Study On The Cellular Functional And Mechanism Of Molecular Markers Associated With Melanoma Malignant Proliferation

Purpose: Melanoma is a kind of malignant tumor derived from the pigment cells in pigmentation area of skin, mucous membrane, eye and central nervous system, with high degree of malignancy and poor prognosis. Conventional treatment approaches, including surgery, radiotherapy, and cytotoxic chemotherapy although to a certain extent, inhibit the occurrence and development of melanoma, but have been used with limited success. However, chemotherapy and radiation therapy can also often elicit serious side effects. Moreover, resistance to both radiotherapy and chemotherapy lead to unfavorable clinical outcomes in most melanoma patients with less than one year of treatment. As a result, it is urgent to develop new agents and new therapeutic approaches to establish an individualized treatment plan, and prolong the survival period and improve the survival rate of patients with melanoma. Aiming at how to find the functional gene which affect malignant proliferation in melanoma, high-throughput(high-throughput) and high content(high-content) cell screening techniques show great potential and is playing an increasingly important role. Via gene manipulation in cancer cells in vitro or in vivo(such as specific inhibition of gene expression or over-expression of gene), the expression of target gene will be down-regulated and up-regulated, and the phenotype and function of cells and the signal transduction pathways will analyzed. Furthermore, the function of gene can be assessed in animal models and clinical tissue specimens to investigate the correlation with cancer development. Methods: In the present study, we compare the whole genome expression profiling in 3 cases of fresh tissues of malignant melanoma and 3 cases of normal skin tissues using microarray-based analysis. For those genes that are up-regulated in melanoma tissuesand the fold change of which is greater than 5-fold, we choose the first 20 gene with fold change more than 5 times and use real time fluorescent quantitative PCR(q PCR) method to verify the expression level of them. For those genes consistent with the microarray results, combining literature screening we get some molecular targets that are important for malignant proliferation in melanoma, we then investigate the mechanism of them in regulating the pathogenesis of melanoma. Using lentivirus-mediated RNAi technology, we knock down the genes in melanoma cell lines. The effect of these genes on cell proliferation was assessed by MTT and plate colony formation assays. Then the cell cycle distribution was analyzed.Cell cycle-related moleculars and cell apoptosis-related moleculars were detected to understand the molecular mechanism of target gene in regulating the proliferation in melanoma. At last, we choose on molecular and investigated its expression on 58 cases of melanoma tissue specimens and 49 cases of mormal skin tissue specimens using Immunohistochemistry analysis. We then analysis the positive expression of this protein and its relationship with clinical factors for melanoma(such as gender, age, metastasis). Results: 1. We got 55 genes that are up-regulated in melanoma tissues and their fold change is greater than 5-fold. We chose 20 genes as candidate genes and their expression was verifiedin 3 cases of fresh tissues of malignant melanoma and 3 cases of normal skin tissues via q PCR analysis. We found that the trend of expression of 15 genes is consistent with the microarray results, that is, the expression of these 15 genes was significantly up-regulated in melanoma tissues than in normal skin tissues(P<0.05). EIF3 D 、MYO6 and ASNS was significantly up-regulated in melanoma tissues than in normal skin tissues. 2. The recombinant lentivirus which expresses sh RNA targeting EIF3 D or MYO6 or ASNS can efficiently infect A375 and A431 cells. After 3 days of lentivirus infection, more than 90% cells express green fluorescent protein(GFP). The expression of target genes in m RNA and protein levels is significantly down-regulated in A375 and A431 cells(p<0.05). These melanoma cells with stable silencing of target genes can be used as models in subsequent functional tests. Results of MTT assay showed that the cell proliferation was significantly impaired after 3 days of lentivirus and the proliferation was inhibited by more than 75% 5 days after of culture(p<0.05). Plate colony formation assay shows that the number of colony per 6-well plate is decreased by more than 80%(p<0.05). Moreover, the cell number in single colony is reduced. PI staining results indicated that the cell cycle distribution was also changed in Lv-sh EIF3 D, Lv-MYO6 or Lv-sh ASNS lentivirus infected cells. EIF3 D mainly regulates the G2/M phase progression, and MYO6 and ASNS mainly regulates the G0/G1 phase progression. Western blot results shows that konck down of EIF3 D resulted in decreased expression of CDK1、CDK2、CDK4 and phosphorylation of p53 and cyclin kinase inhibitor p27. The expression of cyclin B1、cyclin D1 and cell apotosis-related PARP is not significantly changed.3. Immunohistochemistry analysis indicated that the positive expression of EIF3 D was 100% and significantly higher than in normal tissues.However, the positive expression of EIF3 D is not significantly related with the clinical factors(such as gender, age, metastasis). Totally, the results suggest that EIF3D、MYO6、ASNS, especially the molecular EIF3 D plays important roles in the development of melanoma. They may be candidate target for treatment melanoma. Conclusions: Through screening the differential gene via microarray in fresh tissues and function verification in melanoma cell lines, we get three moleculars, EIF3 D, MYO6 and ASNS, that are related to the proliferation of melanoma. are highly specific expression in melanoma tissue. Lentivirus-mediated sh RNA can also effectively and safely silence the expression of EIF3 D or MYO6 or ASNS gene in Skin cancer cells and subsequently significantly inhibits cell proliferation. The study demonstrated the critical involvement of EIF3 D or MYO6 or ASNS in the progression of melanoma cells and depletion of EIF3 D or MYO6 or ASNS could be developed as a possible therapeutic option in the Diagnosis and gene therapy for melanoma. Further investigation should be performed to assess the function of these genes in animals to judge that lentivirus-mediated gene silencing is a potential clinical non-surgical treatment of Skin cancer. We choose EIF3 D as an example and investigate its deep mechanism. We find that EIF3 D regulates the cell proliferation by modulating the expression of cycle related proteins in melanoma. The insights molecular mechanisms of the this molecule that regulate malignant melanoma proliferation remains to be further studied.