摘要
目的和意义
许多研究证实了肿瘤组织中并不是每个肿瘤细胞都具有致瘤性,而是由一部分比例极低的细胞所决定,这部分细胞被称为肿瘤干细胞,并且认为肿瘤干细胞是导致肿瘤发生的“根源”。肿瘤干细胞一个主要特征是具备自我更新能力,它一方面自身分裂一个或两个与母代特性相同的子代细胞,另一方面发生分化产生终末分化肿瘤细胞,这种分裂和分化导致了肿瘤细胞快速扩增。此外,肿瘤干细胞还具有成瘤性以及对化疗放疗的抵抗作用。由于肿瘤干细胞对化疗放疗作用不敏感,导致传统的治疗方式只能杀死终末分化肿瘤细胞,而对肿瘤干细胞作用不明显。治疗后残存的肿瘤干细胞通过不断扩增而导致了肿瘤复发。
针对肿瘤干细胞自我更新、成瘤以及药物抵抗性等特征的治疗方法将有利于“杀灭”肿瘤干细胞,从而根除肿瘤。许多研究已发现在不同器官组织肿瘤干细胞中存在一些特殊功能的基因,这些基因表达或缺失可以调节肿瘤干细胞自我更新能力、放疗和化疗药物敏感性以及在免疫缺陷的小鼠体内成瘤能力。例如增加Let-7在乳腺肿瘤干细胞中的表达,可以降低其分子靶点H-Ras和HMGA2表达,从而可以降低乳腺肿瘤干细胞自我更新能力,并阻滞其成瘤能力,同时也增强了对化疗药物治疗效果。ABCG2是多重耐药基因家族的一种,它在多种肿瘤干细胞中高表达,并且调节其表达可以改变肿瘤干细胞对治疗药物的敏感性。总的而言,这些研究结果提示了联合放疗和化疗药物并针对肿瘤干细胞中特殊基因的分子靶向治疗将是根除肿瘤的一个有效手段
在众多与肿瘤发生相关的基因中,CD24突显出越来越重要的地位。CD24是一种粘蛋白样结构的膜表面分子,由31个氨基酸组成,包含了16个N-和O-连接的糖基化位点。在正常生理条件下,CD24的瞬时表达促进了血液细胞选择性成熟和分化倾向。在胚胎胰腺组织发育过程中,CD24也起到了重要作用。有报道证实,沉默CD24表达可以抑制淋巴前体细胞向B或T细胞发育,从而导致了系统免疫缺失,这说明了CD24在体内胚胎或多种器官成熟过程中发挥了重要作用。近年来,许多研究表明CD24在多种肿瘤与癌前病变组织中高表达,并且与肿瘤的发生发展及预后有着密切联系。我们前期研究结果也表明CD24通过激活MAPK信号途径而促进结肠肿瘤增殖。在脑胶质瘤中,CD24表达缺失促使了肿瘤细胞分化而抑制了肿瘤生长发育,增加了对药物的敏感性。在乳腺癌和胰腺癌中,CD24能维持肿瘤干细胞自我更新和分化潜能的特性。此外,在结肠癌中,CD24表达主要集中在肿瘤干细胞CD133+细胞亚群,在CD133-细胞亚群中表达很少或无表达。然而,CD24对结肠肿瘤干细胞的调控作用及机制目前还不清楚。
本研究采用功能缺失和功能获得性实验,从体外、体外以及mRNA和蛋白水平,探讨了CD24对结肠肿瘤干细胞自我更新,化疗药物抵抗,细胞分化能力以及成瘤能力的调节作用。研究结果不仅可以丰富了结肠肿瘤干细胞理论,更重要是为以肿瘤干细胞为靶标的结肠肿瘤治疗提供了新的思路。
方法与结果
1.建立稳定表达CD24蛋白的细胞克隆
转染CD24重组过表达质粒pcDNA3.1(+)-CD24进入HCT116细胞株并采用1200ug／ml G418进行筛选,建立了CD24表达升高的细胞克隆,并命名为HCT116CD24和空质粒对照细胞克降HCT116vector。采用荧光定量PCR和流式细胞术检测CD24在HCT116CD24和HCT116vector细胞克隆中的表达,结果发现,CD24mRNA水平在HCT116CD24细胞克隆中明显高于HCT116vector(p<0.001),流式细胞术分析结果也提示CD24蛋白表达水平在HCT116CD24细胞克隆中明显升高(p<0.001)。
2.在结肠肿瘤细胞中上调CD24的表达可以增加CD133表达,同时不影响肿瘤细胞上皮属性
采用流式细胞术分析HCT116CD24细胞克隆和HCT116vector细胞克隆中CD133表达和Epcam表达的表达情况,结果证实：与对照组相比,在HCT116CD24细胞克隆中,CD133表达升高了近20倍(p<0.001),而Epcam在两者的表达没有明显差异(p=0.531)。提示CD24表达上调可以增强CD133表达,对Epcam表达无影响,即没有改变结肠肿瘤细胞的上皮属性。
3.上调CD24表达增强了结肠肿瘤细胞对5-FU和奥沙利铂的抵抗性
为探讨CD24表达上调对肿瘤细胞化疗药物敏感性的影响,我们分别选择不同药物浓度的5-FU (0,25μg/ml,50μg/ml,100μg/ml,250μg/ml,500μg/ml)或奥沙利铂(0,5μM, 10μM,20μM,50μM,100μM)作用于HCT116CD24细胞克隆和HCT116vector细胞克隆,采用CCK-8检测细胞活性。结果表明：在5-FU处理后的HCT116CD24细胞克隆中细胞活性明显高于HCT116vector细胞克隆(p<0.001)；在奥沙利铂作用下,HCT116CD24细胞克隆中细胞活性也明显高于HCT116vector细胞克隆(p<0.001)。提示CD24过表达可以增强肿瘤细胞对5-FU和奥沙利铂的抵抗性。
4.在无血清培养体系中,CD24提高了结肠肿瘤干细胞球mammosphere形成比例
在无血清培养体系中,肿瘤干细胞可以形成悬浮"mammosphere",反映了肿瘤干细胞的自我更新能力。无血清体系中包含了DMEM/F12培养基、B27(1:50),20ng/mlFGF-2以及60ng/mlEGF。培养15天后,细胞不再贴壁生长,而是聚集成球悬浮成长。在HCT116CD24组,1000个细胞可以形成约207±11个而HCT116vector组中,1000个细胞则仅能形成26±5个,两组之间有显著差异(p<0.001)。
5.上调CD24表达增强CD133-细胞对5-FU和奥沙利铂抵抗性
采用免疫磁珠分离方法,结合CD133偶联磁珠分离CD133+和CD133-细胞,并且分别转染pcDNA3.1(+)-CD24及pcDNA3.1(+)-vector,分别建立并命名为CD133+/CD24, CD133+/vector, CD133-/CD24和CD133-/vector四组稳定细胞克隆。分别选择不同药物浓度的5-FU (0,25μg/ml,50μg/ml,100μg/ml,250μg/ml, 500μg/ml)或奥沙利铂(0,5μM,10μM,20μM,50μM,100μM)处理48小时后,采用CCK-8检测细胞活性。对于5-FU处理,CD133+／CD24与CD133+/vector之间细胞活性无明显差异(p>0.05)；而CD133-/CD24和CD133-/vector之间细胞活性有明显差异(p<0.001)；此外,CD133-/vector与CD133+/CD24和CD133+/vector之间细胞活性明显下降(p<0.05)。对于奥沙利铂处理后细胞,CD133-/CD24细胞活性明显高于CD133-/vector (p<0.05);而CD133+/CD24与CD133+/vector之间细胞活性也无明显差异(p>0.05)。提示CD24表达升高仅改变了CD133-细胞对化疗药物的敏感性,对CD133+细胞无明显影响。
6.CD24促进了CD133-细胞中DNA合成能力
BrdU作为干细胞DNA合成标记,也能反映了肿瘤干细胞的比例以及自我更新能力,故本研究采用流式细胞术分析各组细胞中BrdU表达。结果表明：在CD133+/CD24与CD133+/vector两组细胞中,BrdU表达水平无明显差异(98.05±1.3%vs 97.75±1.3%,p=0.731)；但在CD133-/CD24和CD133-/vector组之间BrdU表达水平明显差异(27±3.4%Vs.3±0.25%,p<0.001)。提示上调CD24表达后,促使CD133-细胞中有部分细胞获得了结肠肿瘤干细胞特性,而对CD133+细胞无明显影响。
7.上调CD24表达提高CD133-细胞形成mammosphere的能力
在CD133-/CD24组中,1000个细胞在无血清培养体系下可以形成约140±15个mammospheres;但在CD133-/vector组中,1000个细胞仅能形成20±4个mammospheres,两者之间有显著差异(p<0001)。而在CD133+／CD24与CD133+／vector两组中,形成mammosphere的个数没有明显差异(p=0.745)。提示CD24促进肿瘤干细胞的自我更新能力主要是通过促使部分CD133-细胞获得肿瘤干细胞的自我更新能力实现的,而对CD133+细胞的自我更新能力无明显影响。
8.上调CD24表达增强CD133-细胞在裸鼠体内的致瘤能力
将1x106个CD133-/vector和CD133-／CD24细胞分别接种于5周左右的裸鼠背侧皮下,每隔3天测量成瘤体积大小,在30天时处死全部实验裸鼠,取出肿瘤后石蜡包埋、切片用于HE染色,从而鉴定肿瘤特性。CD133-／CD24组移植瘤体积(n=5)随着时间增加而明显增大；但对照组,瘤体体积小,并且增长速度缓慢,两者之间比较有显著性差异(p<0.001)。结果说明了CD24促进了CD133-细胞在裸鼠体内形成瘤体的能力,尽管CD133-/vector细胞中也可形成瘤体,但体积小,而且生长速度缓慢,这可能与采用免疫磁珠分离方法不能完全分离CD133-细胞,在CD133-细胞中存在少量的CD133+细胞。
9.沉默CD24表达诱导结肠肿瘤干细胞凋亡
我们构建了CD24shRNA慢病毒载体,在无血清培养基体系中,利用lipofectaminTM 2000转染CD133+细胞48小时后,采用Hoechst 33342对细胞染色,干扰组lenti-CD24 shRNA组细胞出现明显凋亡,凋亡细胞出现核浓缩,染色增强；同时悬浮细胞数目减少,部分细胞出现脆裂细胞核沉淀于培养皿底部。进一步采用了流式细胞术Annexin V-7AAD双染检测细胞凋亡,结果与染色结果相似,对照组lenti-vector细胞凋亡比例为2.5±0.65%,而lenti-CD24 shRNA组为27+1.4%,两组之间有显著性差异(p<0001)。上述结果表明了在结肠肿瘤干细胞中沉默CD24表达导致了肿瘤干细胞凋亡
10.沉默CD24表达可以减少结肠肿瘤干细胞mammosphere形成
在无血清培养体系中,100个CD133+细胞经过scramble shRNA处理后可以形成大约65+3个mammospheres,但是经过沉默CD24表达后,100个细胞形成nammosphere数目仅为8±2个,两组之间有显著性差异(p<0001)。结果表明在结肠肿瘤CD133+细胞中,沉默CD24表达会导致结肠肿瘤干细胞自我更新能力下降,并且降低了肿瘤干细胞mammosphere形成能力。
11.沉默CD24表达诱导结肠肿瘤干细胞分化
转染lenti-CD24 shRNA或lenti-vector进入无血清培养基中形成的mammosphere中,并进行形态学观察。在lenti-vector组中nammoshere没有明显形态变化,而lenti-CD24 shRNA组中部分mammosphere不再保持原有悬浮球状,逐渐出现分化贴壁,细胞变为长梭状。继而采用细胞免疫荧光染色方法证实了在lenti-CD24 shRNA组中细胞分化标志CD20表达增高,而在lenti-vector表达阴性；OCT-4(调节胚胎干细胞和多种肿瘤干细胞的转录因子)表达却正好相反,在mammosphere中高表达,而在lenti-CD24 shRNA组中表达阴性。提示CD24表达下调可以促进结肠肿瘤干细胞分化,其分子机制可能是通过调节CK20表达和OCT-4表达而改变了肿瘤干细胞特性。
12.沉默CD24表达增强了结肠肿瘤干细胞对化疗药物敏感性
细胞分组情况：采用lipofectaminTM 2000转染lenti-vector进入CD133+细胞作为对照组(lenti-vector);干扰组转染leitiviral CD24 shRNA (lenti-CD24 shRNA);采用lipofectaminTM 2000转染对应体积的PBS作为未处理组。CCK-8检测细胞活性。相对于未处理组,lenti-vector对照组以及lenti-vector+5-FU (500μg/ml)+奥沙利铂(100μM),这三组之间细胞活性没有明显差异；而lenti-CD24 shRNA组中细胞活性显著低于lenti-vector对照组(51.7±3.4% Vs91.5±1.5%,p<0.001)：与lenti-CD24 shRNA+lenti-vector+5-FU (500μg/ml)+奥沙利铂(100μM)相比较,lenti-CD24 shRNA组中细胞活性明显升高(51.7±3.4%Vs 30.6±2.23%,p<0.001)。结果表明在结肠肿瘤干细胞中沉默CD24表达不仅可以导致肿瘤干细胞活性降低,而且可以增强其对化疗药物敏感性,协同化疗药物的治疗效果。
13.沉默CD24表达阻滞了肿瘤干细胞在裸鼠体内成瘤
将1000个结肠肿瘤干细胞mammospheres种植入5周龄左右雌性裸鼠背侧皮下,共15只；当肿瘤生长到15天时,所有成瘤体积大小无明显差异,随机将裸鼠分成3组：无处理组(untreated), lenti-vector组以及lenti-CD24 shRNA,每组5只。Lenti-CD24 shRNA组和lenti-vector组分别瘤体内注射1×103TU／g的lenti-CD24 shRNA和对照lenti-vector,未处理组用等量PBS代替；第22天后再注射同样剂量,最后在第30天处死全部裸鼠,取出肿瘤组织并提取蛋白和用10%福尔马林固定。结果发现注射lenti-CD24 shRNA病毒载体的移植瘤随着时间变化,肿瘤体积改变明显低于未处理组和对照组(p<0.001；p<0.001)；而对照组和未处理组之间肿瘤体积没有明显差别(p=0.306)。我们也检测了移植瘤中CD24和CD133表达水平,与细胞内实验结果相符,CD24和CD133表达水平在lenti-CD24 shRNA病毒载体后明显降低。
14. CD24和CD133共同表达与结直结肠肿瘤转移、分化及生存率相关
为了进一步证实CD24和CD133共同表达与结肠肿瘤患者临床病理学特征之间的关系,我们采用免疫组织化学的方法检测了213例术后结肠肿瘤患者肿瘤组织中CD24和CD133表达,结果显示在结肠肿瘤患者组织中CD24与CD133表达显著相关(r=0.658,p=0.000)；CD24和CD133共同表达与结肠肿瘤患者低分化,淋巴结转移以及远处转移有关,而与性别、年龄、肿瘤分期和生长部位无关。分析其中89例患者100个月的随访资料,CD24和CD133共同表达组100个月的生存率明显低于CD24和CD133共同阴性组(p=0.01)。
结论
综合以上研究结果,我们可以得出以下结论：
1.CD24过表达可以上调结肠肿瘤细胞中CD133的表达,增强结肠肿瘤细胞对化疗药物抵抗性；
2.CD24过表达可以增强结肠肿瘤肿瘤干细胞自我更新的能力；
3.CD24过表达可以促使CD133-细胞获得结肠肿瘤干细胞的特性；
4.沉默CD24表达可以促进结肠肿瘤干细胞凋亡,降低其对化疗药物抵抗性,阻滞细胞体内成瘤能力；
5.CD24和CD133共同表达与结肠肿瘤转移、分化及生存率相关。
因此,我们研究CD24是结肠肿瘤干细胞自我更新、化疗药物抵抗,分化以及成瘤的一个重要调节基因,沉默CD24表达可以有效“杀死”结肠肿瘤干细胞。检测CD24和CD133共同表达可以预测结肠肿瘤患者的生存率。所以CD24可以作为一个有效的结肠肿瘤干细胞治疗的靶标,为针对结肠肿瘤的新的生物治疗药物开发提供了有力的理论依据。
Objective
Traditional models of carcinogenesis posit that cancers might originate in any cell through a series of stochastic genetic events resulting in clonal selection. By contrast, the stem cell model proposes that cancers originate in select cell population from normal tissue stem cells or progenitor cells and that initiating events disrupt the process that regulates stem cell self-renewal,This is so called cancer stem cell or cancer stem-like cell. Self-renewal, one of characteristics of cancer stem cell, is a cell division in which one or both of the resulting daughter cells remains undifferentiated, and retains the ability to give rise to another stem cell with the same capacity to proliferate as the parental cells; In additional, cancer stem cell have the capacity to differentiate, a property that generates the bulk of cells within a tumor. In addition to the property of self-renewal, cancer stem cells might also be resistant to the radiotherapy and chemotherapy, and capacity of tumorigenesis. Because cancer stem cells is not sensitive to chemotherapy and radiotherapy, the current treatments only kill the different lineages of terminally differentiated tumor cells, while no obvious effect on the cancer stem cells. After treatment, residual tumor stem cell with self-renewal has resulted in tumor recurrence.
Then, a new neoadjuvant treatment will be require targeting the cancer stem cell against the self-renewal, tumorigenicity and drug resistance.Many studies have verified that there were several special genes in the cancer stem cell.The presence or absence of the genes in the cancer stem cell can regulate the self-renewal capacity and enhance the sensitivity to radiotherapy and chemotherapy, and block the tumorigenesis in the immunodeficiency mice.For example,inceased let-7 paralleled reduced H-RAS and HMGA2,can reduced proliferation,mammosphere formating,and the proportion of undifferentiated cells in vitro and tumor formation and metastasis in NOD/SCID mice in breast cancer stem cell.ABCG2,one of MDRs (Multiple Resistance Drug family), was expressed in kinds of cancer stem cells. Silencing ABCG2 in cancer stem cell can increased the sensitivity to chemotherapy and radiotherapy. In additional,Blocking interleukin-4 signaling can senditize colorectal cancer stem cells to apoptotic stimuli and increase the in vivo efficacy of cytotoxic therapy.In total, these studies improved outcomes of therapies might require targeting cancer stem cell subgroup.
CD24 was regarded as a prominent role in many genes associated with tumorigensis.The CD24 gene encoded a heavily glycosylated cell surface protein anchored to the membrane by phosphatidylinositol, which consisted of three 31 amino acids with 16 potential O-glycosylation and N-lycosylation sites and was capable of mucin-like characteristics. Under normal physiologic conditions, CD24 transiently facilitated primary hematopoietic cell selection and promoted their progeny maturation during hematopoiesis.It also plays a role during the embryonic development of pancreatic cells. Silencing CD24 expression could inhibit the phenotype transformation in lymphocytes precursors.CD24 played a vital role not only in normal tissue but also in various cancers. Some published literatures have verified that CD24 was related to kinds of human cancer carcinogenesis. Furethermore,cancer stem cells showed the properties of self-renewal and the ability to produce differentiated progeny in dependence of the CD24 expression in breast cancer and panceatic cancer. CD24 also suggested in some studies to contribute to malignant transformation in colorectal cancer and was limited to CD133+ subgroup, but little or no expression in CD 133- cell while cancer cells were isolated. Other document also vertified the results. These data suggested that CD24 promotes tumorigenesis, and acted as a marker of cancer stem cell in some cancers.But,no study searching for markers and function for colorectal cancer stem cells has been preformed to data.
In present study, the role of CD24 in the properties of self-renewal, drug-resistant, and tumorigenicity of colorectal cancer stem cell was investigated. Our study suggested that CD24 was a potential colorectal cancer stem cell marker and a candidate for therapy alone or in combination with chemotherapy drugs in colorectal cancer.
Methods and Results
1. A stable cell line expressing CD24 was established
CD24 recombinant plasmid pcDNA3.1 (+)-CD24, which was constructed using expression vector pcDNA3.1 (+) in previous study, was transfected into HCT116 cells and cultured in medium including G418. After selection, resistant colonies to G418 were chosen and named as HCT116CD24 and HCT116vector. MRNA level and protein expression were assessed by real-time PCR and FCS. Our results showed that the mRNA level and protein expression of CD24 increased significantly compared with control groups (p<0.05).
2. Up-regulating CD24 expression increased the expression of CD133 and EPcam
To investigate the expression of CD133 after CD24 up-regulated, CD133 expression was detected by FCS in HCT116CD24 and HCT116vector cells. Our data showed that CD133 expression increased significantly in HCT116CD24 cells compared with in HCT116vector cells (p<0.001). Epcam was used as a marker of epithelial cell phenotype, there was no change in Epcam expression level and suggested that the cell phenotype still kept (p=0.531).
3. Up-regulating CD24 expression enhanced the resistance to 5-FU and Oxaliplatin
To investigate the effect of CD24 expression on chemotherapy drug, the cell viability was detected by CCK-8 assay after 5-FU and/or Oxaliplatin treated in HCT116CD24 and HCT116vector cells. We results showed that cell viability increased in HCT116CD24cells in a dose-dependent manner after 5-FU treated. The same trend was found in oxaliplatin treated groups. Our data suggested that up-regulating CD24 expression enhanced the resistance to 5-FU and oxaliplatin.
4. Up-regulating CD24 expression induced tumor mammosphere formation
As we all know, tumor mammospheres generation is an in vitro assay of self renewal potency in cancer stem cell. We detected the mammospheres formating in serum-free medium after CD24 recombinant plasmid was transfected.After 15 days, tumor mammospheres in HCT116CD24 group were significantly increased 10 fold than control one (.207±11vs.26±5/1000 cells) Collectively, our data suggested that up-regulating CD24 increased the proportion of cancer stem-like cell in HCT116
5.Up-regualting CD24 regramming CD133- cells into cancer stem-like cell and increased the resistance to 5-FU and Oxaliplatin in CD133- cells subgroup
To further clarify the mechanism which CD24 increased CRC stem-like cell, we isolated CD133+ cell group and CD133" cell group with MACS. CD24 recombinant plasmid was stably transfected respectively into the two groups.The endogenous expression of CD24 is 35% in CD133+ cell group,and no change after overexpression of CD24;In the contrary, there was almost no expression of CD24 in CD133- cell group, but obviously increased after transfection.Moreover,the expression of CD133 also increased in parallel with the expression of CD24.
Consistence with the theory that tumor stem cell was not sensitive to chemodrugs, and CD133- cells was senstive to 5-FU and Oxaliplatin, the results showed that CD133+cells resistance to chemotherapeutic was similar to CD 133+ cells with the treatment of CD24 recombinant plasmid (p>0.05), but the resistance to chemotherapeutic of CD133- cells was obviously enhanced compared to CD133-cells with the treatment of CD24 recombinant plasmid (p<0.05).
6. CD24 increased DNA synthesis in CD133- cell just as cancer stem cell
In addition, the self-renewal of cancer stem-like cells was evaluated using BrdU, which marked cancer stem-like cells. We found that the BrdU positive cells in CD133- cells was increased from 3±0.25% to 27+3.4% after up-regulating CD24 expression. the same results was not see in CD133+ cells and CD133+ cells with the treatment of CD24 recombinant plasmid. There was no change of the the positive cells of BrdU in CD133+cells and CD133+ cells with the treatment of CD24 recombinant plasmid (85±5.4% vs.86±4.3%;p>0.05).
7. CD133- cell can formatting mammosphere after gain-function of CD24
Furthermore, tumor mammospheres was obtained with spheroid culture in serum-free DMEM/F12 medium,containing B27,20ng/ml FGF-2 and 60ng/ml EGF.The mammospheres derived from CD133+ cells was approximatively 830±40.5 in 1000 CD133+ cells, the same as derived from CD 133+ with the treatment of CD24 recombinant plasmid. In the contrary, the mammospheres was increased after overexpression of CD24 in CD133-cells (20±4 vs.140+15, p<0001).
8.CD24 enhanced CD133- cells tumorigenesis in vivo
In accordance with in vitro results, in vivo exnograft tumor further verified that up-regulating CD24 enhanced the tumorigenesis capability of CD133- cells compared with the control (p<0.001). Collectively,these data suggested that Up-regulating CD24 regrammed CD133- cells into cancer stem-like and helped CD133-cells capacible of the charactistic of self-renewal,chemotherapeutic resistance, tumor mammospheres formating,and tumorigenesis.
9. Silencing CD24 resulted in tumor mammosphere differentiation
Given the increasing the proportion of CRC stem-like cell after up-regulation of CD24,we next test the possibility that down-regulating CD24 affects the features of CRC stem cell. CD133+ cell was isolated combination with spheroic culture in serum-free medium. Using the shRNA method with lentiviral vector for knockdown of CD24 expression in CD133+cells,the data showed the treatment of CD24 shRNA could diminish the CD133 expression, and facilitate tumor mammosphere differetiation.Furthermore,tumor mammosphere formating was blocking after CD24 shRNA transfectiong compared with scramble shRNA.Without the treatment of CD24 shRNA,67±10.3% CD133+ cells could format tumor mammospheres;in the contrary, only 13±3.5% CD133+ cells could format mammosphere (p<0.05). There was significantly diffferent between two groups.CK20 indicated cell differetiation,our data shows that CK20 expression increased after the treatment of CD24 shRNA,but OCT-4 decreased.
10. Silencing CD24 expression induced in the cancer stem cell apoptosis
In addtion, we also investigated the role of CD24 in apoptosis. Freshly isolated CD133+ cells, which derived from HCT116, was trasfected respectively with CD24 shRNA and scramble shRNA.Hoechst 33342 staining was used to evaluate the apoptosis morphology. Nuclear enrichment indicated cell apoptosis.There was also significantly increased with CD24 shRNA treatment compared with scramble shRNA treatment.
In a word, these data indicated that CD24 expression played a crucial role in maintaining self-renewal and cancer stem cell properities in CD133+ cells. Since down-regulation of CD24 expression induced in tumor mammospheres differetiation, CD24 shRNA may be a attractive candidate as a potential adjuvant to chemotherapy for colon cancer.
11. Silencing CD24 enhanced the tumor mammmosphere sensitivity to chemotherapy in vitro
To vertify whether CD24 shRNA overcome resistance to chemotherapy in CD133+ cells, using the treatment CD24 shRNA, the results exhibited that the knockdown of CD24 in CD133+ cells can significantly improve the anti-cancer effect in single or combination with 5-FU or oxaliplatin in vitro (F=804.769.p<0.001).Moreover, we detected the expression of ABCG2, one of MDRs, which mainly responsible for cancer stem cell resistance to chemotherapy and irradiation treatment. The ABCG2 expression was reduced-5 fold in the CD24 shRNA than the scramble shRNA.These data suggested that the up-regulating CD24 could effectively strengthen the chemotherapeutic sensitivity of cancer stem cells by regulating ABCG2 expression.
12. Silencing CD24 blocked the tumorigencity of mammosphere in vivo
1000 tumor mammospheres was injected in the nude mice to generate human colorectal cancer.At day 15 and 22, thery were injected with lenti-CD24 shRNA or lenti-scramble shRNA or PBS. In vivo xenograft tumor results confirmed that injection lenti-CD24 shRNA could block the tumorigencity of CD133+ cell or tumor mammmosphere, and CD133 expression was decreased than lenti-scramble shRNA or blank control (F=804.769,P<0.001).. Further, we found that CD24 and CD 133 expression obviously decreased in the xenograft with the treatment of lenti-CD24 shRNA by western blotting.these data collectively suggested that CD24 could act as an effective therapy target.
13. Co-expression of CD24 and CD133 associated with the low grade and metastasis, and predicted poor overall survival in CRC patients
To investigate the correlation between the co-expression of CD24 and CD133 and clinicopathologic feature, immunohistochemical staining was performed in serial sections of CRC paraffin-embedded colorectal cancer specimens. As a result, the CD24 expression was found to be significantly associated with the expression of CD133 (r=0.658,p=0.000),and the co-expression of CD24 and CD133 was dramatically different between positive distal metastasis and negative distal metastasis (p<0.05) compared with the negative expression of CD24 and CD133,the same as positive nodal metastasis (p<0.05),and low tumor grade (p=0.038).These results suggested that co-expression of CD24 and CD 133 was closely correlated with low tumor grade, positive nodal and distal metastasis in colon cancer tissues.
To determine whether the co-expression of CD24 and CD133 was a significant prognostic factor for the survival of patients with surgically resected colorectal cancer, we performed a log-rank test with Kaplan-Meier estimates. Among the 213 patients analyzed,89 patients were followed up. The 100 months overall survival rate was significantly lower with co-expression of CD24 and CD 133 than negative expression in primary colorectal cancer patients (p=0.01). These results clearly indicated that co-expression of CD24 and CD 133 was associated with the 100 months survival of colorectal cancer cases and suggested that co-expression of CD24 and CD 133 might be a biomarker of poor prognosis.
Conclusion
In the present study, using gain of function and loss of functiong,we demonstrated that CD24 expression induced tumor spheres formating,and increased drμg-resistant;Strikingly, CD133-cell subgroup gained cancer stem cell Properities after gain of function of CD24 with BrdU intake. we also showed that Knocking down CD24 resulted in colorectal cancer sphere differentiated,and obviously increased apoptosis. Our data suggested that CD24 mainly maintained the properities of colorectal cancer stem cell by regulating the self-renew, drug-resistant, and tumorigenesis. Collectively, our data suggested that CD24 was a key role in maintaining the properities of CRC stem cell; Strikely, CD24 could regramming the CD133 negative cells into cancer stem cell.Combination with CD133 positive expression, CD24 was correlated with poor overall survival in CRC patients. These data taken together highlight a function of CD24 in CRC stem cell, and indentify CD24 as a potential novel candidate for therapeutic intervention alone or in combination with chemotherapy drugs.
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