雌激素诱导结肠癌细胞凋亡的表观遗传机制研究
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摘要
研究背景
结直肠癌(colorectal cancer, CRC)在肿瘤相关性死亡病例中排在第四位，据统计每年约有608,700人因结直肠癌而死亡。近些年流行病学、临床和实验研究发现雌激素与结直肠癌发病有明显的关系，在过去的几十年里，美国结直肠癌死亡率女性患者下降明显，有分析认为雌激素替代疗法的流行是女性患病率下降的可能原因。目前所使用的结直肠癌化疗药物的治疗效果在年轻女性患者要优于男性患者，这说明雌激素除对女性结直肠癌患病具有保护作用外，在结直肠癌治疗中也有积极的作用，但确切的机制不明。最近研究报道结直肠癌患者癌组织中会出现miRNAs(microRNAs)表达谱的改变，包括miR-135b, miR-96, miR-183, miR-133a, miR-133b, miR-21, miR-31, miR-145,miR-203, miR-223, miR-155等，在结直肠癌中表达明显增加，但是miRNAs改变的原因及表达改变后如何促进结直肠癌的发生、发展，目前仍不清楚。已有研究显示雌激素可通过激活雌激素受体下调乳腺癌MCF-7细胞内miR-21表达，并增加其靶基因的表达。雌激素同样能够影响子宫内膜间质细胞和子宫平滑肌细胞内一些miRNAs的表达。雌激素受体阳性肿瘤中雌激素的表达与miRNA表达有一定关系。因此雌激素对结直肠癌的保护作用可能与雌激素对某些miRNAs表达的调节有关。部分结直肠癌的发生与DNA错配修复功能缺陷有关，在保持遗传稳定方面DNA错配修复(mismatchrepair, MMR)系统发挥着很重要的作用。错配修复基因突变包括遗传和散发性的突变，主要发生在致癌形成作用的早期阶段。通常能够检测出微卫星不稳定(microsatelliteinstability, MSI)或涉及DNA错配修复缺陷的基因主要为MLHI, MSH2, MSH6和PMS2。有研究显示雌激素和微卫星不稳定存在一定的关系，可增强主要的错配修复基因(hMLH1)在结肠上皮细胞的表达。雌激素还可诱导结肠癌细胞凋亡。但雌激素诱导错配修复功能增强和结肠癌细胞凋亡的机制不清。
为了研究雌激素对错配修复基因调节的可能机制，是否有miRNAs参与，可能参与雌激素诱导结肠癌细胞凋亡的靶基因。我们拟进行实验验证结肠癌细胞中雌激素和雌激素受体拮抗剂ICI182,780对细胞内一些miRNAs(miR-31, miR-155, miR-135b,miR-203和miR-223)表达的影响，同时在18例结直肠癌患者组织中评价血清雌激素水平与miRNA和MMR表达的相关性。通过荧光素酶活性实验确定可能参与雌激素诱导结肠癌细胞凋亡的基因。
目的
1.研究结肠癌细胞中miRNA表达与雌激素的关系。
2.研究结肠癌细胞中MMR表达与雌激素的关系。
3.人体组织标本验证雌激素、miRNA和MMR表达的相关性。
4.明确可能参与雌激素诱导结肠癌细胞凋亡的miRNA和靶基因。
研究方法
1.选用三种细胞进行实验，COLO205(细胞内有较高水平的ER-β表达，无ER-α)细胞、SW480细胞(细胞内仅有很低水平的ER-β表达)和MCF-7细胞(细胞内ER-α表达水平较高，而几乎检测不到ER-β表达)。不同浓度雌激素处理细胞流式细胞仪测定凋亡。10nM雌激素处理COLO205细胞0h,6h,12h和24h，提取雌激素处理前后的细胞总RNA，实时定量PCR(RT-qPCR)测定细胞内miR-31, miR-155, miR-135b,miR-203和miR-223的表达水平。细胞预先加入100nM雌激素受体抑制剂6h后，再加入雌激素处理12h，提取雌激素处理前后的细胞总RNA，实时定量PCR(RT-qPCR)测定细胞内miR-31, miR-155和miR-135b的表达。将ER-β质粒转染到SW480细胞内，增加细胞内ER-β的表达。收集转染后24h和48h的细胞，提取蛋白，Western blotting检测转染效果。雌激素处理转染pRST7-ER-β的SW480细胞，检测细胞内miR-31,miR-155和miR-135b的表达。
2. COLO205、SW480和MCF7细胞为研究对象，10nM雌激素处理COLO205细胞0h,6h,12h和24h，RT-qPCR和Western blotting检测细胞内ER-β和MMR表达。预先加入100nM雌激素受体抑制剂6h后，再加入雌激素处理12h，提取雌激素处理前后的细胞总RNA和蛋白，检测细胞内ER-β和MMR表达。雌激素处理MCF-7和SW480细胞12h后，RT-qPCR和Western blotting检测细胞内ER-β和MMR表达。将ER-β质粒转染到SW480细胞内，增加细胞内ER-β的表达后，RT-qPCR检测雌激素对转染pRST7-ER-β的SW480细胞内hMLH1mRNA表达的影响。Western blotting检测雌激素对转染pRST7-ER-β的SW480细胞内hMLH1蛋白表达的影响。
3.共收集18例结直肠癌患者血清样本，结直肠癌和癌旁标本，对雌激素、miRNA和MMR表达的相关性进行分析。
4.转染miR-135b抑制剂到COLO205细胞内，转染24h后收集3h,6h,12h和24h的细胞测定凋亡，提取总RNA测定miR-135b表达。根据在线数据库UCSC(www.geome.ucsc.edu)确定LATS23′UTR，PCR在人基因组扩增LATS23′UTR并克隆到pGL3荧光素报告载体得到pGL3-LATS23’UTR Wt。同时应用QuikChange IIXL Site-Directed Mutagenesis Kit将载体LATS23’UTR序列中的miR-135b结合位点的核心区突变后，克隆到pGL3荧光素报告载体的荧光素酶基因下游得到pGL3-LATS23’UTR Mut。分别将两个载体与miR-135b模拟物或miR-NC共转染HEK-293细胞，pGL3荧光素报告载体为对照，转染24h后，收集细胞裂解液，用双荧光素酶试剂盒(Promega)测定荧光素酶活性。
结果
1.雌激素可诱导COLO205细胞凋亡，并存在一定剂量效应关系。雌激素能够抑制COLO205细胞内miR-31, miR-155和miR-135b的表达，并有时间依赖效应。COLO205细胞预先加入100nM雌激素受体抑制剂6h后，能够拮抗雌激素对细胞内miR-31, miR-155和miR-135b表达的抑制作用。雌激素对SW480和MCF-7细胞内miR-31, miR-155和miR-135b表达无明显抑制作用。ER-β质粒转染到SW480细胞内，经Western blotting检测发现，转染空载组细胞内ER-β蛋白表达量无变化，而转染pRST7-ER-β的SW480细胞内ER-β蛋白表达量明显增加。RT-qPCR结果显示雌激素可抑制转染pRST7-ER-β的SW480细胞内miR-31, miR-155和miR-135b的表达，而对空载组无影响。结果说明雌激素对细胞内某些miRNAs表达的影响，可能是由雌激素受体ER-β介导的。
2. RT-qPCR和Western blotting结果显示，随着雌激素处理时间的增加，细胞内ER-β和hMLH1表达增加，hMSH2表达变化不明显。雌激素受体抑制剂可以抑制雌激素对细胞内ER-β和hMLH1表达的调节。雌激素处理MCF-7和SW480细胞12h后，细胞内hMLH1, hMSH2和ER-β表达无明显变化。将ER-β质粒转染到SW480细胞内，增加细胞内ER-β的表达后，RT-qPCR结果显示雌激素可上调转染pRST7-ER-β的SW480细胞内hMLH1mRNA的表达，而对空载组无影响。Western blotting结果显示雌激素可上调转染pRST7-ER-β的SW480细胞内hMLH1蛋白的表达，而对空载组无影响。雌激素对细胞内hMLH1表达的影响，可能与雌激素受体ER-β有关。
3.血清雌激素水平与miR-31和miR-135b表达有明显的负相关，而与miR-155表达无相关性。我们同样发现，在结肠癌组织中，hMLH1mRNA表达水平与miR-155和miR-135b表达有明显的负相关，而ER-β mRNA表达与血清雌激素水平有明显的正相关性。miR-135b与血清雌激素水平，ER-β和hMLH1表达有相关性。但hMLH1表达与血清雌激素水平无明显相关性。
4. miR-135b抑制剂转染COLO205细胞，随着细胞内miR-135b表达降低，细胞凋亡率明显增加。通过Target Scan和miRanda软件分析预测，荧光素酶活性实验证实miR-135b可以和LATS23’UTR结合。
结论
1.雌激素能够抑制COLO205细胞内miRNAs(miR-31, miR-155和miR-135b)的表达，上调hMLH1的表达，雌激素受体抑制剂能拮抗这一作用。相比于癌旁组织，癌组织中miR-135b表达明显增加。结肠癌组织内miR-135b和ER-β mRNA的表达与患者血清雌激素水平有一定相关性。
2.当多数病人的血清雌激素浓度低于45pg/ml时，结直肠癌组织中hMLH1mRNA的表达与患者血清雌激素水平没有明显相关性。因此雌激素通过雌激素受体对MMR和miRNA的调节作用可能发生在肿瘤形成的早期，在肿瘤形成过程中，随着雌激素水平和/或雌激素受体表达降低miR-31、miR-155和miR-135b表达会逐渐增加，引起错配修复功能不稳，促进结直肠癌发生。
3．雌激素诱导结肠癌细胞凋亡与抑制细胞内miR-135b表达有关，miR-135b的靶基因LATS2可能参与了这一机制。
Background
Colorectal cancer (CRC) is the fourth most common cause of death from cancer,affects around1.2million people world wide, with about608,700fatalities per year. Boththe prevalence and the lifetime risk of CRC are significantly lower in females than in males.This is particularly true when comparing pre-menopausal women to age-matched men. Thesex-specific difference in CRC incidence has been attributed to estrogen, but the exactmechanism is not clear. Since expression alterations of some miRNAs are associated with aseries of genetic events, their expression patterns may either have pro-or anti-cancereffects. Several miRNAs expression alterations have been reported in CRC, includingmiR-135b, miR-96, miR-183, miR-133a, miR-133b, miR-21, miR-31, miR-145, miR-203,miR-223, miR-155. However, the mechanism for miRNA change in CRC is not wellunderstood. Recent studies have indicated that estrogens (especially estradiol E2) canregulate miRNAs expression in human breast cancer cells, endometrial stromal cells, andmyometrial smooth muscle cells. Estrogen receptor (ER) expression is associated withmicroRNA (miRNA) expression in ER-positive tumors. Thus, estrogen’s protect effect maybe related to the regulation of some miRNAs. DNA mismatch repair (MMR) system playsan important role in maintaining genomic stability. The MMR gene alteration includinggenetic and sporadic mutations, commonly occur in the early stages of the neoplasticprocess. MMR dysfunction can cause microsatellite instability (MSI). It has been reportedthat estrogen is associated with MSI. In previous study, estradiol (E2) has shown to havethe ability to induce CRC cells to apoptosis, and to up-regulate major MMR gene (hMLH1)activity in colonic epithelial cells. These findings suggest that estrogen’s anti-colorectalcancer effect may be mediated by MMR function in colonic cells. However, the pathwaythrough which estrogen regulates MMR function is still not well understood.
From our knowledge, studies the direct link between estrogen, miRNA and MMRexpression in cultured cells and patient tissue/serum samples, the relationship of miRNA-target gene in CRC cell apoptosis, have not been done. In this study, we firstevaluated the effects of estrogen and its antagonist ICI182,780on the expression ofmiRNAs (miR-31, miR-135b, miR-155, miR-203and miR-223) and MMR using COLO205,SW480and MCF-7cell lines, followed by examining the association of tissue miRNAexpression, MMR expression and serum E2levels using samples collected from18colorectal cancer patients. Then the miRNA target gene will be confirmed by luciferaseassay.
Objective
1. Studies of direct link between estrogen and miRNA expression in CRC cells.
2. Studies of direct link between estrogen and MMR expression in CRC cells.
3. Studies the association of tissue miRNA expression, MMR expression and serum E2levels using samples collected from18colorectal cancer patients.
4. To check whether miRNAs participates in estrogen-induced apoptosis in culturedcells. Then the target gene will be confirmed.
Materials and methods
1. Studies of direct link between estrogen and miRNA expression in CRC.
COLO205and SW480cells were incubated in different concentrations of estradiol E2for48h;100nM of estrogen receptor antagonist ICI182,780was then added followed byAnnexin-V apoptosis assay. COLO205cells were treated with10nM E2with a finalvolume of0.01%ethanol for0h,6h,12h and24h to determine miRNA expression.SW480and MCF-7cells were treated with EtOH,1nM or10nM E2, for12h alone. Forthe indicated experiments, cells were pretreated with100nM ICI182,780for6h prior toEtOH (control) or E2treatment. SW480cells were transiently transfected with an emptypRST7vector and pRST7-ER-β. After24h of transfection, Western blotting was used toconfirm the transfection efficiency after24h or48h of transfection. After24h oftransfection, SW480(pERβ) and SW480(vector) were treated with EtOH or10nM E2for12h. After incubation, cells were harvested and used for quantitative reversetranscription-PCR (RT-qPCR).
2. Studies of direct link between estrogen and MMR in CRC.
COLO205cells were treated with10nM E2with a final volume of0.01%ethanol for0h,6h,12h and24h to determine mRNA expression, or for0h,24h and48h to determine protein expression. SW480and MCF-7cells were treated with EtOH,1nM or10nM E2for12h to determine mRNA expression, or for0h,24h and48h to determineprotein expression. After24h of transfection, SW480(pERβ) and SW480(vector) weretreated with EtOH or10nM E2for12h. After incubation, cells were harvested and usedfor quantitative reverse transcription-PCR (RT-qPCR) and Western blotting.
3. Studies the association of tissue miRNA expression, MMR expression and serum E2levels using samples collected from18colorectal cancer patients.
A total of18patients9males and9females diagnosed with CRC were enrolled. Thesepatients were randomly selected from patient pool of the hospital’s gastrointestinal clinic;none of the subjects had undergone estrogen replacement therapy. Blood was taken half anhour after colonoscopy for determining the serum E2level. During colonoscopy, four orfive samples from cancer tissues and adjacent non-cancer tissues, respectively, wereobtained and stored in liquid nitrogen. Correlation analyses between serum E2level andmiRNA expression, serum E2level and mRNA expression, and miRNA and mRNAexpression were performed.
4. To check whether miRNAs participates in estrogen-induced apoptosis in culturedcells. Then the target gene will be confirmed.
COLO205were transfected with miR-135b inhibitor, and incubated in differentconcentrations of estradiol E2for0h,6h,12h and24h to determine miRNA expressionand apoptosis. According to the UCSC (www.geome.ucsc.edu), LATS23′UTR wasamplified from the genomic DNA of human by PCR and cloned into luciferase reportergene pGL3-vector.The recombinant pGL3-LATS23’UTR Wt and pGL3-LATS23’UTRMut were respectively transient co-transfected with miR-135b mimic or miRNA NC intoHEK293cells using Lipofectamine. Luciferase activities were measured consecutively.
Rusults
1. After treatment with different concentrations of E2for48h, the apoptotic cells weresignificantly increased in COLO205, and the effect was the strongest at1×10-9M of E2.Estrogen receptor antagonist, ICI182,780, inhibited E2-induced apoptosis in COLO205cells. However, the effect of E2on apoptotic induction was not seen in SW480cells.RT-qPCR results indicated that E2decreased the levels of miR-31, miR-155and miR-135bin a time-dependent manner. ICI182,780reversed E2-induced repression of miR-31, miR-155and miR-135b in COLO205cells. E2did not however affect miRNA expressionin either SW480cells, or MCF-7cells. ER-β protein expression was not up-regulated invector transfected SW480cells, whereas it was up-regulated in pRST7-ER-β transfectedSW480cells after24and48h of transfection by western blot. RT-qPCR results indicatedthat E2decreased the levels of miR-31, miR-155and miR-135b in pRST7-ER-β transfectedSW480cells, but not in vector cells.
2. RT-qPCR and Western blotting showed that E2increased the expression of ER-βand hMLH1, but at less degree of hMSH2, at both mRNA and protein levels in COLO205cells, in a time and dose-dependent manners. These effects were again inhibited byICI182,780. No changes in the expression of hMLH1, hMSH2and ER-β mRNA were seenin MCF-7and SW480cells. RT-qPCR and Western blotting showed that E2increased theexpression of hMLH1, but at less degree of hMSH2, at both mRNA and protein levels inpRST7-ER-β transfected SW480cells.
3. The results indicated that serum E2levels were strongly and negatively correlatedwith miR-31and miR-135b expression, but not for miR-155expression in cancer tissue.We also found that in cancer tissue, hMLH1mRNA levels were negatively correlated withthat of miR-155and miR-135b expression, whereas ER-β mRNA levels were positivelycorrelated with serum E2concentrations. Interestingly, only miR-135b has a significantcorrelation with serum E2levels, ER-β and hMLH1expression. Our previous study showedthat in normal individuals, only when serum E2levels were higher than45pg/ml, a strongpositive correlation of E2with hMLH1gene expression was observed; and there was nosignificant correlation between hMLH1expression and serum E2levels when E2levelswere lower (E2,＜45pg/ml). In the current study of18CRC patients, only two patients’serum E2levels were higher than45pg/ml (a male, E2=69.16pg/ml; a femail, E2=70.18pg/ml).
4. Inhibition of miR-135b expression in COLO205cells, can significantly induced thecells to apoptosis. HEK-293cells were co-transfected with luciferase constructs containingthe wild-type (WT) or mutant (MUT) target site of the LATS23′UTR and miR-135b mimicor miRNA NC for24h. The pGL3-vector was used as a negative control. The luciferaseexpression from the wild type LATS23′UTR was reduced to40%of that of control vectorand mutant.
Conclusions
1. E2inhibited the expressions of miRNAs, and upregulate hMLH1expression inCOLO205cells, which could be reversed by E2antagonist ICI182.780. The expression ofmiR-135b was significantly higher in CRC tissues than in normal tissues, and inverselycorrelated with serum E2level and ER-β mRNA expression in CRC patients’ cancertissues.
2. There was no significant correlation between hMLH1expression and serum E2levelin CRC patients, when more patients’ serum E2levels were lower than45pg/ml.Therefore,the E2regulatory effects on MMR through ER-β and miRNA expression may be animportant event that occurs early in tumorigenesis, and during cancer progression, adecreased estrogen and/or reduced ER-β expression result in the increase of miR-31,miR-155and miR-135b, which contributes to MMR instability and eventually colorectalcancer carcinogenesis.
3. Estrogen might induce apoptosis through the inhibition of miR-135b, whichcontribute to an increase in miR-135b target gene, LATS2(Large Tumor Suppressor2),expression and activity.
结直肠癌(colorectal cancer, CRC)在肿瘤相关性死亡病例中排在第四位，据统计每年约有608,700人因结直肠癌而死亡。近些年流行病学、临床和实验研究发现雌激素与结直肠癌发病有明显的关系，在过去的几十年里，美国结直肠癌死亡率女性患者下降明显，有分析认为雌激素替代疗法的流行是女性患病率下降的可能原因。目前所使用的结直肠癌化疗药物的治疗效果在年轻女性患者要优于男性患者，这说明雌激素除对女性结直肠癌患病具有保护作用外，在结直肠癌治疗中也有积极的作用，但确切的机制不明。最近研究报道结直肠癌患者癌组织中会出现miRNAs(microRNAs)表达谱的改变，包括miR-135b, miR-96, miR-183, miR-133a, miR-133b, miR-21, miR-31, miR-145,miR-203, miR-223, miR-155等，在结直肠癌中表达明显增加，但是miRNAs改变的原因及表达改变后如何促进结直肠癌的发生、发展，目前仍不清楚。已有研究显示雌激素可通过激活雌激素受体下调乳腺癌MCF-7细胞内miR-21表达，并增加其靶基因的表达。雌激素同样能够影响子宫内膜间质细胞和子宫平滑肌细胞内一些miRNAs的表达。雌激素受体阳性肿瘤中雌激素的表达与miRNA表达有一定关系。因此雌激素对结直肠癌的保护作用可能与雌激素对某些miRNAs表达的调节有关。部分结直肠癌的发生与DNA错配修复功能缺陷有关，在保持遗传稳定方面DNA错配修复(mismatchrepair, MMR)系统发挥着很重要的作用。错配修复基因突变包括遗传和散发性的突变，主要发生在致癌形成作用的早期阶段。通常能够检测出微卫星不稳定(microsatelliteinstability, MSI)或涉及DNA错配修复缺陷的基因主要为MLHI, MSH2, MSH6和PMS2。有研究显示雌激素和微卫星不稳定存在一定的关系，可增强主要的错配修复基因(hMLH1)在结肠上皮细胞的表达。雌激素还可诱导结肠癌细胞凋亡。但雌激素诱导错配修复功能增强和结肠癌细胞凋亡的机制不清。
为了研究雌激素对错配修复基因调节的可能机制，是否有miRNAs参与，可能参与雌激素诱导结肠癌细胞凋亡的靶基因。我们拟进行实验验证结肠癌细胞中雌激素和雌激素受体拮抗剂ICI182,780对细胞内一些miRNAs(miR-31, miR-155, miR-135b,miR-203和miR-223)表达的影响，同时在18例结直肠癌患者组织中评价血清雌激素水平与miRNA和MMR表达的相关性。通过荧光素酶活性实验确定可能参与雌激素诱导结肠癌细胞凋亡的基因。
目的
1.研究结肠癌细胞中miRNA表达与雌激素的关系。
2.研究结肠癌细胞中MMR表达与雌激素的关系。
3.人体组织标本验证雌激素、miRNA和MMR表达的相关性。
4.明确可能参与雌激素诱导结肠癌细胞凋亡的miRNA和靶基因。
研究方法
1.选用三种细胞进行实验，COLO205(细胞内有较高水平的ER-β表达，无ER-α)细胞、SW480细胞(细胞内仅有很低水平的ER-β表达)和MCF-7细胞(细胞内ER-α表达水平较高，而几乎检测不到ER-β表达)。不同浓度雌激素处理细胞流式细胞仪测定凋亡。10nM雌激素处理COLO205细胞0h,6h,12h和24h，提取雌激素处理前后的细胞总RNA，实时定量PCR(RT-qPCR)测定细胞内miR-31, miR-155, miR-135b,miR-203和miR-223的表达水平。细胞预先加入100nM雌激素受体抑制剂6h后，再加入雌激素处理12h，提取雌激素处理前后的细胞总RNA，实时定量PCR(RT-qPCR)测定细胞内miR-31, miR-155和miR-135b的表达。将ER-β质粒转染到SW480细胞内，增加细胞内ER-β的表达。收集转染后24h和48h的细胞，提取蛋白，Western blotting检测转染效果。雌激素处理转染pRST7-ER-β的SW480细胞，检测细胞内miR-31,miR-155和miR-135b的表达。
2. COLO205、SW480和MCF7细胞为研究对象，10nM雌激素处理COLO205细胞0h,6h,12h和24h，RT-qPCR和Western blotting检测细胞内ER-β和MMR表达。预先加入100nM雌激素受体抑制剂6h后，再加入雌激素处理12h，提取雌激素处理前后的细胞总RNA和蛋白，检测细胞内ER-β和MMR表达。雌激素处理MCF-7和SW480细胞12h后，RT-qPCR和Western blotting检测细胞内ER-β和MMR表达。将ER-β质粒转染到SW480细胞内，增加细胞内ER-β的表达后，RT-qPCR检测雌激素对转染pRST7-ER-β的SW480细胞内hMLH1mRNA表达的影响。Western blotting检测雌激素对转染pRST7-ER-β的SW480细胞内hMLH1蛋白表达的影响。
3.共收集18例结直肠癌患者血清样本，结直肠癌和癌旁标本，对雌激素、miRNA和MMR表达的相关性进行分析。
4.转染miR-135b抑制剂到COLO205细胞内，转染24h后收集3h,6h,12h和24h的细胞测定凋亡，提取总RNA测定miR-135b表达。根据在线数据库UCSC(www.geome.ucsc.edu)确定LATS23′UTR，PCR在人基因组扩增LATS23′UTR并克隆到pGL3荧光素报告载体得到pGL3-LATS23’UTR Wt。同时应用QuikChange IIXL Site-Directed Mutagenesis Kit将载体LATS23’UTR序列中的miR-135b结合位点的核心区突变后，克隆到pGL3荧光素报告载体的荧光素酶基因下游得到pGL3-LATS23’UTR Mut。分别将两个载体与miR-135b模拟物或miR-NC共转染HEK-293细胞，pGL3荧光素报告载体为对照，转染24h后，收集细胞裂解液，用双荧光素酶试剂盒(Promega)测定荧光素酶活性。
结果
1.雌激素可诱导COLO205细胞凋亡，并存在一定剂量效应关系。雌激素能够抑制COLO205细胞内miR-31, miR-155和miR-135b的表达，并有时间依赖效应。COLO205细胞预先加入100nM雌激素受体抑制剂6h后，能够拮抗雌激素对细胞内miR-31, miR-155和miR-135b表达的抑制作用。雌激素对SW480和MCF-7细胞内miR-31, miR-155和miR-135b表达无明显抑制作用。ER-β质粒转染到SW480细胞内，经Western blotting检测发现，转染空载组细胞内ER-β蛋白表达量无变化，而转染pRST7-ER-β的SW480细胞内ER-β蛋白表达量明显增加。RT-qPCR结果显示雌激素可抑制转染pRST7-ER-β的SW480细胞内miR-31, miR-155和miR-135b的表达，而对空载组无影响。结果说明雌激素对细胞内某些miRNAs表达的影响，可能是由雌激素受体ER-β介导的。
2. RT-qPCR和Western blotting结果显示，随着雌激素处理时间的增加，细胞内ER-β和hMLH1表达增加，hMSH2表达变化不明显。雌激素受体抑制剂可以抑制雌激素对细胞内ER-β和hMLH1表达的调节。雌激素处理MCF-7和SW480细胞12h后，细胞内hMLH1, hMSH2和ER-β表达无明显变化。将ER-β质粒转染到SW480细胞内，增加细胞内ER-β的表达后，RT-qPCR结果显示雌激素可上调转染pRST7-ER-β的SW480细胞内hMLH1mRNA的表达，而对空载组无影响。Western blotting结果显示雌激素可上调转染pRST7-ER-β的SW480细胞内hMLH1蛋白的表达，而对空载组无影响。雌激素对细胞内hMLH1表达的影响，可能与雌激素受体ER-β有关。
3.血清雌激素水平与miR-31和miR-135b表达有明显的负相关，而与miR-155表达无相关性。我们同样发现，在结肠癌组织中，hMLH1mRNA表达水平与miR-155和miR-135b表达有明显的负相关，而ER-β mRNA表达与血清雌激素水平有明显的正相关性。miR-135b与血清雌激素水平，ER-β和hMLH1表达有相关性。但hMLH1表达与血清雌激素水平无明显相关性。
4. miR-135b抑制剂转染COLO205细胞，随着细胞内miR-135b表达降低，细胞凋亡率明显增加。通过Target Scan和miRanda软件分析预测，荧光素酶活性实验证实miR-135b可以和LATS23’UTR结合。
结论
1.雌激素能够抑制COLO205细胞内miRNAs(miR-31, miR-155和miR-135b)的表达，上调hMLH1的表达，雌激素受体抑制剂能拮抗这一作用。相比于癌旁组织，癌组织中miR-135b表达明显增加。结肠癌组织内miR-135b和ER-β mRNA的表达与患者血清雌激素水平有一定相关性。
2.当多数病人的血清雌激素浓度低于45pg/ml时，结直肠癌组织中hMLH1mRNA的表达与患者血清雌激素水平没有明显相关性。因此雌激素通过雌激素受体对MMR和miRNA的调节作用可能发生在肿瘤形成的早期，在肿瘤形成过程中，随着雌激素水平和/或雌激素受体表达降低miR-31、miR-155和miR-135b表达会逐渐增加，引起错配修复功能不稳，促进结直肠癌发生。
3．雌激素诱导结肠癌细胞凋亡与抑制细胞内miR-135b表达有关，miR-135b的靶基因LATS2可能参与了这一机制。
Background
Colorectal cancer (CRC) is the fourth most common cause of death from cancer,affects around1.2million people world wide, with about608,700fatalities per year. Boththe prevalence and the lifetime risk of CRC are significantly lower in females than in males.This is particularly true when comparing pre-menopausal women to age-matched men. Thesex-specific difference in CRC incidence has been attributed to estrogen, but the exactmechanism is not clear. Since expression alterations of some miRNAs are associated with aseries of genetic events, their expression patterns may either have pro-or anti-cancereffects. Several miRNAs expression alterations have been reported in CRC, includingmiR-135b, miR-96, miR-183, miR-133a, miR-133b, miR-21, miR-31, miR-145, miR-203,miR-223, miR-155. However, the mechanism for miRNA change in CRC is not wellunderstood. Recent studies have indicated that estrogens (especially estradiol E2) canregulate miRNAs expression in human breast cancer cells, endometrial stromal cells, andmyometrial smooth muscle cells. Estrogen receptor (ER) expression is associated withmicroRNA (miRNA) expression in ER-positive tumors. Thus, estrogen’s protect effect maybe related to the regulation of some miRNAs. DNA mismatch repair (MMR) system playsan important role in maintaining genomic stability. The MMR gene alteration includinggenetic and sporadic mutations, commonly occur in the early stages of the neoplasticprocess. MMR dysfunction can cause microsatellite instability (MSI). It has been reportedthat estrogen is associated with MSI. In previous study, estradiol (E2) has shown to havethe ability to induce CRC cells to apoptosis, and to up-regulate major MMR gene (hMLH1)activity in colonic epithelial cells. These findings suggest that estrogen’s anti-colorectalcancer effect may be mediated by MMR function in colonic cells. However, the pathwaythrough which estrogen regulates MMR function is still not well understood.
From our knowledge, studies the direct link between estrogen, miRNA and MMRexpression in cultured cells and patient tissue/serum samples, the relationship of miRNA-target gene in CRC cell apoptosis, have not been done. In this study, we firstevaluated the effects of estrogen and its antagonist ICI182,780on the expression ofmiRNAs (miR-31, miR-135b, miR-155, miR-203and miR-223) and MMR using COLO205,SW480and MCF-7cell lines, followed by examining the association of tissue miRNAexpression, MMR expression and serum E2levels using samples collected from18colorectal cancer patients. Then the miRNA target gene will be confirmed by luciferaseassay.
Objective
1. Studies of direct link between estrogen and miRNA expression in CRC cells.
2. Studies of direct link between estrogen and MMR expression in CRC cells.
3. Studies the association of tissue miRNA expression, MMR expression and serum E2levels using samples collected from18colorectal cancer patients.
4. To check whether miRNAs participates in estrogen-induced apoptosis in culturedcells. Then the target gene will be confirmed.
Materials and methods
1. Studies of direct link between estrogen and miRNA expression in CRC.
COLO205and SW480cells were incubated in different concentrations of estradiol E2for48h;100nM of estrogen receptor antagonist ICI182,780was then added followed byAnnexin-V apoptosis assay. COLO205cells were treated with10nM E2with a finalvolume of0.01%ethanol for0h,6h,12h and24h to determine miRNA expression.SW480and MCF-7cells were treated with EtOH,1nM or10nM E2, for12h alone. Forthe indicated experiments, cells were pretreated with100nM ICI182,780for6h prior toEtOH (control) or E2treatment. SW480cells were transiently transfected with an emptypRST7vector and pRST7-ER-β. After24h of transfection, Western blotting was used toconfirm the transfection efficiency after24h or48h of transfection. After24h oftransfection, SW480(pERβ) and SW480(vector) were treated with EtOH or10nM E2for12h. After incubation, cells were harvested and used for quantitative reversetranscription-PCR (RT-qPCR).
2. Studies of direct link between estrogen and MMR in CRC.
COLO205cells were treated with10nM E2with a final volume of0.01%ethanol for0h,6h,12h and24h to determine mRNA expression, or for0h,24h and48h to determine protein expression. SW480and MCF-7cells were treated with EtOH,1nM or10nM E2for12h to determine mRNA expression, or for0h,24h and48h to determineprotein expression. After24h of transfection, SW480(pERβ) and SW480(vector) weretreated with EtOH or10nM E2for12h. After incubation, cells were harvested and usedfor quantitative reverse transcription-PCR (RT-qPCR) and Western blotting.
3. Studies the association of tissue miRNA expression, MMR expression and serum E2levels using samples collected from18colorectal cancer patients.
A total of18patients9males and9females diagnosed with CRC were enrolled. Thesepatients were randomly selected from patient pool of the hospital’s gastrointestinal clinic;none of the subjects had undergone estrogen replacement therapy. Blood was taken half anhour after colonoscopy for determining the serum E2level. During colonoscopy, four orfive samples from cancer tissues and adjacent non-cancer tissues, respectively, wereobtained and stored in liquid nitrogen. Correlation analyses between serum E2level andmiRNA expression, serum E2level and mRNA expression, and miRNA and mRNAexpression were performed.
4. To check whether miRNAs participates in estrogen-induced apoptosis in culturedcells. Then the target gene will be confirmed.
COLO205were transfected with miR-135b inhibitor, and incubated in differentconcentrations of estradiol E2for0h,6h,12h and24h to determine miRNA expressionand apoptosis. According to the UCSC (www.geome.ucsc.edu), LATS23′UTR wasamplified from the genomic DNA of human by PCR and cloned into luciferase reportergene pGL3-vector.The recombinant pGL3-LATS23’UTR Wt and pGL3-LATS23’UTRMut were respectively transient co-transfected with miR-135b mimic or miRNA NC intoHEK293cells using Lipofectamine. Luciferase activities were measured consecutively.
Rusults
1. After treatment with different concentrations of E2for48h, the apoptotic cells weresignificantly increased in COLO205, and the effect was the strongest at1×10-9M of E2.Estrogen receptor antagonist, ICI182,780, inhibited E2-induced apoptosis in COLO205cells. However, the effect of E2on apoptotic induction was not seen in SW480cells.RT-qPCR results indicated that E2decreased the levels of miR-31, miR-155and miR-135bin a time-dependent manner. ICI182,780reversed E2-induced repression of miR-31, miR-155and miR-135b in COLO205cells. E2did not however affect miRNA expressionin either SW480cells, or MCF-7cells. ER-β protein expression was not up-regulated invector transfected SW480cells, whereas it was up-regulated in pRST7-ER-β transfectedSW480cells after24and48h of transfection by western blot. RT-qPCR results indicatedthat E2decreased the levels of miR-31, miR-155and miR-135b in pRST7-ER-β transfectedSW480cells, but not in vector cells.
2. RT-qPCR and Western blotting showed that E2increased the expression of ER-βand hMLH1, but at less degree of hMSH2, at both mRNA and protein levels in COLO205cells, in a time and dose-dependent manners. These effects were again inhibited byICI182,780. No changes in the expression of hMLH1, hMSH2and ER-β mRNA were seenin MCF-7and SW480cells. RT-qPCR and Western blotting showed that E2increased theexpression of hMLH1, but at less degree of hMSH2, at both mRNA and protein levels inpRST7-ER-β transfected SW480cells.
3. The results indicated that serum E2levels were strongly and negatively correlatedwith miR-31and miR-135b expression, but not for miR-155expression in cancer tissue.We also found that in cancer tissue, hMLH1mRNA levels were negatively correlated withthat of miR-155and miR-135b expression, whereas ER-β mRNA levels were positivelycorrelated with serum E2concentrations. Interestingly, only miR-135b has a significantcorrelation with serum E2levels, ER-β and hMLH1expression. Our previous study showedthat in normal individuals, only when serum E2levels were higher than45pg/ml, a strongpositive correlation of E2with hMLH1gene expression was observed; and there was nosignificant correlation between hMLH1expression and serum E2levels when E2levelswere lower (E2,＜45pg/ml). In the current study of18CRC patients, only two patients’serum E2levels were higher than45pg/ml (a male, E2=69.16pg/ml; a femail, E2=70.18pg/ml).
4. Inhibition of miR-135b expression in COLO205cells, can significantly induced thecells to apoptosis. HEK-293cells were co-transfected with luciferase constructs containingthe wild-type (WT) or mutant (MUT) target site of the LATS23′UTR and miR-135b mimicor miRNA NC for24h. The pGL3-vector was used as a negative control. The luciferaseexpression from the wild type LATS23′UTR was reduced to40%of that of control vectorand mutant.
Conclusions
1. E2inhibited the expressions of miRNAs, and upregulate hMLH1expression inCOLO205cells, which could be reversed by E2antagonist ICI182.780. The expression ofmiR-135b was significantly higher in CRC tissues than in normal tissues, and inverselycorrelated with serum E2level and ER-β mRNA expression in CRC patients’ cancertissues.
2. There was no significant correlation between hMLH1expression and serum E2levelin CRC patients, when more patients’ serum E2levels were lower than45pg/ml.Therefore,the E2regulatory effects on MMR through ER-β and miRNA expression may be animportant event that occurs early in tumorigenesis, and during cancer progression, adecreased estrogen and/or reduced ER-β expression result in the increase of miR-31,miR-155and miR-135b, which contributes to MMR instability and eventually colorectalcancer carcinogenesis.
3. Estrogen might induce apoptosis through the inhibition of miR-135b, whichcontribute to an increase in miR-135b target gene, LATS2(Large Tumor Suppressor2),expression and activity.
引文
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[38] Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwideburden of cancer in2008: GLOBOCAN2008. Int J Cancer.2010,127(12):2893-2917
[39] Yang L, Parkin DM, Li L, Chen Y. Time trends in cancer mortality in China:1987–1999. Int J Cancer.2003,106(5):771–83.
[40] Chen WD, Han ZJ, Skoletsky J, Olson J, Sah J, Myeroff L, Platzer P, Lu S, Dawson D,Willis J, Pretlow TP, Lutterbaugh J, Kasturi L, Willson JK, Rao JS, Shuber A,Markowitz SD. Detection in fecal DNA of colon cancer-specific methylation of thenonexpressed vimentin gene.J Natl Cancer Inst.2005,97(15):1124-32.
[41] Olson J, Whitney DH, Durkee K, Shuber AP. DNA stabilization is critical formaximizing performance of fecal DNA-based colorectal cancer tests.Diagn MolPathol.2005,14(3):183-91.
[42] Smith RA, Cokkinides V, Brawley OW. Cancer screening inthe United States,2009. Areview of current American CancerSociety Guidelines and issues in cancer screening.CA Cancer J Clin.2009,59(1):27-41.
[43] Kennelly R, Kavanagh DO, Hogan AM, Winter DC. Oestrogen and the colon: potentialmechanisms for cancer prevention. Lancet Oncol.2008;9(4):385-391.
[44] Bardin A, Boulle N, Lazennec G, Vignon F, Pujol P. Loss of ERbeta expression as acommon step in estrogen-dependent tumor progression. Endocr Relat Cancer2004,11(3):537–51.
[45] Schleipen B, Hertrampf T, Fritzemeier KH, et al. ERβ-specific agonists and genisteininhibit proliferation and induce apoptosis in the large and small intestine.Carcinogenesis.2011,32(11):1675-83.
[46] West NJ, Courtney E, Poullis AP, Leicester RJ. Apoptosis in the colonic crypt,colorectal adenomata, and manipulation by chemoprevetion. Cancer EpidemiolBiomarkers Prev.2009;18(6):1680-7.
[47] Dobrzycka KM, Townson SM, Jiang S, Oesterreich S.Estrogen receptor corepressors--a role in human breast cancer? Endocr Relat Cancer.2003,10(4):517-36.
[48] Hall JM, Couse JF, Korach KS. The Multifaceted Mechanisms of Estradiol andEstrogen Receptor Signaling. J Biol Chem.2001,276(40):36869-72.
[49] Cappelletti V, Miodini P, Di Fronzo G, Daidone MG. Modulation of EstrogenReceptor-Beta Isoforms by Phytoestrogens in Breast Cancer Cells. Int J Oncol.2006,28(5):1185-91.
[50] Weyant MJ, Carothers AM, Mahmoud NN, Bradlow HL, Remotti H, Bilinski RT,Bertagnolli MM.Reciprocal Expression of ERalpha and ERbeta Is Associated withEstrogen-Mediated Modulation of Intestinal Tumorigenesis. Cancer Res,2001,61(6):2547-51.
[51] Caiazza F, Galluzzo P, Lorenzetti S, Marino M.17Beta-estradiol induces ERbetaup-regulation via p38/MAPK activation in colon cancer cells. Biochem Biophys ResCommun.2007,359(1):102-7.
[52] Jin P, Lu XJ, Sheng JQ, Fu L, Meng XM, Wang X, Shi TP, Li SR, Rao J. EstrogenStimulates the Expression of Mismatch Repair Gene hMLH1in Colonic EpithelialCells. Cancer Prev Res,2010;3(8):910-6
[53] Faltejskova P, Svoboda M, Srutova K, Mlcochova J, Besse A, Nekvindova J, Radova L,Fabian P, Slaba K, Kiss I, Vyzula R, Slaby O. Identification and functional screeningof microRNAs highly deregulated in colorectal cancer. J Cell Mol Med.2012,16(11):2655-66.
[54] Kanaan Z, Rai SN, Eichenberger MR, Roberts H, Keskey B, Pan J, Galandiuk S.Plasma miR-21: a potential diagnostic marker of colorectal cancer. Ann Surg.2012,256(3):544-51
[55] Visser S, Yang X.Identification of LATS transcriptional targets in HeLa cells usingwhole human genome oligonucleotide microarray. Gene.2010,449(1-2):22-9.
[56] Visser S, Yang X.LATS tumor suppressor: a new governor of cellular homeostasis.Cell Cycle.2010,9(19):3892-903.
[57] Liang Y, Li Y, Li Z, Liu Z, Zhang Z, Chang S, Wu J. Mechanism of folatedeficiency-induced apoptosis in mouse embryonic stem cells: Cell cyclearrest/apoptosis in G1/G0mediated by microRNA-302a and tumor suppressor geneLats2. Int J Biochem Cell Biol.2012,44(11):1750-60.
[58] Cho WJ, Shin JM, Kim JS, Lee MR, Hong KS, Lee JH, Koo KH, Park JW, Kim KS.miR-372regulates cell cycle and apoptosis of ags human gastric cancer cell linethrough direct regulation of LATS2. Mol Cells.2009,28(6):521-7.
[59] He ZH, Zhang XL, Zhang S, Wu ZJ, Ouyang J. ERβ as a Prognostic Factor forColorectal Cancer Liver Metastasis. J Canc Ther,2012,3(6A):880-887.
[60] Yamashita S, Yamamoto H, Mimori K, Nishida N, Takahashi H, Haraguchi N, TanakaF, Shibata K, Sekimoto M, Ishii H, Doki Y, Mori M. MicroRNA-372is associated withpoor prognosis in colorectal cancer. Oncology.2012,82(4):205-12.
[1] Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwideburden of cancer in2008: GLOBOCAN2008. Int J Cancer2010;127:2893-2917
[2] Chlebowski RT, Wactawski-Wende J, Ritenbaugh C, Hubbell FA, Ascensao J,Rodabough RJ, Rosenberg CA, Taylor VM, Harris R, Chen C, Adams-Campbell LL,White E; Women's Health Initiative Investigators. Estrogen plus Progestin andcolorectal cancer in postmenopausal women. N Engl J Med.2004,350(10):991-1004.
[3] Hendifar A, Yang D, Lenz F, Lurje G, Pohl A, Lenz C, Ning Y, Zhang W, LenzHJ.Gender disparities in metastatic colorectal cancer survival. Clin Cancer Res.2009,15(20):6391-7.
[4] Gravitz L. Chemoprevention: First line of defence. Nature.2011,471(7339): S5-7.
[5]房静远,郑树,姜泊,来茂德,房殿春,韩英,盛剑秋,李景南,中华医学会消化病学分会.中国结直肠肿瘤筛查、早诊早治和综合预防共识意见(二).胃肠病学.2011,16(12):735-744.
[6] Chen GG, Zeng Q, Tse GM. Estrogen and its receptors in cancer. Med Res Rev.2008;28(6):954-974.
[7] Klinge CM. Estrogen Regulation of MicroRNA Expression. Curr Genomics.2009,10(3):169-83
[8] Wada-Hiraike O, Imamov O, Hiraike H, Hultenby K, Schwend T, Omoto Y, Warner M,Gustafsson JA.Role of estrogen receptor beta in colonic epithelium. Proc Natl AcadSci U S A.2006,103(8):2959-64.
[9] Sassen S, Miska EA, Caldas C. MicroRNA: implications for cancer. Virchows Arch.2008,452(1):1-10.
[10] Wickramasinghe NS, Manavalan TT, Dougherty SM, Riggs KA, Li Y, Klinge CM.Estradiol downregulates miR-21expression and increases miR-21target geneexpression in MCF-7breast cancer cells. Nucleic Acids Res.2009;37(8):2584-95
[11] Pan Q, Luo X, Toloubeydokhti T, Chegini N. The expression profile of micro-RNA inendometrium and endometriosis and the influence of ovarian steroids on theirexpression. Mol Hum Reprod.2007,13(11):797-806
[12] Sheng JQ, Fu L, Sun ZQ, Huang JS, Han M, Mu H, Zhang H, Zhang YZ, Zhang MZ,Li AQ, Wu ZT, Han Y, Li SR. Mismatch repair gene mutations in Chinese HNPCCpatients. Cytogenet Genome Res.2008,122(1):22-27.
[13] Ferreira AM, Westers H, Albergaria A, Seruca R, Hofstra RM. Estrogens, MSI andLynch syndrome-associated tumors. Biochim Biophys Acta.2009,1796(2):194-200.
[14] He YQ, Sheng JQ, Ling XL, Fu L, Jin P, Yen L, Rao J. Estradiol regulates miR-135band mismatch repair gene expressions via estrogen receptor-β in colorectal cells. ExpMol Med.2012,44(12):723-732.
[15] Jin P, Lu XJ, Sheng JQ, Fu L, Meng XM, Wang X, Shi TP, Li SR, Rao J. EstrogenStimulates the Expression of Mismatch Repair Gene hMLH1in Colonic EpithelialCells. Cancer Prev Res.2010,3(8):910-6
[16] Faltejskova P, Svoboda M, Srutova K, Mlcochova J, Besse A, Nekvindova J, Radova L,Fabian P, Slaba K, Kiss I, Vyzula R, Slaby O. Identification and functional screeningof microRNAs highly deregulated in colorectal cancer. J Cell Mol Med.2012,16(11):2655-66.
[17] Nagel R, le Sage C, Diosdado B, van der Waal M, Oude Vrielink JA, Bolijn A, MeijerGA, Agami R. Regulation of the adenomatous polyposis coli gene by the miR-135family in colorectal cancer. Cancer Res.2008,68(14):5795-5802.
[18] Douglas EJ, Fiegler H, Rowan A, Halford S, Bicknell DC, Bodmer W, Tomlinson IP,Carter NP. Array comparative genomic hybridization analysis of colorectal cancer celllines and primary carcinomas. Cancer Res,2004,64(14):4817–25.
[19] Necela BM, Carr JM, Asmann YW, Thompson EA. Differential expression ofmicroRNAs in tumors from chronically inflamed or genetic (APC(Min/+)) models ofcolon cancer. PLoS One.2011,6(4):e18501.
[20] Oberg AL, French AJ, Sarver AL, Subramanian S, Morlan BW, Riska SM, BorralhoPM, Cunningham JM, Boardman LA, Wang L, Smyrk TC, Asmann Y, Steer CJ,Thibodeau SN.miRNA expression in colon polyps provides evidence for a multihitmodel of colon cancer. PLoS One.2011;6(6):e20465.
[21] Visser S, Yang X.Identification of LATS transcriptional targets in HeLa cells usingwhole human genome oligonucleotide microarray.Gene.2010,449(1-2):22-9.
[22] Visser S, Yang X.LATS tumor suppressor: a new governor of cellular homeostasis.CellCycle.2010,9(19):3892-903.
[23] Liang Y, Li Y, Li Z, Liu Z, Zhang Z, Chang S, Wu J.Mechanism of folatedeficiency-induced apoptosis in mouse embryonic stem cells: Cell cyclearrest/apoptosis in G1/G0mediated by microRNA-302a and tumor suppressor geneLats2. Int J Biochem Cell Biol.2012,44(11):1750-60.
[24] Cho WJ, Shin JM, Kim JS, Lee MR, Hong KS, Lee JH, Koo KH, Park JW, Kim KS.miR-372regulates cell cycle and apoptosis of ags human gastric cancer cell linethrough direct regulation of LATS2. Mol Cells.2009,28(6):521-7.
[2]房静远,郑树,姜泊,来茂德,房殿春,韩英,盛剑秋,李景南,中华医学会消化病学分会.中国结直肠肿瘤筛查、早诊早治和综合预防共识意见(二).胃肠病学.2011,16(12):735-744.
[3] Chlebowski RT, Wactawski-Wende J, Ritenbaugh C, Hubbell FA, Ascensao J,Rodabough RJ, Rosenberg CA, Taylor VM, Harris R, Chen C, Adams-Campbell LL,White E; Women's Health Initiative Investigators. Estrogen plus Progestin andcolorectal cancer in postmenopausal women. N Engl J Med.2004,350(10):991-1004.
[4] Hendifar A, Yang D, Lenz F, Lurje G, Pohl A, Lenz C, Ning Y, Zhang W, LenzHJ.Gender disparities in metastatic colorectal cancer survival. Clin Cancer Res.2009,15(20):6391-7.
[5] Boland CR, Koi M, Chang DK, Carethers JM. The biochemical basis of microsatelliteinstability and abnormal immunohistochemistry and clinical behavior in Lynchsyndrome: from bench to bedside. Fam Cancer.2008,7(1):41-52.
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[8] Miyamoto T, Shiozawa T, Kashima H, et al. Estrogen up-regulates mismatchrepairactivity in normal and malignant endometrial glandular cells. Endocrinology.2006,147(10):4863-4870.
[9] Garg K, Shih K, Barakat R, Zhou Q, Iasonos A, Soslow RA. Endometrial carcinomasin women aged40years and younger: tumors associated with loss of DNA mismatchrepair proteins comprise a distinct clinicopathologic subset. Am J Surg Pathol.2009,33(12):1869-1877.
[10] Sassen S, Miska EA, Caldas C. MicroRNA: implications for cancer. Virchows Arch.2008,452(1):1-10.
[11] Pan Q, Luo X, Toloubeydokhti T, Chegini N. The expression profile of micro-RNA inendometrium and endometriosis and the influence of ovarian steroids on theirexpression. Mol Hum Reprod.2007,13(11):797-806
[12] Valeri N, Gasparini P, Fabbri M, Braconi C, Veronese A, Lovat F, Adair B, Vannini I,Fanini F, Bottoni A, Costinean S, Sandhu SK, Nuovo GJ, Alder H, Gafa R, Calore F,Ferracin M, Lanza G, Volinia S, Negrini M, McIlhatton MA, Amadori D, Fishel R,Croce CM. Modulation of mismatch repair and genomic stability by miR-155. ProcNatl Acad Sci U S A.2010,107(15):6982-7.
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[18] Wickramasinghe NS, Manavalan TT, Dougherty SM, Riggs KA, Li Y, Klinge CM.Estradiol downregulates miR-21expression and increases miR-21target geneexpression in MCF-7breast cancer cells. Nucleic Acids Res.2009,37(8):2584-95
[19] Pfaffl MW. A new mathematical model for relative quantification in real-timeRT-PCR. Nucleic Acids Res.2001,29(9):e45
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[21] Wada-Hiraike O, Imamov O, Hiraike H, Hultenby K, Schwend T, Omoto Y, Warner M,Gustafsson JA.Role of estrogen receptor beta in colonic epithelium. Proc Natl AcadSci U S A.2006,103(8):2959-64.
[22] Qiu Y, Waters CE, Lewis AE, Langman MJS, Eggo MC. Oestrogen-induced apoptosisin colonocytes expressing oestrogen receptor β. J Endocrinol.2002,174(3):369-377.
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[29] Sarver AL, French AJ, Borralho PM, Thayanithy V, Oberg AL, Silverstein KA,Morlan BW, Riska SM, Boardman LA, Cunningham JM, Subramanian S, Wang L,Smyrk TC, Rodrigues CMP, Thibodeau SN, Steer CJ. Human colon cancer profilesshow differential microRNA expression depending on mismatch repair status and arecharacteristic of undifferentiated proliferative states. BMC Cancer.2009;9:401.
[30] Douglas EJ, Fiegler H, Rowan A, et al. Array comparative genomic hybridizationanalysis of colorectal cancer cell lines and primary carcinomas. Cancer Res,2004;64(14):4817–25.
[31] Nagel R, le Sage C, Diosdado B, van der Waal M, Oude Vrielink JA, Bolijn A, MeijerGA, Agami R. Regulation of the adenomatous polyposis coli gene by the miR-135family in colorectal cancer. Cancer Res.2008,68(14):5795-5802.
[32] Necela BM, Carr JM, Asmann YW, Thompson EA. Differential expression ofmicroRNAs in tumors from chronically inflamed or genetic (APC(Min/+)) models ofcolon cancer. PLoS One.2011,6(4):e18501.
[33] Oberg AL, French AJ, Sarver AL, Subramanian S, Morlan BW, Riska SM, BorralhoPM, Cunningham JM, Boardman LA, Wang L, Smyrk TC, Asmann Y, Steer CJ,Thibodeau SN. miRNA expression in colon polyps provides evidence for a multihitmodel of colon cancer. PLoS One.2011;6(6):e20465.
[34] Xu XM, Qian JC, Deng ZL, Cai Z, Tang T, Wang P, Zhang KH, Cai JP. Expression ofmiR-21, miR-31, miR-96and miR-135b is correlated with the clinical parameters ofcolorectal cancer. Oncol Lett.2012,4(2):339-345.
[35] Shibuya H, Iinuma H, Shimada R, Horiuchi A, Watanabe T. Clinicopathological andprognostic value of microRNA-21and microRNA-155in colorectal cancer. Oncology.2010;79(3-4):313-20.
[36] Slattery ML, Potter JD, Curtin K, Edwards S, Ma KN, Anderson K, Schaffer D,Samowitz WS.Estrogens reduce and withdrawal of estrogens increase risk ofmicrosatellite instability-positive colon cancer. Cancer Res.2001,61(1):126-130.
[37] Topping RP, Wilkinson JC, Scarpinato KD. Mismatch repair protein deficiencycompromises cisplatin-induced apoptotic signaling. J Biol Chem.2009,284(21):14029-39.
[38] Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwideburden of cancer in2008: GLOBOCAN2008. Int J Cancer.2010,127(12):2893-2917
[39] Yang L, Parkin DM, Li L, Chen Y. Time trends in cancer mortality in China:1987–1999. Int J Cancer.2003,106(5):771–83.
[40] Chen WD, Han ZJ, Skoletsky J, Olson J, Sah J, Myeroff L, Platzer P, Lu S, Dawson D,Willis J, Pretlow TP, Lutterbaugh J, Kasturi L, Willson JK, Rao JS, Shuber A,Markowitz SD. Detection in fecal DNA of colon cancer-specific methylation of thenonexpressed vimentin gene.J Natl Cancer Inst.2005,97(15):1124-32.
[41] Olson J, Whitney DH, Durkee K, Shuber AP. DNA stabilization is critical formaximizing performance of fecal DNA-based colorectal cancer tests.Diagn MolPathol.2005,14(3):183-91.
[42] Smith RA, Cokkinides V, Brawley OW. Cancer screening inthe United States,2009. Areview of current American CancerSociety Guidelines and issues in cancer screening.CA Cancer J Clin.2009,59(1):27-41.
[43] Kennelly R, Kavanagh DO, Hogan AM, Winter DC. Oestrogen and the colon: potentialmechanisms for cancer prevention. Lancet Oncol.2008;9(4):385-391.
[44] Bardin A, Boulle N, Lazennec G, Vignon F, Pujol P. Loss of ERbeta expression as acommon step in estrogen-dependent tumor progression. Endocr Relat Cancer2004,11(3):537–51.
[45] Schleipen B, Hertrampf T, Fritzemeier KH, et al. ERβ-specific agonists and genisteininhibit proliferation and induce apoptosis in the large and small intestine.Carcinogenesis.2011,32(11):1675-83.
[46] West NJ, Courtney E, Poullis AP, Leicester RJ. Apoptosis in the colonic crypt,colorectal adenomata, and manipulation by chemoprevetion. Cancer EpidemiolBiomarkers Prev.2009;18(6):1680-7.
[47] Dobrzycka KM, Townson SM, Jiang S, Oesterreich S.Estrogen receptor corepressors--a role in human breast cancer? Endocr Relat Cancer.2003,10(4):517-36.
[48] Hall JM, Couse JF, Korach KS. The Multifaceted Mechanisms of Estradiol andEstrogen Receptor Signaling. J Biol Chem.2001,276(40):36869-72.
[49] Cappelletti V, Miodini P, Di Fronzo G, Daidone MG. Modulation of EstrogenReceptor-Beta Isoforms by Phytoestrogens in Breast Cancer Cells. Int J Oncol.2006,28(5):1185-91.
[50] Weyant MJ, Carothers AM, Mahmoud NN, Bradlow HL, Remotti H, Bilinski RT,Bertagnolli MM.Reciprocal Expression of ERalpha and ERbeta Is Associated withEstrogen-Mediated Modulation of Intestinal Tumorigenesis. Cancer Res,2001,61(6):2547-51.
[51] Caiazza F, Galluzzo P, Lorenzetti S, Marino M.17Beta-estradiol induces ERbetaup-regulation via p38/MAPK activation in colon cancer cells. Biochem Biophys ResCommun.2007,359(1):102-7.
[52] Jin P, Lu XJ, Sheng JQ, Fu L, Meng XM, Wang X, Shi TP, Li SR, Rao J. EstrogenStimulates the Expression of Mismatch Repair Gene hMLH1in Colonic EpithelialCells. Cancer Prev Res,2010;3(8):910-6
[53] Faltejskova P, Svoboda M, Srutova K, Mlcochova J, Besse A, Nekvindova J, Radova L,Fabian P, Slaba K, Kiss I, Vyzula R, Slaby O. Identification and functional screeningof microRNAs highly deregulated in colorectal cancer. J Cell Mol Med.2012,16(11):2655-66.
[54] Kanaan Z, Rai SN, Eichenberger MR, Roberts H, Keskey B, Pan J, Galandiuk S.Plasma miR-21: a potential diagnostic marker of colorectal cancer. Ann Surg.2012,256(3):544-51
[55] Visser S, Yang X.Identification of LATS transcriptional targets in HeLa cells usingwhole human genome oligonucleotide microarray. Gene.2010,449(1-2):22-9.
[56] Visser S, Yang X.LATS tumor suppressor: a new governor of cellular homeostasis.Cell Cycle.2010,9(19):3892-903.
[57] Liang Y, Li Y, Li Z, Liu Z, Zhang Z, Chang S, Wu J. Mechanism of folatedeficiency-induced apoptosis in mouse embryonic stem cells: Cell cyclearrest/apoptosis in G1/G0mediated by microRNA-302a and tumor suppressor geneLats2. Int J Biochem Cell Biol.2012,44(11):1750-60.
[58] Cho WJ, Shin JM, Kim JS, Lee MR, Hong KS, Lee JH, Koo KH, Park JW, Kim KS.miR-372regulates cell cycle and apoptosis of ags human gastric cancer cell linethrough direct regulation of LATS2. Mol Cells.2009,28(6):521-7.
[59] He ZH, Zhang XL, Zhang S, Wu ZJ, Ouyang J. ERβ as a Prognostic Factor forColorectal Cancer Liver Metastasis. J Canc Ther,2012,3(6A):880-887.
[60] Yamashita S, Yamamoto H, Mimori K, Nishida N, Takahashi H, Haraguchi N, TanakaF, Shibata K, Sekimoto M, Ishii H, Doki Y, Mori M. MicroRNA-372is associated withpoor prognosis in colorectal cancer. Oncology.2012,82(4):205-12.
[1] Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwideburden of cancer in2008: GLOBOCAN2008. Int J Cancer2010;127:2893-2917
[2] Chlebowski RT, Wactawski-Wende J, Ritenbaugh C, Hubbell FA, Ascensao J,Rodabough RJ, Rosenberg CA, Taylor VM, Harris R, Chen C, Adams-Campbell LL,White E; Women's Health Initiative Investigators. Estrogen plus Progestin andcolorectal cancer in postmenopausal women. N Engl J Med.2004,350(10):991-1004.
[3] Hendifar A, Yang D, Lenz F, Lurje G, Pohl A, Lenz C, Ning Y, Zhang W, LenzHJ.Gender disparities in metastatic colorectal cancer survival. Clin Cancer Res.2009,15(20):6391-7.
[4] Gravitz L. Chemoprevention: First line of defence. Nature.2011,471(7339): S5-7.
[5]房静远,郑树,姜泊,来茂德,房殿春,韩英,盛剑秋,李景南,中华医学会消化病学分会.中国结直肠肿瘤筛查、早诊早治和综合预防共识意见(二).胃肠病学.2011,16(12):735-744.
[6] Chen GG, Zeng Q, Tse GM. Estrogen and its receptors in cancer. Med Res Rev.2008;28(6):954-974.
[7] Klinge CM. Estrogen Regulation of MicroRNA Expression. Curr Genomics.2009,10(3):169-83
[8] Wada-Hiraike O, Imamov O, Hiraike H, Hultenby K, Schwend T, Omoto Y, Warner M,Gustafsson JA.Role of estrogen receptor beta in colonic epithelium. Proc Natl AcadSci U S A.2006,103(8):2959-64.
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