Silencing Of Notch1Expression By RNA Interference Suppresses Murine Malignant Melanoma Cell Proliferation And Tumor Growth

BackgroundMalignant melanoma (MM) is originated from neural crest melanin cells. The incidence of malignant melanoma increased rapidly in recent decades in the global, and the annual growth rate reached almost3-5%. Malignant melanoma was usually seen in caucasian populations, among them Queensland in Australia and Arizona in the United States have the highest incidence of malignant melanoma. Compare to the white, Asian countries including China and Japan have a lower morbidity. However in recent years, the incidence has a rapidly rising trend. Malignant melanoma is one of the most highly aggressive tumors in the world; it is usually difficult to be detected in early stage and easy to have metastasized distally. Recently it is receive the highest mortality rate of all the skin cancers in developed countries. Its prognostic factors are related with age, gender, position, depth of tumor infiltration, metastasis of lymph node, ulcer and surgery etc. Currently surgical treatment is still the best choice for cure early melanoma, but it has a higher recurrent rate. Patients with advanced metastatic malignant melanoma need to be treated with systemic system, but melanoma cells resists to conventional chemotherapy and radiotherapy, the prognosis is very poor. So seeking a more effective treatment for malignant melanoma is an urgent need.Notch signaling regulates cell fates and plays a fundamental role in cell differentiation, proliferation and apoptosis. Notch consists of four receptors (Notchl to Notch4) and five ligands (Delta-likel,3-4, and Jaggedl-2) in mammals. Notch receptor releases its extracellular domain and transmembrane domain (NICD) after cleaved by TNF-a-converting enzyme (TACE) and y-secretase complex when Notch signaling is activated by receptor-legend binding. Then, NICD translocates into the nucleus and interacts with CSL protein (CBF-1/suppressor of hairless/Lag-1) and mastermind-like (MAML1) protein, activating transcription of Notch target genes. Among the four receptors, Notchl is easily detected and the growth influence role of Notch has mainly been suggested on the basis of Notchl expression studies. It is worth noting that Notch signaling pathway can act as a tumor promoter role or tumor suppressor role in different cancer and different stages development of tumor. In the case of MM, there is strong evidence that Notchl is over activated through immunohistochemistry, and the main effect of Notch signaling appears to be clearly oncogenic and it may represent an early event in melanocytic tumor progression.siRNA technology as immediate area of regulating gene expression in recent years and it is a process of gene silence after transcription. Compared with the traditional tumor treatment, siRNA technology can specificity silence genes expression simply and effectively. Currently, the phenomenon of siRNA targeting Notchl gene inhibiting tumor cell growth and proliferation has been confirmed in glioma, kidney cancer, prostate cancer, pancreatic cancer, acute T lymphocyte leukemia etc.But it still needs to be confirmed by us whether siRNA targeting Notchl gene can down regulation Notch pathways and its downstream signaling to play its role of biology function in malignant melanoma. On the basis of previous study, RNA interference was used to identify the effects of Notchl on the regulation of malignant behaviors in melanoma. Murine melanoma B16F1cells and C57BL/6mice of the same source gene were selected as cell and animal model for in vitro and in vivo experiments. First we silenced Notchl gene in murine melanoma cell line B16F1, then transfect efficiency were analyzed by both RT-PCR and western blot. MTT assay and cell clone assay were used to evaluate the effects of Notchl siRNA-mediated interference on the proliferation of B16F1s in vitro. Transfect murine melanoma cell lines were used to establish in C57BL/6mouse models to observe tumor growth and detective the expression of Notchl protein, proliferation index Ki-67and PCNA proteins, to evaluate the effects of Notchl siRNA-mediated interference on the proliferation of B16F1s in vivo.Objective1. This study was to explore the possibility of proliferation suppression through silenced Notchl gene by siRNA in murine malignant melanoma.2. To explore wheather Notchl could be a target gene for treatment of malignant melanoma and to providing theoretical basis on seeking new ways for treating malignant melanoma.Materials and methods1. Materials1.1Cell and animalMurine malignant melanoma cell line B16F1of low metastatic, was a gift of Dr Yuru Meng (University of Chicago). Female C57BL/6mice,6-8weeks old and weighing approximately18-22g, were purchased from Laboratory Animal Center of Southern Medical University. Animal care and treatment were in accordance with institutional guidelines.1.2Experimental reagentsFetal bovine serum, High-glucose DMEM, LipofectamineTM2000, Notchl siRNA Oligo and negative control, Trizol, PrimeScript(?) RT reagent Kit, SYBR(?) Premix Ex TaqTM, Notchl primers, Goat anti-mouse Notchl polyclonal antibody, RIPA protein lysate, SDS-PAGE gel configuration Kit, MTT kit, DMSO, Rabbit anti-mouse Ki-67monoclonal antibody, Rabbit anti-mouse PCNA polyclonal antibody, Horseradish peroxidase (HRP) labeled secondary antibody anti-Goat or anti-rabbit IgG kit, DAB chromogenic agent, AEC chromogenic agent, Hematoxylin, Anhydrous ethanol,95%Alcohol,1%Hydrochloric acid alcohol etc.1.3Experimental instrumentCarbon dioxide incubator, Clean bench, Optical microscopy, Fluorescence microscope, High-speed refrigerated centrifugal machine, Bench centrifuge machine, Electrophoresis, LightCycler480Realtime PCR System, Paraffin slicing machine,-80℃refrigerator, Culture dishes, Straw, Pipettes, EP tube, Freezing tube, Slides, Coverslips etc.2. Cell cultureMurine malignant melanoma cell line B16F1were cultured as adherent cells in Dulbecco’s modified Eagle media (DMEM) supplemented with10%fetal calf serum. Cells were maintained at37℃in5%CO2in a humidified incubator. Cells were passaged every2-3days by combining trypsin with ethyle-nediaminetetraacetic acid(EDTA) when they reached a confluency of70-80%.3. Experimental procedure3.1Transfer murine malignant melanoma cell lines B16F1with siRNA targeting Notch1The siRNA Oligo targeting Notchl and negative control were designed through siDESIEN website and synthesised by Genepharma, The plasmids were transfected into B16F1cells using LipofectamineTM2000transfection reagent according to the manufacturers’ instructions. Three groups were established in the experiment:A: siNotchl group (B16F1cells transfected with siRNA targeting Notchl); B:siNC group (B16F1cells transfected with scrambled negative control siRNA); C:mock group (Mock transfection with only lipofectamineTM2000in B16F1cells). Transfect efficiency was analyzed by both RT-PCR and western blot.3.2Comparative analysis of growth characteristics for siNotchl transferred B16F1cells in vitroMTT and cell clone assay were used to evaluate the effects of Notchl siRNA-mediated interference on the proliferation of B16F1s in vitro. Calculation for clone formation rate:Number of clones/Number of vaccination cells×100%.3.3Comparative analysis of growth characteristics for siNotchl transferred B16F1 cells in vivoC57BL/6mice models were established with the three groups. To insure stable transfection effect, subcutaneous tumor received corresponding siRNA/PBS injection every3to4days. Mice were observed carefully and xenografted in mice were measured twice a week, tumor volume was calculated using the formula:(lengthxwidth2)/2. All mice were sacrificed humanely after the experiments. Tumor masses were removed for HE staining and immunohistochemical to observe changes in histopathological and to evaluation Notch1, Ki-67and PCNA expression. We calculated the percentage of positive staining cells in total tumor cells as the labeling index.4. Statistical analysisData were analyzes using SPSS13.0. All date were expressed as means±SD(x±s). Results of RT-PCR, western blot analysis, plate clone formation assay and immunohistochemical were assessed using one-way ANOVA. MTT assay and animal tumor volume were analyzed by Factorial analysis of variance square analyze. Differences were considered statistically significant when P<0.05.Results1. Expression of Notchl gene and protein were successfully knockdown by siRNA in B16F1s:Results show that siNotchl resulted in significant inhibition of Notchl mRNA expression compared to siNC group and mock group (P<0.05). In addition, the result of RT-PCR was confirmed by western blot72h after transfection. Expression of Notchl protein has also decreased in siNotchl group transfected with siRNA targeting Notchl compare to siNC group treated with scrambled negative control siRNA or mock group treated with LipofectamineTM2000only (P<0.05), and there is no difference between siNC and mock (P>0.05).2. SiNotchl can suppress cell proliferation and clone formation in malignant melanoma cells B16F1in vitro:MTT assay shows that, siRNA-mediated down regulation of Notchl reduces proliferation in B16F1cells from the second day after transfection. Colony-formation also reveal that the clone formation rate were0.157±0.021in siNotchl groups,0.760±0.030in siNC groups and0.787±0.031in mock groups respectively. Clone formation rate of siNotchl has also decreased in compare to siNC or mock(P<0.05), but siNC and mock group had no difference(P>0.05).3. In vivo antitumor effect of siNotchl:Results shows that tumor growth was remarkably suppressed in mice treated with siNotchl. On the day31, all mice of each group were sacrificed and tumors were removed. The average tumor volume of three groups were1307.947±161.914mm3,2970.683±140.945mm3and3078.987±252.257mm3, siNotchl group was significantly reduced compared with control groups (P<0.05), siNC and mock had no difference(P>0.05). To investigate tumor cell necrosis, HE staining were applied in the treated areas. The tissue of siNotchl group produced numerous inflammatory necrosis microscopically, however richer blood vessels and less necrosis were contained in tumor tissue of siNC and mock group. Immunohistochemical results shows that Notchl was intensely expressed in siNC and mock groups, whereas markedly down-regulated in siNotchl group. In comparison to labeling index in siNC group were (87.20±4.53)%and mock group were (90.36±5.25)%, labeling indexes of siNotchl group were (41.44±6.84)%, and were statistically different (P<0.05). As well as Notchl expression, the expression of Ki-67and PCNA protein were also down-regulated in malignant melanoma tissue in siNotchl group compared to siNC or mock group (P<0.05).Conclusions1. It is possible that the expression of Notchl in murine melanoma cell lines B16F1could knockdown by siRNA transfection technique. siNotchl could suppress tumor growth and cell proliferation in malignant melanoma in vivo and in vivo.2. Notchl is very important for the growth of malignant melanoma cells, which could be an effective target gene for treatment of malignant melanoma, and siNotchl could be an effective way for treatment of malignant melanoma.