Sp1对FHL2转录调控机制的研究
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摘要
目的和意义
FHL2(Four and a half LIM Protein 2)为新确定的癌基因,在胃肠道肿瘤的发生发展过程中起着重要的作用。关于FHL2下游的转录调控已有了一定的报道研究,但对其上游的相关调控尚无文献报道。本研究拟对人新癌基因FHL2 5′端上游进行生物信息学分析以预测其5′端启动子位置和转录调控元件。结合文献,通过软件筛选出转录因子Sp1(Specificity protein 1)为可能的调控因子,明确Sp1与FHL2上游启动子序列的转录调控关系,从而寻找能靶向调控FHL2表达的元件,以期应用于肿瘤的生物靶向治疗。
材料和方法
1、主要材料
Kato-Ⅲ、SW480和LoVo细胞,双荧光素酶报告基因检测试剂盒,核蛋白提取试剂盒,poly(dI-dC),γ-~(32)p,LipofectAMINE2000 Reagent,TRIzol,Sp1抗小鼠单克隆抗体,FHL2抗兔多克隆抗体,Mithramycin A。
2、方法
2.1 FHL2基因5′端上游序列的生物信息学分析
利用NCBI基因组数据库查找FHL2基因全长编码基因。将5′端上游序列用DBTSS软件分析得出转录起始位点及启动子区域,TFsearch软件分析可得出FHL2启动子区的转录调控元件及可能与启动子结合的转录因子。根据转录因子Sp1结合位点使用Primer 5.0分析设计得出4段不同长度启动子的引物。
2.2 FHL2基因启动子荧光素酶报告基因质粒的构建
以SW480细胞中提取的全基因组DNA为模板,使用Hotstart聚合酶扩增FHL2 5′端上游中的4段启动子片段,长度分别为1008bp、881bp、595bp和382bp。扩增产物经PCR产物纯化试剂盒纯化回收,用NheⅠ酶和KpnⅠ酶各10units酶切1小时后,将酶切产物连入酶切后的pGL3-Basic载体(4℃连接16h),转化。转化的菌液铺在含Amp抗性的LB琼脂平板上培养过夜,挑选阳性克隆扩增质粒,经KpnⅠ和NheⅠ双酶切鉴定出阳性克隆,做测序反应鉴定。
2.3 FHL2重组启动子报告基因活性检测
含2 mmol/L谷氨酰氨和10%FBS的1640培养液常规培养各细胞。在转染前1天将2×10~4细胞接种于24孔板,37℃,5%CO_2孵箱培养至次日70%～80%铺满时进行转染。pRL-CMV载体用于标化报告基因活性。每孔分别转入重组质粒0.8μg和pRL-CMV质粒0.008μg,转染4～6 h后,换为完全培养基继续培养,48h后小心吸去孔中的培养基,加入足量的PBS洗涤培养孔,每孔中加入50μl1×PLB(Passive Lysis buffer,裂解液),室温摇动孵育15 min,充分溶解转染细胞,吸取细胞裂解液至1.5 mL离心管中,12000g离心5min,吸取上清液。按照双荧光报告系统的说明书进行操作,分别检测得到萤火虫荧光素酶和海肾荧光素酶的荧光值,二者的比值即为相对荧光表达量(RLU值),代表启动子的活性。每组实验至少重复3次,取平均值。
2.4免疫组织化学
使用免疫组织化学即用型二步法检测胃肠组织中Sp1和FHL2表达及相关情况。兔抗FHL2多克隆抗体为本实验室自备(1:300稀释),小鼠抗Sp1单克隆抗体为Abcom产品(1:200稀释)。所有切片结果采用双盲法,有两个病理科医师独立阅片评分,Sp1阳性表达部位位于细胞核,FHL2阳性表达部位位于细胞核和/或细胞浆,呈棕黄色或棕褐色。每张切片随机选取3～5个不同高倍视野(400×)进行观察。染色结果综合染色强度及阳性细胞百分数两个方面进行半定量分析。指标染色强度判定:无染色为0分,浅黄色为1分,棕黄色为2分,黄褐色为3分。阳性细胞百分数判定:每个视野计数100个细胞,取其平均值。阳性细胞百分数＜5%为0分,5%～20%为1分,20%～60%为2分,＞60%为3分。将染色强度与阳性细胞百分数分值相加即为切片各指标最终得分:0～1分为阴性(-),2分为弱阳性(+),3～4分为阳性(++),5～6分为强阳性(+++)。
2.5细胞核蛋白抽提
首先按照说明书配置PBS/Phosphatase Inhibitors,1×Hypotonic Buffer,Complete Lysis Buffer,存放于冰上;然后将培养皿中细胞用冷PBS洗一次,加入1 ml PBS/Phosphatase Inhibitors;刮下细胞收集于1.5 ml EP管中,3000 rpm,4℃离心5min;弃上清,加入0.25ml 1×低渗缓冲液重悬沉淀,在冰上静置15 min;加入12.5μl Detergent,振荡混匀仪最高速10～20s;4,000g,4℃离心30s;弃上清,往沉淀中加入25μl完全裂解液,立即混匀,置振荡混匀仪150rpm,4℃,30min;振荡混匀仪最高速30s;14,000g,4℃离心15min。小心吸取上清,用BCA蛋白定量试剂盒测定蛋白浓度,根据浓度稀释为2μg/μl,-70℃保存。
2.6凝胶阻滞实验
使用TFsearch软件分析得到转录因子Sp1结合位点,根据结合位点设计结合探针。T4连接酶将5μCi[γ-~(32)p]标记至双链DNA探针,使用纯化柱将未标记的探针去除。先计算所需的Gel Shift Binding 5×Buffer的大致量,加入0.5μg/reaction poly(dI-dC),混匀;标记1.5 ml EP管,依次加入2μl Gel Shift Binding5×Buffer,nuclease-free water,2μg核提取物混匀,室温孵育10min;然后加入相应的γ-~(32)p标记的探针,室温再孵育20min。上样至5%非变性PAGE胶中电泳120V 1h。用保鲜膜包住干燥过的胶,装入片夹,压上胶片,-70℃6～15 hr后显影、定影。
2.7定点突变重组质粒并检测其活性变化
根据说明书设计突变引物,以pLuc-595为模板进行扩增得到Sp1结合位点突变的产物。产物用DpnI酶切(对甲基化及半甲基化位点特异)可将未突变的模板质粒清除,然后转化入Epicurian coli XLl-Blue感受态细胞扩增成功突变的质粒,测序鉴定后命名为pLuc-MT。将突变质粒转染入细胞内24～48h后收集细胞进行双荧光素酶报告基因检测。
2.8 siRNA及MIT抑制Sp1的表达后检测启动子活性变化
根据Sp1 cDNA(NM 138473.2)设计合成Sp1-siRNA片段长度21bp,序列为AAAGCGCUUCAUGAGGAGUGA,阴性对照siRNA(Negative control siRNA)片段为UUCUCCGAACGUGUCACGUTT。按照说明书操作,Kato-Ⅲ、SW480和LoVo细胞种于24孔板过夜,次日使用1μl/孔LipofectAMINE2000 Reagent将siRNA 100 pmol/孔转染入细胞中,同时设置另一组加入MIT(终浓度为1μM),24～48h后收集细胞进行双荧光素酶报告基因检测。
2.9 siRNA及MIT抑制Sp1的表达后检测FHL2 mRNA及蛋白水平变化
使用LipofectAMINE2000 Reagent将siRNA 500 pmol转染入6孔板培养细胞中,同时设置另一组加入MIT(终浓度为1μM),24～48h后收集细胞提取RNA及蛋白,进行RT-PCR及Western Blotting检测FHL2的表达。
2.10数据统计
双荧光素酶报告基因检测、RT-PCR和Western Blotting灰度值分析结果用三次实验结果的(?)±SD表示,两组采用两独立样本t检验;多组间方差齐者采用方差分析,整体比较有显著性差异后多重比较采用LSD法,方差不齐者采用Welch检验分析,整体比较有显著性差异后多重比较采用Games-Howell检验。免疫组化结果癌旁组织和肿瘤组织Sp1和FHL2阳性等级的比较采用非参数检验,Sp1和FHL2两者表达情况采用相关分析。统计处理采用SPSS 13.0软件,检验水准以P＜0.05为差异有统计学意义。
结果
1、FHL2基因启动子荧光素酶报告基因质粒的构建及鉴定
扩增了4段FHL2基因5′端上游启动子序列,大小分别为1008bp、881bp、595bp及382bp。上述片段分别插入到pGL3-Basic载体构建成重组质粒,命名为pLuc-1008、pLuc-881、pLuc-595及pLuc-382。酶切鉴定与荧光素酶基因片段相符,证实载体构建成功。DNA测序报告显示与Genbank中序列完全吻合。
2、胃肠癌细胞株中FHL2重组启动子报告基因活性检测
pGL3-Basic载体含有荧光素酶基因序列,并且载体本身不包含启动子,克隆入启动子后可用荧光素酶定量直观地比较两个启动子的活性及其在各种不同种类细胞系中表达的组织特异性,并以pRL-CMV质粒作为内参以消除转染效率的不同所带来的差异。重组质粒转染不同胃肠癌细胞后,荧光素酶活性RLU值(相对荧光单位)有不同的改变,表明所构建的FHL2基因启动子报告系统有转录活性。4个不同的重组质粒在胃癌Kato-Ⅲ细胞中活性分别为22.34±5.43,20.19±3.85,38.05±2.04和5.25±1.10;肠癌LoVo细胞中活性分别为4.30±0.85,4.65±0.68,9.46±2.31和3.10±0.51;肠癌SW480细胞中活性分别为2.68±0.15,2.02±0.08,3.31±0.23和1.85±0.42。3株肿瘤细胞中均为pLuc-595的RLU值最高(方差分析/Welch整体检验,F/Welch值分别为43.421,20.682和7.345,P值分别为0.000,0.000和0.038)。
3、免疫组织化学验证Sp1表达与FHL2表达成正相关
收集大肠及胃的癌旁粘膜和肿瘤组织石蜡标本进行免疫组织化学检测。Sp1在40例癌旁肠粘膜组织中,表达为阴性的有22例,弱阳性的有8例,阳性的有8例,强阳性的有2例,平均秩为32.98;在44例肠癌组织中,表达为阴性的有12例,弱阳性的有3例,阳性的有16例,强阳性的有13例,平均秩为51.16,表达较癌旁组织增强具有显著性(两等级资料秩和检验,P=0.000);在13例癌旁胃粘膜组织中,表达为阴性的有10例,弱阳性的有3例,无阳性及强阳性表达,平均秩为10.04;在15例胃癌组织中,表达为阴性的有4例,弱阳性的有5例,阳性的有4例,强阳性的有2例,平均秩为18.37,表达较癌旁组织增强具有显著性(两等级资料秩和检验,P=0.000)。FHL2在34例癌旁肠粘膜组织中,表达为阴性的有33例,弱阳性的有1例,无阳性及强阳性表达,平均秩为21.72;在38例肠癌组织中,表达为阴性的有8例,弱阳性的有7例,阳性的有15例,强阳性的有8例,平均秩为49.72,表达较癌旁组织增强具有显著性(两等级资料秩和检验,P=0.000);在12例癌旁胃粘膜组织中,表达为阴性的有10例,无弱阳性表达,阳性的有1例,强阳性的有1例,平均秩为8.79;在16例胃癌组织中,表达为阴性的有1例,弱阳性的有4例,阳性的有7例,强阳性的有4例,平均秩为18.78,表达较癌旁组织增强具有显著性(两等级资料秩和检验,P=0.000)。对同时进行了Sp1和FHL2的表达检测的34例大肠癌病人使用相关分析Spearman检验得到R=0.469,P=0.005,提示Sp1表达与FHL2表达相关,且随着Sp1表达量增高FHL2的表达量也增高。
4、Sp1能与FHL2启动子特异结合
根据TFsearch软件分析得出FHL2启动子上游中Sp1可能的转录结合位点设计了两对探针分别为:探针1:(-485) 5′agagggcccgggcttggaatgt3′(-464),探针2:(-123) 5′tgccaccgcgcccaggcctcgt3′(-102)。以肠癌SW480细胞核提取物进行结合反应,电泳后曝光,标记的探针1+核蛋白抽提物泳道中出现一条特异的结合带,使用50倍未标记的探针1冷竞争反应后条带消失,而探针2泳道中未发现条带。提示探针1能与目的核蛋白中的Sp1特异结合,而探针2不能与转录因子Sp1结合。使用标记的突变探针1进行竞争反应该条带消失,进一步证实了Sp1能与FHL2启动子特异结合。
5、定点突变FHL2重组启动子后报告基因检测活性下降
根据凝胶阻滞实验结果,按照定点突变试剂盒设计突变引物将探针-485agagggcccgggcttggaatgt-464中Sp1位点突变为agaggAccAggTcttggaatgt。突变后的质粒经测序鉴定证实。将pLuc-595和pLuc-MT分别转染入Kato-Ⅲ、SW480和LoVo细胞中比较荧光素酶活性变化。三株细胞中pLuc-MT的RLU值均较pLuc-595的RLU值降低具有显著性(两独立样本t检验,t值分别6.218、42.772和8.377,P值分别为0.003、0.000、0.001)。
6、siRNA及MIT抑制Sp1表达后FHL2重组启动子报告基因检测活性下降
在三株细胞中MIT实验组启动子活性较阴性对照组活性下降均有显著性差异(P均＜0.05)。SW480和LoVo细胞Sp1-siRNA实验组启动子活性较阴性对照组活性下降有显著性差异(P均＜0.05);但Kato-Ⅲ细胞Sp1-siRNA实验组的RLU值较阴性对照组RLU值下降无统计学意义(P＞0.05)。
7、siRNA及MIT抑制Sp1表达后FHL2 mRNA及蛋白表达下降
Kato-Ⅲ、SW480和LoVo三株细胞中的Sp1-siRNA实验组和MIT实验组Sp1 mRNA水平均较阴性对照组中Sp1 mRNA水平显著降低(P均＜0.05),提示设计的Sp1-siRNA能有效抑制Sp1的mRNA表达。实验组FHL2 mRNA水平较阴性对照组FHL2 mRNA水平也有显著性降低(P均＜0.05)。蛋白水平的检测也有类似的结果(P均＜0.05)。
结论
获得了新癌基因FHL2的5′端启动子序列及转录调控信息,根据Sp1结合位点设计克隆的1008bp、881bp、595bp及382bp大小的FHL2启动子片段在胃肠癌细胞中有不同的转录活性,其中以pLuc-595活性最高。临床标本免疫组织化学检测提示Sp1和FHL2在胃肠道肿瘤组织中均较癌旁正常组织高表达,且随着Sp1表达量增高FHL2的表达量也增高,两者在肿瘤组织中的表达量成正相关。通过EMSA进一步证实了Sp1能与FHL2 5′端启动子序列特异的Sp1结合位点结合,突变该Sp1结合位点及抑制Sp1表达后均可发现FHL2转录活性及表达下降。以上结果均提示Sp1在一定程度上参与了FHL2的转录调控,抑制Sp1可下调FHL2的表达,FHL2启动子可能作为靶向性基因治疗的调控元件应用于肿瘤的生物治疗。
BACKGROUD AND OBJECTIVES
As a novel-identified oncogene,FHL2(Four and a half LIM Protein 2) plays important role in the devolpment of gastrointestinal(GI) cancers.It is known that FHL2 being able to modulate the function and/or expression of numerous downstream genes,however,the regulation of FHL2 has been seldomly investigated. By bioinformatical analysis,several putative Sp1(Specificity protein 1) binding elements were found within the 5'-flanking region of FHL2,the purpose of this project is to clarify the effect of Sp1 on transcription regulation of FHL2 gene.
METHODS
1.Cells and cell culture
GI cancer cell line,Kato-Ⅲ,SW480 and LoVo were maintained in 1640(Gibco) supplemented with 10%FBS,100U/ml penicillin.The cells were incubated in a humidified incubator at 37℃with an atmosphere of 5%CO_2.
2.Generation of FHL2 promoter-luciferase constructs.
The genomic DNA was obtained from the SW480.The four insert DNA fragments of the 5-flanking region of the FHL2 gene were amplified by a PCR method using specific primers.The synthetic linkers and restrition sites for both KpnI (GAGGTACC) and NheI(GAGCTAGC) are indicated below.The PCR was performed as follows:95℃for 30min and then 36 cycles of 94℃for 30s(for denaturation),For 55℃for 45s(for annealing),and 72℃for 1min(extension).After the final cycle,an elongation step was carried out at 72℃for 10min.The amplified DNA products were gel-purified using a gel extraction kit(Gel DNA Recovery kit; AXYGEN,USA).For the luciferase reporter gene assays,DNA fragments containing restriction site adapters were digested with either KpnI forward or NheI reverse.The digested DNA fragments were cloned into a firefly luciferase expression vector, pGL3-Basic vector(Promega) which had already been digested with the enzymes KpnI and NheI.The activity of the promoterless vector,pGL3-Basic,which contained no insert,was measured to determine background activity.Each plasmid construct was subcloned into TOP10 bacterial cells,and the plasmids were subsequently isolated and purified.The entire DNA sequences of the cloned fragments were confirmed with gene sequencing.
3.Transient Transfection and Luciferase Assays
For the luciferase assay,cells were plated at a density of approximately 2×10~4 cells in twenty-four wells plate until 70%confluent growth was reached.On the next day,growth medium was removed from cells before transfection.A transfection complex was prepared by mixing 0.8μg of the indicated pGL3-luciferase constructs and 0.008μg pRL-CMV with lipofetamine 2000.Transient transfection was performed according to the instruction of lipofetamine 2000(Invitrogen).Two days later,the cells were harvested;dual-luciferase assay was performed to measure the luciferase activities using a luminometer(Aureon Biosystems) according to the manufacturer's protocol(Promega).Promoter activity was presented as the fold induction of relative luciferase unit(RLU) compared with the basic vector control. RLU= value of firefly luciferase unit/value of renilla luciferase unit.All treatments were triplicated for each single experiment.
4.Immunohistochemistry
Immunohistochemistry was performed to detect Sp1 and FHL2 in situ,antigen retrieval and an indirect immunoperoxidase technique were applied as reported before. The mouse anti-Sp1 monoclonal antibody(dilution 1:200),the rabbit anti-FHL2 polyclonal antibody(dilution 1:300) and the biotin-linked antimouse IgG(Dako, Copenhagen,Denmark) in combination with the ABC complex were used.Normal IgG(Sigma) was used as the isotype controls.
5.Preparation of Cytoplasmic and Nuclear Extract and Western blotting
The cells were resuspended in 400μl of buffer A(containing 10mM Hepes at pH 7.9,1.5mM MgCl_2,10mM KC1,0.5mM DTT,0.5mM PMSF,1μg/ml leupeptin, 1μg/ml aprotinin,and 1μg/ml pepstatin A),lysed with 12.5μl of 10%Nonide P-40, and centrifuged at 12,000g for 10min at 4℃.The supematant was collected and used as the cytoplasmic extracts.The nuclei pellet was re-suspended in 4.0μl of buffer B (20mM Hepes,pH 7.9,containing 1.5mM MgCl_2,450mM NaCl,25%glycerol,0.2 mM EDTA,0.5mM DTT,0.5mM PMSF,1μg/ml leupeptin,1μg/ml aprotinin,1μg/ml pepstatin A),agitated for 60min at 4℃,and the nuclear debris was spun down at 20,000g for 15min.The supernatant(nuclear extract) was collected and stored at-70℃until ready for analysis.Protein concentrations were determined with BCA Protein Assay Kit.
6.Electrophoretic Mobility Shift Assay
Double-strand FHL2 gene specific DNA probes that contained the specific Sp1 binding elements were synthesized.The sequences of sense strands were as follows: (-485)5'- AGAGGGCCCGGGCTTGGAATGT -3'(-464) and(-123) 5'-TGCCAC CGCGCCCAGGCCTCGT -3'(-102).After annealing,double-strand DNA probes were end labeled with 5μCi ofγ-~(32)P -ATP(PerkinElmer Life and Analytical Sciences) using T4 polynucleotide kinase(Promega).For electrophoretic mobility shift assay (EMSA),total reaction mixtures containing 10mmol/L Tris/HCl(pH 7.5),1 mmol/L MgCl_2,0.5mmol/L dithiothreitol,0.5mmol/L ethylenediaminetetraacetic acid, 50mmol/L NaCl,4%glycerol,and 0.5μg of poly(dI-dC)-poly(dI-C)/mL,were incubated with 2μg of nuclear extracts and various unlabeled competing oligonucleotides for 10 minutes at room temperature,followed by addition of 1μl ([0.5-2]×10~5 counts per minute) of the various ~(32)P-end-labeled oligonucleotides. Samples were separated by electrophoresis on 5%nondenaturing polyacrylamide gel, with detection of radioactive bands by autoradiography for 12-16 hours at -70℃.The mutation sequence of the probe was(-485) 5'- AGAGGACCAGGTCTTGGAATGT -3'(-464).
7.siRNA Transfection
The siRNA duplexes consisted of 21 bp oligonucleotides and the sequence of the Sp1-siRNA was as follows:AAAGCGCUUCAUGAGGAGUGA.The GL2 siRNA (UUCUCCGAACGUGUCACGUTT) was used as the negative control.The cells were transfected with siRNA duplexes using LipofectAMINE2000 reagent (Invitrogen) according to the manufacturer's instructions.
8.Reverse-Transcription Polymerase Chain Reaction
RNA was reverse-transcribed to complementary DNA(cDNA) by Thermoscript reverse-transcription polymerase chain reaction(PCR) system(Invitrogen) in accordance with the manufacturer's instructions.PCR was performed using 2μl of resulting cDNA,0.125μl of Hotstart DNA polymerase(Qiagen,Hilden, Germany),forward and reverse primers,and deoxynucleoside triphosphates in a final volume of 25μl.The sequences of the primers were as follows:FHL2 forward:5'-AGAGTTTCATCCCCAAAGACAA -3';FHL2 reverse:5'- AGTTCAGGCAGTA GGCAAAGTC-3';Sp1 forword:5'-CGCTCCCAACTTACAGAA-3';reverse:5'-GCTATTGGCATTGGTGAA -3'.Hotstart PCR was performed for 32 cycles with 95℃denaturation for 30 minutes(first cycle),94℃denaturation for 30 seconds, 53°℃annealing for 30 seconds,and 72℃elongation for 60 seconds and 10 minutes (final cycle).The amplification product was of the expected sizes.
9.Immunoblotting
Cell lysates(30μg) were electrophoresed on denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel(5%stacking gel and 10%separating gel).Proteins were transferred to polyvinylidene difluoride membranes(Perkin Elmer, Fremont,CA).Nonspecific binding was blocked with 10 mmol/L pH 7.6 Tris-HCl buffer saline plus 0.05%Tween-20 containing 5%skim milk.The blots were probed with primary anti-human FHL2 antibody followed by the HRP-conjugated anti-rabbit second antibody.Antigen-antibody complexes were visualized by the enhanced chemiluminescence(ECL) system(PIERCE).
10.Site-Directed Mutagenesis Analysis
The QuikChange Site-Directed Mutagenesis Kit(Stratagene,La Jolla,CA) was used to generate constructs with the mutation of the putative interferon regulatory element(GC box-FHL2).The primers of mutation were as follows:wild-type sequence:agcgtcacgcagagggcccgggcttggaatgtgggagg;mutation sequence:agcgtcacg cagaggaccaggtcttggaatgtgggagg.Briefly,pLuc-595 construct was PCR-amplified in the elongation process using Pfu DNA polymerase with the earlier-described double-strand primers.The incorporation of oligonucleotide primers generates a mutated plasmid containing staggered nicks.The product then is treated with DpnI endonuclease,specific for methylated and hemimethylated DNA,enabling the parental DNA template to be digested(because DNA originating from Escherichia coli usually is dam methylated).The nicked vector DNA carrying the desired mutations was proliferated in Epicurian coli XL1-Blue supercompetent cells.Plasmid DNA was isolated and sequenced to verify the prospected mutated sequence.The mutation plasmid was transfected transiently into cancer cells to Luciferase assay.
11.Statistical Analysis
The data of Luciferase assay,RT-PCR and Western Blotting were expressed as the mean of three independent expression±S.D,evaluated with t-test or one-way ANOVA.Mann-Whitney Test was used to assess the differences of staining intensity. Correlation analyses for quantification of FHL2 and Sp1 positive staining were performed using Spearman Correlation.P＜0.05 was defined as different signicantly. All of these analyses were made using SPSS Version 13(SPSS,Chicago,IL,USA).
RESULTS
1.Positive correlation between Sp1 and FHL2 in colon cancer
To detect Sp1 and FHL2 expression in situ,we collected matched cancer and noncancerous colon tissues under colonoscopy.As determined by IHC,specific nuclear Sp1 protein was high expressed in the carcinoma cells.FHL2 positive signal presented in both the cytoplasm and nucleus of cancer cells but not in the normal colon epithelial cells.These findings demonstrated that Sp1 expression correlated with FHL2 in colon cancer cells.
2.Location of the regulatory promoter of FHL2 gene
To locate the regulatory promoter of FHL2,we used DBTSS software to analyze the promoter seqeunce of FHL2.TF search was applied to find the transcription factor binding site.Several typical and nontypical GC-box were found in the promoter of FHL2 and four different length of gene seqeunces were cloned to investigate the transcription activity of the promoter.Truncated 5'-flanking seqeunces extending up to-997nt of FHL2 gene were inserted in forward orientation upstream of a luciferase reproter gene(pGL3-Basic vector) to generate pLuc constructs pLuc-1008(-997 nt～+10 nt),pLuc881(-870 nt～+10 nt),pLuc595(-584 nt～+10 nt) and pLuc382(-371 nt～+10 nt),the first nucleotide adjacent to the transcription starting site was here difined as 0.Dual luciferase assay revealed that the highest RLU was observed for pLuc-595 in all three cells tested,indicating the presence of cis-enhancing element(s) between -584 to -371nt.However,transfection of the longer FHL2 5'-flanking sequences,pLuc-1008,resulted in a significant decrease in transcription activity,thus implicating the presence of a potential repressor element(s) between -997 and -584nt. All cell lines had a similar pattern of transcription activities with different values for individual constructs.
3.Identification of Sp1-binding sequence in FHL2 promoter
The consensus Sp1 binding sequences comprises GC-rich elements arranged as 5'-(G/T)GGGCGG(G/A)(G/A)(C/T)-3'.Two typical binding elements that located at -485nt to -464nt and -123nt to -102nt were identified within FHL2 5'- flanking region.To test the binding capacity of these sequences,EMSA was performed using the following two-step strategies.First,the capacity of a 50-fold excess of unlabeled oligonucleotides(-485/-464 and -123/-102) was used to compete and block binding to radioactively labeled probes.We found that -485/-464nt but not -123/-102nt oligonucleotide gave rise to specific binding while could be completely blocked by the cold probe.Secondly,a mutated probe with the mutation of Sp1 binding sequence was applied to verify the binding specificity of-485/-464nt probes.We found the site-mutation of Sp1 binding sequence abrogated the binding capacity completely. Therefore,we concluded that a high affinity Sp1 binding element existed in the -485/-464 region of FHL2 gene.
4.Down-regulation of FHL2 transcription activity by inactivation of Sp1 binding activity
To inactivate the binding of Sp1,we had synthesized a Sp1-siRNA to inhibit Sp1 expression.Moreover,mithramycin A(MIT),a novel defined Sp1 inhibitor,was applied to examine its effect on the transcription activity of truncated FHL2 promoter constructs.Kato-Ⅲ,SW480 and LoVo cells were transiently transfected with pLuc-1008,pLuc-881,pLuc-595 and pLuc-382 followed by incubation with control siRNA,Sp1-siRNA or MIT.Cells were lysed and assayed for luciferase activity. Dual luciferase assay showed that Sp1-siRNA and MIT significantly decreased the transcription activity of all constructs,suggesting that Sp1 was essence for FHL2 transcription.In addition,we introduced the gggcccggg to ggAccAggT mutation into pLuc-595 to abrogate Sp1-specific binding activity.We found that the RLU induction of wild type and mutant pLuc-595 constructs were 11.90±1.99 and 4.11±0.86 in Kato-Ⅲcell;7.02±0.03 and 2.31±0.18 in SW480 cells and 7.72±1.17 and 1.65±0.47 in LoVo cells,respectively.Mutation of Sp1 binding sequence decreased the transcription activity of pLuc-595 significantly(P＜0.05 compared with the wild type control).
5.Reduced FHL2 expression by inhibiting Sp1 expresion
At last,we evaluated FHL2 expression in presence or absence of Sp1 suppression by RT-PCR and Western blotting.We found that both Sp1-siRNA and MIT suppressed FHL2 expression.
CONCLUSIONS
1.Sp1 and FHL2 expression were positively correlated in colon cancers with both expressions were upregulated in cancer cells when comparing to normal tissues.
2.Within the 1000bp 5'-fianking region of FHL2 gene,the fragment between -584nt to -371nt possessed high level of transcription activity.
3.A functional Sp1 regulatory element with high affinity was identified at -485nt to -464nt upstream of the transcription starting site that mediated the positive regulation of FHL2 transcription.
FHL2(Four and a half LIM Protein 2)为新确定的癌基因,在胃肠道肿瘤的发生发展过程中起着重要的作用。关于FHL2下游的转录调控已有了一定的报道研究,但对其上游的相关调控尚无文献报道。本研究拟对人新癌基因FHL2 5′端上游进行生物信息学分析以预测其5′端启动子位置和转录调控元件。结合文献,通过软件筛选出转录因子Sp1(Specificity protein 1)为可能的调控因子,明确Sp1与FHL2上游启动子序列的转录调控关系,从而寻找能靶向调控FHL2表达的元件,以期应用于肿瘤的生物靶向治疗。
材料和方法
1、主要材料
Kato-Ⅲ、SW480和LoVo细胞,双荧光素酶报告基因检测试剂盒,核蛋白提取试剂盒,poly(dI-dC),γ-~(32)p,LipofectAMINE2000 Reagent,TRIzol,Sp1抗小鼠单克隆抗体,FHL2抗兔多克隆抗体,Mithramycin A。
2、方法
2.1 FHL2基因5′端上游序列的生物信息学分析
利用NCBI基因组数据库查找FHL2基因全长编码基因。将5′端上游序列用DBTSS软件分析得出转录起始位点及启动子区域,TFsearch软件分析可得出FHL2启动子区的转录调控元件及可能与启动子结合的转录因子。根据转录因子Sp1结合位点使用Primer 5.0分析设计得出4段不同长度启动子的引物。
2.2 FHL2基因启动子荧光素酶报告基因质粒的构建
以SW480细胞中提取的全基因组DNA为模板,使用Hotstart聚合酶扩增FHL2 5′端上游中的4段启动子片段,长度分别为1008bp、881bp、595bp和382bp。扩增产物经PCR产物纯化试剂盒纯化回收,用NheⅠ酶和KpnⅠ酶各10units酶切1小时后,将酶切产物连入酶切后的pGL3-Basic载体(4℃连接16h),转化。转化的菌液铺在含Amp抗性的LB琼脂平板上培养过夜,挑选阳性克隆扩增质粒,经KpnⅠ和NheⅠ双酶切鉴定出阳性克隆,做测序反应鉴定。
2.3 FHL2重组启动子报告基因活性检测
含2 mmol/L谷氨酰氨和10%FBS的1640培养液常规培养各细胞。在转染前1天将2×10~4细胞接种于24孔板,37℃,5%CO_2孵箱培养至次日70%～80%铺满时进行转染。pRL-CMV载体用于标化报告基因活性。每孔分别转入重组质粒0.8μg和pRL-CMV质粒0.008μg,转染4～6 h后,换为完全培养基继续培养,48h后小心吸去孔中的培养基,加入足量的PBS洗涤培养孔,每孔中加入50μl1×PLB(Passive Lysis buffer,裂解液),室温摇动孵育15 min,充分溶解转染细胞,吸取细胞裂解液至1.5 mL离心管中,12000g离心5min,吸取上清液。按照双荧光报告系统的说明书进行操作,分别检测得到萤火虫荧光素酶和海肾荧光素酶的荧光值,二者的比值即为相对荧光表达量(RLU值),代表启动子的活性。每组实验至少重复3次,取平均值。
2.4免疫组织化学
使用免疫组织化学即用型二步法检测胃肠组织中Sp1和FHL2表达及相关情况。兔抗FHL2多克隆抗体为本实验室自备(1:300稀释),小鼠抗Sp1单克隆抗体为Abcom产品(1:200稀释)。所有切片结果采用双盲法,有两个病理科医师独立阅片评分,Sp1阳性表达部位位于细胞核,FHL2阳性表达部位位于细胞核和/或细胞浆,呈棕黄色或棕褐色。每张切片随机选取3～5个不同高倍视野(400×)进行观察。染色结果综合染色强度及阳性细胞百分数两个方面进行半定量分析。指标染色强度判定:无染色为0分,浅黄色为1分,棕黄色为2分,黄褐色为3分。阳性细胞百分数判定:每个视野计数100个细胞,取其平均值。阳性细胞百分数＜5%为0分,5%～20%为1分,20%～60%为2分,＞60%为3分。将染色强度与阳性细胞百分数分值相加即为切片各指标最终得分:0～1分为阴性(-),2分为弱阳性(+),3～4分为阳性(++),5～6分为强阳性(+++)。
2.5细胞核蛋白抽提
首先按照说明书配置PBS/Phosphatase Inhibitors,1×Hypotonic Buffer,Complete Lysis Buffer,存放于冰上;然后将培养皿中细胞用冷PBS洗一次,加入1 ml PBS/Phosphatase Inhibitors;刮下细胞收集于1.5 ml EP管中,3000 rpm,4℃离心5min;弃上清,加入0.25ml 1×低渗缓冲液重悬沉淀,在冰上静置15 min;加入12.5μl Detergent,振荡混匀仪最高速10～20s;4,000g,4℃离心30s;弃上清,往沉淀中加入25μl完全裂解液,立即混匀,置振荡混匀仪150rpm,4℃,30min;振荡混匀仪最高速30s;14,000g,4℃离心15min。小心吸取上清,用BCA蛋白定量试剂盒测定蛋白浓度,根据浓度稀释为2μg/μl,-70℃保存。
2.6凝胶阻滞实验
使用TFsearch软件分析得到转录因子Sp1结合位点,根据结合位点设计结合探针。T4连接酶将5μCi[γ-~(32)p]标记至双链DNA探针,使用纯化柱将未标记的探针去除。先计算所需的Gel Shift Binding 5×Buffer的大致量,加入0.5μg/reaction poly(dI-dC),混匀;标记1.5 ml EP管,依次加入2μl Gel Shift Binding5×Buffer,nuclease-free water,2μg核提取物混匀,室温孵育10min;然后加入相应的γ-~(32)p标记的探针,室温再孵育20min。上样至5%非变性PAGE胶中电泳120V 1h。用保鲜膜包住干燥过的胶,装入片夹,压上胶片,-70℃6～15 hr后显影、定影。
2.7定点突变重组质粒并检测其活性变化
根据说明书设计突变引物,以pLuc-595为模板进行扩增得到Sp1结合位点突变的产物。产物用DpnI酶切(对甲基化及半甲基化位点特异)可将未突变的模板质粒清除,然后转化入Epicurian coli XLl-Blue感受态细胞扩增成功突变的质粒,测序鉴定后命名为pLuc-MT。将突变质粒转染入细胞内24～48h后收集细胞进行双荧光素酶报告基因检测。
2.8 siRNA及MIT抑制Sp1的表达后检测启动子活性变化
根据Sp1 cDNA(NM 138473.2)设计合成Sp1-siRNA片段长度21bp,序列为AAAGCGCUUCAUGAGGAGUGA,阴性对照siRNA(Negative control siRNA)片段为UUCUCCGAACGUGUCACGUTT。按照说明书操作,Kato-Ⅲ、SW480和LoVo细胞种于24孔板过夜,次日使用1μl/孔LipofectAMINE2000 Reagent将siRNA 100 pmol/孔转染入细胞中,同时设置另一组加入MIT(终浓度为1μM),24～48h后收集细胞进行双荧光素酶报告基因检测。
2.9 siRNA及MIT抑制Sp1的表达后检测FHL2 mRNA及蛋白水平变化
使用LipofectAMINE2000 Reagent将siRNA 500 pmol转染入6孔板培养细胞中,同时设置另一组加入MIT(终浓度为1μM),24～48h后收集细胞提取RNA及蛋白,进行RT-PCR及Western Blotting检测FHL2的表达。
2.10数据统计
双荧光素酶报告基因检测、RT-PCR和Western Blotting灰度值分析结果用三次实验结果的(?)±SD表示,两组采用两独立样本t检验;多组间方差齐者采用方差分析,整体比较有显著性差异后多重比较采用LSD法,方差不齐者采用Welch检验分析,整体比较有显著性差异后多重比较采用Games-Howell检验。免疫组化结果癌旁组织和肿瘤组织Sp1和FHL2阳性等级的比较采用非参数检验,Sp1和FHL2两者表达情况采用相关分析。统计处理采用SPSS 13.0软件,检验水准以P＜0.05为差异有统计学意义。
结果
1、FHL2基因启动子荧光素酶报告基因质粒的构建及鉴定
扩增了4段FHL2基因5′端上游启动子序列,大小分别为1008bp、881bp、595bp及382bp。上述片段分别插入到pGL3-Basic载体构建成重组质粒,命名为pLuc-1008、pLuc-881、pLuc-595及pLuc-382。酶切鉴定与荧光素酶基因片段相符,证实载体构建成功。DNA测序报告显示与Genbank中序列完全吻合。
2、胃肠癌细胞株中FHL2重组启动子报告基因活性检测
pGL3-Basic载体含有荧光素酶基因序列,并且载体本身不包含启动子,克隆入启动子后可用荧光素酶定量直观地比较两个启动子的活性及其在各种不同种类细胞系中表达的组织特异性,并以pRL-CMV质粒作为内参以消除转染效率的不同所带来的差异。重组质粒转染不同胃肠癌细胞后,荧光素酶活性RLU值(相对荧光单位)有不同的改变,表明所构建的FHL2基因启动子报告系统有转录活性。4个不同的重组质粒在胃癌Kato-Ⅲ细胞中活性分别为22.34±5.43,20.19±3.85,38.05±2.04和5.25±1.10;肠癌LoVo细胞中活性分别为4.30±0.85,4.65±0.68,9.46±2.31和3.10±0.51;肠癌SW480细胞中活性分别为2.68±0.15,2.02±0.08,3.31±0.23和1.85±0.42。3株肿瘤细胞中均为pLuc-595的RLU值最高(方差分析/Welch整体检验,F/Welch值分别为43.421,20.682和7.345,P值分别为0.000,0.000和0.038)。
3、免疫组织化学验证Sp1表达与FHL2表达成正相关
收集大肠及胃的癌旁粘膜和肿瘤组织石蜡标本进行免疫组织化学检测。Sp1在40例癌旁肠粘膜组织中,表达为阴性的有22例,弱阳性的有8例,阳性的有8例,强阳性的有2例,平均秩为32.98;在44例肠癌组织中,表达为阴性的有12例,弱阳性的有3例,阳性的有16例,强阳性的有13例,平均秩为51.16,表达较癌旁组织增强具有显著性(两等级资料秩和检验,P=0.000);在13例癌旁胃粘膜组织中,表达为阴性的有10例,弱阳性的有3例,无阳性及强阳性表达,平均秩为10.04;在15例胃癌组织中,表达为阴性的有4例,弱阳性的有5例,阳性的有4例,强阳性的有2例,平均秩为18.37,表达较癌旁组织增强具有显著性(两等级资料秩和检验,P=0.000)。FHL2在34例癌旁肠粘膜组织中,表达为阴性的有33例,弱阳性的有1例,无阳性及强阳性表达,平均秩为21.72;在38例肠癌组织中,表达为阴性的有8例,弱阳性的有7例,阳性的有15例,强阳性的有8例,平均秩为49.72,表达较癌旁组织增强具有显著性(两等级资料秩和检验,P=0.000);在12例癌旁胃粘膜组织中,表达为阴性的有10例,无弱阳性表达,阳性的有1例,强阳性的有1例,平均秩为8.79;在16例胃癌组织中,表达为阴性的有1例,弱阳性的有4例,阳性的有7例,强阳性的有4例,平均秩为18.78,表达较癌旁组织增强具有显著性(两等级资料秩和检验,P=0.000)。对同时进行了Sp1和FHL2的表达检测的34例大肠癌病人使用相关分析Spearman检验得到R=0.469,P=0.005,提示Sp1表达与FHL2表达相关,且随着Sp1表达量增高FHL2的表达量也增高。
4、Sp1能与FHL2启动子特异结合
根据TFsearch软件分析得出FHL2启动子上游中Sp1可能的转录结合位点设计了两对探针分别为:探针1:(-485) 5′agagggcccgggcttggaatgt3′(-464),探针2:(-123) 5′tgccaccgcgcccaggcctcgt3′(-102)。以肠癌SW480细胞核提取物进行结合反应,电泳后曝光,标记的探针1+核蛋白抽提物泳道中出现一条特异的结合带,使用50倍未标记的探针1冷竞争反应后条带消失,而探针2泳道中未发现条带。提示探针1能与目的核蛋白中的Sp1特异结合,而探针2不能与转录因子Sp1结合。使用标记的突变探针1进行竞争反应该条带消失,进一步证实了Sp1能与FHL2启动子特异结合。
5、定点突变FHL2重组启动子后报告基因检测活性下降
根据凝胶阻滞实验结果,按照定点突变试剂盒设计突变引物将探针-485agagggcccgggcttggaatgt-464中Sp1位点突变为agaggAccAggTcttggaatgt。突变后的质粒经测序鉴定证实。将pLuc-595和pLuc-MT分别转染入Kato-Ⅲ、SW480和LoVo细胞中比较荧光素酶活性变化。三株细胞中pLuc-MT的RLU值均较pLuc-595的RLU值降低具有显著性(两独立样本t检验,t值分别6.218、42.772和8.377,P值分别为0.003、0.000、0.001)。
6、siRNA及MIT抑制Sp1表达后FHL2重组启动子报告基因检测活性下降
在三株细胞中MIT实验组启动子活性较阴性对照组活性下降均有显著性差异(P均＜0.05)。SW480和LoVo细胞Sp1-siRNA实验组启动子活性较阴性对照组活性下降有显著性差异(P均＜0.05);但Kato-Ⅲ细胞Sp1-siRNA实验组的RLU值较阴性对照组RLU值下降无统计学意义(P＞0.05)。
7、siRNA及MIT抑制Sp1表达后FHL2 mRNA及蛋白表达下降
Kato-Ⅲ、SW480和LoVo三株细胞中的Sp1-siRNA实验组和MIT实验组Sp1 mRNA水平均较阴性对照组中Sp1 mRNA水平显著降低(P均＜0.05),提示设计的Sp1-siRNA能有效抑制Sp1的mRNA表达。实验组FHL2 mRNA水平较阴性对照组FHL2 mRNA水平也有显著性降低(P均＜0.05)。蛋白水平的检测也有类似的结果(P均＜0.05)。
结论
获得了新癌基因FHL2的5′端启动子序列及转录调控信息,根据Sp1结合位点设计克隆的1008bp、881bp、595bp及382bp大小的FHL2启动子片段在胃肠癌细胞中有不同的转录活性,其中以pLuc-595活性最高。临床标本免疫组织化学检测提示Sp1和FHL2在胃肠道肿瘤组织中均较癌旁正常组织高表达,且随着Sp1表达量增高FHL2的表达量也增高,两者在肿瘤组织中的表达量成正相关。通过EMSA进一步证实了Sp1能与FHL2 5′端启动子序列特异的Sp1结合位点结合,突变该Sp1结合位点及抑制Sp1表达后均可发现FHL2转录活性及表达下降。以上结果均提示Sp1在一定程度上参与了FHL2的转录调控,抑制Sp1可下调FHL2的表达,FHL2启动子可能作为靶向性基因治疗的调控元件应用于肿瘤的生物治疗。
BACKGROUD AND OBJECTIVES
As a novel-identified oncogene,FHL2(Four and a half LIM Protein 2) plays important role in the devolpment of gastrointestinal(GI) cancers.It is known that FHL2 being able to modulate the function and/or expression of numerous downstream genes,however,the regulation of FHL2 has been seldomly investigated. By bioinformatical analysis,several putative Sp1(Specificity protein 1) binding elements were found within the 5'-flanking region of FHL2,the purpose of this project is to clarify the effect of Sp1 on transcription regulation of FHL2 gene.
METHODS
1.Cells and cell culture
GI cancer cell line,Kato-Ⅲ,SW480 and LoVo were maintained in 1640(Gibco) supplemented with 10%FBS,100U/ml penicillin.The cells were incubated in a humidified incubator at 37℃with an atmosphere of 5%CO_2.
2.Generation of FHL2 promoter-luciferase constructs.
The genomic DNA was obtained from the SW480.The four insert DNA fragments of the 5-flanking region of the FHL2 gene were amplified by a PCR method using specific primers.The synthetic linkers and restrition sites for both KpnI (GAGGTACC) and NheI(GAGCTAGC) are indicated below.The PCR was performed as follows:95℃for 30min and then 36 cycles of 94℃for 30s(for denaturation),For 55℃for 45s(for annealing),and 72℃for 1min(extension).After the final cycle,an elongation step was carried out at 72℃for 10min.The amplified DNA products were gel-purified using a gel extraction kit(Gel DNA Recovery kit; AXYGEN,USA).For the luciferase reporter gene assays,DNA fragments containing restriction site adapters were digested with either KpnI forward or NheI reverse.The digested DNA fragments were cloned into a firefly luciferase expression vector, pGL3-Basic vector(Promega) which had already been digested with the enzymes KpnI and NheI.The activity of the promoterless vector,pGL3-Basic,which contained no insert,was measured to determine background activity.Each plasmid construct was subcloned into TOP10 bacterial cells,and the plasmids were subsequently isolated and purified.The entire DNA sequences of the cloned fragments were confirmed with gene sequencing.
3.Transient Transfection and Luciferase Assays
For the luciferase assay,cells were plated at a density of approximately 2×10~4 cells in twenty-four wells plate until 70%confluent growth was reached.On the next day,growth medium was removed from cells before transfection.A transfection complex was prepared by mixing 0.8μg of the indicated pGL3-luciferase constructs and 0.008μg pRL-CMV with lipofetamine 2000.Transient transfection was performed according to the instruction of lipofetamine 2000(Invitrogen).Two days later,the cells were harvested;dual-luciferase assay was performed to measure the luciferase activities using a luminometer(Aureon Biosystems) according to the manufacturer's protocol(Promega).Promoter activity was presented as the fold induction of relative luciferase unit(RLU) compared with the basic vector control. RLU= value of firefly luciferase unit/value of renilla luciferase unit.All treatments were triplicated for each single experiment.
4.Immunohistochemistry
Immunohistochemistry was performed to detect Sp1 and FHL2 in situ,antigen retrieval and an indirect immunoperoxidase technique were applied as reported before. The mouse anti-Sp1 monoclonal antibody(dilution 1:200),the rabbit anti-FHL2 polyclonal antibody(dilution 1:300) and the biotin-linked antimouse IgG(Dako, Copenhagen,Denmark) in combination with the ABC complex were used.Normal IgG(Sigma) was used as the isotype controls.
5.Preparation of Cytoplasmic and Nuclear Extract and Western blotting
The cells were resuspended in 400μl of buffer A(containing 10mM Hepes at pH 7.9,1.5mM MgCl_2,10mM KC1,0.5mM DTT,0.5mM PMSF,1μg/ml leupeptin, 1μg/ml aprotinin,and 1μg/ml pepstatin A),lysed with 12.5μl of 10%Nonide P-40, and centrifuged at 12,000g for 10min at 4℃.The supematant was collected and used as the cytoplasmic extracts.The nuclei pellet was re-suspended in 4.0μl of buffer B (20mM Hepes,pH 7.9,containing 1.5mM MgCl_2,450mM NaCl,25%glycerol,0.2 mM EDTA,0.5mM DTT,0.5mM PMSF,1μg/ml leupeptin,1μg/ml aprotinin,1μg/ml pepstatin A),agitated for 60min at 4℃,and the nuclear debris was spun down at 20,000g for 15min.The supernatant(nuclear extract) was collected and stored at-70℃until ready for analysis.Protein concentrations were determined with BCA Protein Assay Kit.
6.Electrophoretic Mobility Shift Assay
Double-strand FHL2 gene specific DNA probes that contained the specific Sp1 binding elements were synthesized.The sequences of sense strands were as follows: (-485)5'- AGAGGGCCCGGGCTTGGAATGT -3'(-464) and(-123) 5'-TGCCAC CGCGCCCAGGCCTCGT -3'(-102).After annealing,double-strand DNA probes were end labeled with 5μCi ofγ-~(32)P -ATP(PerkinElmer Life and Analytical Sciences) using T4 polynucleotide kinase(Promega).For electrophoretic mobility shift assay (EMSA),total reaction mixtures containing 10mmol/L Tris/HCl(pH 7.5),1 mmol/L MgCl_2,0.5mmol/L dithiothreitol,0.5mmol/L ethylenediaminetetraacetic acid, 50mmol/L NaCl,4%glycerol,and 0.5μg of poly(dI-dC)-poly(dI-C)/mL,were incubated with 2μg of nuclear extracts and various unlabeled competing oligonucleotides for 10 minutes at room temperature,followed by addition of 1μl ([0.5-2]×10~5 counts per minute) of the various ~(32)P-end-labeled oligonucleotides. Samples were separated by electrophoresis on 5%nondenaturing polyacrylamide gel, with detection of radioactive bands by autoradiography for 12-16 hours at -70℃.The mutation sequence of the probe was(-485) 5'- AGAGGACCAGGTCTTGGAATGT -3'(-464).
7.siRNA Transfection
The siRNA duplexes consisted of 21 bp oligonucleotides and the sequence of the Sp1-siRNA was as follows:AAAGCGCUUCAUGAGGAGUGA.The GL2 siRNA (UUCUCCGAACGUGUCACGUTT) was used as the negative control.The cells were transfected with siRNA duplexes using LipofectAMINE2000 reagent (Invitrogen) according to the manufacturer's instructions.
8.Reverse-Transcription Polymerase Chain Reaction
RNA was reverse-transcribed to complementary DNA(cDNA) by Thermoscript reverse-transcription polymerase chain reaction(PCR) system(Invitrogen) in accordance with the manufacturer's instructions.PCR was performed using 2μl of resulting cDNA,0.125μl of Hotstart DNA polymerase(Qiagen,Hilden, Germany),forward and reverse primers,and deoxynucleoside triphosphates in a final volume of 25μl.The sequences of the primers were as follows:FHL2 forward:5'-AGAGTTTCATCCCCAAAGACAA -3';FHL2 reverse:5'- AGTTCAGGCAGTA GGCAAAGTC-3';Sp1 forword:5'-CGCTCCCAACTTACAGAA-3';reverse:5'-GCTATTGGCATTGGTGAA -3'.Hotstart PCR was performed for 32 cycles with 95℃denaturation for 30 minutes(first cycle),94℃denaturation for 30 seconds, 53°℃annealing for 30 seconds,and 72℃elongation for 60 seconds and 10 minutes (final cycle).The amplification product was of the expected sizes.
9.Immunoblotting
Cell lysates(30μg) were electrophoresed on denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel(5%stacking gel and 10%separating gel).Proteins were transferred to polyvinylidene difluoride membranes(Perkin Elmer, Fremont,CA).Nonspecific binding was blocked with 10 mmol/L pH 7.6 Tris-HCl buffer saline plus 0.05%Tween-20 containing 5%skim milk.The blots were probed with primary anti-human FHL2 antibody followed by the HRP-conjugated anti-rabbit second antibody.Antigen-antibody complexes were visualized by the enhanced chemiluminescence(ECL) system(PIERCE).
10.Site-Directed Mutagenesis Analysis
The QuikChange Site-Directed Mutagenesis Kit(Stratagene,La Jolla,CA) was used to generate constructs with the mutation of the putative interferon regulatory element(GC box-FHL2).The primers of mutation were as follows:wild-type sequence:agcgtcacgcagagggcccgggcttggaatgtgggagg;mutation sequence:agcgtcacg cagaggaccaggtcttggaatgtgggagg.Briefly,pLuc-595 construct was PCR-amplified in the elongation process using Pfu DNA polymerase with the earlier-described double-strand primers.The incorporation of oligonucleotide primers generates a mutated plasmid containing staggered nicks.The product then is treated with DpnI endonuclease,specific for methylated and hemimethylated DNA,enabling the parental DNA template to be digested(because DNA originating from Escherichia coli usually is dam methylated).The nicked vector DNA carrying the desired mutations was proliferated in Epicurian coli XL1-Blue supercompetent cells.Plasmid DNA was isolated and sequenced to verify the prospected mutated sequence.The mutation plasmid was transfected transiently into cancer cells to Luciferase assay.
11.Statistical Analysis
The data of Luciferase assay,RT-PCR and Western Blotting were expressed as the mean of three independent expression±S.D,evaluated with t-test or one-way ANOVA.Mann-Whitney Test was used to assess the differences of staining intensity. Correlation analyses for quantification of FHL2 and Sp1 positive staining were performed using Spearman Correlation.P＜0.05 was defined as different signicantly. All of these analyses were made using SPSS Version 13(SPSS,Chicago,IL,USA).
RESULTS
1.Positive correlation between Sp1 and FHL2 in colon cancer
To detect Sp1 and FHL2 expression in situ,we collected matched cancer and noncancerous colon tissues under colonoscopy.As determined by IHC,specific nuclear Sp1 protein was high expressed in the carcinoma cells.FHL2 positive signal presented in both the cytoplasm and nucleus of cancer cells but not in the normal colon epithelial cells.These findings demonstrated that Sp1 expression correlated with FHL2 in colon cancer cells.
2.Location of the regulatory promoter of FHL2 gene
To locate the regulatory promoter of FHL2,we used DBTSS software to analyze the promoter seqeunce of FHL2.TF search was applied to find the transcription factor binding site.Several typical and nontypical GC-box were found in the promoter of FHL2 and four different length of gene seqeunces were cloned to investigate the transcription activity of the promoter.Truncated 5'-flanking seqeunces extending up to-997nt of FHL2 gene were inserted in forward orientation upstream of a luciferase reproter gene(pGL3-Basic vector) to generate pLuc constructs pLuc-1008(-997 nt～+10 nt),pLuc881(-870 nt～+10 nt),pLuc595(-584 nt～+10 nt) and pLuc382(-371 nt～+10 nt),the first nucleotide adjacent to the transcription starting site was here difined as 0.Dual luciferase assay revealed that the highest RLU was observed for pLuc-595 in all three cells tested,indicating the presence of cis-enhancing element(s) between -584 to -371nt.However,transfection of the longer FHL2 5'-flanking sequences,pLuc-1008,resulted in a significant decrease in transcription activity,thus implicating the presence of a potential repressor element(s) between -997 and -584nt. All cell lines had a similar pattern of transcription activities with different values for individual constructs.
3.Identification of Sp1-binding sequence in FHL2 promoter
The consensus Sp1 binding sequences comprises GC-rich elements arranged as 5'-(G/T)GGGCGG(G/A)(G/A)(C/T)-3'.Two typical binding elements that located at -485nt to -464nt and -123nt to -102nt were identified within FHL2 5'- flanking region.To test the binding capacity of these sequences,EMSA was performed using the following two-step strategies.First,the capacity of a 50-fold excess of unlabeled oligonucleotides(-485/-464 and -123/-102) was used to compete and block binding to radioactively labeled probes.We found that -485/-464nt but not -123/-102nt oligonucleotide gave rise to specific binding while could be completely blocked by the cold probe.Secondly,a mutated probe with the mutation of Sp1 binding sequence was applied to verify the binding specificity of-485/-464nt probes.We found the site-mutation of Sp1 binding sequence abrogated the binding capacity completely. Therefore,we concluded that a high affinity Sp1 binding element existed in the -485/-464 region of FHL2 gene.
4.Down-regulation of FHL2 transcription activity by inactivation of Sp1 binding activity
To inactivate the binding of Sp1,we had synthesized a Sp1-siRNA to inhibit Sp1 expression.Moreover,mithramycin A(MIT),a novel defined Sp1 inhibitor,was applied to examine its effect on the transcription activity of truncated FHL2 promoter constructs.Kato-Ⅲ,SW480 and LoVo cells were transiently transfected with pLuc-1008,pLuc-881,pLuc-595 and pLuc-382 followed by incubation with control siRNA,Sp1-siRNA or MIT.Cells were lysed and assayed for luciferase activity. Dual luciferase assay showed that Sp1-siRNA and MIT significantly decreased the transcription activity of all constructs,suggesting that Sp1 was essence for FHL2 transcription.In addition,we introduced the gggcccggg to ggAccAggT mutation into pLuc-595 to abrogate Sp1-specific binding activity.We found that the RLU induction of wild type and mutant pLuc-595 constructs were 11.90±1.99 and 4.11±0.86 in Kato-Ⅲcell;7.02±0.03 and 2.31±0.18 in SW480 cells and 7.72±1.17 and 1.65±0.47 in LoVo cells,respectively.Mutation of Sp1 binding sequence decreased the transcription activity of pLuc-595 significantly(P＜0.05 compared with the wild type control).
5.Reduced FHL2 expression by inhibiting Sp1 expresion
At last,we evaluated FHL2 expression in presence or absence of Sp1 suppression by RT-PCR and Western blotting.We found that both Sp1-siRNA and MIT suppressed FHL2 expression.
CONCLUSIONS
1.Sp1 and FHL2 expression were positively correlated in colon cancers with both expressions were upregulated in cancer cells when comparing to normal tissues.
2.Within the 1000bp 5'-fianking region of FHL2 gene,the fragment between -584nt to -371nt possessed high level of transcription activity.
3.A functional Sp1 regulatory element with high affinity was identified at -485nt to -464nt upstream of the transcription starting site that mediated the positive regulation of FHL2 transcription.
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