| Background and purposeThe Yin Yang 1(YY1)transcription factor has been identified to target a plethora of potential target genes,which are important for cell proliferation and differentiation.Although the role that YY1 plays in different human types of cancer have been reported,its biological and mechanistic significance in melanoma has not been well defined.Yin Yang 1(YY1)is a ubiquitously distributed transcription factor belonging to the GLI-Kruppel class of zinc finger proteins that is involved in repressing and activating a diverse number of promoters.YY1 is involved in approximately 10%of the total mammalian genes,the expression levels of YY1 must be tightly monitored for the survival of cells and organisms.Accordingly,abnormal YY1 protein levels have been shown to affect the clinical behavior of several cancer types.Melanoma is the deadliest form of skin cancer,characterized by a rapid progression,metastasis to regional lymph nodes and distant organs as well as a limited efficiency of therapeutics.It is readily curable if diagnosed at an early stage,however a large percentage of melanomas arise without association with premalignant nevi.This leads to ineffective early detection and results in approximately 10%of patients presenting with metastatic disease upon first diagnosis.Studies have repeatedly showed that in the majority of the tumors studied,YY1 transcript levels are significantly higher than in the relative normal counterparts for each cancer type analyzed.Elevated YY1 level were consecutively found in ovarian cancer,breast cancer,cervical cancer,and osteosarcoma.However,there is limited information available as to the involvement of YY1 in melanoma.In this study,elevated YY1 levels were observed in patients with melanoma,compared with benign nevi and normal tissue controls.Furthermore,we found that the increased YY1 was associated with melanoma metastasis state and tumor stage.More importantly,we identified miR-9 as a down-stream target of YY1,and both YY1 and miR-9 are functionally important in regulating melanoma growth and progression.Additionally,RYBP was shown to be a direct target of miR-9 through binding to its 3’ UTR,thus forming a YY1～miR-9～RYBP axis.Objective:Given the fundamental nature of YY1 in different human cancers,we decided to study the role of YY1 in human melanoma.In this study,we performed RT-qPCR analysis to detect YY1 mRNA levels in benign nevus and melanoma specimens.We examined the effects of YY1 levels on the proliferation,differentiation,migration and invasion of malignant melanoma cells in vitro by regulating the differential expression of YY1 in malignant melanoma cell lines.We further identified the expression regulation in YY1 melanoma,and to elucidate its functional significance in regulating the growth and development of melanoma.In addition,RYBP,in which the PYG protein physically interacts with YY1,was identified as a novel target for miR-9.We also found that YY1 can affect RYBP levels in melanoma cells,resulting in the regulation of YY1-miR-9～RYBP.Further revealing the preliminary molecular mechanism of YY1 play a regulatory role in melanoma,providing a preliminary theoretical basis for exploring new targets for melanoma diagnosis and treatment.Method:1.We first analyzed the expression of YY1 mRNA in 14 benign sputum and 24 melanoma specimens by RT-qPCR,including 15 metastatic melanomas.The relationship between the parameters between the clinical pathology and age,gender,TNM staging.2.The relative levels of YY1 mRNA in normal tissues and melanoma cell lines(WM852,WM1791C,WM8,WM209,FO-1,WM983A,WM793 and Daju);3.Immunoblotting analysis was performed to detect the expression of YY1 in normal melanocyte strains(in normal melanocyte strains(HEM)and malignant melanoma cell lines(WM852,WM1791C,WM8,WM209,FO-1,WM983A,WM793 and Daju).At the same time,a melanoma cell strain suitable for the experiment was screened,and a cell line stably expressing YY1 in low level was established by transfecting siRNA.4.CCK8 and colony formation assays examined the effect of YY1 on the proliferation of melanoma cells in vitro.Transwell migration and invasion assays examined the effect of YY1 on migration and invasion of melanoma cells in vitro.5.ChIP-q-PCR analysis confirmed that in the melanoma cells,the YY1 site is only present in the upstream gene sequences of miR-9-1 and miR-9-3,and YY1 inhibits miR-9 expression in melanoma cells,overexpression of miR-9 in melanoma cells can inhibit the proliferation,metastasis and invasion of malignant cells.6.The luciferase reporter assay was used to detect the signal molecules miR-9 and RYBP of YY1 target in melanoma cells.si-YYl and miR-9 inhibitors were added during the experiment.Immunoblotting analysis was performed to test the changes in the expression levels of signaling pathway-related proteins.7.Immunoblotting assay:The protein levels of RYBP in WM1791C and WM209 cells which were transfected with mimic control or miR-9 mimics were analyzed,and miR-9 inhibitors were applied immediately after application of si-YY1 to compare RYBP protein levels.8.The t-test and a non-parametric test were undertaken to analyze the data.A two-sided P-valueof less than 0.05 was considered statistically significant.All statistical computations wereperformed using SPSS(SPSS Inc.,Chicago.IL,USA).and we use SPSS to analyze the data.Results1.In 14 normal pigmented nevi tissues and 24 malignant melanoma tissues,YY1 mRNA was highly expressed in malignant melanoma tissues;and YY1 expression was significantly positively correlated with TNM stage;metastatic melanoma compared with the primary tissues,the level of YY1 in metastatic melanoma is higher than in primary melanoma.No correlation was observed between YAP1 expression and age,gender,location or tumor size.The expression of YY1 in malignant melanoma cell lines(WM852,WM1791C,WM8 and WM209)was significantly higher than that of normal melanocyte strains.progression by modulating the miR-9～RYBP axis.2.Immunoblotting techniques were used to detect a significant increase in endogenous YAP1 protein levels in the melanoma and GC cell lines(WM852,WM1791C,WM8 and WM209)relative to the four normal tissue controls.The WM1791C and WM209 melanoma cell lines were finally screened for subsequent signaling pathway studies.Transfected siRNA to establish a stable and low expression of YY1 cell line,YY1 specific siRNA and control siRNA were transfected into WM1791C and WM209 cells,and the siRNA YY1 was confirmed to be low expression level by RT-qPCR.3.YY1 specific siRNA was transfected into WM1791C and WM209 cells,and YY1 levels were accurately determined by RT-qPCR.Compared to WM1791Ca and WM209 cells that were not transfected with siRNA,cells transfected with siRNA,WM1791C and WM209 cells,showed significant proliferation reduction.At the same time,S phase cells in WM1791C and WM209 cells transfected with siRNA were reduced by 20%and 18%,respectively.Therefore,inhibition of YY1 can effectively inhibit the proliferation of tumor cells and the cycle of cells in vitro.By using CCK-8 for the Si-YY1 or si-control-treated WM1791C and WM209 cells,the proliferation of cells after low expression of YY1 was measured,and the proliferative ability was significantly inhibited.4.YY1 enhances the ability of WM1791C and WM209 cells to migrate and invasion in vitro.WM1791C and WM209 cells transfected with si-YYl and siRNA were used to analyze cell transfer and invasion ability.The carcinogenic YY1 is inhibited,resulting in a significant reduction in cell migration during the repair process of artificial damage on laminar cell flow.At the same time,Transwell experiments also showed that the migration and invasion of WM1791C cells were inhibited after low expression of YY1.5.The inhibition of YY1 doubled the content of miR-9,indicating that YY1 achieves high expression of miR-9 by transcriptional repression in melanoma cells.Comparing the expression levels of miR-9 in normal tissues,benign nevi and malignant melanoma,it was found that melanoma tissue was significantly reduced compared with normal tissues.miR-9 also differs in primary and metastatic melanoma,with a markedly high expression in primary melanoma tissue.The expression of miR-9 gradually decreased with tumor stage,and the expression of miR-9 was compared between different melanoma cell lines and normal controls.The level of miR-9 was negatively correlated with tumor metastasis and different tumor development stages.6.The over-expression of miR-9 in melanoma cells inhibits proliferation,metastasis and invasion of tumor cells.The levels of miR-9 in WM852,WM1791C,WM8 and WM209 were all significantly reduced.The miR-9 levels in WM1791C and WM209 cells transfected with miR-9 were 200-fold.The CCK-8 proliferation assay showed that cell growth and circulatory system were inhibited after miR-9 transfection,indicating miR-9 inhibited the metastasis of tumor cells.In vitro invasion experiments of transwell cells confirmed that WM11791C cells transfected with miR-9 showed a significant decrease in cell invasion ability7.The pMIR-reporter plasmid was constructed and transfected into 293T cells using miR-9 mimic and control group mimic,respectively.Luciferase reporter experiments confirmed that miR-9 significantly reduced the luciferase intensity of a wide range of RYBP reporter genes,and immunoblot analysis in WM1791C,WM209 cells showed that RYBP protein was reduced in cells transfected with miR-9 mimic double.YY1 negatively regulates miR-9 and miR-9 to inhibit RYBP expression,and a decrease in YY1 level leads to a significant decrease in RYBP levels.Si-YY1 and miR-9 inhibitors were injected into WM1791C cells,si-YY1 reduced RYBP protein levels by a factor of two,and miR-9 inhibitors were applied immediately after application of si-YY1,resulting in RYBP protein compared to the control group.The increase in level indicates that YY1 regulates RYBP by regulating miR-9.It was finally confirmed that RYBP can regulate the proliferation,metastasis and invasion of melanoma cells,and further confirmed the existence of the regulatory axis of YY1-miR-9～RYBP.Conclusion:Main Innovative Points 1.Studies have repeatedly showed that in the majority of the tumors studied,YY1 transcript levels are significantly higher than in the relative normal counterparts for each cancer type analyzed.For the first time in this study,we analyzedthe expression and effect of YY1 in melanoma.2.We estimated that YY1 as a transcriptional repressor of miR-9 and a promoter of melanoma growth and progression by modulating the miR-9～RYBP axis.It is essential to identify additional regulators and molecules that play critical roles in melanoma growth and progression,which could serve as the potential points of intervention for future therapies. |
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