcDNA3.0-KiSS-l plasmid were directly injected into subcutaneously of nude mice, formed the tumours after 5-10 days. pcDNA3.0-KiSS-l plasmid were injected three times one week for tow weeks). To examine the difference of expression of KiSS-1 and MMP-9 mRNA in three groups in molecular biology level. Analysis the effect that KiSS-1 gene suppress the metastasis of endometrial carcinoma.Results1. The positivity and relative content of KiSS-lmRNA in patients with endometrial carcinoma(37.5%, 46.83 + 2.66) are all lower than in patients with EIN(80%, 56.75 + 1.94) and normal endometrium(83.3%, 56.79 + 1.62). The positivity of KISS-1 mRNA in patients with endometrial carcinoma correlates with their clinical stage, myometrial invasion and lymph node metastasis(P< 0.05). There is no difference in the positivity of MMP-9 mRNA in patients with endometrial carcinoma (46.9%), EIN(40%) and normal endometrium (25%), but the relative content of MMP-9mRNA in patients with endometrial carcinoma is higher than in patients with normal endometrium. The positivity of MMP-9mRNA in patients with endometrial carcinoma correlates with their clinical stage and lymph node metastasis(p<0.05). The loss of KiSS-lmRNA expression correlates negatively with the expression of MMP-9mRNA in patients with endometrial carcinoma.2. Using system, we observed the stable expression of KiSS-1 mRNA and the significant difference in cells, expression between transfected and non-transfected cell lines. The expression of MMP-9mRNA and metastasis ability of the transfected cell lines decreased significantly. High expression...