Tamoxifen通过下调miR-200家族表达促进子宫内膜癌细胞上皮间质转化的机制研究
摘要
TAMOXIFEN(他莫昔芬,TAM)作为雌激素竞争拮抗剂能够与乳腺细胞的雌激素受体结合,对依赖雌激素生长的乳腺癌细胞起到抑制的作用,它已经被应用治疗雌激素受体阳性的乳腺癌患者。但是研究表明,TAM在子宫组织中可以表现出拟雌激素的作用,从而诱发组织中肿瘤的发生。例如,雌激素是诱发子宫内膜癌的一个关键因素,虽然TAM拟雌激素的作用对于绝经期的妇女可以有效的维持骨量,但是却能够提高子宫内膜癌的发病率,诱发子宫内膜发生增生、息肉、癌、肉瘤等多种病理改变。
MicroRNA (miRNA)是由约22个核苷酸组成的非编码单链RNA,它们能够在转录后水平通过与靶mRNA 3'非编码翻译区碱基形成特异性互补配对,抑制靶基因的翻译或降低mRNA的稳定性,从而沉默或干扰靶基因的表达。近些年来miRNA被发现在胚胎的发生和肿瘤调控等复杂的生理过程中发挥了重要的作用。
miR 200在调控上皮间质变(EMT)过程发挥了重要的作用,影响肿瘤的发生和转移。miR 200家族能够通过直接抑制靶基因ZEB1和ZEB2来上调E cadherin的表达,从而抑制了EMT的发生。在本研究中,我们探索了miR 200这个EMT过程中发挥重要作用的miRNA分子在TAM作用的子宫内膜癌细胞系中的作用及其相关调控机制。
首先,我们发现子宫内膜癌细胞系ECC 1在TAM的作用下发生了EMT的转化。ECC 1细胞是一类雌激素依赖的子宫内膜癌细胞系。我们通过实时定量的方法验证,ECC 1在TAM的刺激下,EMT相关的标记分子的mRNA水平发生了明显的变化,出现了向间质细胞转化的趋势。划痕试验和细胞侵袭试验也验证细胞在TAM的作用下,细胞的迁移和侵袭能力有明显的增强。
为探索miRNAs在TAM诱导子宫内膜癌细胞发生EMT过程中的作用,我们用miRNAs表达谱芯片检测了TAM刺激ECC 1细胞前后有明显差异的miRNAs。发现了4种miRNAs显著的上调,19种miRNAs显著的下调。其中miR 200b分子已被证实在乳腺癌等肿瘤中能够促进EMT,并在这些肿瘤的发生和转移过程中发挥了重要的作用。为阐明TAM诱导子宫内膜癌细胞发生EMT的分子机制,我们把miR 200分子作为后续实验的研究对象,深入研究了miR 200与ECC1发生EMT的关系及miR 200自身的调控机制。
根据生物信息学软件TargetScan预测ZEB2为miR 200家族的靶基因,我们通过双荧光素酶报告实验证实miR 200b能够结合ZEB2的3' UTR。在ECC 1细胞中过表达miR 200b能够上调E cadherin和下调Snail和N cadherin等EMT重要基因的表达。miR 200b能够明显抑制TAM刺激的ECC 1细胞的侵袭能力。
我们接下来阐明了TAM刺激ECC 1细胞调控miR 200b 200a 429启动子的相关机制。首先我们克隆出miR 200b 200a 429启动子的荧光素报告载体,利用生物信息学软件TESS对miR 200b 200a 429的启动子序列进行了分析,发现了启动子区域有c Myc, c Myb, sp1等重要转录因子的结合位点。我们进一步用实验表明c Myc能够特异性的结合miR 200b 200a 429的启动子并对其活性有明显的抑制作用。而且我们发现TAM能够上调ECC 1细胞中c Myc的表达。
综上所述,我们确定了miR 200家族作为一个重要的因子在TAM诱发子宫内膜癌变中可能发挥了重要的作用。TAM通过上调c Myc来抑制miR 200的表达,miR 200表达的下降导致靶基因ZEB2的上调,转录因子ZEB2上调引起E cadherin的下调,使细胞间的粘附作用减弱,促进肿瘤细胞的转移和侵袭。本研究证实了TAM能够促进子宫内膜细胞发生EMT,阐明了miR 200在此过程中的作用机制,揭示了c Myc对miR 200的直接调控作用,明确了TAM c Myc miR 200靶基因这一信号传导通路,为深入理解TAM诱发子宫内膜癌的分子机制提供了新的线索。
TAM as an antagonist to estradiol (E2) in estrogen receptor (ER) positive breast tumors can prevent breast cancer in high risk women. However, it shows partial estrogen like actions in other target tissues. The partial estrogen like actions make beneficial effect on bones in postmenopausal women, but the effect of TAM on uterus is well known to raise the incidences of endometrial cancer. Endometrial carcinoma is the most common gynaecological malignancy in which estradiol has been identified as a classic aetiological factor. Endometrial pathologies associated with TAM use include hyperplasia, polyps, carcinomas and sarcomas.
MicroRNAs (miRNAs) are small noncoding RNAs ( 22n)that regulate gene expression by repressing target messenger RNAs (mRNAs) via specific base pairing interactions in 3'untranslated regions (3' UTRs). miRNAs repress gene expression by interfering with mRNA stability or protein translation. Recently miRNAs have been identified in regulating complex physiological processes such as embryogenesis and oncogenesis. miR 200 family has been found to play a central role in the regulation of the epithelial to mesenchymal transition (EMT) process during cancer progression and metastasis. The increase of the miR 200 family intracellular levels inhibits EMT by up regulation of E cadherin expression through direct targeting of its ZEB1 and ZEB2 transcriptional repressors. In this study, we tested the hypothesis that miR 200 family , an important miRNA during EMT process ,is regulated by Tamoxifen in ECC 1 endometrial cancer cells.
We aimed to determine whether ECC 1 cells treated by TAM that have undergone an EMT ,which is an oestrogen responsive endometrial carcinoma cell line . We investigated the effect of Tamoxifen on the expression of this epithelial marker by RT PCR analysis when the ECC 1 cells after treating with TAM. The ECC 1 cells acquired a mesenchymal mRNA expression profile. We used wound healing assay and transwell assay to confirm that TAM induces EMT process in ECC 1 cells.
We performed miRNAs microarray to investigate miRNAs expression profiles in ECC 1 cells and ECC 1 cells treated with TAM. The study showed significantly increased expression of four miRNAs and down regulation of nineteen miRNAs in ECC 1 cells treated with TAM compared with ECC 1 cells. Interestingly, miR 200b plays an important role in EMT, so we investigated if miR 200 is regulated by TAM in ECC 1 cells .
The transcription factor ZEB2 has been suggested to be target of miR 200 family, According to TargetScan 4.1 (http://www.targetscan.org). We could show that overexpression of miR 200b led to reduced expression of ZEB2, Snail and N cadherin genes in ECC 1 cells,and increased expression of E cadherin simultaneously. As predicted, miR 200b had the strongest inhibitory effect on ZEB2 expression, as shown by mRNA and protein levels.Overexpression of miR 200b resulted in ~80% reduction in luciferase activity in ECC 1 cells transfected with wild type construct of human ZEB2 3' UTR . Furthermore, we evaluated the role of miR 200 suppression of ZEB2 expression on invasion in ECC 1 cells.Overexpression of miR 200b resulted in a significant derease in the number of invading cells which were treated with TAM.
We investigated whether TAM regulates miR 200b 200a 429 gene expression through a region encompassing 1574 to +120 bp relative to the putative TSS region previously reported to function as a promoter of miR 200b 200a 429. Using TESS (http://www.cbil.upenn.edu/cgi bin/tess/tess) to analyze the 110/+19 promoter region, we found some important transcription factor binding sites such as c Myc, c Myb and Sp1. Previous study showed that extensive reprogramming of the miRNAs transcriptome by Myc contributes to tumorigenesis.So we supposed that c Myc may play an important role in regulating miR 200b 200a 429 promoter in ECC 1 cells.We found that c Myc inhibited the activity of the promoter in ECC 1 cells, we also observed that TAM can increase expression of c Myc in ECC 1 cells.
In conclusion, we have identified miR 200 as an important factor that TAM mediates their carcinogenic roles in the uterus, our findings also illustrate the loss of miR 200 in regulating tumor cell EMT process. We have identified miR 200 that are regulated by tamxifen that by increasing c Myc expression in the endometrial cancer cells. Our study showed that TAM can induce EMT in ECC 1 cells, which revealed that c Myc directly regulates miR 200 expression . It is clear that miR200 regulates ZEB1/ZEB2/E Cadherin through TAM c Myc pathway.And our results provide new insights into molecular mechanism of TAM induced endometrial carcinoma.
MicroRNA (miRNA)是由约22个核苷酸组成的非编码单链RNA,它们能够在转录后水平通过与靶mRNA 3'非编码翻译区碱基形成特异性互补配对,抑制靶基因的翻译或降低mRNA的稳定性,从而沉默或干扰靶基因的表达。近些年来miRNA被发现在胚胎的发生和肿瘤调控等复杂的生理过程中发挥了重要的作用。
miR 200在调控上皮间质变(EMT)过程发挥了重要的作用,影响肿瘤的发生和转移。miR 200家族能够通过直接抑制靶基因ZEB1和ZEB2来上调E cadherin的表达,从而抑制了EMT的发生。在本研究中,我们探索了miR 200这个EMT过程中发挥重要作用的miRNA分子在TAM作用的子宫内膜癌细胞系中的作用及其相关调控机制。
首先,我们发现子宫内膜癌细胞系ECC 1在TAM的作用下发生了EMT的转化。ECC 1细胞是一类雌激素依赖的子宫内膜癌细胞系。我们通过实时定量的方法验证,ECC 1在TAM的刺激下,EMT相关的标记分子的mRNA水平发生了明显的变化,出现了向间质细胞转化的趋势。划痕试验和细胞侵袭试验也验证细胞在TAM的作用下,细胞的迁移和侵袭能力有明显的增强。
为探索miRNAs在TAM诱导子宫内膜癌细胞发生EMT过程中的作用,我们用miRNAs表达谱芯片检测了TAM刺激ECC 1细胞前后有明显差异的miRNAs。发现了4种miRNAs显著的上调,19种miRNAs显著的下调。其中miR 200b分子已被证实在乳腺癌等肿瘤中能够促进EMT,并在这些肿瘤的发生和转移过程中发挥了重要的作用。为阐明TAM诱导子宫内膜癌细胞发生EMT的分子机制,我们把miR 200分子作为后续实验的研究对象,深入研究了miR 200与ECC1发生EMT的关系及miR 200自身的调控机制。
根据生物信息学软件TargetScan预测ZEB2为miR 200家族的靶基因,我们通过双荧光素酶报告实验证实miR 200b能够结合ZEB2的3' UTR。在ECC 1细胞中过表达miR 200b能够上调E cadherin和下调Snail和N cadherin等EMT重要基因的表达。miR 200b能够明显抑制TAM刺激的ECC 1细胞的侵袭能力。
我们接下来阐明了TAM刺激ECC 1细胞调控miR 200b 200a 429启动子的相关机制。首先我们克隆出miR 200b 200a 429启动子的荧光素报告载体,利用生物信息学软件TESS对miR 200b 200a 429的启动子序列进行了分析,发现了启动子区域有c Myc, c Myb, sp1等重要转录因子的结合位点。我们进一步用实验表明c Myc能够特异性的结合miR 200b 200a 429的启动子并对其活性有明显的抑制作用。而且我们发现TAM能够上调ECC 1细胞中c Myc的表达。
综上所述,我们确定了miR 200家族作为一个重要的因子在TAM诱发子宫内膜癌变中可能发挥了重要的作用。TAM通过上调c Myc来抑制miR 200的表达,miR 200表达的下降导致靶基因ZEB2的上调,转录因子ZEB2上调引起E cadherin的下调,使细胞间的粘附作用减弱,促进肿瘤细胞的转移和侵袭。本研究证实了TAM能够促进子宫内膜细胞发生EMT,阐明了miR 200在此过程中的作用机制,揭示了c Myc对miR 200的直接调控作用,明确了TAM c Myc miR 200靶基因这一信号传导通路,为深入理解TAM诱发子宫内膜癌的分子机制提供了新的线索。
TAM as an antagonist to estradiol (E2) in estrogen receptor (ER) positive breast tumors can prevent breast cancer in high risk women. However, it shows partial estrogen like actions in other target tissues. The partial estrogen like actions make beneficial effect on bones in postmenopausal women, but the effect of TAM on uterus is well known to raise the incidences of endometrial cancer. Endometrial carcinoma is the most common gynaecological malignancy in which estradiol has been identified as a classic aetiological factor. Endometrial pathologies associated with TAM use include hyperplasia, polyps, carcinomas and sarcomas.
MicroRNAs (miRNAs) are small noncoding RNAs ( 22n)that regulate gene expression by repressing target messenger RNAs (mRNAs) via specific base pairing interactions in 3'untranslated regions (3' UTRs). miRNAs repress gene expression by interfering with mRNA stability or protein translation. Recently miRNAs have been identified in regulating complex physiological processes such as embryogenesis and oncogenesis. miR 200 family has been found to play a central role in the regulation of the epithelial to mesenchymal transition (EMT) process during cancer progression and metastasis. The increase of the miR 200 family intracellular levels inhibits EMT by up regulation of E cadherin expression through direct targeting of its ZEB1 and ZEB2 transcriptional repressors. In this study, we tested the hypothesis that miR 200 family , an important miRNA during EMT process ,is regulated by Tamoxifen in ECC 1 endometrial cancer cells.
We aimed to determine whether ECC 1 cells treated by TAM that have undergone an EMT ,which is an oestrogen responsive endometrial carcinoma cell line . We investigated the effect of Tamoxifen on the expression of this epithelial marker by RT PCR analysis when the ECC 1 cells after treating with TAM. The ECC 1 cells acquired a mesenchymal mRNA expression profile. We used wound healing assay and transwell assay to confirm that TAM induces EMT process in ECC 1 cells.
We performed miRNAs microarray to investigate miRNAs expression profiles in ECC 1 cells and ECC 1 cells treated with TAM. The study showed significantly increased expression of four miRNAs and down regulation of nineteen miRNAs in ECC 1 cells treated with TAM compared with ECC 1 cells. Interestingly, miR 200b plays an important role in EMT, so we investigated if miR 200 is regulated by TAM in ECC 1 cells .
The transcription factor ZEB2 has been suggested to be target of miR 200 family, According to TargetScan 4.1 (http://www.targetscan.org). We could show that overexpression of miR 200b led to reduced expression of ZEB2, Snail and N cadherin genes in ECC 1 cells,and increased expression of E cadherin simultaneously. As predicted, miR 200b had the strongest inhibitory effect on ZEB2 expression, as shown by mRNA and protein levels.Overexpression of miR 200b resulted in ~80% reduction in luciferase activity in ECC 1 cells transfected with wild type construct of human ZEB2 3' UTR . Furthermore, we evaluated the role of miR 200 suppression of ZEB2 expression on invasion in ECC 1 cells.Overexpression of miR 200b resulted in a significant derease in the number of invading cells which were treated with TAM.
We investigated whether TAM regulates miR 200b 200a 429 gene expression through a region encompassing 1574 to +120 bp relative to the putative TSS region previously reported to function as a promoter of miR 200b 200a 429. Using TESS (http://www.cbil.upenn.edu/cgi bin/tess/tess) to analyze the 110/+19 promoter region, we found some important transcription factor binding sites such as c Myc, c Myb and Sp1. Previous study showed that extensive reprogramming of the miRNAs transcriptome by Myc contributes to tumorigenesis.So we supposed that c Myc may play an important role in regulating miR 200b 200a 429 promoter in ECC 1 cells.We found that c Myc inhibited the activity of the promoter in ECC 1 cells, we also observed that TAM can increase expression of c Myc in ECC 1 cells.
In conclusion, we have identified miR 200 as an important factor that TAM mediates their carcinogenic roles in the uterus, our findings also illustrate the loss of miR 200 in regulating tumor cell EMT process. We have identified miR 200 that are regulated by tamxifen that by increasing c Myc expression in the endometrial cancer cells. Our study showed that TAM can induce EMT in ECC 1 cells, which revealed that c Myc directly regulates miR 200 expression . It is clear that miR200 regulates ZEB1/ZEB2/E Cadherin through TAM c Myc pathway.And our results provide new insights into molecular mechanism of TAM induced endometrial carcinoma.
引文
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