核转录因子E2F6通过β联蛋白信号通路调控恶性黑素瘤细胞增殖和转移的机制研究

An investigation into the mechanisms underlying the regulatory effect of the E2F6 transcription factor on proliferation and metastasis of malignant melanoma cells through β-catenin signaling pathway

目的研究核转录因子E2F6在人恶性黑素瘤组织和细胞系中的表达,及其对恶性黑素瘤细胞A375增殖、迁移和侵袭的影响。方法收集重庆市中医院2012年1月至2017年12月皮肤科确诊的50例皮肤恶性黑素瘤和30例色素痣冻存组织及石蜡切片。通过qRT-PCR分析E2F6 mRNA在人恶性黑素瘤和色素痣组织及7株恶性黑素瘤细胞系(HM、A375、WM451、WM35、SK-MEL-1、Hs-695T、MDA-MB-435s)和色素痣细胞中的表达,免疫组化和Western印迹检测E2F6、β联蛋白在人恶性黑素瘤组织中的表达。采用脂质体转染法将E2F6抑制质粒和对照质粒转染至A375细胞,通过qRT-PCR和Western印迹验证E2F6基因的敲减效率。通过CCK8、软琼脂平板克隆实验、Transwell迁移和侵袭实验、3D细胞培养实验检测E2F6基因敲减对A375细胞增殖、迁移和侵袭的影响,流式细胞仪检测细胞周期和凋亡率,通过Western印迹检测总β联蛋白、活化β联蛋白、c-Myc和细胞周期蛋白D1等水平。两组间比较采用t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验;采用Pearson相关系数分析皮肤恶性黑素瘤中E2F6和β联蛋白表达的相关性。结果7株恶性黑素瘤细胞系E2F6 mRNA相对表达水平均高于色素痣细胞(均P<0.001)。qRT-PCR显示,皮肤恶性黑素瘤组织中E2F6 mRNA的相对表达(0.00055±0.00017)高于色素痣组织(0.00018±0.00009,t=3.22,P<0.001)。免疫组化、Western印迹显示,皮肤恶性黑素瘤组织中E2F6的相对表达水平高于色素痣组织(均P<0.001),而β联蛋白的相对表达水平低于色素痣组(均P<0.001)。相关性分析显示,恶性黑素瘤组织中E2F6蛋白与β联蛋白的表达呈负相关(免疫组化:r=-0.56,Western印迹:r=-0.63,均P<0.01)。敲减A375细胞E2F6基因后,E2F6抑制组E2F6 mRNA、蛋白相对水平低于对照组(t=3.38、2.76,P<0.001)。CCK-8实验显示,继续培养后48 h,E2F6抑制组细胞增殖能力低于对照组(t=4.58,P<0.01);软琼脂平板实验显示,E2F6抑制组细胞相对克隆比低于对照组(t=2.26,P<0.001);迁移实验显示,E2F6抑制组穿出小室细胞数(165±23)低于对照组(376±22,t=3.14,P<0.01);侵袭实验显示,E2F6抑制组穿出小室细胞数(96±11)低于对照组(315±31,t=2.12,P<0.01);3D细胞培养实验显示,E2F6抑制组细胞形态发生明显变化,侵袭性伪足消失。流式细胞仪检测显示,E2F6抑制组G0-G1期细胞比例、细胞凋亡率均高于对照组(均P<0.001)。Western印迹显示,E2F6抑制组β联蛋白水平、活化β联蛋白水平及其下游靶基因蛋白c-Myc、细胞周期蛋白D1水平均低于对照组(P<0.001),P21蛋白水平高于对照组(P<0.001);E2F6抑制组上皮-间质转化相关分子波形蛋白、神经钙黏着蛋白水平低于对照组(P<0.001),而上皮钙黏着蛋白水平高于对照组(P<0.001)。结论核转录因子E2F6在恶性黑素瘤中高表达,敲减A375细胞E2F6基因可能通过拮抗β联蛋白信号抑制细胞增殖、迁移和侵袭。

Objective To determine the expression of the E2F6 transcription factor in human malignant melanoma tissues and cell lines,and to evaluate the effect of E2F6 on proliferation,migration and invasion of a malignant melanoma cell line A375.Methods Frozen tissues and paraffin-embedded tissue sections were collected from 50 cases of cutaneous malignant melanoma and 30 cases of pigmented nevus in Department of Dermatology,Chongqing Traditional Chinese Medicine Hospital from January 2012 to December 2017.Quantitative reverse transcription-PCR(qRT-PCR)was performed to determine the mRNA expression of E2F6 in the malignant melanoma and pigmented nevus tissues,as well as in 7 malignant melanoma cell lines(HM,A375,WM451,WM35,SK-MEL-1,Hs-695T and MDA-MB-435s)and pigmented nevus cells,and immunohistochemical study and Western blot analysis were conducted to determine the protein expression of E2F6 andβ-catenin in the malignant melanoma tissues.An E2F6-inhibiting plasmid and a control plasmid were separately transfected into A375 cells by using a liposome-mediated transfection method,and the E2F6 gene-knockdown efficiency was verified by qRT-PCR and Western blot analysis.Cell counting kit-8(CCK8)assay,soft-agar plate cloning assay,Transwell migration and invasion assays and 3D cell culture assay were conducted to evaluate the effect of E2F6 gene knockdown on the proliferation,migration and invasion of A375 cells,flow cytometry was performed to detect the cell cycle and apoptosis rate,and Western blot analysis was conducted to determine the protein expression of totalβ-catenin,activatedβ-catenin,c-Myc and cyclin D1.The comparison between two groups was carried out by t test,the comparison among several groups by one-way analysis of variance,and multiple comparisons by least significant difference t test;Pearson correlation coefficient was used to analyze the correlation between E2F6 andβ-catenin expression in cutaneous malignant melanoma.Results The E2F6 mRNA expression was significantly higher in the 7 malignant melanoma cell lines than in the pigmented nevus cells(all P<0.001).qRT-PCR showed that the relative mRNA expression of E2F6 was significantly higher in the cutaneous malignant melanoma tissues(0.00055±0.00017)than in the pigmented nevus tissues(0.00018±0.00009,t=3.22,P<0.001).Both the immunohistochemical study and Western blot analysis showed significantly increased E2F6 protein expression,but decreasedβ-catenin protein expression in the cutaneous malignant melanoma tissues compared with the pigmented nevus tissues(all P<0.001).Correlation analysis showed that E2F6 protein expression was negatively correlated withβ-catenin expression in the malignant melanoma tissues(immunohistochemical study:r=-0.56,Western blot analysis:r=-0.63,both P<0.01).After knockdown of the E2F6 gene in A375 cells,the mRNA and protein expression of E2F6 was significantly lower in the E2F6 inhibition group than in the control group(t=3.38,2.76 respectively,both P<0.001).CCK8 assay showed that the cellular proliferative ability was significantly lower in the E2F6 inhibition group than in the control group(t=4.58,P<0.01)48 hours after transfection;soft-agar plate cloning assay showed that the colony-formation ratio was significantly lower in the E2F6 inhibition group than in the control group(t=2.26,P<0.001);Transwell migration and invasion assays showed that the number of cells crossing the chamber was significantly lower in the E2F6 inhibition group(165±23,96±11 respectively)than in the control group(376±22,315±31,t=3.14,2.12,respectively,both P<0.01);3D cell culture assay showed that the cell morphology markedly changed,and the invasive pseudopodia disappeared in the E2F6 inhibition group.Flow cytometry revealed that the proportion of cells at G0-G1 phase and apoptosis rate were significantly higher in the E2F6 inhibition group than in the control group(both P<0.001).Western blot analysis showed significantly decreased protein expression ofβ-catenin,activatedβ-catenin and its downstream target proteins c-Myc and cyclin D1,but significantly increased protein expression of P21 in the E2F6 inhibition group compared with the control group(all P<0.001);additionally,the E2F6 inhibition group showed significantly decreased protein expression of epithelial-mesenchymal transition-related molecules vimentin and N-cadherin,but significantly increased expression of E-cadherin compared with the control group(all P<0.001).Conclusions The E2F6 transcription factor is highly expressed in malignant melanoma.Knockdown of the E2F6 gene in A375 cells can inhibit cell proliferation,migration and invasion by antagonizing theβ-catenin signaling pathway.