Effect Of NFIB On Proliferation,Invasion And Metastasis Of Malignant Melanoma And Its Mechanism

[Objective] To investigate the effect and preliminary mechanism of NFIB involved in malignant melanocytes.[Methods] The expression of NFIB in human melanoma was detected by immunohistochemistry(IHC)assay,the bioinformatics database was used to explore the relationship between survival rate of Melanoma patients and gene expression of NFIB;the knockdown and overexpression of NFIB gene in melanoma cell lines were constructed by packaging NFIB-specific si RNA lentivirus(Lv-si NFIB)and NFIB c DNA lentivirus(NFIB-c DNA),Real time PCR and Western blot(WB)confirmed its effectiveness;CCK8 method,plate clone formation experiment,Transwell chamber experiment and scratch test to test effect of NFIB gene on proliferation,invasion and migration of melanoma cells;TGF-β induces EMT in malignant melanoma,basing on this model studies to study the effect of NFIB on the morphology in EMT of malignant melanoma.the EMT-related protein expression were detected by Western Blot.[Results] 1.The expression of NFIB was significantly higher in melanoma tissues than benign nevus tissues and normal skin tissues(p <0.001).After further verification in the database of bioinformatics,NFIB in melanoma tissues is higher than that in benign pigmented nevus and normal skin tissues,which is consistent with the results of immunohistochemistry in the specimens.Patients with high expression of NFIB gene suggest poor prognosis.2.CCK8 experiments and plate colony formation experiments showed that the ability of proliferation in si NFIB-transfected melanoma cells was decreased(p < 0.01)and the number of clone formation rates were decreased(p < 0.01).Transwell chamber experiments and scratch tests explored that the number of invasion and migration of si NFIB-transfected melanoma cells were depressed(p < 0.05).3.After TGF-β1 stimulation,the cells of NFIB-si Nc group changed from paving stone-like morphology to spindle-shaped cells,and the gap between cells and cells was significantly widened.The number of cells in the same visual field decreased,which was consistent with cell morphology during EMT.Compared with the TGF-β1 group,the NFIB-si RNA group melanoma cells showed paving stones,the cells were evenly arranged,and the number of cells in the same field of view increased.4.WB assay showed that the expression levels of E-cadherin(p < 0.01)and ZO-1(p < 0.05)were elevated in both NFIB-si RNA groups,while the expression levels of N-cadherin and vimentin were concurrently diminished(p <0.001),which were in accordance to the tendency of EMT transformation.5.WB assay showed that the expression of ZEB1 protein in melanoma cell line overexpressing NFIB was higher than that in the control group(p < 0.01),and the expression of interstitial phenotype markers such as vimentin and N-cadherin was increased(p < 0.01).The expression of epithelial phenotype markers ZO-1 and E-cadherin was decreased(p < 0.01).Meanwhile the expression levels of ZEB-1 was decreased in both NFIB-si RNA groups(p < 0.01).[Conclusions] This study found that NFIB is generally highly expressed in melanoma tissues and melanoma patients with high expression of NFIB suggest a poor prognosis.In addition,NFIB has the ability to promote melanoma cell proliferation,invasion,metastasis.And NFIB may mediate the EMT in melanoma cells by ZEB1,but its mechanism needs further investigate.