Expression And Significance Of Heparanase, MMP2 And TIMP2 In Malignant Melanoma

Malignant melanoma is a highly malignant tumor and often primarily affects the skin and mucous membrane, widely infiltrates epidermis, dermis, subcutaneous tissue, underlying soft tissue or the mucous membrane, even the neighboring organ(or organization), and spreads through the blood and lymph systems to other parts of the body. Malignant melanoma is the third most common skin cancer and represents from 3% to 5% of cutaneous malignancies, and the mortality rate is extremely high. Invasion and metastasis of tumor cells were the main causes of cancer patients' death, therefore, the mechanisms for the metastasis and invasion of malignant melanoma have recently become the focus of intensive research activity.The major barrier for invading tumour cells is the basement membrane (BM) that surrounds the vessels, and the extracellular matrix (ECM) which forms a scaffold in tissues to hold cells together. Degradation of BM and ECM is an essential step in tumour invasion and metastasis. The BM and ECM are composed of an interlocking network of proteins and complex carbohydrates:(1)Structure protein:Including collagen, laminin, fibronectin and vitreous body union element and so on; (2)Glycosaminoglycan: The heparin sulfate proteoglycans(HSPGs) is it's principal constituent. The research mostly concentrates on the matrix metalloproteinase(MMP), the serine Proteinase and the cysteine Proteinase that are the substrate for structure protein in the past more than 10 years. Now it is believed that the major carbohydrate is heparan sulphate (HS), which acts as the glue to maintain the integrity of the BM and ECM. The enzyme responsible for cleaving HS, heparanase(Hpa), has been shown to play a key role in the degradation of the BM and ECM, and its activity strongly correlates with the metastatic capacity of tumour cells. In despite of there have been a lot of reports about the relation between the Hpa or MMP2 or TIMP2 and the invasion and metastasis of tumor, there has not been one report about combination of the three targets with cutaneous malignant melanoma (CMM). It has not been reported that the study of the expression of MMP2 and TIMP2 in tumor transfected by heparanase antisense oligodeoxynucleotide(Hpa-ASODN) and carried by nude mice. For further discussing the relationship among Hpa, MMP2, TIMP2 and malignant melanoma's metastasis, seeking the target of early detecting the invasion and metastasis of malignant melanoma and seeking the effective method of suppressing malignant melanoma's invasion and metastasis, this study detects the expression of Hpa, MMP2 and TIMP2 in the 30 cases of malignant melanoma tissues, 30 cases of melanocytic nevus tissues and 15 cases of normal skin tissues with immunohistochemical techniques. Using the A375 cells cultured by Hpa-ASODN, then injecting them into the subcutaneous of nude mice, constructing the animal model of malignant melanoma in nude mice, detecting the expression of MMP2 and TIMP2 that relate to the Hpa in the nude mice, through above test, we try to describe that the expression of Hpa, MMP2 and TIMP2 in malignant melanoma are associated with invasion and metastasis and the expression of MMP2 and TIMP2 in tumors carried by nude mice are down-regulated by Hpa-ASODN. Those results provide the basic theory to explore the invasion and metastasis of malignant melanoma. This experiment is divided into two parts. Part one: Expression and Significance of Heparanase, MMP2 andTIMP2 in Malignant Melanoma, melanocytic nevus and normal skinMaterial and methods1. Using the technology of immunohistochemistry SP to detect the expression of heparanase, MMP2 and TIMP2 in the 30 cases of malignant melanoma, 30 cases of melanocytic nevus and 15 cases of normal skin tissue.2. Statistical analysis: All the statistical analyses were performed using SPSS 10.0 for windows, the count information calculated the positive rate, the comparison of positive rates used the Chi-square.α=0.05 was considered to be of statistical significance.Result1. Expression of HpaIn cutaneous malignant melanoma, melanocytic nevus and normal skin tissue, The positive expression rate of Hpa were 63.33%(19/30), 6.67%(2/30) and 0(0/15) respectively. There was a significant difference of Hpa positive between cutaneous malignant melanoma and melanocytic nevus (x²₁=21.172, .P=0.000); there was a significant difference of Hpa positive between cutaneous malignant melanoma and normal skin tissue(x²₂=27.805, P=0.000); there was no significant difference of Hpa positive between melanocytic nevus and normal skin tissue.2. Expression of MMP2In cutaneous malignant melanoma, melanocytic nevus and normal skin tissue, The positive expression rate of MMP2 were 70.00% (21/30), 13.33% (4/30) and 0(0/15) respectively. There was a significant difference of MMP2 positive between cutaneous malignant melanoma and melanocytic nevus(x²₁=19.817, ,P=0.000); there was a significant difference of MMP2 positive between cutaneous malignant melanoma and normal skin tissue(x²₂=19.688, P=0.000); there was no significant difference of MMP2 positive between melanocytic nevus and normal skin tissue.3. Expression of TIMP2In cutaneous malignant melanoma, melanocytic nevus and normal skin tissue, The positive expression rate of TIMP2 were 60.00%(18/30), 6.67%(2/30) and 0(0/15) respectively. There was a significant difference of TIMP2 positive between cutaneous malignant melanoma and melanocytic nevus (x²₁=19.200, P=0.000); there was a significant difference of TIMP2 positive between cutaneous malignant melanoma and normal skin tissue (x²₂=15.000, .P=0.000); there was no significant difference of TIMP2 positive between melanocytic nevus and normal skin tissue.4. Correlation of Hpa, MMP2 and TIMP2These samples were obtained as 3 sections cut from tumours embedded in wax blocks, and detected the expression of Hpa, MMP2 and TIMP2. Eighteen cases were found positive in Hpa, MMP2 and TIMP2. Among them, the ratio of MMP2-positive and Hpa-positive were 90.48%( 19/21), the ratio of Hpa-positive and TIMP2-positive were 94.74%(18/19), the ratio of MMP2-positive and TIMP2-positive were 85.71%(18/21). These results demonstrated a positive correlation between Hpa and MMP2, r=0.860, P=0.000; a positive correlation between Hpa and TIMP2, r=0.932, p=0.000; a positive correlation between MMP2 and TIMP2, r=0.802, P= 0.000.Part two: The study on the incidence of heparanase antisense oligodeoxynucleotides for the expression of MMP2 and TIMP2 intumor carried by nude miceMaterial and method1.A375 cells were transfected by one Hpa-ASODN and one non-sense oligonucleotide(N-ODN) throught using cationic liposome-mediated method for 48 hours, after injecting the subcutaneous of nude mice, the growth of tumors were observed for seven weeks.2.The effects of heparanase antisense oligodeoxynucleotides on the expression of MMP2 and TIMP2 in the tumor carried by nude mice were observed by immunohistochemistry SP.3.Statistical analysis: All the statistical analyses were performed using SPSS 10.0 forwindows. Enumeration date were expressed by standard deviation( X±S). The mean of more groups used the ANVOA. a=0.05 was considered to be of statistical significance.Results1.A375 cells transfected by ASODNs were cultivated in vivo respectively and were injected into the sub-skin of nude mice, tumors carried by nude mice were found at 7-9 days.2.The expression of MMP2 and TIMP2 protein in transplanted tumor were inhibited by three different levels heparanase antisense oligodeoxynucleotids, the inhibitory effects of 30μmol/L ASODN group were the most effective. It was significant difference of the two groups (P<0.05).3.The inhibitory effects to MMP2 protein expression were stronger than that to TIMP2 protein expression; Significant difference were found between N-ODN transfected group, non-transfected group and experimental group(P<0.05).Conclusion1 .The expression of Hpa and MMP2 in cutaneous malignant melanoma(CMM) were more higher than that in melanocytic nevus and normal skin tissue.2. Significant correlations were observed between Hpa and MMP2. It suggested that Hpa and MMP2 promoted and cordinate each other in the invasion and metastasis of CMM;3. Significant correlations were observed among TIMP2, MMP2 and Hpa. It suggested that the expression of TIMP2 was tightly regulated by the expression of MMP2 and Hpa in cutaneous malignant melanoma invasion and metastasis.4. It was found that Hpa-ASODN significantly inhibited the expression of MMP2 and TIMP2 in tumors carried by nude mice.A375 cell...