CPSF6调控Notch信号通路促进破骨细胞分化及乳腺癌骨转移

CPSF6 regulates Notch signal pathway to promote osteoclast differentiation and bone metastasis of breast cancer

目的:探讨剪切及多聚腺苷酸化特异因子6(cleavage and polyadenylation specificity factor,CPSF6)在乳腺癌骨转移中的作用及机制。方法:采用实时荧光定量PCR(RT-qPCR)、免疫印迹法(Western blotting,WB)、免疫组织化学染色(immunohistochemical staining,IHC)分析12例乳腺癌骨转移患者转移灶及20例乳腺癌患者原发灶CPSF6表达情况。采用细胞实验和体内转移模型探索CPSF6对肿瘤细胞生长和转移能力的影响,及CPSF6敲低对乳腺癌细胞骨转移能力的影响。分析乳腺癌细胞通过CPSF6重塑细胞外微环境的能力,并检验CPSF6对下游靶基因的调控作用。结果:CPSF6在乳腺癌患者转移灶中表达水平显著增高。CPSF6与乳腺癌细胞的增殖和迁移能力正相关。在231-BM细胞中敲低CPSF6后,其分泌的细胞上清降低破骨细胞前体细胞向成熟细胞分化的能力。CPSF6敲低的细胞在小鼠体内形成溶骨性病灶的能力显著降低(P<0.01),而且转移灶外周的破骨细胞数量显著下降(P<0.01)。CPSF6缺失能下调肿瘤细胞内Notch通路配体JAG1的表达水平,导致微环境中破骨前体细胞Notch通路失活并抑制下游靶基因。结论:乳腺癌骨转移灶中存在CPSF6基因过度表达。CPSF6通过调控Notch信号通路,促进肿瘤组织周围破骨细胞的成熟和溶骨活性,从而导致乳腺癌的溶骨性骨转移。

Objective:To explore the function and mechanism of cleavage and polyadenylation factor 6(CPSF6)in breast cancer bone metastasis.Methods:The expression of CPSF6 was analyzed in 12 patients with bone metastasis of breast cancer and 20 patients with primary breast cancer by real-time fluorescent quantitative PCR(RT-qRCR),Western blotting(WB)and immunohistochemical staining(IHC).To explore the effect of CPSF6 on the growth and metastatic ability of tumor cells through cell experiments.To explore the effect of CPSF6 knockout on bone metastatic ability of breast cancer cells by in vivo metastasis model.To further explore the ability of breast cancer cells to reshape the extracellular microenvironment through CPSF6,and to examine the regulatory effect of CPSF6 on downstream target genes.Results:The expression level of CPSF6 in metastatic foci of breast cancer patients was significantly increased.CPSF6 is positively correlated with the proliferation and migration of breast cancer cells.After knocking down CPSF6 in 231-BM cells,the supernatant secreted by CPSF6 decreased the ability of osteoclast progenitor cells to differentiate into mature cells,and the number of osteoclasts around the metastatic focus decreased significantly in vivo.Knock-down CPSF6 can down-regulate the expression of Notch pathway ligand JAG1 in tumor cells,which leads to the inactivation of Notch pathway and inhibition of downstream target genes in osteoclast progenitor cells in microenvironment.Conclusions:CPSF6,-dependent Notch signal pathway,and finally produce osteolytic lesions.CPSF6 is highly expressed in bone metastatic foci.CPSF6 promotes the maturation rate and osteolytic activity of osteoclasts around tumor tissues by regulating the Notch signaling pathway,thus promoting the osteolytic bone metastasis of breast cancer.