CD24通过MAPK通路诱导大肠癌细胞增殖的研究
摘要
目的和意义
CD24是迎过糖基磷脂酰肌醇(GPI)锚定在细胞膜上的高度糖基化的蛋白分子,具有多个N-或O-连接的糖基化位点和P-选择素结合位点。近年研究提示:CD24在乳腺癌、胆管癌、胃癌、前列腺癌及大肠癌等多种实体瘤中高表达,且与肿瘤的发生发展及预后密切相关。CD24的表达不仅促进乳腺癌细胞与细胞外基质的黏附、迁移和侵袭,而且,体外和体内实验都提示CD24促进肿瘤细胞的生长和增殖。然而,CD24促进增殖的机制还不明确。因此,本课题旨在研究CD24在大肠癌增殖中的作用,并从体内和体外两方面探讨CD24促进大肠癌细胞增殖的机制。这对于明确大肠癌发生发展分子机制,为大肠癌的早期诊断和个体化治疗寻找新的分子标志都具有重要的意义。
材料和方法
1、主要材料
SW1116、SW480、SW620、HCT8、LoVo及Colo205大肠癌细胞株,真核细胞表达质粒pcDNA3.1(+),Lipofectamine~(TM) 2000,G418,CD24(C-20)、Raf-1(C-12)、p-Raf-1(Ser338)、p-ERK(E-4)、ERK1/2(137F5)、p38 MAPK、phospho-p38 MAPK、SAPK/JNK、phospho-SAPK/JNK(Thr183/Tyr185)等抗体,MEK1/2抑制剂U0126,p38 MAPK抑制剂SB203580,106例大肠癌及癌旁正常黏膜的石蜡包埋组织标本。
2、主要方法
2.1构建CD24表达质粒
从GeneBank查询完整的人CD24 cDNA序列(no.NM_013230),按照cDNA设计引物,上游引物5'-ta-ggtacc act atgggcagagcaatgg包含了酶切位点EcoRI,下游引物5'-ccg gaattc cg ttaagagtagagatg包含了酶切位点KpnI,目的片段长度为265bp。以人正常肠组织cDNA文库为模板,PCR扩增CD24目的片段,按赛百盛“easy-do”PCR体系的说明加样,反应条件为94℃预变性5min,然后进入以下36个循环:94℃变性30s,54℃退火30s,72℃延伸1min;循环完毕,72℃延伸10min。PCR产物和原始质粒纯化,EcoRI和KpnI 37℃双酶切,酶切产物纯化后进行连接反应,连接产物转化入感受态细胞,挑取菌落扩增、鉴定。
2.2 RT-PCR检测CD24 mRNA水平
按Invitrogen的Trizol的说明书提取细胞总mRNA,使用Fermentas的逆转录试剂盒,按说明书将mRNA逆转录为cDNA,PCR扩增CD24目的片段,按赛百盛“easy-do”PCR体系加样,反应条件同上,退火温度为56℃,1.2%琼脂糖凝胶电泳检测PCR产物。
2.3流式细胞术检测CD24蛋白表达水平
制备浓度为5×10~6/ml的细胞悬液,5%正常兔血清封闭,按1:100加入CD24单克隆抗体,冰上孵育60min,洗涤;按1:200加入兔抗小鼠的FITC标记的二抗,冰上孵育40min,洗涤,重悬,上机检测。
2.4重组质粒转染SW480细胞并建立稳定表达CD24的细胞克隆株
六孔板中细胞生长至70%左右融合时,按Invitrogen公司Lipofectamine2000~(TM)产品说明书将质粒导入SW480细胞,转染48h后改用含600μg/ml G418、10%FBS的1640培养基连续筛选,挑取单克隆后继续扩大培养,用KF-PCR和流式细胞术鉴定目的基因(CD24)的表达。
2.5 Western blotting
提取细胞总蛋白,BCA法测定蛋白浓度,按《分子克隆》第三版的配方配制10%蛋白电泳分离胶和5%积层胶,蛋白变性上样,上样量30μg,恒压100V电泳约3h,2mA/cm~2,60min恒流半干电转膜,5%的脱脂奶粉/TBS-T室温封闭1h,5%的脱脂奶粉/TBS-T 1:500稀释相应的一抗,与PVDF膜4℃孵育过夜,TBS-T洗涤,同样方法1:5000稀释HRP标记二抗,室温1h,TBS-T洗涤后,ECL化学发光法检测,Quantity one(Bio-Rad)软件半定量分析。
2.6 CCK-8法检测细胞增殖
接种处理好的单细胞悬液于96孔板,每孔100μl含2000(增殖实验)或3000(增殖抑制实验)个细胞的RPMI-1640完全培养基,每组设6个复孔,不同处理时间后,加入CCK-8溶液10μl,继续培养2h后在450nm波长下,酶联免疫分析仪测定各孔光吸收值,记录结果,以时间为横轴,光吸收值为纵轴绘制细胞生长曲线,数据以mean±SD表示。
2.7免疫组织化学(SABC法)检测CD24、p-ERK1/2和p-p38 MAPK在大肠癌组织中的表达情况
大肠癌组织标本的连续切片(4μm)进行脱蜡水化,0.01M枸橼酸钠缓冲溶液(pH 6.0)微波抗原修复10min,3%过氧化氢灭活内源性过氧化物酶20min,正常山羊或兔血清封闭30min,抗体稀释液稀释后的一抗(约50μl),4℃过夜,1×PBS-T冲洗,生物素化二抗与一抗孵育室温15min,HRP偶联的亲和素(SABC试剂)和生物素化的二抗室温孵育10min,DAB显色1min,苏木素复染细胞核,盐酸酒精分化,梯度酒精脱水、二甲苯透明、中性树脂封片、镜检。所有结果由两个病理科高级医师独立阅片评分。
2.8统计学分析
结果均经SPSS 13.0软件统计分析。流式细胞术实验结果取三次结果的平均值,Western blotting和RT-PCR进行灰度扫描后将实验组换算成对照组的相对百分数进行统计,比较采用方差分析(one-way ANOVA),多重比较采用LSD法;增殖实验和增殖抑制实验的结果取三次实验结果中的一次作为统计结果,比较采用析因设计的方差分析;免疫组化各分子染色强度(等级资料)与性别、年龄和肿瘤部位之间的关系分析采用非参数秩和检验,而表达强度与Duke's分期和肿瘤分级之间的关系分析采用Spearman秩相关检验;免疫组化染色的两分子之间的相关性采用Spearman秩相关检验;所有的统计结果以P＜0.05作为有统计学意义;参数比较前进行方差齐性检验,存方差齐性的基础上进行比较。
结果
1、成功构建CD24的重组表达质粒
对重组质粒进行双酶切(EcoRI和KpnI),得到260bp左右的片段,与预计片段大小一致,而空载体没有相应条带;质粒测序证实重组质粒插入片段与Genebank上的序列完全一致。这说明CD24的表达质粒构建成功。
2、CD24在不同的大肠癌细胞株中存在差异表达
RT-PCR从mRNA水平分析CD24在不同大肠癌细胞中的表达情况。结果提示,CD24在SW620和Colo205细胞中高表达,在SW1116细胞中中度表达,而在SW480、HCT8和LoVo细胞中几乎没有表达。因为CD24是膜蛋白,故应用流式细胞术从蛋白质水平检测CD24的表达,其结果和RT-PCR的结果一致。这说明CD24在不同大肠癌细胞株中存在差异表达。
3、建立稳定表达CD24蛋白的细胞克隆
经过30天左右的浓度为600μg/ml的G418的筛选,细胞克隆首先通过RT-PCR鉴定,结果提示,CD24 mRNA水平在克隆1和4的增加有显著性意义(F=152.323,P=0.000)。为进一步证明CD24在蛋白水平的表达,对SW480细胞、转染空载体的细胞、克隆1和4细胞进行了流式细胞术的分析。转染pcDNA3.1(+)空载体的细胞被命名为SW480~(vec),克隆1和4分别被命名为SW480~(CD24+1)和SW480~(CD24+4)。结果提示,CD24在SW480~(CD24+1)和SW480~(CD24+4)的表达量分别是35.00±1.31和51.93±2.71,比SW480和SW480~(vec)细胞具有显著性增加(F=532.400,P=0.000)。
4、CD24过表达引起大肠癌细胞的增殖率显著性增加
SW480、SW480~(vec)和SW480~(CD24+1)细胞接种于96孔板中,分别于接种后24、48、72、96h测定细胞增殖情况。结果提示,与SW480~(vec)和SW480细胞相比,SW480~(CD24+1)细胞的增殖具有显著性增加(F=34.540,P=0.000)。这说明CD24的过表达诱导大肠癌细胞的增殖。
5、CD24的过表达激活ERK1/2、p38 MAPK和Raf-1,而对JNK1/2的活性没有影响
为了阐明CD24促进增殖的机制,我们检测了MAPK家族中关键分子的蛋白水平和磷酸化水平的变化。结果提示,在SW480~(CD24+1)和SW480~(CD24+4)细胞中,ERK1/2和p38 MAPK的磷酸化水平比在SW480和SW480~(vec)细胞中的具有显著性增加(分别是F=24.117,P=0.000和F=41.267,P=0.000),而JNK1/2激酶的活性无显著性差异。研究提示,Raf-1是调节ERK1/2激酶的上游分子。为了明确CD24对ERK1/2激酶的上游分子的影响,我们检测了Raf-1蛋白水平和磷酸化水平的变化。结果提示,Raf-1蛋白表达量和活性均具有显著性增加(F=101.988,P=0.000;F=37.580,P=0.002)。这些结果说明CD24对Raf-ERK通路和p38 MAPK激酶起重要的调节作用。
6、抑制ERK1/2和p-p38 MAPK的活性可抑制CD24诱导的大肠癌细胞增殖
在SW480~(CD24+1)细胞中分别加入不同浓度(0μM、5μM、10μM、20μM)的MEK1/2抑制剂U0126或p38抑制剂SB203580,分别于处理后24h和72h进行检测观察细胞的活性。首先通过Western bloting验证,ERK1/2和p38 MAPK的激活均被抑制剂所抑制。在抑制剂处理后24h,SW480~(CD24+1)细胞增殖无显著性降低(U0126和SB203580处理组分别为F=1.293,P=0.201和F=2.969,P=0.057);在72h时,细胞增殖均具有显著性降低(U0126和SB203580处理组分别为F=23.59,P=0.000和F=8.428,P=0.001)。为了进一步明确U0126和SB203580对细胞增殖的抑制作用是否具有协同效应,我们用U0126和SB203580同时处理SW480~(CD24+1)细胞,结果提示,二者的抑制作用没有协同作用。这些结果提示抑制ERK1/2和p-p38 MAPK的活性可抑制CD24诱导的癌细胞增殖。
7、CD24在大肠癌组织中高表达,表达强度与大肠癌的分期和分级呈正相关关系
CD24在大肠癌组织中表现为膜表达和浆表达,而在癌旁正常黏膜中几乎没有表达,极少数病例中可见轻度膜表达。CD24膜表达的阳性率为34.9%,表达强度与肿瘤的分级呈负相关(r=13.741,P=0.038),而与肿瘤的进展程度关系不密切。CD24浆表达的阳性率为89.6%,表达强度与肿瘤的进展程度(Duke's分期)(r=0.269,P=0.005)和肿瘤的分级(r=0.235,P=0.015)呈正相关,而与患者的性别、年龄及肿瘤的部位关系不密切。这说明CD24在大肠癌的发生发展中起重要的作用
8、p-ERK1/2和p-p38在大肠癌组织中高表达,表达强度与大肠癌Duke's分期及分化程度关系不密切
p-ERK1/2在大肠癌组织中表现为细胞浆表达,而在癌旁正常黏膜组织中没有表达。p-ERK1/2浆表达阳性率为84.0%,表达强度在男性患者高于女性患者(z=-2.454,P=0.014),与肿瘤的进展程度(Duke's分期)(r=0.125,P=0.203)和肿瘤的分级(r=0.016,P=0.874)无相关关系,与患者的年龄和肿瘤的发生部位关系也不密切。
p-p38 MAPK在大肠癌组织中表现为浆表达和核表达,而在癌旁正常黏膜中没有表达。p-p38 MAPK核表达的阳性率为24.5%,浆表达的阳性率为78.3%,表达强度与肿瘤的进展程度(Duke's分期)(r=0.150,P=0.125)和分级(r=0.071,P=0.472)无相关关系,与患者的年龄、性别、肿瘤的部位关系也不密切。这提示p-ERK1/2和p38 MAPK激酶在大肠癌中处于激活状态,可能参与了大肠癌的生物学功能。
9、在大肠癌组织中,ERK1/2和p38 MAPK的激活与CD24的浆表达呈正相关关系
为了明确CD24和p-ERK1/2、p-p38 MAPK在组织中的关系,我们对106例大肠癌组织标本进行连续切片,同时对三个分子进行染色分析。结果提示,p-ERK1/2的表达和CD24浆表达呈正相关关系(r=0.571,P=0.000),p-p38 MAPK的浆表达和CD24浆表达也呈正正相关关系(r=0.528,P=0.000)。这提示在大肠癌组织中,CD24和p-ERK1/2、p-p38 MAPK同样存在紧密的联系,进一步证实了体外实验的结果。
结论
本研究通过体外细胞学实验提示,CD24在大肠癌细胞株中存在差异表达,CD24促进大肠癌细胞的增殖,同时激活Raf-ERK通路和p38 MAPK激酶,而且,抑制ERK1/2和p38 MAPK激酶的活性后,大肠癌细胞的增殖也受到抑制。体内实验的结果提示,在大肠癌组织中CD24高表达,表达强度随肿瘤的分期和分级的增加逐渐升高。p-ERK1/2、p-p38在大肠癌组织中也明显高表达,表达强度与CD24浆表达强度呈正相关关系。这些结果提示CD24在大肠癌的发生发展中起重要作用;CD24促进大肠癌的生长、增殖;而在CD24促进大肠癌的增殖的过程中,ERK1/2通路和p38激酶的活化是必要的。CD24和MAPK之间的相互作用可能是大肠癌生长、增殖的重要分子机制,明确它们之间的调控关系对于完善大肠癌发病机制的信号传导通路理论,并为新的分子靶标的确立提供实验数据,进而对于大肠癌的早期诊断和生物化个体化治疗都具有重要的意义。
Objective
CD24 is a glycosylphosphatidylinositol(GPI)-anchored membrane protein with several potential O-or N-linked glycosylation sites.CD24 was initially described to be up-regulated in breast cancer and later confirmed to be overexpressed in human cancers such as prostate cancer and cholangiocarcinoma,suggesting that this gene may play a key role in tumorigenesis.Its expression is usually associated with poor prognosis.In colorectal cancer(CRC),CD24 was commonly up-regulated and cytoplasmic CD24 expression remained a significant independent prognostic marker. Down-regulation of CD24 expression induced growth inhibition in CRC,and this effect was augmented in combination with classic chemotherapies.Taken together, CD24 does play an important role in tumor development and progression.However, the underlying mechanisms of how CD24 contributes to the progression of malignant tumor remain largely unclear.In our present study,we determined the role of CD24 in the proliferation of CRC cells and attempted to elucidate the potential mechanism in this process.
Materials and methods
Reagents and meterials
SW1116,SW480,SW620,HCT8,LoVo and Colo205 colorectal cell lines, MEK1/2 inhibitor U0126,p38 MAPK inhibitor SB203580,G418,ERK1/2(137F5) antibody,p38 MAPK antibody,SAPK/JNK antibody,phospho-SAPK/JNK (Thr183/Tyr185) antibody and phospho-p38 MAPK antibody,CD24(C-20) polyclonal antibody,Raf-1(C-12) antibody,p-Raf-1(Ser 338) antibody,p-ERK(E-4) antibody,formalin-fixed and paraffin-embedded tissue samples from 106 primary human CRCs.
Construction of the CD24 expression plasmid and stable transfection
A full-length human CD24 cDNA corresponding to bases 111 to 353 of Genbank accession(no.NM_013230) was amplified by PCR from normal intestinal cDNA library.The primer pairs for CD24 were 5'-TAGGTACCACTATGGGCAGAGCAA TGG-3'(F) and 5'-CCGGAATTCCGTTAAGAGTAGAGATGC-3'(R).The PCR product was cloned into the pcDNA3.1(+) vector(Invitrogen) to create the pcDNA3.1(+)-CD24 plasmid.The orientation of the insert and the sequence of cDNA were verified by sequencing.Cultured SW480 cells were transfected with the recombinant plasmids and the empty vector using the lipofectamine 2000 reagent (Invitrogen,Life Technologies) according to the manufacturer's instructions. Transfectants were selected in medium containing 600μg/ml G418.After selection and isolation of stably transfected clones,the clones were analyzed for CD24 expression using RT-PCR and flow cytometry.
Reverse transcription-polymerase chain reaction(RT-PCR)
Total RNA was extracted from cells using Trizol(Invitrogen).RNA samples(3μg) were subjected to reverse transcription using a RevertAid~(TM) First Strand cDNA Synthesis Kit(Fermentas).The PCR was initiated by 5 min incubation at 94℃,ended after a 10 min extension at 72℃,36 cycles for denaturation at 94℃for 30s, annealing at 55℃,and extension 72℃for 1 min using PCR kit(SBS Genetech Co. Ltd.,Beijing,China). Flow cytometry
Cells were harvested and resuspended in PBS/5%FBS in a density of 2×10~5/ml. CD24 monoclonal antibodies(Ab-1,Neomarker) were applied at 5μg/ml(1:100). After incubation for 60 min on ice,the cells were washed with PBS/5%FBS twice and incubated for a further 40 min with fluorescein isothiocyanate(FITC)-conjugated secondary antibody on ice.The cells were washed with PBS/5%FBS twice again and analyzed by a Flow Cytometer.As negative controls,cells were stained with both isotype-matched control antibodies.
Western blotting
Cells lysed with ice-cold lysis buffer and PMSF.Protein samples(30μg) were separated by 12%acrylamide gel using a Bio-Rad Mini-ProteanⅢsystem(100V for 3h).Proteins were transferred to PVDF membranes in 2.0mA/cm~2 for 2h in transfer buffer.Following transferring,the membranes were blocked for 1 h at room temperature with 5%skimmed milk powder in 0.05%TBS-T.Blots were then incubated at room temperature with primary antibodies in 2%BSA dissolved in TBS-T(1:500 dilution).Primary antibodies were removed and the blots were extensively washed with TBS-T for three times.Blots were then incubated for 1 h at room temperature with the proper horseradish peroxidase(HRP)-conjugated secondary antibodies(1:5000 dilution).Then,blots were extensively washed as above and detected by using the Enhanced ECL System.Actin was also detected as internal control.All experiments were repeated twice and blots were analyzed using quantity one 1-D analysis software(Bio-Rad).
Proliferation assays
Cell growth was evaluated using a WST-8 assay(Beyotime Institute Biotech, Shanghai,China).The cells were seeded into the 96-well culture plates at a density of 2000 cells,and the medium was replaced with 100μl of fresh medium and 10μl of WST-8 reagent in each well at 24,48,72 and 96h,respetively.After the plate was returned to incubator for 1-2h,the absorbency of A450 was measured using an enzyme-linked immunosorbent assay plate reader.
Immunohistochemical analysis
Representive 4μM sections were prepared,deparaffinized,and rehydrated in xylene, graded alcohol,and water.Antigen retrieval was enhanced by microwaving the slides in 0.01M citrate buffer(pH 6.0) for a total of 10min.Endogenous peroxidase activity was quenched by treatment with 3%hydrogen peroxide for 30 min,followed by incubation with goat or rabbit serum for 30min.The primary antibodies were applied and incubated overnight at 4℃.Subsequent steps utilized the UltraSensitive~(TM) SP kit (Maxin-bio,Fuzhou,China) according to the manufacture's instructions.Peroxidase activity was visualized with diaminobenzidine and slides were counterstained with haematoxylin,dehydrated and mounted.For negative controls,blocking solution was added instead of the primary antibody.All slides were assessed by two pathologists independently and blinded to the case.
Statistical analysis.
Data were compiled with the software package SPSS 13.0.Factorial analysis ANOVA was employed to determine whether the results of proliferation assays had statistical significance.The non-parametric Kruskal-Wallis or Mann-Whitney tests were applied to test the significance of differences among groups of clinicopathological parameters,whereas correlations between expression of proteins were evaluated by the Spearman rank-order correlation coefficient.The level of significance was defined as P＜0.05.
Result
Expression of CD24 was cell type-dependent in colorectal cancer cell lines
Flow cytometry was used to detect CD24 expression at protein level in the colorectal cancer cell lines.Of six cell lines,SW1116 maintained moderate expression of CD24,SW620 and Colo205 contained higher expression,but expression was scarcely observed in SW480,HCT8,and LoVo cell lines.To investigate CD24 mRNA at the level of transcription,a standard RT-PCR experiment described in methods was performed.The result was consistent with Flow cytometric analysis.These results showed Expression of CD24 was cell type-dependent in colorectal cancer cell lines.
Overexpression of CD24 induced proliferation in vitro
CD24 recombinant plasmid,pcDNA3.1(+)-CD24,was constructed using expression vector pcDNA3.1(+),and was then stably transfected into the SW480 cells. Two clones,named as SW480~(CD24+1) and SW480~(CD24+4),exhibited dramatically increased expression of CD24 at mRNA transcripts level compared with the parental and vector control cells.Expression of CD24 at protein level in the clones was confirmed by Flow cytometric analysis.The growth ability of SW480~(CD24+1) cells in vitro was assessed by WST-8 assay,and the result showed that these cells proliferated more quickly than SW480~(vec) cells,which were transfected with the vector alone,and the parental SW480 cells.
Overexpression of CD24 induced activation of ERK1/2,Raf-1 and p38 MAPK,but it had no effect on JNK1/2
To elucidate the potiential mechanisms of CD24-induced proliferation,we examined the activation of MAPK pathways.The rusults showed that ERK1/2 and p38 MAPK were activated in the SW480~(CD24+1) and SW480~(CD24+4) cells compared with SW480~(vec) cells and the parental SW480 cells.However,no activation of JNK1/2 was observed.To determine the role of CD24 in Raf-ERK pathway,we further detected the activity of Raf-1,the upstream ERK1/2 activator,and our data showed that Raf-1 protein levels and kinase activity were elevated after the SW480 cells overexpressed with CD24.
Suppressing the activation of ERK1/2 and p38 MAPK abrogated CD24-induced proliferation in the SW480~(CD24+1) cells
To further evaluated the contribution of the ERK1/2 and p38 MAPK in the CD24-induced proliferation,we inhibited their activations using specific pharmacological inhibitors.The starved SW480~(CD24+1) cells were treated either with inhibitor of the upstream ERK1/2 activator MEK1/2,U0126,or the p38 MAPK inhibitor SB203580.DMSO was used as control.The cells were treated separately with 5,10 and 20μM inhibitors and observed at 24 and 72h after treatment followed by WST-8 assay for cell viability.Meantime,parallel cultures were analyzed for total phosphor-tyrosine content by western blotting.Our data showed that the activation of ERK1/2 and p38 MAPK were inhibited,and there were significant decreases of cell viability in the cells treated with different concentration U0126 and SB203580 after 72h.
Cytoplasmic CD24 expression increased with tumor stage and grade
CD24 showed a membranous and a cytoplasmic immunostainings in colorectal tissue.For cytoplasmic CD24 staining,89.6%cases showed positive and 45.3%cases were moderately or strongly positive,while its expression in adjacent normal mucosa was either absent or barely detectable on cell membrane.There was a significantly positive correlation between cytoplasmic CD24 expression and tumor stage and grade, while no correlation was observed between cytoplasmic CD24 expression and age, gender,localization,and tumor grade.For CD24 membranous staining,only 34.9% was classified as positive.CD24 membranous expression showed a significantly negative correlation with tumor grade,but no association with other clinicopathologic parameters.
Activations of the ERK1/2 and p38 MAPK strongly correlated with cytoplasmic CD24 expression in human CRC tissue
To investigate the correlations among ERK1/2,p38 MAPK and CD24 in vivo, immmunohistochemical analysis was performed.Immunostaining of p-ERK1/2 was cytoplasmic,while p-p38 MAPK revealed strong cytoplasmic and partly nuclear staining.Cytoplasmic expression of p-ERK1/2 and p-p38 MAPK was observed in 84.0%and in 78.3%cases,respectively.In adjacent normal mucosa,the staining of both molecules was weak or absent.Cytoplasmic expression of both molecules had no significant correlation with tumor stage and grade.Nuclear p-p38 MAPK staining were classified as 24.5%,which had no significant correlation and trend with any clinicopathological parameters.Expression of p-ERK1/2 correlated with cytoplasmic p-p38 MAPK expression in CRCs(r=0.780,P＜0.001).More importantly,our data showed that strong correlations were found between cytoplasmic CD24 expression and immounostainings of ERK1/2 or p38 MAPK in CRCs(r=0.571,P＜0.001; r=0.528,P＜0.001;respectiveely),suggesting that activations of the ERK1/2 and p38 MAPK strongly correlated with cytoplasmic CD24 expression in human CRC tissue.
Conclusion
In the present study,we evaluated the role of CD24 in the proliferation of CRC cells and the potential mechanisms.Our data showed that increased CD24 expression was cell type-dependent and occurred in 89.6%of human CRCs,and cytoplasmic CD24 expression increased with tumor stage and grade.CD24 could induced proliferation of CRC cells in vitro.In addition,we showed,for the first time, CD24-induced proliferation was dependent on activation of the ERK1/2 and p38 MAPK in CRC.Moreover,activation of ERK1/2 and p38 MAPK also appears to be strongly correlated with CD24 expression in CRC tissue.Our results suggest that CD24 plays a key role in the proliferation of CRC.Furthermore,CD24-dependent MAPK pathway activation is required for CRC cell proliferation.Our finding provided a novel interpretation for the mechanism of CD24-induced proliferation in CRC.The linkage of CD24 and MAPK pathway may unravel a novel mechanism in regulation of proliferation of CRCs.
CD24是迎过糖基磷脂酰肌醇(GPI)锚定在细胞膜上的高度糖基化的蛋白分子,具有多个N-或O-连接的糖基化位点和P-选择素结合位点。近年研究提示:CD24在乳腺癌、胆管癌、胃癌、前列腺癌及大肠癌等多种实体瘤中高表达,且与肿瘤的发生发展及预后密切相关。CD24的表达不仅促进乳腺癌细胞与细胞外基质的黏附、迁移和侵袭,而且,体外和体内实验都提示CD24促进肿瘤细胞的生长和增殖。然而,CD24促进增殖的机制还不明确。因此,本课题旨在研究CD24在大肠癌增殖中的作用,并从体内和体外两方面探讨CD24促进大肠癌细胞增殖的机制。这对于明确大肠癌发生发展分子机制,为大肠癌的早期诊断和个体化治疗寻找新的分子标志都具有重要的意义。
材料和方法
1、主要材料
SW1116、SW480、SW620、HCT8、LoVo及Colo205大肠癌细胞株,真核细胞表达质粒pcDNA3.1(+),Lipofectamine~(TM) 2000,G418,CD24(C-20)、Raf-1(C-12)、p-Raf-1(Ser338)、p-ERK(E-4)、ERK1/2(137F5)、p38 MAPK、phospho-p38 MAPK、SAPK/JNK、phospho-SAPK/JNK(Thr183/Tyr185)等抗体,MEK1/2抑制剂U0126,p38 MAPK抑制剂SB203580,106例大肠癌及癌旁正常黏膜的石蜡包埋组织标本。
2、主要方法
2.1构建CD24表达质粒
从GeneBank查询完整的人CD24 cDNA序列(no.NM_013230),按照cDNA设计引物,上游引物5'-ta-ggtacc act atgggcagagcaatgg包含了酶切位点EcoRI,下游引物5'-ccg gaattc cg ttaagagtagagatg包含了酶切位点KpnI,目的片段长度为265bp。以人正常肠组织cDNA文库为模板,PCR扩增CD24目的片段,按赛百盛“easy-do”PCR体系的说明加样,反应条件为94℃预变性5min,然后进入以下36个循环:94℃变性30s,54℃退火30s,72℃延伸1min;循环完毕,72℃延伸10min。PCR产物和原始质粒纯化,EcoRI和KpnI 37℃双酶切,酶切产物纯化后进行连接反应,连接产物转化入感受态细胞,挑取菌落扩增、鉴定。
2.2 RT-PCR检测CD24 mRNA水平
按Invitrogen的Trizol的说明书提取细胞总mRNA,使用Fermentas的逆转录试剂盒,按说明书将mRNA逆转录为cDNA,PCR扩增CD24目的片段,按赛百盛“easy-do”PCR体系加样,反应条件同上,退火温度为56℃,1.2%琼脂糖凝胶电泳检测PCR产物。
2.3流式细胞术检测CD24蛋白表达水平
制备浓度为5×10~6/ml的细胞悬液,5%正常兔血清封闭,按1:100加入CD24单克隆抗体,冰上孵育60min,洗涤;按1:200加入兔抗小鼠的FITC标记的二抗,冰上孵育40min,洗涤,重悬,上机检测。
2.4重组质粒转染SW480细胞并建立稳定表达CD24的细胞克隆株
六孔板中细胞生长至70%左右融合时,按Invitrogen公司Lipofectamine2000~(TM)产品说明书将质粒导入SW480细胞,转染48h后改用含600μg/ml G418、10%FBS的1640培养基连续筛选,挑取单克隆后继续扩大培养,用KF-PCR和流式细胞术鉴定目的基因(CD24)的表达。
2.5 Western blotting
提取细胞总蛋白,BCA法测定蛋白浓度,按《分子克隆》第三版的配方配制10%蛋白电泳分离胶和5%积层胶,蛋白变性上样,上样量30μg,恒压100V电泳约3h,2mA/cm~2,60min恒流半干电转膜,5%的脱脂奶粉/TBS-T室温封闭1h,5%的脱脂奶粉/TBS-T 1:500稀释相应的一抗,与PVDF膜4℃孵育过夜,TBS-T洗涤,同样方法1:5000稀释HRP标记二抗,室温1h,TBS-T洗涤后,ECL化学发光法检测,Quantity one(Bio-Rad)软件半定量分析。
2.6 CCK-8法检测细胞增殖
接种处理好的单细胞悬液于96孔板,每孔100μl含2000(增殖实验)或3000(增殖抑制实验)个细胞的RPMI-1640完全培养基,每组设6个复孔,不同处理时间后,加入CCK-8溶液10μl,继续培养2h后在450nm波长下,酶联免疫分析仪测定各孔光吸收值,记录结果,以时间为横轴,光吸收值为纵轴绘制细胞生长曲线,数据以mean±SD表示。
2.7免疫组织化学(SABC法)检测CD24、p-ERK1/2和p-p38 MAPK在大肠癌组织中的表达情况
大肠癌组织标本的连续切片(4μm)进行脱蜡水化,0.01M枸橼酸钠缓冲溶液(pH 6.0)微波抗原修复10min,3%过氧化氢灭活内源性过氧化物酶20min,正常山羊或兔血清封闭30min,抗体稀释液稀释后的一抗(约50μl),4℃过夜,1×PBS-T冲洗,生物素化二抗与一抗孵育室温15min,HRP偶联的亲和素(SABC试剂)和生物素化的二抗室温孵育10min,DAB显色1min,苏木素复染细胞核,盐酸酒精分化,梯度酒精脱水、二甲苯透明、中性树脂封片、镜检。所有结果由两个病理科高级医师独立阅片评分。
2.8统计学分析
结果均经SPSS 13.0软件统计分析。流式细胞术实验结果取三次结果的平均值,Western blotting和RT-PCR进行灰度扫描后将实验组换算成对照组的相对百分数进行统计,比较采用方差分析(one-way ANOVA),多重比较采用LSD法;增殖实验和增殖抑制实验的结果取三次实验结果中的一次作为统计结果,比较采用析因设计的方差分析;免疫组化各分子染色强度(等级资料)与性别、年龄和肿瘤部位之间的关系分析采用非参数秩和检验,而表达强度与Duke's分期和肿瘤分级之间的关系分析采用Spearman秩相关检验;免疫组化染色的两分子之间的相关性采用Spearman秩相关检验;所有的统计结果以P＜0.05作为有统计学意义;参数比较前进行方差齐性检验,存方差齐性的基础上进行比较。
结果
1、成功构建CD24的重组表达质粒
对重组质粒进行双酶切(EcoRI和KpnI),得到260bp左右的片段,与预计片段大小一致,而空载体没有相应条带;质粒测序证实重组质粒插入片段与Genebank上的序列完全一致。这说明CD24的表达质粒构建成功。
2、CD24在不同的大肠癌细胞株中存在差异表达
RT-PCR从mRNA水平分析CD24在不同大肠癌细胞中的表达情况。结果提示,CD24在SW620和Colo205细胞中高表达,在SW1116细胞中中度表达,而在SW480、HCT8和LoVo细胞中几乎没有表达。因为CD24是膜蛋白,故应用流式细胞术从蛋白质水平检测CD24的表达,其结果和RT-PCR的结果一致。这说明CD24在不同大肠癌细胞株中存在差异表达。
3、建立稳定表达CD24蛋白的细胞克隆
经过30天左右的浓度为600μg/ml的G418的筛选,细胞克隆首先通过RT-PCR鉴定,结果提示,CD24 mRNA水平在克隆1和4的增加有显著性意义(F=152.323,P=0.000)。为进一步证明CD24在蛋白水平的表达,对SW480细胞、转染空载体的细胞、克隆1和4细胞进行了流式细胞术的分析。转染pcDNA3.1(+)空载体的细胞被命名为SW480~(vec),克隆1和4分别被命名为SW480~(CD24+1)和SW480~(CD24+4)。结果提示,CD24在SW480~(CD24+1)和SW480~(CD24+4)的表达量分别是35.00±1.31和51.93±2.71,比SW480和SW480~(vec)细胞具有显著性增加(F=532.400,P=0.000)。
4、CD24过表达引起大肠癌细胞的增殖率显著性增加
SW480、SW480~(vec)和SW480~(CD24+1)细胞接种于96孔板中,分别于接种后24、48、72、96h测定细胞增殖情况。结果提示,与SW480~(vec)和SW480细胞相比,SW480~(CD24+1)细胞的增殖具有显著性增加(F=34.540,P=0.000)。这说明CD24的过表达诱导大肠癌细胞的增殖。
5、CD24的过表达激活ERK1/2、p38 MAPK和Raf-1,而对JNK1/2的活性没有影响
为了阐明CD24促进增殖的机制,我们检测了MAPK家族中关键分子的蛋白水平和磷酸化水平的变化。结果提示,在SW480~(CD24+1)和SW480~(CD24+4)细胞中,ERK1/2和p38 MAPK的磷酸化水平比在SW480和SW480~(vec)细胞中的具有显著性增加(分别是F=24.117,P=0.000和F=41.267,P=0.000),而JNK1/2激酶的活性无显著性差异。研究提示,Raf-1是调节ERK1/2激酶的上游分子。为了明确CD24对ERK1/2激酶的上游分子的影响,我们检测了Raf-1蛋白水平和磷酸化水平的变化。结果提示,Raf-1蛋白表达量和活性均具有显著性增加(F=101.988,P=0.000;F=37.580,P=0.002)。这些结果说明CD24对Raf-ERK通路和p38 MAPK激酶起重要的调节作用。
6、抑制ERK1/2和p-p38 MAPK的活性可抑制CD24诱导的大肠癌细胞增殖
在SW480~(CD24+1)细胞中分别加入不同浓度(0μM、5μM、10μM、20μM)的MEK1/2抑制剂U0126或p38抑制剂SB203580,分别于处理后24h和72h进行检测观察细胞的活性。首先通过Western bloting验证,ERK1/2和p38 MAPK的激活均被抑制剂所抑制。在抑制剂处理后24h,SW480~(CD24+1)细胞增殖无显著性降低(U0126和SB203580处理组分别为F=1.293,P=0.201和F=2.969,P=0.057);在72h时,细胞增殖均具有显著性降低(U0126和SB203580处理组分别为F=23.59,P=0.000和F=8.428,P=0.001)。为了进一步明确U0126和SB203580对细胞增殖的抑制作用是否具有协同效应,我们用U0126和SB203580同时处理SW480~(CD24+1)细胞,结果提示,二者的抑制作用没有协同作用。这些结果提示抑制ERK1/2和p-p38 MAPK的活性可抑制CD24诱导的癌细胞增殖。
7、CD24在大肠癌组织中高表达,表达强度与大肠癌的分期和分级呈正相关关系
CD24在大肠癌组织中表现为膜表达和浆表达,而在癌旁正常黏膜中几乎没有表达,极少数病例中可见轻度膜表达。CD24膜表达的阳性率为34.9%,表达强度与肿瘤的分级呈负相关(r=13.741,P=0.038),而与肿瘤的进展程度关系不密切。CD24浆表达的阳性率为89.6%,表达强度与肿瘤的进展程度(Duke's分期)(r=0.269,P=0.005)和肿瘤的分级(r=0.235,P=0.015)呈正相关,而与患者的性别、年龄及肿瘤的部位关系不密切。这说明CD24在大肠癌的发生发展中起重要的作用
8、p-ERK1/2和p-p38在大肠癌组织中高表达,表达强度与大肠癌Duke's分期及分化程度关系不密切
p-ERK1/2在大肠癌组织中表现为细胞浆表达,而在癌旁正常黏膜组织中没有表达。p-ERK1/2浆表达阳性率为84.0%,表达强度在男性患者高于女性患者(z=-2.454,P=0.014),与肿瘤的进展程度(Duke's分期)(r=0.125,P=0.203)和肿瘤的分级(r=0.016,P=0.874)无相关关系,与患者的年龄和肿瘤的发生部位关系也不密切。
p-p38 MAPK在大肠癌组织中表现为浆表达和核表达,而在癌旁正常黏膜中没有表达。p-p38 MAPK核表达的阳性率为24.5%,浆表达的阳性率为78.3%,表达强度与肿瘤的进展程度(Duke's分期)(r=0.150,P=0.125)和分级(r=0.071,P=0.472)无相关关系,与患者的年龄、性别、肿瘤的部位关系也不密切。这提示p-ERK1/2和p38 MAPK激酶在大肠癌中处于激活状态,可能参与了大肠癌的生物学功能。
9、在大肠癌组织中,ERK1/2和p38 MAPK的激活与CD24的浆表达呈正相关关系
为了明确CD24和p-ERK1/2、p-p38 MAPK在组织中的关系,我们对106例大肠癌组织标本进行连续切片,同时对三个分子进行染色分析。结果提示,p-ERK1/2的表达和CD24浆表达呈正相关关系(r=0.571,P=0.000),p-p38 MAPK的浆表达和CD24浆表达也呈正正相关关系(r=0.528,P=0.000)。这提示在大肠癌组织中,CD24和p-ERK1/2、p-p38 MAPK同样存在紧密的联系,进一步证实了体外实验的结果。
结论
本研究通过体外细胞学实验提示,CD24在大肠癌细胞株中存在差异表达,CD24促进大肠癌细胞的增殖,同时激活Raf-ERK通路和p38 MAPK激酶,而且,抑制ERK1/2和p38 MAPK激酶的活性后,大肠癌细胞的增殖也受到抑制。体内实验的结果提示,在大肠癌组织中CD24高表达,表达强度随肿瘤的分期和分级的增加逐渐升高。p-ERK1/2、p-p38在大肠癌组织中也明显高表达,表达强度与CD24浆表达强度呈正相关关系。这些结果提示CD24在大肠癌的发生发展中起重要作用;CD24促进大肠癌的生长、增殖;而在CD24促进大肠癌的增殖的过程中,ERK1/2通路和p38激酶的活化是必要的。CD24和MAPK之间的相互作用可能是大肠癌生长、增殖的重要分子机制,明确它们之间的调控关系对于完善大肠癌发病机制的信号传导通路理论,并为新的分子靶标的确立提供实验数据,进而对于大肠癌的早期诊断和生物化个体化治疗都具有重要的意义。
Objective
CD24 is a glycosylphosphatidylinositol(GPI)-anchored membrane protein with several potential O-or N-linked glycosylation sites.CD24 was initially described to be up-regulated in breast cancer and later confirmed to be overexpressed in human cancers such as prostate cancer and cholangiocarcinoma,suggesting that this gene may play a key role in tumorigenesis.Its expression is usually associated with poor prognosis.In colorectal cancer(CRC),CD24 was commonly up-regulated and cytoplasmic CD24 expression remained a significant independent prognostic marker. Down-regulation of CD24 expression induced growth inhibition in CRC,and this effect was augmented in combination with classic chemotherapies.Taken together, CD24 does play an important role in tumor development and progression.However, the underlying mechanisms of how CD24 contributes to the progression of malignant tumor remain largely unclear.In our present study,we determined the role of CD24 in the proliferation of CRC cells and attempted to elucidate the potential mechanism in this process.
Materials and methods
Reagents and meterials
SW1116,SW480,SW620,HCT8,LoVo and Colo205 colorectal cell lines, MEK1/2 inhibitor U0126,p38 MAPK inhibitor SB203580,G418,ERK1/2(137F5) antibody,p38 MAPK antibody,SAPK/JNK antibody,phospho-SAPK/JNK (Thr183/Tyr185) antibody and phospho-p38 MAPK antibody,CD24(C-20) polyclonal antibody,Raf-1(C-12) antibody,p-Raf-1(Ser 338) antibody,p-ERK(E-4) antibody,formalin-fixed and paraffin-embedded tissue samples from 106 primary human CRCs.
Construction of the CD24 expression plasmid and stable transfection
A full-length human CD24 cDNA corresponding to bases 111 to 353 of Genbank accession(no.NM_013230) was amplified by PCR from normal intestinal cDNA library.The primer pairs for CD24 were 5'-TAGGTACCACTATGGGCAGAGCAA TGG-3'(F) and 5'-CCGGAATTCCGTTAAGAGTAGAGATGC-3'(R).The PCR product was cloned into the pcDNA3.1(+) vector(Invitrogen) to create the pcDNA3.1(+)-CD24 plasmid.The orientation of the insert and the sequence of cDNA were verified by sequencing.Cultured SW480 cells were transfected with the recombinant plasmids and the empty vector using the lipofectamine 2000 reagent (Invitrogen,Life Technologies) according to the manufacturer's instructions. Transfectants were selected in medium containing 600μg/ml G418.After selection and isolation of stably transfected clones,the clones were analyzed for CD24 expression using RT-PCR and flow cytometry.
Reverse transcription-polymerase chain reaction(RT-PCR)
Total RNA was extracted from cells using Trizol(Invitrogen).RNA samples(3μg) were subjected to reverse transcription using a RevertAid~(TM) First Strand cDNA Synthesis Kit(Fermentas).The PCR was initiated by 5 min incubation at 94℃,ended after a 10 min extension at 72℃,36 cycles for denaturation at 94℃for 30s, annealing at 55℃,and extension 72℃for 1 min using PCR kit(SBS Genetech Co. Ltd.,Beijing,China). Flow cytometry
Cells were harvested and resuspended in PBS/5%FBS in a density of 2×10~5/ml. CD24 monoclonal antibodies(Ab-1,Neomarker) were applied at 5μg/ml(1:100). After incubation for 60 min on ice,the cells were washed with PBS/5%FBS twice and incubated for a further 40 min with fluorescein isothiocyanate(FITC)-conjugated secondary antibody on ice.The cells were washed with PBS/5%FBS twice again and analyzed by a Flow Cytometer.As negative controls,cells were stained with both isotype-matched control antibodies.
Western blotting
Cells lysed with ice-cold lysis buffer and PMSF.Protein samples(30μg) were separated by 12%acrylamide gel using a Bio-Rad Mini-ProteanⅢsystem(100V for 3h).Proteins were transferred to PVDF membranes in 2.0mA/cm~2 for 2h in transfer buffer.Following transferring,the membranes were blocked for 1 h at room temperature with 5%skimmed milk powder in 0.05%TBS-T.Blots were then incubated at room temperature with primary antibodies in 2%BSA dissolved in TBS-T(1:500 dilution).Primary antibodies were removed and the blots were extensively washed with TBS-T for three times.Blots were then incubated for 1 h at room temperature with the proper horseradish peroxidase(HRP)-conjugated secondary antibodies(1:5000 dilution).Then,blots were extensively washed as above and detected by using the Enhanced ECL System.Actin was also detected as internal control.All experiments were repeated twice and blots were analyzed using quantity one 1-D analysis software(Bio-Rad).
Proliferation assays
Cell growth was evaluated using a WST-8 assay(Beyotime Institute Biotech, Shanghai,China).The cells were seeded into the 96-well culture plates at a density of 2000 cells,and the medium was replaced with 100μl of fresh medium and 10μl of WST-8 reagent in each well at 24,48,72 and 96h,respetively.After the plate was returned to incubator for 1-2h,the absorbency of A450 was measured using an enzyme-linked immunosorbent assay plate reader.
Immunohistochemical analysis
Representive 4μM sections were prepared,deparaffinized,and rehydrated in xylene, graded alcohol,and water.Antigen retrieval was enhanced by microwaving the slides in 0.01M citrate buffer(pH 6.0) for a total of 10min.Endogenous peroxidase activity was quenched by treatment with 3%hydrogen peroxide for 30 min,followed by incubation with goat or rabbit serum for 30min.The primary antibodies were applied and incubated overnight at 4℃.Subsequent steps utilized the UltraSensitive~(TM) SP kit (Maxin-bio,Fuzhou,China) according to the manufacture's instructions.Peroxidase activity was visualized with diaminobenzidine and slides were counterstained with haematoxylin,dehydrated and mounted.For negative controls,blocking solution was added instead of the primary antibody.All slides were assessed by two pathologists independently and blinded to the case.
Statistical analysis.
Data were compiled with the software package SPSS 13.0.Factorial analysis ANOVA was employed to determine whether the results of proliferation assays had statistical significance.The non-parametric Kruskal-Wallis or Mann-Whitney tests were applied to test the significance of differences among groups of clinicopathological parameters,whereas correlations between expression of proteins were evaluated by the Spearman rank-order correlation coefficient.The level of significance was defined as P＜0.05.
Result
Expression of CD24 was cell type-dependent in colorectal cancer cell lines
Flow cytometry was used to detect CD24 expression at protein level in the colorectal cancer cell lines.Of six cell lines,SW1116 maintained moderate expression of CD24,SW620 and Colo205 contained higher expression,but expression was scarcely observed in SW480,HCT8,and LoVo cell lines.To investigate CD24 mRNA at the level of transcription,a standard RT-PCR experiment described in methods was performed.The result was consistent with Flow cytometric analysis.These results showed Expression of CD24 was cell type-dependent in colorectal cancer cell lines.
Overexpression of CD24 induced proliferation in vitro
CD24 recombinant plasmid,pcDNA3.1(+)-CD24,was constructed using expression vector pcDNA3.1(+),and was then stably transfected into the SW480 cells. Two clones,named as SW480~(CD24+1) and SW480~(CD24+4),exhibited dramatically increased expression of CD24 at mRNA transcripts level compared with the parental and vector control cells.Expression of CD24 at protein level in the clones was confirmed by Flow cytometric analysis.The growth ability of SW480~(CD24+1) cells in vitro was assessed by WST-8 assay,and the result showed that these cells proliferated more quickly than SW480~(vec) cells,which were transfected with the vector alone,and the parental SW480 cells.
Overexpression of CD24 induced activation of ERK1/2,Raf-1 and p38 MAPK,but it had no effect on JNK1/2
To elucidate the potiential mechanisms of CD24-induced proliferation,we examined the activation of MAPK pathways.The rusults showed that ERK1/2 and p38 MAPK were activated in the SW480~(CD24+1) and SW480~(CD24+4) cells compared with SW480~(vec) cells and the parental SW480 cells.However,no activation of JNK1/2 was observed.To determine the role of CD24 in Raf-ERK pathway,we further detected the activity of Raf-1,the upstream ERK1/2 activator,and our data showed that Raf-1 protein levels and kinase activity were elevated after the SW480 cells overexpressed with CD24.
Suppressing the activation of ERK1/2 and p38 MAPK abrogated CD24-induced proliferation in the SW480~(CD24+1) cells
To further evaluated the contribution of the ERK1/2 and p38 MAPK in the CD24-induced proliferation,we inhibited their activations using specific pharmacological inhibitors.The starved SW480~(CD24+1) cells were treated either with inhibitor of the upstream ERK1/2 activator MEK1/2,U0126,or the p38 MAPK inhibitor SB203580.DMSO was used as control.The cells were treated separately with 5,10 and 20μM inhibitors and observed at 24 and 72h after treatment followed by WST-8 assay for cell viability.Meantime,parallel cultures were analyzed for total phosphor-tyrosine content by western blotting.Our data showed that the activation of ERK1/2 and p38 MAPK were inhibited,and there were significant decreases of cell viability in the cells treated with different concentration U0126 and SB203580 after 72h.
Cytoplasmic CD24 expression increased with tumor stage and grade
CD24 showed a membranous and a cytoplasmic immunostainings in colorectal tissue.For cytoplasmic CD24 staining,89.6%cases showed positive and 45.3%cases were moderately or strongly positive,while its expression in adjacent normal mucosa was either absent or barely detectable on cell membrane.There was a significantly positive correlation between cytoplasmic CD24 expression and tumor stage and grade, while no correlation was observed between cytoplasmic CD24 expression and age, gender,localization,and tumor grade.For CD24 membranous staining,only 34.9% was classified as positive.CD24 membranous expression showed a significantly negative correlation with tumor grade,but no association with other clinicopathologic parameters.
Activations of the ERK1/2 and p38 MAPK strongly correlated with cytoplasmic CD24 expression in human CRC tissue
To investigate the correlations among ERK1/2,p38 MAPK and CD24 in vivo, immmunohistochemical analysis was performed.Immunostaining of p-ERK1/2 was cytoplasmic,while p-p38 MAPK revealed strong cytoplasmic and partly nuclear staining.Cytoplasmic expression of p-ERK1/2 and p-p38 MAPK was observed in 84.0%and in 78.3%cases,respectively.In adjacent normal mucosa,the staining of both molecules was weak or absent.Cytoplasmic expression of both molecules had no significant correlation with tumor stage and grade.Nuclear p-p38 MAPK staining were classified as 24.5%,which had no significant correlation and trend with any clinicopathological parameters.Expression of p-ERK1/2 correlated with cytoplasmic p-p38 MAPK expression in CRCs(r=0.780,P＜0.001).More importantly,our data showed that strong correlations were found between cytoplasmic CD24 expression and immounostainings of ERK1/2 or p38 MAPK in CRCs(r=0.571,P＜0.001; r=0.528,P＜0.001;respectiveely),suggesting that activations of the ERK1/2 and p38 MAPK strongly correlated with cytoplasmic CD24 expression in human CRC tissue.
Conclusion
In the present study,we evaluated the role of CD24 in the proliferation of CRC cells and the potential mechanisms.Our data showed that increased CD24 expression was cell type-dependent and occurred in 89.6%of human CRCs,and cytoplasmic CD24 expression increased with tumor stage and grade.CD24 could induced proliferation of CRC cells in vitro.In addition,we showed,for the first time, CD24-induced proliferation was dependent on activation of the ERK1/2 and p38 MAPK in CRC.Moreover,activation of ERK1/2 and p38 MAPK also appears to be strongly correlated with CD24 expression in CRC tissue.Our results suggest that CD24 plays a key role in the proliferation of CRC.Furthermore,CD24-dependent MAPK pathway activation is required for CRC cell proliferation.Our finding provided a novel interpretation for the mechanism of CD24-induced proliferation in CRC.The linkage of CD24 and MAPK pathway may unravel a novel mechanism in regulation of proliferation of CRCs.
引文
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[2]Springer T, Galfre G, Secher DS, et al.Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens.Eur J Immunol.1978;8:539-51.
[3]Kay R, Rosten P M, Humphries R K.CD24, a signal transducer modulating B cell activation responses, is a very short pep tide with a glycosyl phosphatidylinositol membrane anchor.JImmunol.1991;147:1412-6.
[4]Kristiansenl G, Sammar M, Altevogt P.Tumour biological aspects of CD24, a mucin-like adhesion molecule.J Mol Histo.2004;35:255-62.
[5]Aigner B, Sthoeger Z, Fogel M, et al.CD24, a Mucin-Type Glycoprotein, Is a Ligand for P-Selectin on Human Tumor Cells.Blood.1997; 89: 3385-95.
[6]Aigner S, Ramos C, Hafezi-Moghadam A, et al.CD24 mediates rolling of breast carcinoma cells on P-selectin.FASEB J.1998;12:1241-51.
[7]Friederichs J, Zeller Y, Hafezi-Moghadam A, et al.The CD24/P-selectin Binding Pathway Initiates Lung Arrest of Human A125 Adenocarcinoma Cells.Cancer Res.2000;60:6714-22.
[8]Tomoko T, Nobutaka K, Kenichi M, et al.Pre-B Cell Antigen Receptor-Mediated Signal Inhibits CD24-Induced Apoptosis in Human Pre-B Cells.J Immunol.2003;170:252-60.
[9]Toyo S, Nobutaka K, Tomoko T, et al.CD24 Induces Apoptosis in Human B Cells Via the Glycolipid-Enriched Membrane Domains/Rafts-Mediated Signaling System.J Immunol.2001;166:5567-77.
[10]Li O, Zheng P, Liu Y CD24 Expression on T Cells Is Required for Optimal T Cell Proliferation in Lymphopenic Host.J Exp Med.2004;200:1083-9.
[11]Li O, Chang X, Zhang H, et al.Massive and destructive T cell response to homeostatic cue in CD24-deficient lymphopenic hosts.J Exp Med.2006;203:1713-20.
[12]Nieoullon V, Belvindrah R, Rougon G, et al.mCD24 regulates proliferation of neuronal committed precursors in the subventricular zone.Mol Cell Neurosci.2005;28:462-74.
[13]Nieoullon V, Belvindrah R , Rougon G, et al.Mouse CD24 is required for homeostatic cell renewal.Cell Tissue Res.2007;329:457-67.
[14]Kristiansen G, Winzer K, Mayordomo E, et al.CD24 Expression Is a New Prognostic Marker in Breast Cancer.Clin Cancer Res.2003;9:4906-13.
[15]Agrawal S,Kuvshinoff B,Khoury T,et al.CD24 Expression is an Independent Prognostic Marker in Cholangiocarcinoma.J Gastrointest Surg.2007;11:445-51.
[16]Weichert W,Denkert C,Burkhard M,et al.Cytoplasmic CD24 Expression in Colorectal Cancer Independently Correlates with Shortened Patient Survival.Clin Cancer Res.2005;11:6574-81.
[17]Chou Y,Jeng Y,Lee T,et al.Cytoplasmic CD24 Expression Is a Novel Prognostic Factor in Diffuse-Type Gastric Adenocarcinoma.Ann Surg Oncol.2007;14:2748-58.
[18]Sano A,Kato H,Sakurai S,et al.CD24 expression is a novel prognostic factor in esophageal squamous cell carcinoma.Ann Surg Oncol.2009;16:506-14.
[19]Choi Y,Kim S,Shin Y,et al.Cytoplasmic CD24 expression in advanced ovarian serous borderline tumors.Gynecol Oncol.2005:97:379-86.
[20]Huang L,Hsu H.Cloning and Expression of CD24 Gene in Human Hepatocellular Carcinoma:A Potential Early Tumor Marker Gene Correlates with p53 Mutation and Tumor Differentiation.Cancer Res.1995;55:4717-21.
[21]Kristiansen G,Pilarsky C,Pervan J,et al.CD24 Expression Is a Significant Predictor of PSA Relapse and Poor Prognosis in Low Grade or Organ Confined Prostate Cancer.Prostate.2004;58:183-92.
[22]Schostak M,Krausel H,Miller K,et al.Quantitative real-time RT-PCR of CD24 mRNA in the detection of prostate cancer.BMC Urology.2006;6:7.
[23]Sagiv E,Memeo L,Karin A,et al.CD24 Is a New Oncogene,Early at the Multistep Process of Colorectal Cancer Carcinogenesis.Gastroenterology.2006;131:630-9.
[24]Baumann P.Cremers N,Kroese F,et al.CD24 Expression Causes the Acquisition of Multiple Cellular Properties Associated with Tumor Growth and Metastasis.Cancer Res.2005;65:10783-93.
[25]Smith S,Oxford G,Wu Z,et al.The Metastasis-Associated Gene CD24 Is Regulated by Ral GTPase and Is a Mediator of Cell Proliferation and Survival in Human Cancer.Cancer Res.2006;66:1917-22.
[26]Sagiv E,Starr A,Rozovski U,et al.Targeting CD24 for treatment of colorectal and pancreatic cancer by monoclonal antibodies or small interfering RNA.Cancer Res.2008;68:2803-12.
[27]Kim H,Kim J,Lee K,et al.Isolation of CD24(high) and CD24(low/-) cells from MCF-7: CD24 expression is positively related with proliferation, adhesion and invasion in MCF-7.Cancer Lett.2007;258:98-108.
[28]Simons K, Ikonen E.Functional rafts in cell membranes.Nature.1997;387:569-72.
[29]Johnson G, Lapadat R.Mitogen-Activated Protein Kinase Pathways Mediated by ERK, JNK,and p38 Protein Kinases.Science.2002;298:1911-2.
[30]Tsuchiya T, Tsuno NH, Asakage M, et al.Apoptosis induction by p38 MAPK inhibitor in human colon cancer cells.Hepatogastroenterology.2008;55:930-5.
[31]Hui L, Bakiri L, Mairhorfer A, et al.p38a suppresses normal and cancer cell proliferation by antagonizing the JNK-c-Jun pathway.Nat Genet.2007;39:741-9.
[32]Ventura JJ, Tenbaum S, Perdiguero E, et al.p38alpha MAP kinase is essential in lung stem and progenitor cell proliferation and differentiation.Nat Genet.2007;39:750-8.
[33]Tournier C, Hess P, Yang DD, et al.Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway.Science.2000;288:870-4.
[34]Sammar M, Gulbins E, Hilbert K, et al.Mouse CD24 as a Signaling Molecule for Integrin-Mediated Cell Binding: Functional and Physical Association with src Kinases.Biochem Bioph Res Co.1997;234:330-4.
[35]Zarn J, Zimmermann S, Pass M, et al.Association of CD24 with the Kinase c-fgr in a Small Cell Lung Cancer Cell Line and with the Kinase lyn in an Erythroleukemia Cell Line.BiochemBioph Res Co.1996;225:384-91.
[36]Tateno M, Nishida Y, Yamada T.Regulation of JNK by Src During Drosophila Development.Science.2000;287:324-7.
[37]Ciuffini L, Castellani L, Salvati E, et al.Delineating v-Src downstream effector pathways in transformed myoblasts.Oncogene.2008;27:528-39.
[38]Scapoli L, Ramos-Nino M, Martinelli M, et al.Src-dependent ERK5 and Src/EGFR-dependent ERK1/2 activation is required for cell proliferation by asbestos.Oncogene.2004;23:805-13.
[39]Fukushima T, Tezuka T, Shimomura T, et al.Silencing of insulin-like growth factor-binding protein-2 in human glioblastoma cells reduces both invasiveness and expression of progression-associated gene CD24.J Biol Chem.2007;282:18634-44.
[40]Walz G, Aruffo A, Kolanus W, et al.Recognition by ELAM-1 of the sialyl-Lex determinant on myeloid and tumor cells.Science.1990;250:1132-5.
[41]A igner S, RuppertM, Hubbe M, et al.Heat stable antigen (mouse CD24) supports myeloid cell binding to endothelial and p latelet P-selectin.Int Immunol.1995;7:1557-65.
[42]Chang L,Karin M.Mammalian MAP kinase signaling cascades.Nature.2001;410:37-40.
[43]Beeram M,Patnaik A,Rowinsky EK.Raf:a strategic target for therapeutic development against cancer.J Clin Oncol.2005;23:6771-90.
[44]Leicht DT,Balan V,Kaplun A,et al.Raf kinases:Function,regulation and role in human cancer.Biochimica et Biophysica Acta.2007;1773:1196-212.
[45]Stokoe D,Macdonald SG,Cadwallader K,et al.Activation of Raf as a result of recruitment to the plasma membrane.Science.1994;264:1463-7.
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