USF1下调结直肠癌中FHL2基因转录调控的机制研究
摘要
研究背景:
FHL2(four and a half LIM domain protein 2)是一种多功能蛋白。不同的亚细胞器中FHL2可能参与不同的生物学过程。FHL2能够与integrin、presenilin-2以及电压门控K~+通道受体相互作用,从而与信号转导蛋白(包括ERK2、FAPp175和腺苷酸环化酶)发生相互作用,影响转录因子和辅助因子的信号传导,进而影响基因表达。FHL2还可以调节剪接、DNA复制和修复过程。另外,FHL2可以与结构蛋白如β-actinin、肌动蛋白和肌联蛋白结合。
最初发现FHL2基因在肌原细胞中表达,但是在恶性横纹肌肉瘤细胞中下调。后来的研究发现FHL2除了在骨骼肌和平滑肌中不表达,在脑、肝脏和肺中低表达之外,在其余组织中均表达,而且与肿瘤关系密切。在鳞癌、恶性胶质瘤、黑色素瘤、髓系白血病、宫颈癌、结直肠癌、肺癌、肝癌和肾癌中FHL2的转录活性明显增高,而在淋巴细胞白血病、Burkitt's中FHL2的转录活性极低甚至缺乏。肺癌标本中FHL2重度核染,而在正常肺组织中FHL2无表达,且高表达FHL2的非癌病人1年生存率明显低于不表达FHL2的病人。与正常卵巢组织相比,在人卵巢癌的上皮细胞FHL2表达增高。与正常肝脏组织相比,10例肝癌标本中8例标本的FHL2mRNA表达水平明显增高。
本课题组的前期工作已经完成以下内容:1、胃肠道肿瘤组织中FHL2的表达明显高于配对的正常组织。反义FHL2稳定转染胃结肠肿瘤细胞后发现细胞变长或者成为梭状,胞浆变长或者成为树枝状且核浆比例变小。反义FHL2可以诱导CEA、E-cadherin和F-actin的成熟,且抑制包括cox-2、survivin、c—jun和hTERT等癌基因的表达,抑制AP-1和hTERT的启动子活性。抑制FHL2可以抑制血清依赖、锚定依赖和非依赖的细胞生长,并且抑制裸鼠成瘤实验中肿瘤的形成。因此抑制FHL2基因可以诱导胃肠道肿瘤细胞的分化并且抑制胃肠道肿瘤的形成;2、免疫组化发现FHL2蛋白在结直肠癌组织中高度表达,但其表达程度与癌症病理分级无明显相关;3、已经成功制备并鉴定了兔抗人的FHL2多克隆抗体。
FHL2由4个半LIM结构域组成,其中半个LIM结构域位于N端,属于LIM蛋白。人类FHL2基因定位在人染色体2q12-q14,由7个外显子组成,其中前3个是非编码序列。对该基因的启动子进行生物信息学分析,发现其启动子序列上存在着多个潜在的转录因子结合位点,包括p53、SRF、Nkx2.5、MEF-2、E2F、E-box、AP1等。尽管特定转录因子的实际作用还未完全明确,但有证据支持p53、SRF、Nkx2.5、MEF-2、E2F、AP1等参与了FHL2基因的转录。虽然在FHL2启动子中发现含有多个E-box位点,但至今尚无关于E-box位点介导FHL2转录水平调控机制研究的报道。根据启动子序列预测软件,我们推测USF1可能通过结合FHL2基因启动子上的E-box位点,从而调控FHL2基因的转录。
E-box位点是USF1、USF2、TFE3等多个bHLH-zip(basic helix-loop-helixleucine zipper)转录因子家族成员的结合位点。1985年,Sawadogo等人首次从Hela细胞核内提取物中部分纯化出一种称为USF(上游刺激因子,后来命名为USF1)的序列特异性转录因子,并在体外实验发现,加入USF可以使腺病毒Majorlate promoter的转录活性增强10～20倍。随后的研究证实了USF是进化高度保守的bHLH-zip转录因子家族的成员,能通过结合DNA上的E-box位点(核心序列为5'CANNTG 3')发挥作用。
USF是细胞内普遍存在的转录因子,其细胞特异性的表达是由于与之发生相互作用的转录调节因子及其转录后激活和翻译后修饰均具有细胞特异性。USF1具有类似于Myc蛋白的bHLH-LZ结构,可竞争性结合相同的靶DNA序列,从而拮抗Myc促进细胞增殖与转化的功能,为正常胚胎发育所必需。Hela细胞中过表达USF可引起生长抑制,然而由于Saos-2细胞缺乏USF的转录活性,在该细胞中并未发现该现象。6种乳腺癌细胞中3种USF转录活性缺失。USF转录活性在肿瘤细胞中缺失或者降低,这可能与E-box位点的突变或者甲基化修饰而终止蛋白质-DNA的相互作用,以及翻译后修饰和蛋白质-蛋白质的相互作用的改变,而抑制USF的转录活性,最终下调P53的表达。DNA损伤时,激活的USF1可通过调节P53和BRCA2基因参与DNA的修复。在永生化的肿瘤细胞中USF1通过与hTERT结合,参与肿瘤的进程。
因此,我们推测USF1可能通过结合FHL2基因启动子上的E-box位点,从而调控FHL2基因的转录。
研究目的:
探讨USF1在结直肠癌发生中的作用及其对FHL2基因的转录调控作用与机制。
研究内容、方法:
一、利用RT-PCR和Western blot方法检测不同的人胃肠道肿瘤细胞中USF1的表达情况;
二、利用免疫组化方法检测不同分期的人结直肠肿瘤组织中USF1的表达情况;
三、利用EMSA和ChIP方法检测USF1蛋白与FHL2基因启动子的结合情况;
四、利用基因克隆技术构建FHL2启动子的真核表达载体,利用定点突变技术构建FHL2启动子的突变载体;
五、利用双荧光素酶报告基因系统检测定点突变FHL2启动子的E-box位点后,突变载体荧光素酶活性的变化;
六、利用基因克隆技术构建并鉴定重组质粒pcDNA3.1(+)-USF1;
七、利用RT-PCR和Western blot方法检测过表达或者干扰USF1后FHL2的表达情况;
八、利用双荧光素酶报告基因系统检测过表达或者干扰USF1后FHL2启动子的荧光素酶活性。
结果:
一、USF1在不同的人胃肠道肿瘤细胞株中具有不同的表达水平8株人胃肠道肿瘤细胞均表达USF1蛋白。USF1在蛋白水平的表达量以SW480、SW620和LoVo细胞最高,而在KATOⅢ细胞中的含量最低;USF1在mRNA水平的表达量以SW480、SW620、LoVo和HCT116细胞最高,而在KATOⅢ细胞中的含量最低。
二、USF1在人结直肠肿瘤组织的表达显著增强USF1在人正常结直肠黏膜中蛋白呈低表达;在有不典型增生的人腺瘤组织中,USF1的表达相对于正常人结直肠黏膜开始增强;而在人结直肠癌组织中其表达显著增强,差别有统计学意义(P=0.0018)。USF1蛋白的表达在不同Dukes'分期、不同分化程度的结直肠癌组织之间无明显差异(P＞0.05)。
三、USF1对FHL2基因的转录具有调控作用
1、USF1能够与FHL2启动子的上游E-box位点特异性结合EMSA实验证实了SW480细胞的核蛋白与-817bp/-812bp处E-box位点结合形成了DNA-核蛋白复合物,且竞争性探针可以抑制此复合物的形成。ChIP实验证实了SW480细胞中USF1蛋白能与FHL2基因DNA结合。
2、定点突变FHL2启动子的E-box位点后,突变载体的荧光素酶活性较未突变组明显升高
双荧光素酶报告基因系统证实在SW480细胞和LoVo细胞中4个FHL2基因启动子报告载体的转录活性不同,其中pluc595的荧光素酶活性值最高。
对FHL2启动子区域-817/-812bp处的E-box进行定点突变,使其由CAGCTG序列变为TAATTG,从而破坏E-box位点的序列,使其不能与USF1结合。定点突变FHL2启动子后,突变载体荧光素酶活性较未突变组明显升高(p＜0.05)。
3、构建重组质粒pcDNA3.1(+)-USF1,并筛选出USF1高表达的SW480细胞和LoVo细胞,将其命名为SW480USF1和LoVoUSF1。
4、在SW480USF1和LoVoUSF1中,FHL2表达水平显著降低(P＜0.05)。
5、在SW480细胞和LoVo细胞中采用USF1 siRNA干扰USF1的表达后,FHL2表达水平显著增高(P＜0.05)。
6、在SW480USF1和LoVoUSF1中,FHL2基因启动子的转录活性下降。
7、在SW480细胞和LoVo细胞中,采用USF1 siRNA干扰USF1的表达后,FHL2基因启动子的转录活性上调。
结论:
1、USF1在结直肠癌组织中高度表达,但其表达程度与结直肠癌病理分期、分化程度无明显相关;
2、USF1蛋白同FHL2基因启动子具有直接相互作用;
3、USF1对内源性FHL2表达存在着负性调控作用;
4、USF1对FHL2启动子存在反式抑制作用。
Background
FHL2(4-1/2 LIM protein 2),also known as DRAL(down-regulated in rhabdomyosarcoma LIM protein),was initially cloned by its abundant expression in the human heart.It is the second member of a small family of proteins with 4-1/2 LIM domains.The acronym LIM is derived from the names of 3 transcription factors, Lin-11,Isl-1,and Mec-3,in which such a domain was first identified.
FHL2 expression is abundant in the heart,but FHL2 is also observed in several other organs,such as brain,liver,lung,cortex et al.FHL2 is not expressed in spleen, thymus,blood leukocytes,skin,skeletal muscle or the stromal smooth muscle cells of the prostate.It exerts different functions in different types of cells.We first showed that FHL2 expression is elevated in gastrointestinal cancer cell lines and cancer tissues.We firstly showed that FHL2 expression is elevated in gastrointestinal cancer cell lines and cancer tissues.
The human FHL2 gene maps to 2q12-q14 and consists of seven exons,of which the first three are non-coding.Computer analysis of the promoter reveals a plethora of putative transcription factor binding sites p53,Nkx2.5,SRF,MEF-2,E-box et al. Although the actual contribution of particular transcription factors has not been scrutinized,some evidence exists that p53,Nkx2.5,SRF,MEF-2 may be involved in the transcription of the FHL2 gene.Yet there is no evidence that E-box regulates the FHL2 gene expression.We speculate that USF1 bind to E-box motif of FHL2 gene promoter by computer analysis of the promoter.
USF is a group of basic helix-loop-helix(bHLH) transcription factors encoded by two distinct genes whose products correspond to 43 kDa USF1 and 44 kDa USF2. USF in the expression of several tissue-specific or developmentally regulated genes, including human growth 2,hormone,mouse metallothionein I,rat gamma,fibrinogen and Xenopus TFⅢA Cox-2、Osteopontin、IL-10、PAI-1、BRCA2、surfactant protein A、calcyclin、MHC I gene、OPN、BMP-4、TGF-β_2、CyclinB1、TGF-βⅡreceptor et al.
Objective
1.To investigate the role of USF 1 in human colorectal cancer.
2.To indentify the role of USF1 in the regulation of FHL2 gene Transcription.
Methods
1.RT-PCR and immunoblotting methods were performed to detect USF 1 expression in two gastric cancer and six colorectal cancer cell lines.
2.USF1 expression was assessed by Immunohistochemistry in human colorectal cancer tissues.
3.USF1 binding to the FHL2 promoter were indentified by EMSA and ChIP assay in the SW480 human colorectal cancer cell line in vivo and in vitro.
4.The activity of FHL2 gene promoter and the mutant FHL2 gene promoter were investigated by dual luciferase reporter assay.
5.Genetic manipulation was performed to up-regulate of knock down expression of USF1,and followed by RT-PCR,western blot and dual luciferase repoter assay to research the effect of USF1 genes on FHL2.
Result
1.All of the gastric cancer cells and six colon cancer cells expressed differernt level of USF1.
2.The role of USF1 in human colorectal cancer progression was assessed by immunohistochemistry.USF1 immunoactivity was lower in normal colorectal mucosa.USF1 expression increased in adenoma with atypical hyperplasia.An even stronger expression of USF1 was found in all of colorectal cancer samples.In colorectal cancer on different Dukes'stage,USF1 expression was no difference.These data demonstrated that upregulation of USF1 protein occurs in most colorectal cancer tissues,implying that USF1 may play a role in colorectal cancer progression.
3.EMSA showed that USF1 recognized the E-box motif located within -817/-812 bp of the FHL2 promoter.ChIP revealed in vivo USF1 binded to the FHL2 promoter in the SW480 human colorectal cancer cell line.
4.The E-Box mutation increased the FHL2 promoter activity.
5.Overexpression of USF1 down-regulated the expression of FHL2 and the activity of FHL2 gene promoter.Transfection of USF1-siRNA enhanced the expressions of FHL2 and the activity of FHL2 gene promoter.
Conclusion
1.USF1 expression had no correlaion with colorectal cancer progression.
2.USF1 protein interacts with FHL2 promoter in the SW480 human colorectal cancer.
3.USF1 and FHL2 expressions had negative correlation with colon cancer cells.
4.USF1 mediated the down-regulation of FHL2 transcription.
FHL2(four and a half LIM domain protein 2)是一种多功能蛋白。不同的亚细胞器中FHL2可能参与不同的生物学过程。FHL2能够与integrin、presenilin-2以及电压门控K~+通道受体相互作用,从而与信号转导蛋白(包括ERK2、FAPp175和腺苷酸环化酶)发生相互作用,影响转录因子和辅助因子的信号传导,进而影响基因表达。FHL2还可以调节剪接、DNA复制和修复过程。另外,FHL2可以与结构蛋白如β-actinin、肌动蛋白和肌联蛋白结合。
最初发现FHL2基因在肌原细胞中表达,但是在恶性横纹肌肉瘤细胞中下调。后来的研究发现FHL2除了在骨骼肌和平滑肌中不表达,在脑、肝脏和肺中低表达之外,在其余组织中均表达,而且与肿瘤关系密切。在鳞癌、恶性胶质瘤、黑色素瘤、髓系白血病、宫颈癌、结直肠癌、肺癌、肝癌和肾癌中FHL2的转录活性明显增高,而在淋巴细胞白血病、Burkitt's中FHL2的转录活性极低甚至缺乏。肺癌标本中FHL2重度核染,而在正常肺组织中FHL2无表达,且高表达FHL2的非癌病人1年生存率明显低于不表达FHL2的病人。与正常卵巢组织相比,在人卵巢癌的上皮细胞FHL2表达增高。与正常肝脏组织相比,10例肝癌标本中8例标本的FHL2mRNA表达水平明显增高。
本课题组的前期工作已经完成以下内容:1、胃肠道肿瘤组织中FHL2的表达明显高于配对的正常组织。反义FHL2稳定转染胃结肠肿瘤细胞后发现细胞变长或者成为梭状,胞浆变长或者成为树枝状且核浆比例变小。反义FHL2可以诱导CEA、E-cadherin和F-actin的成熟,且抑制包括cox-2、survivin、c—jun和hTERT等癌基因的表达,抑制AP-1和hTERT的启动子活性。抑制FHL2可以抑制血清依赖、锚定依赖和非依赖的细胞生长,并且抑制裸鼠成瘤实验中肿瘤的形成。因此抑制FHL2基因可以诱导胃肠道肿瘤细胞的分化并且抑制胃肠道肿瘤的形成;2、免疫组化发现FHL2蛋白在结直肠癌组织中高度表达,但其表达程度与癌症病理分级无明显相关;3、已经成功制备并鉴定了兔抗人的FHL2多克隆抗体。
FHL2由4个半LIM结构域组成,其中半个LIM结构域位于N端,属于LIM蛋白。人类FHL2基因定位在人染色体2q12-q14,由7个外显子组成,其中前3个是非编码序列。对该基因的启动子进行生物信息学分析,发现其启动子序列上存在着多个潜在的转录因子结合位点,包括p53、SRF、Nkx2.5、MEF-2、E2F、E-box、AP1等。尽管特定转录因子的实际作用还未完全明确,但有证据支持p53、SRF、Nkx2.5、MEF-2、E2F、AP1等参与了FHL2基因的转录。虽然在FHL2启动子中发现含有多个E-box位点,但至今尚无关于E-box位点介导FHL2转录水平调控机制研究的报道。根据启动子序列预测软件,我们推测USF1可能通过结合FHL2基因启动子上的E-box位点,从而调控FHL2基因的转录。
E-box位点是USF1、USF2、TFE3等多个bHLH-zip(basic helix-loop-helixleucine zipper)转录因子家族成员的结合位点。1985年,Sawadogo等人首次从Hela细胞核内提取物中部分纯化出一种称为USF(上游刺激因子,后来命名为USF1)的序列特异性转录因子,并在体外实验发现,加入USF可以使腺病毒Majorlate promoter的转录活性增强10～20倍。随后的研究证实了USF是进化高度保守的bHLH-zip转录因子家族的成员,能通过结合DNA上的E-box位点(核心序列为5'CANNTG 3')发挥作用。
USF是细胞内普遍存在的转录因子,其细胞特异性的表达是由于与之发生相互作用的转录调节因子及其转录后激活和翻译后修饰均具有细胞特异性。USF1具有类似于Myc蛋白的bHLH-LZ结构,可竞争性结合相同的靶DNA序列,从而拮抗Myc促进细胞增殖与转化的功能,为正常胚胎发育所必需。Hela细胞中过表达USF可引起生长抑制,然而由于Saos-2细胞缺乏USF的转录活性,在该细胞中并未发现该现象。6种乳腺癌细胞中3种USF转录活性缺失。USF转录活性在肿瘤细胞中缺失或者降低,这可能与E-box位点的突变或者甲基化修饰而终止蛋白质-DNA的相互作用,以及翻译后修饰和蛋白质-蛋白质的相互作用的改变,而抑制USF的转录活性,最终下调P53的表达。DNA损伤时,激活的USF1可通过调节P53和BRCA2基因参与DNA的修复。在永生化的肿瘤细胞中USF1通过与hTERT结合,参与肿瘤的进程。
因此,我们推测USF1可能通过结合FHL2基因启动子上的E-box位点,从而调控FHL2基因的转录。
研究目的:
探讨USF1在结直肠癌发生中的作用及其对FHL2基因的转录调控作用与机制。
研究内容、方法:
一、利用RT-PCR和Western blot方法检测不同的人胃肠道肿瘤细胞中USF1的表达情况;
二、利用免疫组化方法检测不同分期的人结直肠肿瘤组织中USF1的表达情况;
三、利用EMSA和ChIP方法检测USF1蛋白与FHL2基因启动子的结合情况;
四、利用基因克隆技术构建FHL2启动子的真核表达载体,利用定点突变技术构建FHL2启动子的突变载体;
五、利用双荧光素酶报告基因系统检测定点突变FHL2启动子的E-box位点后,突变载体荧光素酶活性的变化;
六、利用基因克隆技术构建并鉴定重组质粒pcDNA3.1(+)-USF1;
七、利用RT-PCR和Western blot方法检测过表达或者干扰USF1后FHL2的表达情况;
八、利用双荧光素酶报告基因系统检测过表达或者干扰USF1后FHL2启动子的荧光素酶活性。
结果:
一、USF1在不同的人胃肠道肿瘤细胞株中具有不同的表达水平8株人胃肠道肿瘤细胞均表达USF1蛋白。USF1在蛋白水平的表达量以SW480、SW620和LoVo细胞最高,而在KATOⅢ细胞中的含量最低;USF1在mRNA水平的表达量以SW480、SW620、LoVo和HCT116细胞最高,而在KATOⅢ细胞中的含量最低。
二、USF1在人结直肠肿瘤组织的表达显著增强USF1在人正常结直肠黏膜中蛋白呈低表达;在有不典型增生的人腺瘤组织中,USF1的表达相对于正常人结直肠黏膜开始增强;而在人结直肠癌组织中其表达显著增强,差别有统计学意义(P=0.0018)。USF1蛋白的表达在不同Dukes'分期、不同分化程度的结直肠癌组织之间无明显差异(P＞0.05)。
三、USF1对FHL2基因的转录具有调控作用
1、USF1能够与FHL2启动子的上游E-box位点特异性结合EMSA实验证实了SW480细胞的核蛋白与-817bp/-812bp处E-box位点结合形成了DNA-核蛋白复合物,且竞争性探针可以抑制此复合物的形成。ChIP实验证实了SW480细胞中USF1蛋白能与FHL2基因DNA结合。
2、定点突变FHL2启动子的E-box位点后,突变载体的荧光素酶活性较未突变组明显升高
双荧光素酶报告基因系统证实在SW480细胞和LoVo细胞中4个FHL2基因启动子报告载体的转录活性不同,其中pluc595的荧光素酶活性值最高。
对FHL2启动子区域-817/-812bp处的E-box进行定点突变,使其由CAGCTG序列变为TAATTG,从而破坏E-box位点的序列,使其不能与USF1结合。定点突变FHL2启动子后,突变载体荧光素酶活性较未突变组明显升高(p＜0.05)。
3、构建重组质粒pcDNA3.1(+)-USF1,并筛选出USF1高表达的SW480细胞和LoVo细胞,将其命名为SW480USF1和LoVoUSF1。
4、在SW480USF1和LoVoUSF1中,FHL2表达水平显著降低(P＜0.05)。
5、在SW480细胞和LoVo细胞中采用USF1 siRNA干扰USF1的表达后,FHL2表达水平显著增高(P＜0.05)。
6、在SW480USF1和LoVoUSF1中,FHL2基因启动子的转录活性下降。
7、在SW480细胞和LoVo细胞中,采用USF1 siRNA干扰USF1的表达后,FHL2基因启动子的转录活性上调。
结论:
1、USF1在结直肠癌组织中高度表达,但其表达程度与结直肠癌病理分期、分化程度无明显相关;
2、USF1蛋白同FHL2基因启动子具有直接相互作用;
3、USF1对内源性FHL2表达存在着负性调控作用;
4、USF1对FHL2启动子存在反式抑制作用。
Background
FHL2(4-1/2 LIM protein 2),also known as DRAL(down-regulated in rhabdomyosarcoma LIM protein),was initially cloned by its abundant expression in the human heart.It is the second member of a small family of proteins with 4-1/2 LIM domains.The acronym LIM is derived from the names of 3 transcription factors, Lin-11,Isl-1,and Mec-3,in which such a domain was first identified.
FHL2 expression is abundant in the heart,but FHL2 is also observed in several other organs,such as brain,liver,lung,cortex et al.FHL2 is not expressed in spleen, thymus,blood leukocytes,skin,skeletal muscle or the stromal smooth muscle cells of the prostate.It exerts different functions in different types of cells.We first showed that FHL2 expression is elevated in gastrointestinal cancer cell lines and cancer tissues.We firstly showed that FHL2 expression is elevated in gastrointestinal cancer cell lines and cancer tissues.
The human FHL2 gene maps to 2q12-q14 and consists of seven exons,of which the first three are non-coding.Computer analysis of the promoter reveals a plethora of putative transcription factor binding sites p53,Nkx2.5,SRF,MEF-2,E-box et al. Although the actual contribution of particular transcription factors has not been scrutinized,some evidence exists that p53,Nkx2.5,SRF,MEF-2 may be involved in the transcription of the FHL2 gene.Yet there is no evidence that E-box regulates the FHL2 gene expression.We speculate that USF1 bind to E-box motif of FHL2 gene promoter by computer analysis of the promoter.
USF is a group of basic helix-loop-helix(bHLH) transcription factors encoded by two distinct genes whose products correspond to 43 kDa USF1 and 44 kDa USF2. USF in the expression of several tissue-specific or developmentally regulated genes, including human growth 2,hormone,mouse metallothionein I,rat gamma,fibrinogen and Xenopus TFⅢA Cox-2、Osteopontin、IL-10、PAI-1、BRCA2、surfactant protein A、calcyclin、MHC I gene、OPN、BMP-4、TGF-β_2、CyclinB1、TGF-βⅡreceptor et al.
Objective
1.To investigate the role of USF 1 in human colorectal cancer.
2.To indentify the role of USF1 in the regulation of FHL2 gene Transcription.
Methods
1.RT-PCR and immunoblotting methods were performed to detect USF 1 expression in two gastric cancer and six colorectal cancer cell lines.
2.USF1 expression was assessed by Immunohistochemistry in human colorectal cancer tissues.
3.USF1 binding to the FHL2 promoter were indentified by EMSA and ChIP assay in the SW480 human colorectal cancer cell line in vivo and in vitro.
4.The activity of FHL2 gene promoter and the mutant FHL2 gene promoter were investigated by dual luciferase reporter assay.
5.Genetic manipulation was performed to up-regulate of knock down expression of USF1,and followed by RT-PCR,western blot and dual luciferase repoter assay to research the effect of USF1 genes on FHL2.
Result
1.All of the gastric cancer cells and six colon cancer cells expressed differernt level of USF1.
2.The role of USF1 in human colorectal cancer progression was assessed by immunohistochemistry.USF1 immunoactivity was lower in normal colorectal mucosa.USF1 expression increased in adenoma with atypical hyperplasia.An even stronger expression of USF1 was found in all of colorectal cancer samples.In colorectal cancer on different Dukes'stage,USF1 expression was no difference.These data demonstrated that upregulation of USF1 protein occurs in most colorectal cancer tissues,implying that USF1 may play a role in colorectal cancer progression.
3.EMSA showed that USF1 recognized the E-box motif located within -817/-812 bp of the FHL2 promoter.ChIP revealed in vivo USF1 binded to the FHL2 promoter in the SW480 human colorectal cancer cell line.
4.The E-Box mutation increased the FHL2 promoter activity.
5.Overexpression of USF1 down-regulated the expression of FHL2 and the activity of FHL2 gene promoter.Transfection of USF1-siRNA enhanced the expressions of FHL2 and the activity of FHL2 gene promoter.
Conclusion
1.USF1 expression had no correlaion with colorectal cancer progression.
2.USF1 protein interacts with FHL2 promoter in the SW480 human colorectal cancer.
3.USF1 and FHL2 expressions had negative correlation with colon cancer cells.
4.USF1 mediated the down-regulation of FHL2 transcription.
引文
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