Clusterin is a gene-specific target of microRNA-21 in head and neck squamous cell carcinoma.
Clusterin is a gene-specific target of microRNA-21 in head and neck squamous cell carcinoma.
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DOI：
10.1158/1078-0432.ccr-13-2675
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发表时间：
2014-02-15
期刊：
影响因子：
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通讯作者：
Califano J
中科院分区：
文献类型：
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作者：
Mydlarz W;Uemura M;Ahn S;Hennessey P;Chang S;Demokan S;Sun W;Shao C;Bishop J;Krosting J;Mambo E;Westra W;Ha P;Sidransky D;Califano J
MicroRNA-21(miRNA-21)具有原癌特性，但在头颈部鳞状细胞癌(HNSCC)中尚未发现miRNA-21的特异性靶点。进一步研究miRNA-21及其特定靶点对于理解HNSCC生物学至关重要。使用定制的miRNA微阵列研究了10例HNSCC和10例正常粘膜的miRNA表达谱。对13例HNSCC和5例正常粘膜原发组织进行了mRNA表达谱芯片分析。为了确定miRNA-21的下游靶点，对瞬时转染后的口腔角质形成细胞进行了基因芯片分析。在16例HNSCC和15例正常粘膜样本中，通过RT-qPCR验证miRNA和mRNA的表达。微阵列和生物信息学分析相结合，以确定潜在的基因靶点。体外试验观察了miRNA-21及其特定基因靶点的功能和相互作用。MiRNA-21在HNSCC中表达上调，并刺激细胞生长。综合分析确定Clusterin(CLU)是一个潜在的miRNA-21基因靶点。在正常和HNSCC细胞系中强制表达miRNA-21后，CLU表达下调。含有CLU 3‘非编码区的荧光素酶的活性被miRNA-21的异位表达所抑制。CLU在原发HNSCC中表达下调，并与miRNA-21过表达相关。CLU变异体1(CLU-1)是HNSCC中主要的剪接变异体，其生长抑制作用被miRNA-21过表达所逆转。CLU是HNSCC中致癌miRNA-21的一个特异的功能靶点。ClU-1亚型是miRNA-21靶向的主要生长抑制变异体。
MicroRNA-21 (miRNA-21) has proto-oncogenic properties, though no miRNA-21 specific targets have been found in head and neck squamous cell carcinoma (HNSCC). Further study of miRNA-21 and its specific targets is essential to understanding HNSCC biology. miRNA expression profiles of 10 HNSCC and 10 normal mucosa samples were investigated using a custom miRNA microarray. 13 HNSCC and 5 normal mucosa primary tissue specimens underwent mRNA expression microarray analysis. To identify miRNA-21 downstream targets, oral keratinocyte cells were subjected to microarray analysis after miRNA-21 transient transfection. miRNA and mRNA expression were validated by RT-qPCR in a separate cohort of 16 HNSCC and 15 normal mucosal samples. Microarray and bioinformatics analyses were integrated to identify potential gene targets. In vitro assays looked at the function and interaction of miRNA-21 and its specific gene targets. miRNA-21 was upregulated in HNSCC and stimulated cell growth. Integrated analyses identified Clusterin (CLU) as a potential miRNA-21 gene target. CLU was downregulated after forced expression of miRNA-21 in normal and HNSCC cell lines. The activity of a luciferase construct containing the 3’UTR of CLU was repressed by the ectopic expression of miRNA-21. CLU was also downregulated in primary HNSCC and correlated with miRNA-21 over-expression. CLU variant 1 (CLU-1) was the predominant splice variant in HNSCC, and showed growth suppression function that was reversed by miRNA-21 over-expression. CLU is a specific, functional target of oncogenic miRNA-21 in HNSCC. CLU-1 isoform is the predominant growth suppressive variant targeted by miRNA-21.