骨硬化蛋白在葡萄膜黑色素瘤发生和发展中的作用及其生物学机制

Role of sclerostin in the occurrence and development of uveal melanoma and its biological mechanism

目的探讨骨硬化蛋白(SOST)和WNT/CTNNB1信号通路对人葡萄膜黑色素瘤(UM)细胞细胞周期、迁移和侵袭的影响及其相关机制。方法分别收集20例上皮细胞型UM组织和16例梭形细胞型UM组织进行免疫组织化学染色检测SOST、Wnt-1和Catenin beta-1蛋白含量。选择3株人UM组织来源的细胞系OCM-1(原发梭型)、Mum-2B(转移上皮型)和Mum-2C(转移梭型),分为空白对照组、空质粒组和SOST siRNA组,其中空白对照组不转染质粒、空质粒组用SOST阴性对照质粒转染,SOST siRNA组用含SOST siRNA转染。转染24 h后,采用实时荧光定量PCR和Western blot分别检测SOST、CTNNB1、WNT蛋白家族1(WNT1)、CCND1、基质金属蛋白酶(MMP)2和MMP9的mRNA和相应蛋白表达水平;采用Transwell方法测定转染后细胞的侵袭和迁移能力;采用流式细胞术检测转染后各组细胞周期分布。取BALB/c nude雌性小鼠9只采用随机数字表法随机分为OCM-1组、OCM-1空载体组和SOST shRNA组,分别接种未感染慢病毒的OCM-1、感染空载慢病毒的OCM-1以及感染SOST shRNA慢病毒的OCM-1,观察SOST沉默对小鼠原位成瘤的影响。通过免疫共沉淀实验探讨OCM-1细胞SOST与低密度脂蛋白受体相关蛋白(LRP)-5/6蛋白相互作用。结果免疫组织化学染色结果发现恶性程度较低的梭形细胞型UM组织中SOST表达水平高于上皮细胞型UM组织,Wnt-1和Catenin beta-1表达水平明显低于上皮细胞型UM组织,差异均有统计学意义(均P<0.01)。实时荧光定量PCR结果显示,各细胞系中SOST siRNA组SOST mRNA相对表达量明显低于相应空质粒组,CCND1、WNT1及MMP9 mRNA相对表达量明显高于空质粒组,差异均有统计学意义(均P<0.05);OCM-1和Mum-2C细胞系中,SOST siRNA组CTNNB1 mRNA相对表达量明显高于空质粒组,差异均有统计学意义(均P<0.01)。Western blot结果显示,SOST siRNA组SOST蛋白相对表达量低于空质粒组,Wnt-1、Catenin beta-1、cyclin-D1、MMP2、MMP9蛋白相对表达量高于空质粒组,差异均有统计学意义(均P<0.01)。Transwell实验结果显示,各细胞系中SOST siRNA组的细胞侵袭和迁移能力明显强于空白对照组和空质粒组,差异均有统计学意义(均P<0.01)。流式细胞术显示SOST siRNA组G1期细胞比例以及G1/S期比值均明显低于空白对照组和空质粒组,差异均有统计学意义(均P<0.01)。OCM-1组、OCM-1空载体组和SOST shRNA组眼球体积分别为(42.7±4.6)、(49.0±22.9)和(135.2±32.7)mm3,总体比较差异有统计学意义(F=19.963,P<0.01),其中SOST shRNA组小鼠眼球较OCM-1组和OCM-1空载体组大,差异均有统计学意义(均P<0.05)。免疫共沉淀结果显示,SOST可分别与LRP-5和LRP-6相互结合发生相互作用。结论沉默SOST可促进UM细胞的侵袭和迁移,并使其处于分裂期的比例升高,可以促进肿瘤在裸鼠眼内的生长。SOST可能是通过与膜上受体LRP-5/LRP-6相互作用进而调节WNT/CTNNB1信号通路来发挥这一功能。

Objective To investigate the effects of sclerostin(SOST)and WNT/CTNNB1 signaling pathway on the cell cycle,migration and invasion of human uveal melanoma(UM)cells and its related mechanism.MethodsUM tissues from 20 cases of epithelioid UM and 16 cases of spindle cell type UM were collected.The contents of SOST,Wnt-1 and Catenin beta-1 proteins in the collected tissues were detected by immunohistochemical staining.Three human UM tissue derived cell lines OCM-1(primary spindle cell type),Mum-2B(metastatic epithelioid)and Mum-2C(metastatic spindle cell type)were selected and divided into three groups,blank control group not transfected,empty vector group transfected with SOST negative control vector and SOST siRNA group transfected with SOST siRNA.After 24-hour transfection,the mRNA and protein expression levels of SOST,CTNNB1,WNT protein family 1(WNT1),CCND1,matrix metalloproteinase(MMP)2 and MMP9 were detected by real-time fluorescence quantitative PCR and Western blot,respectively.The invasion and migration ability of the transfected cells were measured by transwell method,and the cell cycle distribution was detected by flow cytometry.Another 9 female BALB/c nude mice were selected and randomized into OCM-1 group,OCM-1 empty vector group and SOST shRNA group,inoculated with OCM-1 without lentivirus infection,OCM-1 with blank lentivirus infection and OCM-1 with SOST shRNA lentivirus infection,respectively.Six weeks after inoculation,the in situ formation of tumor was observed.The interaction between SOST and low density lipoprotein receptor related protein(LRP)-5/6 in OCM-1 cells was explored by co-immunoprecipitation assay.The study protocol was approved by the Ethics Committee of Tianjin Medical University Eye Hospital(2018KY[L]-20).ResultsImmunohistochemical staining results showed that the SOST expression level was higher and the expression levels of Wnt-1 and Catenin beta-1 were lower in spindle cell type UM tissues than in epithelioid UM tissues,and the differences were all statistically significant(all at P<0.01).The real-time fluorescence quantitative PCR results showed that the relative expression of SOST mRNA was significantly lower and the relative expressions of CCND1,WNT1 and MMP9 mRNA were significantly higher in SOST siRNA groups than in corresponding empty vector groups in the three cell lines(all atP<0.05).In OCM-1 and Mum-2C cell lines,the relative expressions of CTNNB1 mRNA were significantly higher in SOST siRNA groups than in empty vector groups(all atP<0.01).Western blot results showed that the relative expression of SOST protein was significantly lower and the relative expressions of Wnt-1,Catenin beta-1,cyclin-D1,MMP2 and MMP9 proteins were significantly higher in SOST siRNA groups than in empty vector groups(all atP<0.01).Transwell assay showed that the cell invasion and migration ability of SOST siRNA group was significantly higher than that of blank control group and empty vector group in the three cell lines(all atP<0.01).Flow cytometry showed that the proportion of G1-phase cells and the G1/S-phase ratio were significantly lower in SOST siRNA group than in blank control groups and empty vector groups(all atP<0.01).The eyeball volume of OCM-1 group,OCM-1 empty vector group and SOST shRNA group was(42.7±4.6),(49.0±22.9)and(135.2±32.7)mm3,respectively,showing a significant overall difference(F=19.963,P<0.01).The eyeball volume of SOST shRNA group was larger than that of OCM-1 group and OCM-1 empty vector group,and the differences were statistically significant(both atP<0.05).Co-immunoprecipitation results showed that SOST could interact with LRP-5 and LRP-6 by binding to them,respectively.ConclusionsSilencing SOST can promote the invasion and migration of UM cells,and increase the proportion of UM cells in the division phase.Silencing SOST can promote tumor growth in eyes of nude mice.SOST may play this function by interacting with the membrane receptor LRP-5/LRP-6 and then regulating the WNT/CTNNB1 signal pathway.