Sox2在神经母细胞瘤中的表达及其对神经母细胞瘤干细胞生物学特性的影响
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摘要
研究背景与目的
神经母细胞瘤(neuroblastoma,NB)是小儿最常见的颅外恶性实体肿瘤,占小儿所有恶性肿瘤的10%,恶性程度高,多数患儿预后不良。尽管近年来手术结合化疗的综合疗法在治疗NB方面取得了很大的进步,但是其发病率和死亡率仍然较高。因此,深入研究NB的发生、发展机理至关重要。神经母细胞瘤是一种胚胎性肿瘤,胚胎期神经嵴细胞发育分化受阻是神经母细胞瘤发生的主要原因,亦是衡量神经母细胞瘤恶性程度及影响预后的主要危险因素。Sox2是一种胚胎干细胞转录调控基因,是调控胚胎发育的关键因子,在早期胚胎发育过程中对维持胚胎干细胞多潜能性和自我更新的能力具有关键性的调控作用。故我们试图探索Sox2基因在神经母细胞瘤中的表达及其对神经母细胞瘤干细胞生物学特性的影响,探讨其在神经母细胞瘤发生发展过程中所起的作用。材料与方法
1.Sox2在神经母细胞瘤中的表达：采用RT-PCR、Real-time PCR、Western blot及免疫组织化学等方法检测Sox2在神经母细胞瘤组织和瘤旁组织中的表达情况,并结合临床资料,分析其表达与临床病理参数的关系。
2.Sox2对神经母细胞瘤干细胞生物学特性的影响：采用RT-PCR检测Sox2在神经母细胞瘤BE(2)-C细胞中的表达情况。利用慢病毒载体感染BE(2)-C细胞构建Sox2高表达[BE(2)-C-Sox2]和低表达[BE(2)-C-shRNA]细胞,然后采用CCK-8活细胞数检测和流式细胞仪细胞周期分析,探讨Sox2对BE(2)-C细胞增殖能力的影响。维甲酸(RA)和5-溴2’-去氧尿甙(BrdU)干预细胞诱导分化后采用Western blot检测细胞标志蛋白的表达差异,分析Sox2对BE(2)-C细胞分化能力的影响。平板克隆和软琼脂克隆形成实验检测细胞克隆形成数目和克隆形成率,分析Sox2对BE(2)-C细胞克隆形成能力的影响。裸鼠成瘤实验分析Sox2对BE(2)-C细胞体内成瘤能力的影响。
3.基因芯片检测Sox2下调时神经母细胞瘤干细胞基因表达谱变化：应用基因表达谱芯片检测BE(2)-C细胞和BE(2)-C-shRNA细胞基因表达谱情况,筛选差异基因,并采用Real-time PCR检测验证相关差异表达的基因。
结果
1.Sox2在神经母细胞瘤中的表达：RT-PCR结果显示在神经母细胞瘤组织中可检测到Sox2基因mRNA水平表达。免疫组织化学染色结果显示Sox2主要在肿瘤细胞核中表达,而瘤旁组织中表达呈阴性。Real-time PCR及Western blot结果均显示神经母细胞瘤组织中Sox2的表达水平明显高于瘤旁组织,差异有统计学意义(P<0.05)。经统计分析发现Sox2在高分期肿瘤中的表达水平高于低分期肿瘤(P<0.05),且未经化疗的肿瘤其Sox2的表达水平高于经化疗的肿瘤(P<0.05)。Sox2在神经母细胞瘤中的表达水平与性别、年龄、肿瘤部位、大小、病理分型等参数无相关性(均P>0.05)。
2.Sox2对神经母细胞瘤干细胞生物学特性的影响：RT-PCR结果显示在神经母细胞瘤BE(2)-C细胞系中可检测到Sox2基因mRNA水平表达。利用慢病毒载体感染BE(2)-C细胞成功构建Sox2高表达[BE(2)-C-Sox2]和低表达[BE(2)-C-shRNA]稳定细胞。CCK-8活细胞数检测显示72h时BE(2)-C-Sox2细胞的AV值(1.465±0.046)高于BE(2)-C细胞(1.01±0.018),差异有统计学意义(P<0.05)；而BE(2)-C-shRNA细胞的AV值(0.912±0.020)低于BE(2)-C细胞(1.201±0.018),差异有统计学意义(P<0.05)。流式细胞仪细胞周期分析显示BE(2)-C-Sox2组S期细胞百分比(55.2±4.3%)高于BE(2)-C组细胞(38.6±3.5%),BE(2)-C-shRNA组S期细胞百分比(21.8±5.7%)低于BE(2)-C组细胞,三组组间比较差异有统计学意义(P<0.05),而三组细胞G0/G1期细胞百分比的比较情况呈现出相反的结果；BE(2)-C-Sox2、BE(2)-C及BE(2)-C-shRNA组的细胞增殖指数分别为65.2±5.6%、36.3±2.8%、51.7±4.6%,三组组间比较差异有统计学意义(P<0.05)。RA或BrdU干预后Western blot检测细胞标志蛋白(Peripherin、NF-68、Vimentin、S-100)显示,经RA或BrdU干预的BE(2)-C-Sox2细胞其标志蛋白表达量低于经同种药物干预的BE(2)-C细胞和空载体细胞(P<0.05)；而经RA或BrdU干预的BE(2)-C-shRNA细胞其标志蛋白表达量高于经同种药物干预的BE(2)-C细胞和空载体细胞(P<0.05)。平板克隆形成实验显示BE(2)-C-Sox2细胞的克隆形成数目(103±18)和克隆形成率(20.6%)均高于BE(2)-C细胞(75±12,15%),而BE(2)-C-shRNA细胞的克隆形成数目(42±7)和克隆形成率(8.4%)均低于BE(2)-C细胞,三组组间比较差异有统计学意义(P<0.05)。软琼脂克隆形成实验未成功,细胞均死亡。裸鼠成瘤实验显示BE(2)-C-Sox2接种组肿瘤的生长速度和体积均大于BE(2)-C接种组,而BE(2)-C-shRNA接种组肿瘤的生长速度和体积均低于BE(2)-C接种组,三组组间比较差异有统计学意义(P<0.05)。
3.基因芯片检测Sox2下调时神经母细胞瘤干细胞基因表达谱变化：Sox2基因表达下调时基因表达谱芯片筛得差异表达的基因有596条,其中差异显著的基因有FMN1、GFRA2、HIST1H2BK、CARTPT、AHNAK2、PLP2、TSPAN8、TIMP2、EPO和PRR11等。经Real-time PCR验证,证实芯片筛选的结果可靠。
结论
1.Sox2在神经母细胞瘤(组织和细胞)中存在表达,其表达水平与肿瘤的临床分期有一定相关性,与性别、年龄等参数无明显相关性。化疗药物对Sox2的表达有抑制作用。
2.Sox2促进神经母细胞瘤干细胞的增殖、克隆形成和体内成瘤等生物学特性,但对其分化有一定的抑制作用,提示Sox2在神经母细胞瘤的发生发展过程中具有重要作用。
3.Sox2表达下调时基因表达谱芯片筛选得相关差异表达的基因有：FMN1、GFRA2.HIST1H2BK、CARTPT、AHNAK2、PLP2、TSPAN8、TIMP2、EPO和PRR11等。芯片结果尚有待于进一步分析和研究。
Backgrounds and objective
Neuroblastoma (NB) is the most common extracranial solid pediatric tumor and accounts for10%of childhood cancers. Although major advances have been made in surgery and chemotherapy of NB, the morbidity and mortality remain high. So far, the molecular mechanisms responsible for the pathogenesis of NB remain elusive, thus researches on the genisis and progress of NB are of great importance. NB is an embryonic tumor, and the inhibition of embryonal neural crest cell differentiation is the main reason of the genisis of NB and major risk factor of its manignancy and prognosis. Sox2is a transcription factor expressed in both embryonic and adult stem cells. It plays a pivotal role in the maintenance of self-renewal and differentiation potential. In this study, we explore the expression of Sox2in human NB and the relationship between the expression levels of Sox2and various clinicopathological parameters. Meanwhile, the potential functions of Sox2on the genesis and progress of NB are also explored in this study.
Materials and Methods
1. The research of Sox2expression in human NB:RT-PCR, Real-time PCR, Western blot analysis and immunohistochemical staining were employed to detect the expression of Sox2in human NB, and the relationship between Sox2expression and clinical data was assessed.
2. The effects of Sox2on the biological characteristics of neuroblastoma stem cell:RT-PCR was performed to detect the expression of Sox2in human neuroblastoma BE(2)-C cell. Sox2-overexpressed and mRNA knockdown stem cell lines were established by infecting BE(2)-C cell via lentivirus vectors. Cell counting kit-8(CCK-8) assay and flow cytometry were applied to test the effect of Sox2on the proliferative ability. Western blot analysis was used to detect the marker proteins after drug interference in order to analyze the effect of Sox2on differentiation properties. Plate colony-forming assay and tumorigenicity assay were performed to analyze the effect of Sox2on the malignant potential.
3. Gene chip detect the gene expression profiling changes in down-regulating Sox2neuroblastoma stem cell:The gene expression profiles of BE(2)-C-shRNA and BE(2)-C cells were investigated with Genechip Human Gene1.OST. Differentially expressed genes were analyzed by data analyses. Real-time PCR was performed to verify the gene chip results
Results
1. The research of Sox2expression in human NB:Sox2mRNA was detected in NB tissues by RT-PCR. The immunohistochemical staining showed that Sox2was primarily localized in the nuclei of NB cells, not observed in paracancerous cells. Using Real-time PCR, we found that Sox2mRNA relative expression levels was significantly higher in tumor tissues than in the adjacent non-cancerous tissues (P<0.05), and the relative expression levels in stage Ⅲ and Ⅳ NB were higher compared with those in stage Ⅰ and Ⅱ NB (P<0.05). Sox2expression was significantly decreased in the chemotherapy subgroup as compared with that of the non-chemotherapy subgroup in stage Ⅲ and Ⅳ tumors (P<0.05). Western blot analysis confirmed the results at the protein level.
2. The effects of Sox2on the biological characteristics of neuroblastoma stem cell:Sox2mRNA was detected in human neuroblastoma BE(2)-C cell by RT-PCR. Sox2-overexpressed[BE(2)-C-Sox2] and mRNA knockdown[BE(2)-C-shRNA] stem cell lines were successfully established by infecting BE(2)-C cell via lentivirus vectors. BE(2)-C-Sox2cell showed relatively higher cell proliferative number than BE(2)-C cell (P<0.05) in CCK-8assay. The percentage of cells in S phase and PLI index in BE(2)-C-Sox2group was higher than BE(2)-C group (P<0.05) in flow cytometry assay. BE(2)-C-Sox2cell showed higher colony-forming number and efficiency (P<0.05), speed of tumorigenesis and volumes of tumors in vivo than BE(2)-C and BE(2)-C-shRNA cells (P<0.05), and exhibited decreased expression levels of other type cell marker proteins than BE(2)-C and BE(2)-C-shRNA cells (P<0.05). Conversely, down-regulation of Sox2showed an inverse result.
3. Gene chip detect the gene expression profiling changes in down-regulating Sox2neuroblastoma stem cell:596differentially expressed genes were screened by Genechip Human Gene1.0ST. The significantly differentially expressed genes were FMN1, GFRA2, HIST1H2BK, CARTPT, AHNAK2, PLP2, TSPAN8, TIMP2, EPO, PRR11and etc. Real-time PCR verified the gene chip results.
Conclusions
1. Sox2is expressed in human neuroblastoma and neuroblastoma stem cell, and its expression correlates to the clinical stage of NB, but not other clinicopathological parameters including patient gender and age, tumor size, location and histological classification. Moreover, Sox2expression can be inhibited by chemotherapy.
2. Sox2plays a key role in the abilities of proliferation, differentiation, colony-forming and tumorigenesis of neuroblastoma.
3. Differentially expressed genes via gene chip in down-regulating Sox2neuroblastoma stem cell are FMN1, GFRA2, HIST1H2BK, CARTPT, AHNAK2, PLP2, TSPAN8, TIMP2, EPO, PRR11and etc. Further analyses and research are needed to explore the data.
神经母细胞瘤(neuroblastoma,NB)是小儿最常见的颅外恶性实体肿瘤,占小儿所有恶性肿瘤的10%,恶性程度高,多数患儿预后不良。尽管近年来手术结合化疗的综合疗法在治疗NB方面取得了很大的进步,但是其发病率和死亡率仍然较高。因此,深入研究NB的发生、发展机理至关重要。神经母细胞瘤是一种胚胎性肿瘤,胚胎期神经嵴细胞发育分化受阻是神经母细胞瘤发生的主要原因,亦是衡量神经母细胞瘤恶性程度及影响预后的主要危险因素。Sox2是一种胚胎干细胞转录调控基因,是调控胚胎发育的关键因子,在早期胚胎发育过程中对维持胚胎干细胞多潜能性和自我更新的能力具有关键性的调控作用。故我们试图探索Sox2基因在神经母细胞瘤中的表达及其对神经母细胞瘤干细胞生物学特性的影响,探讨其在神经母细胞瘤发生发展过程中所起的作用。材料与方法
1.Sox2在神经母细胞瘤中的表达：采用RT-PCR、Real-time PCR、Western blot及免疫组织化学等方法检测Sox2在神经母细胞瘤组织和瘤旁组织中的表达情况,并结合临床资料,分析其表达与临床病理参数的关系。
2.Sox2对神经母细胞瘤干细胞生物学特性的影响：采用RT-PCR检测Sox2在神经母细胞瘤BE(2)-C细胞中的表达情况。利用慢病毒载体感染BE(2)-C细胞构建Sox2高表达[BE(2)-C-Sox2]和低表达[BE(2)-C-shRNA]细胞,然后采用CCK-8活细胞数检测和流式细胞仪细胞周期分析,探讨Sox2对BE(2)-C细胞增殖能力的影响。维甲酸(RA)和5-溴2’-去氧尿甙(BrdU)干预细胞诱导分化后采用Western blot检测细胞标志蛋白的表达差异,分析Sox2对BE(2)-C细胞分化能力的影响。平板克隆和软琼脂克隆形成实验检测细胞克隆形成数目和克隆形成率,分析Sox2对BE(2)-C细胞克隆形成能力的影响。裸鼠成瘤实验分析Sox2对BE(2)-C细胞体内成瘤能力的影响。
3.基因芯片检测Sox2下调时神经母细胞瘤干细胞基因表达谱变化：应用基因表达谱芯片检测BE(2)-C细胞和BE(2)-C-shRNA细胞基因表达谱情况,筛选差异基因,并采用Real-time PCR检测验证相关差异表达的基因。
结果
1.Sox2在神经母细胞瘤中的表达：RT-PCR结果显示在神经母细胞瘤组织中可检测到Sox2基因mRNA水平表达。免疫组织化学染色结果显示Sox2主要在肿瘤细胞核中表达,而瘤旁组织中表达呈阴性。Real-time PCR及Western blot结果均显示神经母细胞瘤组织中Sox2的表达水平明显高于瘤旁组织,差异有统计学意义(P<0.05)。经统计分析发现Sox2在高分期肿瘤中的表达水平高于低分期肿瘤(P<0.05),且未经化疗的肿瘤其Sox2的表达水平高于经化疗的肿瘤(P<0.05)。Sox2在神经母细胞瘤中的表达水平与性别、年龄、肿瘤部位、大小、病理分型等参数无相关性(均P>0.05)。
2.Sox2对神经母细胞瘤干细胞生物学特性的影响：RT-PCR结果显示在神经母细胞瘤BE(2)-C细胞系中可检测到Sox2基因mRNA水平表达。利用慢病毒载体感染BE(2)-C细胞成功构建Sox2高表达[BE(2)-C-Sox2]和低表达[BE(2)-C-shRNA]稳定细胞。CCK-8活细胞数检测显示72h时BE(2)-C-Sox2细胞的AV值(1.465±0.046)高于BE(2)-C细胞(1.01±0.018),差异有统计学意义(P<0.05)；而BE(2)-C-shRNA细胞的AV值(0.912±0.020)低于BE(2)-C细胞(1.201±0.018),差异有统计学意义(P<0.05)。流式细胞仪细胞周期分析显示BE(2)-C-Sox2组S期细胞百分比(55.2±4.3%)高于BE(2)-C组细胞(38.6±3.5%),BE(2)-C-shRNA组S期细胞百分比(21.8±5.7%)低于BE(2)-C组细胞,三组组间比较差异有统计学意义(P<0.05),而三组细胞G0/G1期细胞百分比的比较情况呈现出相反的结果；BE(2)-C-Sox2、BE(2)-C及BE(2)-C-shRNA组的细胞增殖指数分别为65.2±5.6%、36.3±2.8%、51.7±4.6%,三组组间比较差异有统计学意义(P<0.05)。RA或BrdU干预后Western blot检测细胞标志蛋白(Peripherin、NF-68、Vimentin、S-100)显示,经RA或BrdU干预的BE(2)-C-Sox2细胞其标志蛋白表达量低于经同种药物干预的BE(2)-C细胞和空载体细胞(P<0.05)；而经RA或BrdU干预的BE(2)-C-shRNA细胞其标志蛋白表达量高于经同种药物干预的BE(2)-C细胞和空载体细胞(P<0.05)。平板克隆形成实验显示BE(2)-C-Sox2细胞的克隆形成数目(103±18)和克隆形成率(20.6%)均高于BE(2)-C细胞(75±12,15%),而BE(2)-C-shRNA细胞的克隆形成数目(42±7)和克隆形成率(8.4%)均低于BE(2)-C细胞,三组组间比较差异有统计学意义(P<0.05)。软琼脂克隆形成实验未成功,细胞均死亡。裸鼠成瘤实验显示BE(2)-C-Sox2接种组肿瘤的生长速度和体积均大于BE(2)-C接种组,而BE(2)-C-shRNA接种组肿瘤的生长速度和体积均低于BE(2)-C接种组,三组组间比较差异有统计学意义(P<0.05)。
3.基因芯片检测Sox2下调时神经母细胞瘤干细胞基因表达谱变化：Sox2基因表达下调时基因表达谱芯片筛得差异表达的基因有596条,其中差异显著的基因有FMN1、GFRA2、HIST1H2BK、CARTPT、AHNAK2、PLP2、TSPAN8、TIMP2、EPO和PRR11等。经Real-time PCR验证,证实芯片筛选的结果可靠。
结论
1.Sox2在神经母细胞瘤(组织和细胞)中存在表达,其表达水平与肿瘤的临床分期有一定相关性,与性别、年龄等参数无明显相关性。化疗药物对Sox2的表达有抑制作用。
2.Sox2促进神经母细胞瘤干细胞的增殖、克隆形成和体内成瘤等生物学特性,但对其分化有一定的抑制作用,提示Sox2在神经母细胞瘤的发生发展过程中具有重要作用。
3.Sox2表达下调时基因表达谱芯片筛选得相关差异表达的基因有：FMN1、GFRA2.HIST1H2BK、CARTPT、AHNAK2、PLP2、TSPAN8、TIMP2、EPO和PRR11等。芯片结果尚有待于进一步分析和研究。
Backgrounds and objective
Neuroblastoma (NB) is the most common extracranial solid pediatric tumor and accounts for10%of childhood cancers. Although major advances have been made in surgery and chemotherapy of NB, the morbidity and mortality remain high. So far, the molecular mechanisms responsible for the pathogenesis of NB remain elusive, thus researches on the genisis and progress of NB are of great importance. NB is an embryonic tumor, and the inhibition of embryonal neural crest cell differentiation is the main reason of the genisis of NB and major risk factor of its manignancy and prognosis. Sox2is a transcription factor expressed in both embryonic and adult stem cells. It plays a pivotal role in the maintenance of self-renewal and differentiation potential. In this study, we explore the expression of Sox2in human NB and the relationship between the expression levels of Sox2and various clinicopathological parameters. Meanwhile, the potential functions of Sox2on the genesis and progress of NB are also explored in this study.
Materials and Methods
1. The research of Sox2expression in human NB:RT-PCR, Real-time PCR, Western blot analysis and immunohistochemical staining were employed to detect the expression of Sox2in human NB, and the relationship between Sox2expression and clinical data was assessed.
2. The effects of Sox2on the biological characteristics of neuroblastoma stem cell:RT-PCR was performed to detect the expression of Sox2in human neuroblastoma BE(2)-C cell. Sox2-overexpressed and mRNA knockdown stem cell lines were established by infecting BE(2)-C cell via lentivirus vectors. Cell counting kit-8(CCK-8) assay and flow cytometry were applied to test the effect of Sox2on the proliferative ability. Western blot analysis was used to detect the marker proteins after drug interference in order to analyze the effect of Sox2on differentiation properties. Plate colony-forming assay and tumorigenicity assay were performed to analyze the effect of Sox2on the malignant potential.
3. Gene chip detect the gene expression profiling changes in down-regulating Sox2neuroblastoma stem cell:The gene expression profiles of BE(2)-C-shRNA and BE(2)-C cells were investigated with Genechip Human Gene1.OST. Differentially expressed genes were analyzed by data analyses. Real-time PCR was performed to verify the gene chip results
Results
1. The research of Sox2expression in human NB:Sox2mRNA was detected in NB tissues by RT-PCR. The immunohistochemical staining showed that Sox2was primarily localized in the nuclei of NB cells, not observed in paracancerous cells. Using Real-time PCR, we found that Sox2mRNA relative expression levels was significantly higher in tumor tissues than in the adjacent non-cancerous tissues (P<0.05), and the relative expression levels in stage Ⅲ and Ⅳ NB were higher compared with those in stage Ⅰ and Ⅱ NB (P<0.05). Sox2expression was significantly decreased in the chemotherapy subgroup as compared with that of the non-chemotherapy subgroup in stage Ⅲ and Ⅳ tumors (P<0.05). Western blot analysis confirmed the results at the protein level.
2. The effects of Sox2on the biological characteristics of neuroblastoma stem cell:Sox2mRNA was detected in human neuroblastoma BE(2)-C cell by RT-PCR. Sox2-overexpressed[BE(2)-C-Sox2] and mRNA knockdown[BE(2)-C-shRNA] stem cell lines were successfully established by infecting BE(2)-C cell via lentivirus vectors. BE(2)-C-Sox2cell showed relatively higher cell proliferative number than BE(2)-C cell (P<0.05) in CCK-8assay. The percentage of cells in S phase and PLI index in BE(2)-C-Sox2group was higher than BE(2)-C group (P<0.05) in flow cytometry assay. BE(2)-C-Sox2cell showed higher colony-forming number and efficiency (P<0.05), speed of tumorigenesis and volumes of tumors in vivo than BE(2)-C and BE(2)-C-shRNA cells (P<0.05), and exhibited decreased expression levels of other type cell marker proteins than BE(2)-C and BE(2)-C-shRNA cells (P<0.05). Conversely, down-regulation of Sox2showed an inverse result.
3. Gene chip detect the gene expression profiling changes in down-regulating Sox2neuroblastoma stem cell:596differentially expressed genes were screened by Genechip Human Gene1.0ST. The significantly differentially expressed genes were FMN1, GFRA2, HIST1H2BK, CARTPT, AHNAK2, PLP2, TSPAN8, TIMP2, EPO, PRR11and etc. Real-time PCR verified the gene chip results.
Conclusions
1. Sox2is expressed in human neuroblastoma and neuroblastoma stem cell, and its expression correlates to the clinical stage of NB, but not other clinicopathological parameters including patient gender and age, tumor size, location and histological classification. Moreover, Sox2expression can be inhibited by chemotherapy.
2. Sox2plays a key role in the abilities of proliferation, differentiation, colony-forming and tumorigenesis of neuroblastoma.
3. Differentially expressed genes via gene chip in down-regulating Sox2neuroblastoma stem cell are FMN1, GFRA2, HIST1H2BK, CARTPT, AHNAK2, PLP2, TSPAN8, TIMP2, EPO, PRR11and etc. Further analyses and research are needed to explore the data.
引文
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