Interaction with SP1, but not binding to the E-box motifs, is responsible for BHLHE40/DEC1-induced transcriptional suppression of CLDN1 and cell invasion in MCF-7 cells
Interaction with SP1, but not binding to the E-box motifs, is responsible for BHLHE40/DEC1-induced transcriptional suppression of CLDN1 and cell invasion in MCF-7 cells
复制标题
与 SP1 相互作用，但不与 E-box 基序结合，导致 BHLHE40/DEC1 诱导的 CLDN1 转录抑制和 MCF-7 细胞中的细胞侵袭
DOI：
10.1002/mc.22829
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发表时间：
2018
影响因子：
4.6
通讯作者：
Liu Yang
中科院分区：
文献类型：
--
作者：
Zheng Qin;Wang Chen;Wang Liang;Zhang Di;Liu Nan;Ming Xiaocui;Zhou Haitao;Guli Qiere;Liu Yang
碱性螺旋-环-螺旋家族成员e40（BHLHE 40）位于3p26.1，通过抑制时钟基因和时钟控制基因的表达，作为昼夜节律的转录抑制因子。最近的研究表明，BHLHE 40可能参与调节肿瘤细胞的进展。但BHLHE 40调控肿瘤细胞侵袭和转移的机制尚不清楚.我们的体外试验表明，BHLHE 40促进肿瘤细胞侵袭，而通过siRNA沉默BHLHE 40抑制MCF-7细胞的肿瘤细胞侵袭。BHLHE 40抑制CLDN 1、CLDN 4和CDH 1的mRNA和蛋白表达，促进SNAI 1和SNAI 2的表达。报告基因分析表明BHLHE 40抑制CLDN 1的转录，但不是通过直接结合CLDN 1启动子中的E‐box基序。进一步的研究表明BHLHE 40通过阻止SP1与CLDN 1启动子区域内的特定基序之间的相互作用来抑制CLDN 1的转录。BHLHE 40不能进一步抑制SP1 siRNA转染后的CLDN 1反式激活，即BHLHE 40诱导的CLDN 1抑制依赖于SP1。此外，我们的数据表明，SP1是CLDN 1转录的主要调节因子，它与位于转录起始位点上游-233至-61 bp的特定基序结合。免疫沉淀和共定位数据揭示了BHLHE 40和SP1之间的相互作用。通过构建缺失突变体，我们发现BHLH和橙子区域对于BHLHE 40-SP1相互作用都是必需的。BHLHE 40可能作为一种抑制性核辅因子或招募其他抑制性辅因子来抑制SP1介导的CLDN 1反式激活。这些结果表明，BHLHE 40促进细胞侵袭，并可能用作乳腺癌预防和治疗的新靶点。
Basic helix‐loop‐helix family member e40 (BHLHE40) is located in 3p26.1 and acts as a transcriptional repressor of the circadian rhythm by suppressing the expression of the clock genes and clock‐controlled genes. Recent research indicated that BHLHE40 may be involved in regulating tumor cell progression. However the mechanism by which BHLHE40 regulates the invasion andmetastasisof tumor cells is unclear. Our in vitro assays showed that BHLHE40 promoted tumor cell invasion while BHLHE40 silencing by siRNA suppressed tumor cell invasion of MCF‐7 cells. BHLHE40 suppressed the mRNA and protein expression ofCLDN1 CLDN4andCDH1and promoted the expression ofSNAI1andSNAI2. Reporter assays demonstrated that BHLHE40 suppressedCLDN1transcription but not through direct binding to the E‐box motifs in theCLDN1promoter. Further studies demonstrated BHLHE40 suppressedCLDN1transcription by preventing the interaction between SP1 and a specific motif within the promoter region ofCLDN1. BHLHE40 could not further suppressCLDN1transactivation after SP1 siRNA transfection that is, BHLHE40‐induced suppression ofCLDN1relied on SP1. Furthermore our data indicated that SP1 was a major regulator ofCLDN1transcription by binding to a specific motif that was located at −233 to −61 bp upstream of the transcription start site. Immunoprecipitation and co‐localization data revealed an interaction between BHLHE40 and SP1. By constructing deletion mutants we found that the BHLH and Orange regions are both essential for the BHLHE40‐SP1 interaction. BHLHE40 probably acts as an inhibitory nuclear cofactor or perhaps recruits other inhibitory cofactors to inhibit the SP1‐mediatedCLDN1transactivation. These results suggest that BHLHE40 facilitates cell invasion and may be used as a novel target for breast cancer prevention and treatment.