The Expression And Function Of ADM In Human Melanoma

Malignant melanomar, which is the fastest-growing incidence ofcancer,received widespread attention in recent years and its clinical studiesalso progress rapidly. It is highly malignant melanoma, easy recurrence andmetastasis, poor prognosis.Studies have shown that5-year survival rate is lessthan60%in patients with>3mm about tumor thickness and less than5%,whose average age is only for6-9months, after a distant metastasis.Therefore, Comprehensive and in-depth study on the mechanism and thedevelopment of melanoma, seeking to find the index which can effectivelypredict the biological behavior, is of great significance in its early diagnosis,treatment and prognosis.Adrenomedullin (ADM), which was foundin the Organization of pheochromocytoma by Japan scholars in1993, is theendogenous Vasoactive Peptides.Miller MJ observed that it was highexpression of ADM in more than90%of patients with the cancer in lungs,intestines, breast, ovarian, prostate, brain, cartilage, blood and othertissue-derived. And he also pointed out that ADM played an important role inthe occurrence and development of cancer. Untill now, there is no reportADM expression in melanoma and it may play a role in biology about it. So, itneed further study.Part one：Expression of ADM in malignant melanomaObjective:To investigate the expression of ADM protein in human malignantmelanoma, cutaneous naevi and A375．Methods:It is detected with SP immunohistochemistry about the expression ofADM in cell lines A375,fibroblast of human and tissue of malignantmelanoma and cutaneous naevi. Results:The positive expression of ADM was found12cases(80％)in15casesmalignant melanoma and13cases(43.3%) in30cases of cutaneous naevi，with a significant difference(P<0.05)．The expression of ADM significantlyincrease in A375but in fibroblast the expression of ADM are weak.ConclusionsThe expression of ADM significantly increase in malignant melanomasuggest that ADM may correlate close with the tumorigenesis and progress ofmalignant melanoma.Part two: The function of ADM in malignant melanomaObjective:To investigate the influence for malignant melanoma after interferenceADM gene by siRNA.Methods:1.It is the experimental group that treated with the most effective ADM-siRNA, cells transfected non-special sequence as negative controlgroup,cells treated with nothing as blank control group.2.Select the most effective siRNA by qRT-PCR and SPImmunohistochemistry method.3.MTT was adopted to evaluate the influence on the cell proliferation.4.Apoptosis rate was determined with AnnexinV-EGFP/PI doublestaining and cell cycle with PI staining was detected by flow cytometry.5.Cell invasiveness was determined by Matrigel Transwell in vitroinvasion assay.Results:1.The siRAN-2was the most effective siRNA. The interferenceefficiency of siRAN-1,siRAN-2were15%，76%,50%by qRT-PCR.2. ADM-specific siRNA-2inhibited strongly ADM protein expression inA375cell lines. The expression of ADM in the group which transfected ADMsiRNA-2was lowest than that in other groups by SP Immunhistochemistry. There was no significantly difference in ADM expression between blankcontrol group and negative control group (P＞0.05).3. The proliferation of A375which knocked out ADM by siRNA withMTT assay: After transfection24、48hours, average absorbance ofexperimental group was0.389±0.0375、0.469±0.0330，which was significantlylower than that in blank control group(0.574±0.0733、0.685±0.0154) andnegative control group (0.548±0.0376、0.676±0.0374), with a significantdifference (P<0.05).There was no significantly difference between blankcontrol group and negative control group(P＞0.05).4. The early apoptosis rate of A375cell line after knocked out ADM bysiRNA showed (20.20±6.045)%in experimental group was higher thanthat of blank control group (1.67±0.340)%and negatigve controlgroup(4.59±1.294)%,with a significant difference(P<0.05).5. The percentages of cells in G2/M phase were significantly increased insiRNA-transfected cells(11.360±1.224)%than that of blank control groupcells(1.02±0.990)%and negatigve control group cells(4.150±1.032)%(P<0.05), There was no statistical difference between blank control groupcells and negatigve control group cells (P>0.05).6.The number of invasion cells was43.750±3.772in experimental groupafter knocked out ADM by siRNA significantly lower in cells compared withthe un-transfected cells70.500±2.723and blank vector transfectant67.500±3.795，with a significant difference(P<0.05). There was no statisticaldifference between un-transfected cells and blank vectortransfectants(P>0.05).MTT assay showed that the OD value of invasion cellswas obviously lower in experimental group cells0.185±0.055than that ofblank control group cells0.363±0.006and negatigve control group0.361±0.086,with a significant difference(P<0.05). There was no statisticaldifference between blank control group and negatigve control group (P>0.05).Conclusions1. ADM siRNA can effectively knock out the ADM gene and inhibit thegrowth of human melanoma cells (A375) 2. ADM siRNA can promote the apoptosis of A3753. ADM siRNA can inhibit the proliferation of siRNA cells via G2/Marrest and induction of cell apoptosis in vitro4. ADM siRNA suppresses the invasive ability of malignant melanomacells in vitro...