PDF(Pubmed)
Abstract：
BACKGROUND: While melanomas commonly harbor losses of 9p21, on which CDKN2A resides, the presence of additional tumor suppressor elements at this locus is incompletely characterized. Here we assess the expression levels and functional role of microRNA-876-3p (miR-876), whose gene also maps to 9p21.
METHODS: Expression of miR-876 was assessed in human tissues and cell lines using quantitative miRNA reverse transcriptase polymerase chain reaction (qRT-PCR). MIR876 copy number was determined in The Cancer Genome Atlas (TCGA) melanoma cohort. The consequences of regulation of miR-876 expression were assessed on melanoma cell colony formation, migration, invasion, apoptosis, cell cycle progression, and drug sensitivity in culture, and on in vivo tumor growth in a xenograft model. Genome-wide transcriptomic changes induced by miR-876 overexpression were determined using RNA sequencing (RNA-Seq).
RESULTS: miR-876 expression was significantly decreased in primary melanoma samples when compared with nevi, and in human melanoma cell lines when compared with human melanocytes. Analysis of the TCGA cohort revealed deletions in MIR876 in > 50% of melanomas. miR-876 overexpression resulted in decreased melanoma cell colony formation, migration, and invasion, which was accompanied by cell cycle arrest and increased apoptosis. Intra-tumoral injections of miR-876 significantly suppressed melanoma growth in vivo. RNA-Seq analysis of miR-876-treated tumors revealed downregulation of several growth-promoting genes, along with upregulation of tumor suppressor genes, which was confirmed by qRT-PCR analysis. Computational analyses identified MAPK1 (or ERK2) as a possible target of miR-876 action. Overexpression of miR-876 significantly suppressed luciferase expression driven by the MAPK1/ERK2 3\' UTR, and resulted in decreased ERK protein expression in melanoma cells. MAPK1/ERK2 cDNA overexpression rescued the effects of miR-876 on melanoma colony formation. miR-876 overexpression sensitized melanoma cells to treatment with the BRAF inhibitor vemurafenib.
CONCLUSIONS: These studies identify miR-876 as a distinct tumor suppressor on 9p21 that is inactivated in melanoma and suggest miR-876 loss as an additional mechanism to activate ERK and the mitogen activated protein kinase (MAPK) pathway in melanoma. In addition, they suggest the therapeutic potential of combining miR-876 overexpression with BRAF inhibition as a rational therapeutic strategy for melanoma.
METHODS: Expression of miR-876 was assessed in human tissues and cell lines using quantitative miRNA reverse transcriptase polymerase chain reaction (qRT-PCR). MIR876 copy number was determined in The Cancer Genome Atlas (TCGA) melanoma cohort. The consequences of regulation of miR-876 expression were assessed on melanoma cell colony formation, migration, invasion, apoptosis, cell cycle progression, and drug sensitivity in culture, and on in vivo tumor growth in a xenograft model. Genome-wide transcriptomic changes induced by miR-876 overexpression were determined using RNA sequencing (RNA-Seq).
RESULTS: miR-876 expression was significantly decreased in primary melanoma samples when compared with nevi, and in human melanoma cell lines when compared with human melanocytes. Analysis of the TCGA cohort revealed deletions in MIR876 in > 50% of melanomas. miR-876 overexpression resulted in decreased melanoma cell colony formation, migration, and invasion, which was accompanied by cell cycle arrest and increased apoptosis. Intra-tumoral injections of miR-876 significantly suppressed melanoma growth in vivo. RNA-Seq analysis of miR-876-treated tumors revealed downregulation of several growth-promoting genes, along with upregulation of tumor suppressor genes, which was confirmed by qRT-PCR analysis. Computational analyses identified MAPK1 (or ERK2) as a possible target of miR-876 action. Overexpression of miR-876 significantly suppressed luciferase expression driven by the MAPK1/ERK2 3\' UTR, and resulted in decreased ERK protein expression in melanoma cells. MAPK1/ERK2 cDNA overexpression rescued the effects of miR-876 on melanoma colony formation. miR-876 overexpression sensitized melanoma cells to treatment with the BRAF inhibitor vemurafenib.
CONCLUSIONS: These studies identify miR-876 as a distinct tumor suppressor on 9p21 that is inactivated in melanoma and suggest miR-876 loss as an additional mechanism to activate ERK and the mitogen activated protein kinase (MAPK) pathway in melanoma. In addition, they suggest the therapeutic potential of combining miR-876 overexpression with BRAF inhibition as a rational therapeutic strategy for melanoma.
摘要：
背景：虽然黑色素瘤通常损失9p21，CDKN2A位于其上，在该位点存在其他肿瘤抑制因子的特征不完全。在这里，我们评估了microRNA-876-3p（miR-876）的表达水平和功能作用，其基因也映射到9p21。
方法:使用定量miRNA逆转录酶聚合酶链反应(qRT-PCR)在人组织和细胞系中评估miR-876的表达。在癌症基因组图谱(TCGA)黑素瘤队列中确定MIR876拷贝数。miR-876表达的调控对黑色素瘤细胞集落形成的影响进行了评估，迁移,入侵,凋亡，细胞周期进程，和培养中的药物敏感性，以及异种移植模型中的体内肿瘤生长。使用RNA测序(RNA-Seq)测定由miR-876过表达诱导的全基因组转录组变化。
结果：与痣相比，miR-876在原发性黑色素瘤样本中的表达显着降低，与人类黑素细胞相比，在人类黑素瘤细胞系中。对TCGA队列的分析显示MIR876在>50%的黑素瘤中缺失。miR-876过表达导致黑素瘤细胞集落形成减少，迁移,和入侵,伴随着细胞周期停滞和细胞凋亡增加。肿瘤内注射miR-876显著抑制体内黑色素瘤生长。miR-876处理的肿瘤的RNA-Seq分析揭示了几种生长促进基因的下调，随着肿瘤抑制基因的上调，通过qRT-PCR分析证实。计算分析将MAPK1(或ERK2)鉴定为miR-876作用的可能靶标。miR-876的过表达显著抑制了由MAPK1/ERK23'UTR驱动的荧光素酶表达，并导致黑色素瘤细胞中ERK蛋白表达降低。MAPK1/ERK2cDNA过表达拯救了miR-876对黑色素瘤集落形成的影响。miR-876过表达使黑色素瘤细胞对BRAF抑制剂vemurafenib治疗敏感。
结论：这些研究确定miR-876是9p21上的一种独特的肿瘤抑制因子，在黑色素瘤中失活，并提示miR-876丢失是激活ERK和促分裂原活化蛋白激酶（MAPK）途径的另一种机制。此外,他们提出了miR-876过表达与BRAF抑制相结合作为黑色素瘤合理治疗策略的治疗潜力.
方法:使用定量miRNA逆转录酶聚合酶链反应(qRT-PCR)在人组织和细胞系中评估miR-876的表达。在癌症基因组图谱(TCGA)黑素瘤队列中确定MIR876拷贝数。miR-876表达的调控对黑色素瘤细胞集落形成的影响进行了评估，迁移,入侵,凋亡，细胞周期进程，和培养中的药物敏感性，以及异种移植模型中的体内肿瘤生长。使用RNA测序(RNA-Seq)测定由miR-876过表达诱导的全基因组转录组变化。
结果：与痣相比，miR-876在原发性黑色素瘤样本中的表达显着降低，与人类黑素细胞相比，在人类黑素瘤细胞系中。对TCGA队列的分析显示MIR876在>50%的黑素瘤中缺失。miR-876过表达导致黑素瘤细胞集落形成减少，迁移,和入侵,伴随着细胞周期停滞和细胞凋亡增加。肿瘤内注射miR-876显著抑制体内黑色素瘤生长。miR-876处理的肿瘤的RNA-Seq分析揭示了几种生长促进基因的下调，随着肿瘤抑制基因的上调，通过qRT-PCR分析证实。计算分析将MAPK1(或ERK2)鉴定为miR-876作用的可能靶标。miR-876的过表达显著抑制了由MAPK1/ERK23'UTR驱动的荧光素酶表达，并导致黑色素瘤细胞中ERK蛋白表达降低。MAPK1/ERK2cDNA过表达拯救了miR-876对黑色素瘤集落形成的影响。miR-876过表达使黑色素瘤细胞对BRAF抑制剂vemurafenib治疗敏感。
结论：这些研究确定miR-876是9p21上的一种独特的肿瘤抑制因子，在黑色素瘤中失活，并提示miR-876丢失是激活ERK和促分裂原活化蛋白激酶（MAPK）途径的另一种机制。此外,他们提出了miR-876过表达与BRAF抑制相结合作为黑色素瘤合理治疗策略的治疗潜力.
