Effects Of PPO Gene Silencing By SiRNA On Proliferation And Apoptosis On Human Malignant Melanoma Cells A375

Objective:Malingant melanoma was a kind of malignant cancer derived from neural crest melanocytes.Its incidence rate was ranking the third in the malignant skin canner(6.8%-20%)[1].It was the malignant cancer which cause death most easily. Malingant melanoma metastasis was higher and earlier than the other canner, so the prognosis is serious. Early diagnosis and proper staging was the key treatment to malignant melanoma. Complete surgical removal of lesions was the best treatment options for the early and noninvasive lesions. For advanced patients, the main treatments were chemotherapy and immune therapy. Chemotherapy drugs can be used alone or combined with other therapy [2]. However, all the treatments were not satisfactory and immunotherapy is still in experimental stage.PPO (proliferation and phosphorylation oncogene) gene was a new canner gene. Liu Siyuan[3] found it in the 1p36 Region by cloning technology. The gene number is KIAA0090. Its DNA length is about 36000bp,and cDNA length is about 6022bp. The new canner gene has a total of 25 exons and encodes 993 amino acids. The gene had been expressed in a variety of malignant cancer tissues and cells. They were closely related to cells proliferation and can phosphorylate MEK1 / 2 and ERK2 in the MAPK pathway .So we name the gene as the PPO (proliferation and phosphorylation oncogene).RNA interference (RNAi) was a new method of cancer treatments in the genetic level. RNA interference was a kind of gene silence induced by double-stranded RNA (ds-RNA). With the evolution of malignant melanoma, some patients resisted with chemotherapy. RNA interference technology was a new avenue for the treatment of the malignant melanoma. By using the cDNA sequences of PPO gene fragments, Ya-Ling Liu [4]immunized female BALB / c mice. Prepared spleen cells of the mice for feeder cells, Fused myeloma cells and spleen cells. Then cloned the hybridoma antibody. Determined the Ig subclass of the hybridoma antibody. They injected hybridoma cells into the abdominal cavity of the mice. Then extracted the ascites and purified the antibody. In this way finished, producing PPO antibody .Then they detected the expression of PPO gene with western blot, and PPO antibodies were detected. The results showed that, PPO line was located in the 140×103 near the membrane. Western blotting clearly showed that the PPO antibody specificity can be used to detect malignant tumors. PPO gene and gene-related protein were over expression in A375 cells. We can inhibited the expression of PPO gene-related protein in the MAPK pathway inhibited the growth and proliferation of MM in cell level need for research.Method:1 cell cultureThe A375 cells were cultured in DMEM serum medium (including penicillin 100U/ml, streptomycin 100U/ml, PH7.2) containing 10% fetal bovine and placed in the incubator at 37℃with saturated humidity and 5% CO2.2 The differentiation and morphology of cell were observed by inverted microscope.3 Observed the adenovirus transfection efficiency,the differentiation and morphoiogical change of A375 cells by fluorescence microscope when MOI (the number of virus / cell number) = 5,10,20,50,100.4 Detected the expressions of mRNA of PPO after transfection at 48 hours by RT-PCR.5 After transfection 1 day, 3 days, 5 days, 7 days,when MOI = 50,detected the proliferation of A375 cells by MTT. The cells were divided into negative control group adding no drugs, empty retroviral group adding Adrs-5, experimental group adding the same amount of PPO.6 When MOI = 50,detected the early apoptotic by flow cytometry technique 1 day, 3 days, 5 days, 7 days after transfection.7 Statistic analysis: the data was analyzed by statistical software SPSS13.0 for windows, Based on the One-way ANOVA with a = 0.05 as significant differences standards.Results:1 The human A375 cells without adenovirus transfected were spindle or polygonal, and grew adhesively with even distribution and almost the same size. They were strong proliferation and rare karyopyknosis.2 When MOI = 5,10,20,50,100 the transfection efficency was found to be about 50%,67%,69%,70%,70% respectively,when 24 hours after transfection.When MOI = 50 the transfection efficiency is the best .The transfection did not change any more with the MOI increased. So took MOI = 50 in the following check the following transfection experiments. Some A375 cells were found in varying degrees of shrinkage, cracking, and poor state of cell growth, some cells became long and narrow. While the negative control group and empty retroviral cells were distributed with the same size and strong growth. We found only a few off the wall cells.3 Detected of PPO gene mRNA of A375 cells by RT-PCR after transfection 48 hours. The mRNA of experimental group was less expressed than that transfection of the negative control group, which showed that adenovirus has been successfully transfected .A375 cells and the PPO gene was silenced partly .4 To detect growth inhibition rate of A375 cells by MTT: The difference of absorbency among the three groups was not significant (p> 0.05) 1 days, 2 days and 3 days after transfection. There were not significant difference between the control group and empty retroviral group 5 days, 7 days after transfection (p>0.05). The absorbency of experimental group was significantly higher than control group and empty retroviral group 5 days, 7 days after transfection (p<0.05) Conclusion:1 PPO adenovirus vector successfully transfected into human A375 malignant melanoma cells.2 The level of PPO gene expression could be reduced after transfection by adenovirus.3 Bedown PPO gene could inhibit the growth of A375 cell proliferation.4 PPO adenovirus vector transfected A375 melanoma cells can induce early apoptosis in A375 cells.