| Melanoma is a malignant tumor originating from skin or mucosal pigmented cells.According to statistics,the annual morbidity of melanoma in the world is increasing by 3 to 8 percent.Melanoma is the leading cause of death from skin malignant tumors among white people in the West,and it is also one of the diseases seriously affecting the life and health of our people.According to the location,melanoma can be divided into cutaneous melanoma,mucosal melanoma choroidal melanoma,etc.Cutaneous melanoma is the most common subtype.In western countries,cutaneous melanoma mostly occurs in sun-related areas.In our country,skin melanoma mostly occurs at the tip of the finger,under the nail,or at the sole of the foot.Melanoma is characterized by occult onset,rapid progression and high metastasis rate.The exact cause of melanoma is still being explored.Melanoma is insensitive to radiotherapy and chemotherapy.Surgery is the main treatment.Biotherapy is inefficient and limited in clinical application due to serious side effects and biosafety.Molecular targeting therapy and immunotherapy have achieved good results in recent years.Due to the complexity of pathogenic molecular mechanism,there is no specific drug that can cover and cure melanoma.The pathogenesis of melanoma needs to be further explored in order to find effective and specific therapeutic targets for its treatment.Retrospective studies showed that most melanoma patients,especially those with branch melanoma,had a history of pigmented nevus for several years or even twenty or thirty years before the onset of melanoma.Pigmented lesions have not changed for many years.Recently,it was found that the growth of pigmented lesions was accelerated,the margin of protuberance was irregular,the surface appeared blistering and damage,and the ulcer healed poorly.At this time,pathological biopsy confirmed malignant melanoma.Therefore,the analysis of gene expression difference between pigmented nevi and melanomas is helpful to find the pathogenic factors of melanoma.We used bioinformatics to select the Affymetrix company' s expression profile chip in GEO database for analysis.For the mRNA expression of melanoma,we downloaded and analyzed GEO data numbered GSE 46517.The chip raw data of 31 primary melanoma tissue and 9 tissue samples of pigmented nevi were downloaded.We use Affy and Limma in R language to standardize and analyze our data by matched T test,and then filter all the probe results according to the criteria of P value<0.05 and FC?1.5.After annotation,we get all the different genes,among which DDX39A gene is one of them.DDX39 protein,49KDa,is synthesized by coding DDX39A gene.DDX39 is a member of the DEAD box RNA helicases family.The DEAD box protein family consists of many members,including DDX1,DDX3,DDX5,DDX17,DDX39 etc.Some members of the DEAD box protein family are thought to be involved in the tyrogenesis,spermatogenesis,cell growth and division.There have been numerous reports indicating that DEAD box proteins are involved in processes that are key to cellular proliferation and/or neoplastic transformation,including the growth of tumor cells,cell cycle control,apoptosis,transshipment or removal of RNA,recombination of cytoskeleton or cell migration and signal transduction,etc.It also plays oncogenic and tumor-suppressive roles in different tumors.Up-regulation of DDX39 protein has recently been found in various human tumor tissues or cells.For example,lung squamous cell carcinoma,gastrointestinal stromal tumors,pancreatic cancer,prostate cancer,hepatocellular carcinoma,malignant mesothelioma.It has also been reported that DDX39 protein is down-regulated in some tumors,such as bladder urothelial carcinoma infiltrating muscle layer,and colorectal cancer,which indicates that DDX39 protein may plays oncogenic and tumor-suppressive roles in different cancer.Although DDX39 has been reported to regulate the progression of many tumors,but its role in tumor development and regulatory mechanisms has not been understood well.There is no report on the study of DDX39 protein in melanoma.Here we studied the role of DDX39 in melanoma growth,migration,invasion,and metastasis and analyzed the prognostic value of DDX39 in melanoma.We hope to provide useful molecular markers for the treatment and prognosis of melanoma.The research contents mainly include the following three parts:Part ? Down-regulation of DDX39 protein impact on the proliferation,invasion and migration of melanoma cellsObjective:Investigate the effects of down-regulation of DDX39 protein on the proliferation,invasion and migration of melanoma cells,and to study the biological role of DDX39 protein in melanoma cells.Methods:DDX39 protein was down-regulated by RNA interference mediated by lentivirus.Both melanoma cells A375 and SK-MEL28 were infected with lentiviruses expressing DDX39 RNAi(shDDX39A group)and negative control lentiviruses(shCtrl group).MTT assay was used to detect the effect of down-regulation of DDX39 protein on cell proliferation.Flow cytometry was used to detect the effect of down-regulation of DDX39 protein on cell cycle and cell activity.Transwell chamber model was used to detect the effect of down-regulation of DDX39 protein on cell migration and invasion ability.Results:HCS cell count experiment showed that the fold change of day5/dayl of the A375 cell number was 8.51±0.44,the DDX39-interference(shDDX39A)group was 2.58 ± 0.05,with a statistical difference(P<0.01).The fold change of day5/dayl of the SKMEL28 cell number was 5.02±0.14,the DDX39-interference(shDDX39A)group was 3.30±0.04,with a statistical difference(P<0.01).The MTT assay showed that t the fold change of day5/dayl of the A375 cell number was 4.66 ± 0.16,the DDX39-interference(shDDX39A)group was 2.56±0.09,with a statistical difference(P<0.001).The fold change of day5/day1 of the SKMEL28 cell number was 2.69 ±0.04,the DDX39-interference(shDDX39A)group was 1.72±0.04,with a statistical difference(P<0.001).The results of cell cycle showed that after 5 days of A375 cell culture,the proportion of cells in the control group was 23.79%±0.14%in the G2/M phase,and the proportion of the shDDX39A group in the G2/M phase was 25.28%±0.54%,with a statistical difference(P<0.05);The results of cell cycle showed that after 5 days of SKMEL28 cell culture,the proportion of cells in the control group was 20.79%±0.41%in the G2/M phase,and the proportion of the shDDX39A group in the G2/M phase was 23.78%±0.97%,with a statistical difference(P<0.05);In the capase3/7 activity experiment,the fluorescence intensity value of the shCtrl group was 8727.33±254.58,and the fluorescence intensity value of the shDDX39A group was 13902.00±486.72 after 5 days of A375 cell culture,with a statistical difference(P<0.01).After 5 days of SKMEL28 cell culture,the fluorescence intensity value of the shCtrl group was 15260.30 ± 259.50,the fluorescence intensity value for the shDDX39A group were 21447.30 ± 605.48,with a statistical difference(P<0.01).Transwell migration experiment showed that the number of cells in the lower chamber of the A375 cell line shCtrl group was 121±1.29,and the number of cells in the shDDX39A group in the lower chamber was 26 ± 1.39;The number of cells in the lower chamber of the SK-MEL-28 cell line shCtrl group was 131±5.42,and the number of cells in the shDDX39A group to the lower chamber was 78±5.27.Compared with the control group,the mobility of the shDDX39A group was significantly lower(P<0.01).The invasion test showed that the number of cells in the lower chamber was 37±2.27 in the A375 cell line shCtrl group,7±0.5 in the shDDX39A group,62 ± 4.35 cells in the SK-MEL-28 cell line shCtrl group and 19±0.22 cells in the shDDX39A group.Compared with the control group,the mobility of shDDX39A group was significantly lower than that of the control group(P<0.01).Conclusions:Down regulation of DDX39 protein can significantly inhibit the proliferation of melanoma cells,which is due to cell cycle arrest,increase cell apoptosis,reduce cell invasion and migration ability.Part ? Down-regulation of DDX39 protein impact on the proliferation,invasion and migration of melanoma cells.Objective:To study the role and molecular mechanism of DDX39 in promoting the proliferation,invasion and migration of melanoma cells.Methods:Melanoma cell line A375 was separately infected with Lentivirus vector expressing DDX39 RNAi(shDDX39A group)and irrelevant sequence(shCtrl group).The expression levels of 22 proteins in two groups were examined by Western blot.shDDX39A group cells were reinfection with Lentivirus vector over-expressing down-regulated proteins to observe the effects on cell proliferation,migration and invasion.We hope o screen out the key proteins in the downstream signal transduction pathway that may act on DDX39.We can further study on the molecular mechanism of DDX39 promoting melanoma cell growth,invasion and migration.Results:Compared with shCtrl group,Western-Blot assay showed that six proteins were down-regulated in shDDX39A group.They are Snail,MYC,CDH2,P38mapk,AKT1 and NFKB,respectively.The over expressed lentivirus vector was designed and packaged to re-infect A375 cells in shDDX39A group.HCS cell counting test screened out Snail might be the positive protein for functional recovery test A375 cells were divided into three groups:NC+NC group(normal target cell A375 with negative control lentivirus vector),KD+NC group(normal target cell A375 with DDX39A gene shRNA lentivirus vector and over expression negative lentivirus vector)and KD+OE group(normal target cell A375 with DDX39A gene shRNA lentivirus vector and SNAI1 over expression lentivirus vector).HCS cell counting test showed that compared with NC+NC group,the proliferation fold of day,compared with dayl decreased in KD+NC group(6.02±0.57 vs 2.21 ±0.15,p<0.05),and compared with NC+NC group,the proliferation fold of day5 compared with day1 increased in KD+OE group(2.21±0.19 vs 4.19±0.23,p<0.05).MTT showed that compared with NC+NC group,the proliferation fold of day5 compared with day1 decreased in KD+NC group(6.72±0.07 vs 2.93±0.05,p<0.001),and compared with NC+NC group,the proliferation fold of day5 compared with day1 increased in KD+OE group(2.93±0.05 vs 4.91±0.05,p<0.001).The Caspase3/7 activity experiment showed that the activity of Caspase3/7 of apoptotic cells in KD+NC group was higher than that in NC+NC group(6348 ± 248.6 vs 12381.67 ± 138.87,P<0.001),and the activity of Caspase3/7 of apoptotic cells in KD+OE group was lower than that in NC+KD group(6408.67 ± 371.30 vs 12381.67 ± 138.87,P<0.001).Transwell migration experiment showed that the number of cells in the lower chamber of the NC+NC group was109±7.22,and the number of cells in the NC+KD group in the lower chamber was 48±1.60,and the number of cells in the lower chamber of KD+OE group was 91±0.39.It suggest that compared with NC+NC group cells,the migration rate of NC+KD group cells decreased(P<0.001),and compared with NC+KD group cells,the migration rate of KD+OE group cells increased(P<0.001).Transwell invasion experiment showed that the number of cells in the lower chamber of the NC+NC group was113±3.56,and the number of cells in the NC+KD group in the lower chamber was 30±5.41,and the number of cells in the lower chamber of KD+OE group was 106±3.46.It suggest that compared with NC+NC group cells,the invasion ability of NC+KD group cells decreased(P<0.001),and compared with NC+KD group cells,the invasion ability of KD+OE group cells increased(P<0.001).Conclusions:Snail signal transduction pathway may be one of the pathways in which DDX39 promotes the growth,invasion and metastasis of melanoma.Part ? The expression of DDX39 protein and its relationship with clinicopathological features and prognosis in melanoma tissue.Objective:The study aimed to investigate DDX39 expression in melanoma and nevus pigmentosus,the expression of DDX39,protein in primary and recurrent metastatic tumors,the relationship with clinicopathological features and prognosis in melanoma tissue.Analyze the relationship of Snail and DDX39 in melanoma tissue.Methods:51 paraffin specimens were collected from patients with melanoma who underwent surgery in our hospital.Fifteen of them also collected Matched Metastasis and recurrence tissues.In addition,paraffin-embedded tissues from patients with nevus pigmentosus were collected as control.Compare the expression of protein DDX39 in melanoma and pigmented nevus tissues,as well as in primary and recurrence/metastasis of tumors by Immunohistochemical methods.Analyze the relationship of clinicopathological features,DDX39 and prognosis in melanoma tissue.Analyze the relationship of Snail and DDX39 in melanoma tissue.Data analysis by SPSS 25.0 statistical software.Results:DDX39 protein is mainly localized in the nucleus and a small amount in the cytoplasm.The expression of DDX39 protein in melanoma tissue was significantly higher than that in pigmented nevus tissue(p<0.05).The relationship between DDX39 protein and clinicopathological features of melanoma showed that the expression of DDX39 protein was positively correlated with the AJCC clinical stage and Breslow depth of melanoma(p<0.05).The expression of DDX39 and Snail protein in 15 cases of melanoma and its matched recurrent and metastatic tissues was analyzed by Immunohistochemical method.The results showed that the expression of DDX39 and Snail protein were increased in recurrent and metastatic tissues(P<0.05).The relationship among DDX39 protein,clinicopathological parameters and prognosis of patients was studied by Kaplan-Meier method.It was found that DDX39 expression,the AJCC clinical stage,Breslow thickness and Clark grade were significantly negative correlated with the prognosis of melanoma(P<0.05).Patients with high expression of DDX39 protein have poor prognosis,DFS time significantly shortened(P<0.05).The OS time of patients with higher expression of DDX39 protein was shorter than that of patients with lower expression of DDX39 protein,but there was no statistical significance.Conclusions:The expression of DDX39 protein in melanoma tissue was significantly higher than that in pigmented nevus tissue.The expression of DDX39 and Snail protein was positively correlated with Breslow thickness and AJCC clinical stage.The expression of DDX39 and Snail protein in metastasis and recurrence of melanoma was higher,suggesting DDX39 might play a role in promoting the migration and invasion potential of melanoma.The higher expression of DDX39,the worse prognosis of melanoma patients. |
PDF Full Text Request