CD99+/mCD99L2-调控H/RS细胞与B淋巴瘤细胞转化蛋白质组学研究及相关通路分析
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摘要
研究背景和目的
霍奇金淋巴瘤(Hodgkin lymphoma, HL)是恶性淋巴瘤的一大类型,具有特征性的肿瘤细胞H/RS(Hodgkin/Reed-Sternberg)细胞,但H/RS细胞一般仅占肿瘤组织的极少部分(<1%),其余是大量以T淋巴细胞为主的炎性反应背景细胞,且经常发生变异,造成诊断上的困难。要把握H/RS的本质,就必须清楚H/RS细胞是如何发生、发展,与背景细胞之间究竟存在怎样复杂的相互关系,而要从根本上探究这些问题,迫切需要类似人HL病理特征的实验动物模型。目前虽然存在诸多人源性H/RS细胞株,但成瘤实验罕见报道,因为研究显示运用人H/RS细胞株时往往需要免疫严重缺陷的实验鼠如SCIDBNX或NOG鼠才能成瘤,但是越是免疫缺陷则越不能反映该肿瘤具有丰富免疫背景及炎症特性。因此构建鼠源性的H/RS细胞来构建鼠源性的动物模型成为研究的一个长期设想。
Kuppers等研究者发现H/RS细胞来源于生发中心的残疾B细胞,认为生发中心B细胞在成熟过程中,一部分细胞经历了有利的突变选择,被T辅助及滤泡树突状细胞所选择,经过反复的增生、突变和选择,阳性选择细胞分化为浆细胞及记忆B细胞；而另一部分GCB细胞经过不利的突变,如反义突变、自身反应性的获得等成为功能上残缺的所谓前凋亡细胞,并经历了程序性细胞死亡
一些残缺的前凋亡细胞丢失了被抗原选择的能力,从而没有经历正常的凋亡过程,成为H/RS前体细胞。H/RS细胞在克隆过程中其B细胞表面标志部分或全部消失,出现CD15、CD30特征性抗原标记物,成为H/RS细胞的生物学特性之一。由于H/RS细胞免疫表型及受体的改变,使其逃避免疫监视和细胞凋亡而得以生存及克隆性增殖。人CD99是一个糖基化跨膜蛋白,研究发现CD99的下调与人H/RS细胞的形成有关。本研究组前期通过脂质体转染CD99基因表达阴性的HL细胞株L428,筛选稳定表达CD99的L428-CD99细胞亚系,初步证实L428-CD99细胞亚系重现部分B细胞特征；同时发现鼠源性CD99(mouse CD99antigen-like2, mCD99L2)和CD99高度同源,通过慢病毒ShRNA质粒LV-mCD99L2转染内源性(?)CD99L2基因表达阳性的B淋巴瘤细胞株A20,筛选稳定表达干扰质粒的LV-mCD99L2-A20细胞亚系,初步证实有些LV-mCD99L2-A20细胞和人H/RS细胞有相似特征。本研究拟在构建稳定表达人CD99基因的L428亚系和稳定干扰鼠CD99基因的鼠B淋巴瘤A20细胞亚系的基础上,利用荧光差异凝胶双向电泳和质谱分析技术分别比较两种处理前后的细胞株蛋白表达差异,分离与鉴定与人、鼠CD99相互作用的靶蛋白,通过生物信息学研究分析方法得到的人、鼠两组数据,筛选出在细胞转化中起重要作用的蛋白,分析验证其功能及可能涉及的信号通路,为进一步阐明InCD99L2相互作用蛋白在构建H/RS细胞模型和类人HL动物模型中的作用和意义提供理论依据。
研究内容与方法
一、L428-CD99和LV-mCD99L2-A20细胞亚系的鉴定及分析
1.L428-CD99细胞亚系的鉴定
对前期构建的稳定过表达CD99基因的L428-CD99克隆株反复传代培养,利用RT-PCR、实时荧光定量RT-PCR、Western blot检测(?)CD99mRNA和蛋白表达；通过光镜观察、细胞计数和鬼笔环肽染色检测CD99基因过表达对L428细胞形态大小以及细胞骨架蛋白的影响；利用MTT实验检测CD99基因过表达对L428细胞增殖能力的影响；利用免疫细胞化学和流式细胞检测相关抗原标记分析CD99基因上调对L428细胞分化相关蛋白的影响。
2. LV-mCD99L2-A20细胞亚系的鉴定
对前期筛选出的稳定干扰1(?)CD99L2基因表达的LV-mCD99L2-A20细胞株反复传代培养,利用载体上通用引物进行PCR检测干扰载体整合至LV-mCD99L2-A20细胞情况；利用RT-PCR检测目的基因片段mCD99L2的mRNA表达,通过实时荧光定量RT-PCR检测mCD99L2基因的干扰效率；镜下观察观察mCD99L2干扰对A20细胞形态大小的影响,利用MTT实验检测]nCD99L2干扰对A20细胞增殖能力的影响。
二、CD99+/mCD99L2-调控H/RS细胞与B淋巴瘤细胞转化的蛋白质组学分析
运用荧光差异双向凝胶电泳技术分别对人鼠两组转化细胞(人源性L428-CD99细胞／空载体转染的L428-CTR细胞和鼠源性LV-mCD99L2-A20和空载体转染的LV-Gus-A20细胞)进行蛋白分离得到差异蛋白凝胶电泳图,结合软件分析和手工筛选,与制备胶匹配后,挖取了差异蛋白质点,采用灵敏且高通量的肽质量指纹图谱蛋白质鉴定方法鉴定差异蛋白,进一步采用GOfact开放软件进行在线聚类分析(http://www.hupo.org.cn/gofact),对所得差异蛋白所参与的生物过程、所参与构成的细胞组件、所介导的分子功能进行注释,分析和细胞转化相关的蛋白。
三、CD99+/mCD99L2-调控H/RS细胞与B淋巴瘤细胞转化的差异蛋白筛选及初步验证
1.筛选差异蛋白并分析
通过生物信息学检索预测CD99和]mCD99L2相互作用的蛋白,结合双向荧光凝胶电泳和质谱分析得到的人鼠两组差异蛋白,筛选分析人CD99+/鼠mCD99L2-调控H/RS细胞与B淋巴瘤细胞转化的差异蛋白。
2.筛选蛋白在人细胞、组织上的表达验证
利用实时荧光定量RT-PCR、免疫细胞化学、Western blot和免疫荧光共聚焦检测筛选差异蛋白在人H/RS细胞转化细胞(L428-CD99和L428-CTR)和病理组织中的mRNA和蛋白表达。
3.筛选蛋白在鼠源性细胞上的表达验证
利用免疫细胞化学、Western blot检测筛选差异蛋白在LV-mCD99L2-A20和LV-Gus-A20细胞中表达。
四、差异蛋白Septin-2在L428细胞和B淋巴瘤细胞初步功能验证及相关信号通路分析
1、Septin-2蛋白与H/RS细胞和B淋巴细胞调控因子的关系
信息学分析、western blot检测验证Septin-2与调控因子nf-kappaB和c-Myb的关系。
2、Septin-2在H/RS细胞和B淋巴瘤细胞转化中初步功能验证
瞬时干扰L428细胞中Septin-2表达,通过镜下观察、荧光共聚焦检测、CCK8实验和流式细胞检测Septin-2基因干扰对L428细胞形态、细胞骨架蛋白、细胞增殖能力和免疫表型的影响。
3、Septin-2在H/RS细胞和B淋巴瘤细胞转化中可能参与的信号通路分析
采用Western blot检测H/RS细胞L428稳定过表达CD99基因前后及瞬时干扰Septin-2基因前后相关通路蛋白的表达；结合蛋白组学分析结果,通过生物信息学和文献资料分析Septin-2在CD99调控H/RS细胞向B淋巴瘤细胞转化中可能参与的信号调控通路。
结果
一、L428-CD99和LV-mCD99L2-A20细胞亚系的鉴定及分析
1.L428-CD99细胞亚系的鉴定
(1) RT-PCR,实时荧光定量RT-PCR和Western blot检测显示,与空载体组比较,L428-CD99细胞亚系CD99基因和蛋白表达均增高。
(2)培养细胞镜下形态观察、HE染色后计数,L428-CD99细胞亚系细胞体积变小,差异具有显著性(t=7.131,P=0.018)；鬼笔环肽染色发现,与对照组相比,L428-CD99细胞骨架发生重建。
(3)MTT检测发现,CD99基因过表达组较空载体组增殖减慢,差异具有显著性(F=773.374,P=0.000)；
(4)免疫细胞化学和流式细胞检测发现,CD99基因过表达后,与对照组相比,CD30、CD15和MUM1表达降低,CD10、CD19、CD79α、BCL-6、PAX5和CD38表达增高,而CD20和CD138的表达没有明显改变。
2. LV-mCD99L2-A20细胞亚系的鉴定
(1)PCR检测干扰载体稳定整合于LV-mCD99L2-A20细胞基因组,RT-PCR和实时荧光定量RT-PCR检测LV-mCD99L2-A20细胞中目的基因片段mCD99L2的nRNA表达稳定降低,mCD99L2基因的干扰效率约51%。
(2)传代培养细胞于倒置显微镜下和HE染色发现,LV-mCD99L2-A20细胞细胞体积明显增大,出现类人H/RS细胞的巨大细胞；MTT实验检测mCD99L2干扰使A20细胞增殖能力下降,差异具有显著性(F=77.452,P=0.000)。
二、CD99+/mCD99L2-调控H/RS细胞与B淋巴瘤细胞转化的蛋白质组学分析
1.人源性转化细胞组荧光差异双向凝胶电泳和质谱分析
人源性转化细胞组通过荧光差异双向凝胶电泳和质谱分析得到38个差异蛋白,与L428-CD99细胞而言正相关的蛋白21个,负相关的蛋白17个。其中RhoGDP-dissociation inhibitor2(GDIR2)和Septin-2(SEPT2)参与细胞骨架；DNAmismatch repair protein Msh2(MSH2), Hematopoietic lineage cell-specific protein(HS1)参与细胞分化；Rho GDP-dissociation inhibitor2(GDIR2)、Prostaglandin E synthase3(TEBP)、Prohibitin(PHB)、Hematopoietic lineage cell-specific protein(HS1)、Sorcin (SORCN)和GEM-interacting protein (GMIP)参与信号通路；Prohibitin (PHB)、Heat shock protein beta-1(HSPB1)和Hematopoietic lineage cell-specific protein (HS1)参与调节基因表达。
2.鼠源性转化细胞组荧光差异双向凝胶电泳和质谱分析
鼠源性转化细胞组通过荧光差异双向凝胶电泳和质谱分析得到41个差异蛋白,其中与LV-mCD99L2-A20细胞而言正相关的蛋白21个,负相关的蛋白20个。其中参与细胞骨架的蛋白有Actin, cytoplasmic1(ACTB)、Septin-2(SEPT2)、 PDZ and LIM domain protein1(PDLI1), Stathmin (STMN1);参与细胞分化的蛋白有stathmin (STMN1)、ADP-ribosylation factor-like protein6(ARL6);参与信号通路的蛋白有Ran GTPase-activating protein1(RAGP1)、 ADP-ribosylation factor-like protein6(ARL6);参与调节基因表达的蛋白有Eukaryotic translation initiation factor4E (IF4E)、Nucleophosmin (NPM)、60kDa heat shock protein, mitochondrial (CH60)、PDZ and LIM domain protein1(PDLI1)、Hypermethylated in cancer2protein (HIC2)> Poly(U)-binding-splicing factor PUF60(GCP60)等。
三、CD99+/mCD99L2-调控H/RS细胞与B淋巴瘤细胞转化差异蛋白筛选及验证
1.通过生物信息学检索和人鼠两组转化细胞差异蛋白的分析比较,选取骨架蛋白Septin-2和Stathmin作为验证的目标蛋白。
2.实时荧光定量RT-PCR.免疫细胞和细胞化学、Western blot(?)和荧光共聚焦检测显示Septin-2和Stathmin的nRNA水平、蛋白水平在人转化细胞组L428和L428-CD99中存在差异表达,蛋白水平在组织标本中也存在差异,Septin-2和CD99表达负相关,Stathmin(?)和CD99表达正相关。
3.免疫细胞化学、Western blot检测显示Septin-2和Stathmin在鼠源性转化细胞A20和LV-mCD99L2-A20中存在差异表达,与人源性转化细胞上表达一致。
四、差异蛋白Septin-2在L428细胞和B淋巴瘤细胞初步功能验证及相关信号通路分析
1. Septin-2上游调控因子NF-kappaB1、c-Myb和XBP-1也是促进H/RS细胞形成和B细胞分化的调控因子。Western blot检测显示NF-kappaB1和c-Myb参与H/RS细胞和B淋巴细胞的转化,Septin-2的表达受nf-kappaB、c-Myb调控,与nf-kappaB表达呈正相关,与c-Myb表达呈负相关；nf-kappaB和c-Myb不受Septin-2的调控。
2.倒置显微镜下连续观察和荧光共聚焦检测显示L428瞬时干扰SEPT2后细胞骨架发生重建,细胞伪足状突起消失。
3.CCK8试验显示与空载体组L428-cn细胞相比,L428-sisept2细胞株生长较缓慢,差异具有显著性(F=204.927,P=0.000)。
4.流式细胞检测结果显示L428细胞瞬时干扰Septin-2后细胞的部分抗原标记出现了小幅改变,H/RS细胞特征性抗原标记CD30和CD15表达下降,B细胞抗原标记CD19表达上升,生发中心标记CD10和早期浆细胞标记CD38表达也有上升。
5. Western blot检测发现,干扰L428中Septin-2和过表达L428中CD99表达,RhoA蛋白表达都明显降低；结合RhoGDI2、HS1、Stathmin等差异蛋白和信息学检索,提出CD99调控H/RS细胞和B淋巴瘤细胞转化过程中,可能通过上调RhoGDI2来抑制Rho Family GTPases信号通路中的Rho GTP酶,主要是抑制了RhoA活性,从而抑制细胞骨架GTP结合蛋白Septin-2的表达,促使骨架重建并抑制了细胞的增殖活性；同时Septin-2也能通过调节RhoA蛋白参与B细胞受体通路,和HS1共同平衡转化细胞的骨架重组,参与调节B细胞转录因子和B细胞抗原的形成分化。另一骨架蛋白Stathmin可能起到协同改变细胞骨架和形态作用。
结论
1.前期构建L428-CD99和LV-mCD99L2-A20细胞亚系反复传代培养并验证CD99+/mCD99L2-调控了H/RS细胞与B淋巴瘤细胞的转化；
2.人H/RS细胞株L428过表达CD99后得到38个差异蛋白,21个表达上调,17个表达下调；鼠源性B淋巴瘤细胞株A20敲低mCD99L2表达后得到41个差异蛋白,21个表达上调,20个表达下调；部分蛋白参与细胞分化、信号通路、骨架改变和基因表达调节等功能,和细胞转化密切相关；
3.骨架蛋白Septin-2和Stathmin在人鼠两组转化细胞中皆存在差异表达,Septin-2与CD99表达负相关,Stathmin与CD99表达正相关；
4.初步验证了Septin-2在人H/RS细胞转化中通过骨架重建参与细胞形态改变,降低细胞增殖活性,参与H/RS细胞向B淋巴瘤细胞转化过程中的抗原分化；分析其可能主要通过Rho Family GTPases信号通路和B细胞受体的信号通路发挥作用。
创新之处
1.本研究通过蛋白质组学技术筛选CD99+/mCD99L2-调控了H/RS细胞与B淋巴瘤细胞转化的差异蛋白,分析出一批和转化密切相关的蛋白；
2.初步验证Septin-2的功能并分析相关作用信号通路,初步提出CD99调控H/RS细胞向B淋巴瘤细胞转化的分子机制。
Backgroud
Hodgkin's lymphoma (HL) is one category of malignant lymphomas, with characteristic Hodgkin/Reed-Sternberg (H/RS) tumorous cells. But the proportion of H/RS cells is of little and less than1%of the whole bulk of the tumor, with the remanent predominant T cell background of reactive inflammation. The frequent variation of the tumors further strengthen the diagnostic dilemma. To master a series problems including the intrinsic qulity of the H/RS cells, the mechanism of the origination and development of H/RS cell, the complicated relationship between H/RS cells and the background must be elucidated. To explore these promlems from the root, a suitable animal model with similar characteristic pathologic feature of HL is required. Though a large quantity of human originated H/RS exist, their tumorigenesis of H/RS cell in animal is rarely reported. Research indicates that successful tumorigenesis of H/RS cell in animals restricted to the severe immunodeficient mice like SCID, BNX, NOG. But the more severe the immunodeficiency of the animal, the less the tumorigenesis can reveal the character of the tumor with a background of abundant immunity and inflammation. It is our a long-term vision to built animal model with H/RS cells of mouse.
Kiippers et al. revealed that H/RS cell originated from crippled B cells in the germinal center. The research speculated that some cells underwent a favored mutation selection, and accepted by T helper cells and follicular dendritic cells. With a repeating process of proliferation, mutation and selection, the positively selected cells differentiated to plasma cells and memory B cells. The other group, however underwent an unfavorable mutation, like negative mutation, automatic reactive acquisition, and became the so called functionally crippled pro-apoptotic cells, and underwent a programmatic cell death. Some crippled pro-apoptotic cells lost their superficial markers partly or completely, and presented the characteristic antigen marker CD15and CD30, as one of the biological character of H/RS cell. Because of the change of immune phenotype and receptor of H/RS cell, the cell escaped the immune surveillance and cell apoptosis, survived and proliferated in clone.
The human CD99is a glycosylated transmembrane protein. Research showed that the down-regulation of CD99was related to the forming of human H/RS. Our research group has previously transfected the CD99gene into the CD99negative HL cell line L428by lipidosome transfection, and screened out the CD99stably expressing cell suline L428-CD99. The preliminary studies indicated that L428-CD99cell subline reappeared with some of the B cell features. We further found that the mouse originated CD99(mouse CD99antigen-like2, mCD99L2) was highly homologous with that of human. Letivirus ShRNA vector was applied the transfect the endogenous mCD99L2gene positive B lymphoma cell line A20, and screened out the interfering vector stably expressing cell subline LV-mCD99L2-A20, and preliminarily proved that some of the LV-mCD99L2-A20cells owned similar character with that of human H/RS cells.
Our research group have previously constructed subseries of L428cell line with stable overexpression of CD99gene and A20cell line with low mCD99L2gene. On this basis,2D-DIGE and Mass Spectrometry will be applied to compare the protein expression difference between the two cells lines before and after their treatment. The target protein that interact with human and mouse CD99will be isolated and indentified. From the data obtained from the bioinformatics research and analysis of both the group of human and mouse, we expect to screen out the important proteins that functioning during the cell transformation, analyze and prove their functions and the possible related signal transduction pathway. Our study may provide the role and a theoretical basis for interacting protein with mCD99L2in building H/RS cell model and HL animal model.
Objective
Contents and methods
1. Identification of L428-CD99cell subline and LV-mCD99L2-A20cell subline.
(1) Identification of L428-CD99cell subline
The previously constructed L428-CD99clone cell line with stable expressiom of CD99gene was contiously subcultured, and the mRNA and protein expression of CD99was measured by real-time PCR, Western blot and confocal microscope.The effect of CD99low expression on proliferation, celluar size and cytoskeletal proteins was detected by MTT, HE staining and phalloidin staining.The diognosis markers were detected by immunocytochemistry and flow cytometry.
(2) Identification of LV-mCD99L2-A20cell subline
The previously screened out LV-mCD99L2-A20cell line with stably interfering expressiom of mCD99L2was contiously subcultured. The general primers for amplification of the vector gene were applied for PCR detection of the situation of the interfering vector that integrating into LV-mCD99L2-A20cells. The mRNA and protein expression of mCD99L2was measured by real-time PCR, Western blot.The effect of mCD99L2overexpression on morphological changes and proliferation was detected by HE staining and MTT.
2.Proteomic analysis of CD99+/mCD99L2-regulating the transformation between H/RS and B lymphoma cells
Differential proteome analysis of upregulating human CD99or downregulating mouse CD99Like2gene during translation between Hodgkin/Reed-Sternberg and B lymphocyte cell was conducted using two-dimensional differfntial in gel electrophoresis combined with matrix-assisted laser desorption/time of fight (MALDITOF)mass spectrometry. Further, cluster analysis was carried out using the ways included the software Gofact (http://www.hupo.org.cn/gofact), annotation of differentially expressed proteins with the proceeds of the involved biological processes, composition of cell components and molecular function.
3. The screening and primary identification of differentially expressed proteins in CD99+/mCD99L2-regulating the transformation between H/RS and B lymphoma cells
(1) Screening and analysis of differently expressed proteins
Bioinformatics network was employed to predict interactive proteins with CD99and mCD99L2before2-D DIGE and mass spectral analysis to screen the differentially expressed proteins in human CD99+/mice mCD99L2-regulating the transformation between H/RS and B cell lymphoma cells
(2) Expression verification of targeted proteins in human cells and tissues
mRNA and expression of targeted proteins in L428-CD99and L428-CTR were tested by RT-PCR, immunocytochemistry, Western blot and Confocal Immunofluorescence Microscopy
(3) Expression verification of targeted proteins in mice cells
Expression of targeted Proteins in LV-mCD99L2-A20with stable RNA interference of mCD99L2and LV-Gus-A20cells transfected with empty vector were examined by immunocytochemistry and Western blot.
4. Primary biofunction verification and signal transduction analysis of differently expressed Septin-2in L428cells and B lymphoma cells
(1) The interaction between Septin-2and regulating factors of H/RS cells and B lymphocytes
The interaction between Septin-2and nf-kappaB and c-Myb was examined by Bioinformatics and Western Blot
(2) The biofunction verification of Septin-2in H/RS-B cell lymphoma cells transformation
Microscopy and Fluorescent Phalloidin Staining of Septin-2interfered L428cells were performed to test the effect of Septin-2RNA interference on cellular morphology and cytoskeletal protein. CCK-8experiment and flow cytometry was conducted to test the effect of Septin-2RNA interference on cell proliferation and immunophenotype of L428cells.
(3) The analysis of the possible role of Septin-2in signal transduction of H/RS-B cell lymphoma cells transformation
Signal cascase proteins were tested by Western blot before and after stable RNA interference of CD99and transient interference of Septin-2. Combined with bioinformatics, proteomics and literature data, the role of Septin-2in the signal transduction pathway of CD99regulating H/RS to transform into B cell lymphoma was analyzed.
Results
1. Identification and analysis of cell fraction of L428-CD99and LV-mCD99L2 -A20cells
(1) Iidentification of cell fraction of L428-CD991) RT-PCR, Real-Time Reverse Transcription PCR and Western Blot showed the increase CD99gene and its expression in L428-CD99compared to empty vector
2) Significant difference was found (t=7.131, P=0.018) in cell volume of L428-CD99microscopically, phalloidin staining revealed cytoskeleton remodelling in L428-CD99compared with control group.
3) MTT Assay showed cells with CD99over expression has a decreased cell proliferation(F=773.374, P=0.000),compared with empty vector
4) Flow cytometry (FCM) and immunohistochemistry (IHC) showed that in comparison with control group, cells with CD99over expression significantly reduced the expression of CD30, CD15and MUM1, promoted the expression of CD10、CD19、CD79α、BCL-6、PAX5and CD38, but had no impact on CD20and CD138.
(2) Iidentification of cell fraction of LV-mCD99L2-A20
1) PCR confirmed the stable intergration of interference vector into LV-mCD99L2-A20genomem, RT-PCR and Real-Time RT-PCR showed the mRNA copy numbers of mCD99L2in LV-mCD99L2-A20is reduced with an efficient rate of51%.
2) Reverse microscope and HE staining demonstrated that the cell volume of the subcultureed LV-mCD99L2-A20increased significantly, presented with giant cells the resembling human H/RS cells. MTT assays revealed RNA interence of mCD99L2significantly supressed the proliferation of A20.
2.Proteomics of CD99+/mCD99L2-regulating the transformation between H/RS and B lymphoma cells.
(1)2-D DIGE and mass spectral analysis of human tumor derived cell lines harvested38differently expressed proteins,21of which was positivly correlated with L428-CD99and17had negative correlation. Rho GDP-dissociation inhibitor2(GDIR2) and Septin-2(SEPT2) are related to cytoskeleton organization; DNA mismatch repair protein Msh2(MSH2), Hematopoietic lineage cell-specific protein are related to cell differentiation; Rho GDP-dissociation inhibitor2(GDIR2)、 Prostaglandin E synthase3(TEBP)、Prohibitin (PHB)、Hematopoietic lineage cell-specific protein (HS1)、Sorcin (SORCN) and GEM-interacting protein (GMIP) are related to signal transduction; Prohibitin(PHB)、Heat shock protein beta-1(HSPB1)和Hematopoietic lineage cell-specific protein (HS1) are related to regulation of gene expression
(2)2-D DIGE and mass spectral analysis of cell lines derived from murine tumor targeted42proteins,21and20of them were positively and negatively correlated with LV-mCD99L2-A20, respectively. Actin, cytoplasmic1(ACTB)、Septin-2(SEPT2)、 PDZ and LIM domain protein1(PDLI1)、Stathmin (STMN1) are related to cytoskeleton organization; stathmin(STMN1) and ADP-ribosylation factor-like protein6(ARL6)are related to cell differentiation; Ran GTPase-activating protein1(RAGP1)、 ADP-ribosylation factor-like protein6(ARL6) are related to signal transduction; Eukaryotic translation initiation factor4E (IF4E)、Nucleophosmin (NPM)、60kDa heat shock protein, mitochondrial (CH60)、PDZ and LIM domain protein1(PDLI1)、 Hypermethylated in cancer2protein (HIC2)、Poly(U)-binding-splicing factor PUF60(GCP60) are related to regulation of gene expression.
3. Screening and identification of the differential expressing proteins in the process of CD99+/mCD99L2regulating the transformation between H/RS and B lymphoma cells
(1) Bioinformatics retrieving and differential protomics analysis of humanity and mouse transforming cell lines pinpointed Septin-2and Stathmin as the targeted protein for further investigation.
(2) Real time RT-PCR, IHC, ICC, WB and Confocol showed significant difference between L428and L428-CD99in terms of mRNA and proteins expression of Septin-2and Stathmin. Septin-2negatively correlated with CD99expression while Stathmin positively correlated with CD99expression.
(3) ICC and WB detection indicated that differential expression of Septin-2and Stathmin existed between mouse originated transforming cell A20andLV-mCD99L2-A20, while they were consistent with that of humanity transformed cells.
4. Biofunction verification and signal transduction pathways analysis of Septin-2in L428and B lymphoma cells
(1) The Septin-2upstream regulating factors NF-kappaB1, c-Myb and XBP-1were also the regulating factors in promoting H/RS formation and B lymphocyte differentiation. WB showed that NF-kappaB1and c-Myb were involved in H/RS-B cell lymphoma cells transformation. The expression of Septin-2was regulated by nf-kappaB and c-Myb. Septin-2expression was positively correlated with that of nf-kappa B and negatively correlated with that of c-Myb, while the expression nf-kappa and c-Myb were not modulated by Septin-2.
(2) Consecutive observation with inverse microscope and confocol assay showed that L428with transcient RNA interference of SEPT2presented with remodeling of cell structure and vanishing of pseudo foot process.
(3) CCK8experiment revealed the reducing of cell proliferation of L428-sisept2compared with that of L428-cn (F=204.927, P=0.000).
(4) FCM showed partial changes of antigen phenotype in L428after RNAi of Septin-2. The expression of characteristic markers CD30and CD15reduced while the B cell marker CD19increased. The expression of germinal center marker CD10and early stage marker of plasma cell CD38also increased.
(5) WB tests showed RNAi of SEPT2and over-expression of CD99in L428was related to the reduction of RhoA expression. Combined with of differential protein expression analysis and bioinformatic retrieving of RhoGDI2, HS1and Stathmin, we suggested that CD99regulating H/RS cell and B lymphoma cell transformation by up-regulating RhoGDI2for the inhibition of Rho GTPases in the Rho Family GTPases signal transtdution pathway. It mainly inhibited the activity of RhoA as the way to inhibit the expression of cell structure GTP binding protein Septin-2, promote the remodeling of cell structure and inhibit cell proliferation. Meanwhile, Septin-2also participated in the B cell receptor pathway by regulating RhoA protein, remodeling the transformed cell structure through co-balancing with HS1, participated in regulating B cell transcript factor and B cell antigen differentiation with the help of Stathmin..
Conclusion
1. The formerly constructed cell subline L428-CD99and LV-mCD99L2-A20are confirmed by consecutive subculture and assay that CD99+/mCD99L2regulates the transformation between H/RS cell and B cell lymphoma.
2.Among the38targeted proteins identified in CD99over-expressed L428,21are up-regulated and17down-regulated. Among the41proteins selected from mCD99L2dow-regulated A20,22are upregulated and20are down-regulated. Some of targeted proteins are involved in the process of cell differentiation, signal transduction and gene transcription regulation.
3. Septin-2and Stathmin are differently expressed in both human and mice derived cells. Septin-2negatively correlated with CD99while Stathmin positively correlated with CD99.
4. Primary conclusion can be drawn that Septin-2is involved in cellular morphological change by participating structure remodeling in H/RS cells transformation, reducing cell proliferation and plays an important role in antigen differentiation process in H/RS-B lymphocyte cells transformation by Rho Family GTPases signal cascade and B cell receptor signal pathway.
Discoveries and innovations
1. Based on previously established cell subline with the character that CD99+/mCD99L2-regulates H/RS-B lymphocyte cells transformation with cell models of L428-CD99and LV-mCD99L2-A20, this study further investigate the role of different expression of Septin-2and Stathmin in transfoming cells. Protomics detection reveals the related proteins in cell differentiation, signal transduction,gene transcription regulation and cell transformation.
2. This study provided Preliminary verification that in the process CD99+/mCD99L2-regulating the transformation between H/RS and B lymphoma cells, Septin-2may suppress cell proliferation by regulating cell structure remodeling and morphological change, as well as participation in antigen differentiation. While CD99regulates H/RS to lymphocyte transformation, Septin-2participates in ho Family GTPases and BCR signal transduction pathways.
霍奇金淋巴瘤(Hodgkin lymphoma, HL)是恶性淋巴瘤的一大类型,具有特征性的肿瘤细胞H/RS(Hodgkin/Reed-Sternberg)细胞,但H/RS细胞一般仅占肿瘤组织的极少部分(<1%),其余是大量以T淋巴细胞为主的炎性反应背景细胞,且经常发生变异,造成诊断上的困难。要把握H/RS的本质,就必须清楚H/RS细胞是如何发生、发展,与背景细胞之间究竟存在怎样复杂的相互关系,而要从根本上探究这些问题,迫切需要类似人HL病理特征的实验动物模型。目前虽然存在诸多人源性H/RS细胞株,但成瘤实验罕见报道,因为研究显示运用人H/RS细胞株时往往需要免疫严重缺陷的实验鼠如SCIDBNX或NOG鼠才能成瘤,但是越是免疫缺陷则越不能反映该肿瘤具有丰富免疫背景及炎症特性。因此构建鼠源性的H/RS细胞来构建鼠源性的动物模型成为研究的一个长期设想。
Kuppers等研究者发现H/RS细胞来源于生发中心的残疾B细胞,认为生发中心B细胞在成熟过程中,一部分细胞经历了有利的突变选择,被T辅助及滤泡树突状细胞所选择,经过反复的增生、突变和选择,阳性选择细胞分化为浆细胞及记忆B细胞；而另一部分GCB细胞经过不利的突变,如反义突变、自身反应性的获得等成为功能上残缺的所谓前凋亡细胞,并经历了程序性细胞死亡
一些残缺的前凋亡细胞丢失了被抗原选择的能力,从而没有经历正常的凋亡过程,成为H/RS前体细胞。H/RS细胞在克隆过程中其B细胞表面标志部分或全部消失,出现CD15、CD30特征性抗原标记物,成为H/RS细胞的生物学特性之一。由于H/RS细胞免疫表型及受体的改变,使其逃避免疫监视和细胞凋亡而得以生存及克隆性增殖。人CD99是一个糖基化跨膜蛋白,研究发现CD99的下调与人H/RS细胞的形成有关。本研究组前期通过脂质体转染CD99基因表达阴性的HL细胞株L428,筛选稳定表达CD99的L428-CD99细胞亚系,初步证实L428-CD99细胞亚系重现部分B细胞特征；同时发现鼠源性CD99(mouse CD99antigen-like2, mCD99L2)和CD99高度同源,通过慢病毒ShRNA质粒LV-mCD99L2转染内源性(?)CD99L2基因表达阳性的B淋巴瘤细胞株A20,筛选稳定表达干扰质粒的LV-mCD99L2-A20细胞亚系,初步证实有些LV-mCD99L2-A20细胞和人H/RS细胞有相似特征。本研究拟在构建稳定表达人CD99基因的L428亚系和稳定干扰鼠CD99基因的鼠B淋巴瘤A20细胞亚系的基础上,利用荧光差异凝胶双向电泳和质谱分析技术分别比较两种处理前后的细胞株蛋白表达差异,分离与鉴定与人、鼠CD99相互作用的靶蛋白,通过生物信息学研究分析方法得到的人、鼠两组数据,筛选出在细胞转化中起重要作用的蛋白,分析验证其功能及可能涉及的信号通路,为进一步阐明InCD99L2相互作用蛋白在构建H/RS细胞模型和类人HL动物模型中的作用和意义提供理论依据。
研究内容与方法
一、L428-CD99和LV-mCD99L2-A20细胞亚系的鉴定及分析
1.L428-CD99细胞亚系的鉴定
对前期构建的稳定过表达CD99基因的L428-CD99克隆株反复传代培养,利用RT-PCR、实时荧光定量RT-PCR、Western blot检测(?)CD99mRNA和蛋白表达；通过光镜观察、细胞计数和鬼笔环肽染色检测CD99基因过表达对L428细胞形态大小以及细胞骨架蛋白的影响；利用MTT实验检测CD99基因过表达对L428细胞增殖能力的影响；利用免疫细胞化学和流式细胞检测相关抗原标记分析CD99基因上调对L428细胞分化相关蛋白的影响。
2. LV-mCD99L2-A20细胞亚系的鉴定
对前期筛选出的稳定干扰1(?)CD99L2基因表达的LV-mCD99L2-A20细胞株反复传代培养,利用载体上通用引物进行PCR检测干扰载体整合至LV-mCD99L2-A20细胞情况；利用RT-PCR检测目的基因片段mCD99L2的mRNA表达,通过实时荧光定量RT-PCR检测mCD99L2基因的干扰效率；镜下观察观察mCD99L2干扰对A20细胞形态大小的影响,利用MTT实验检测]nCD99L2干扰对A20细胞增殖能力的影响。
二、CD99+/mCD99L2-调控H/RS细胞与B淋巴瘤细胞转化的蛋白质组学分析
运用荧光差异双向凝胶电泳技术分别对人鼠两组转化细胞(人源性L428-CD99细胞／空载体转染的L428-CTR细胞和鼠源性LV-mCD99L2-A20和空载体转染的LV-Gus-A20细胞)进行蛋白分离得到差异蛋白凝胶电泳图,结合软件分析和手工筛选,与制备胶匹配后,挖取了差异蛋白质点,采用灵敏且高通量的肽质量指纹图谱蛋白质鉴定方法鉴定差异蛋白,进一步采用GOfact开放软件进行在线聚类分析(http://www.hupo.org.cn/gofact),对所得差异蛋白所参与的生物过程、所参与构成的细胞组件、所介导的分子功能进行注释,分析和细胞转化相关的蛋白。
三、CD99+/mCD99L2-调控H/RS细胞与B淋巴瘤细胞转化的差异蛋白筛选及初步验证
1.筛选差异蛋白并分析
通过生物信息学检索预测CD99和]mCD99L2相互作用的蛋白,结合双向荧光凝胶电泳和质谱分析得到的人鼠两组差异蛋白,筛选分析人CD99+/鼠mCD99L2-调控H/RS细胞与B淋巴瘤细胞转化的差异蛋白。
2.筛选蛋白在人细胞、组织上的表达验证
利用实时荧光定量RT-PCR、免疫细胞化学、Western blot和免疫荧光共聚焦检测筛选差异蛋白在人H/RS细胞转化细胞(L428-CD99和L428-CTR)和病理组织中的mRNA和蛋白表达。
3.筛选蛋白在鼠源性细胞上的表达验证
利用免疫细胞化学、Western blot检测筛选差异蛋白在LV-mCD99L2-A20和LV-Gus-A20细胞中表达。
四、差异蛋白Septin-2在L428细胞和B淋巴瘤细胞初步功能验证及相关信号通路分析
1、Septin-2蛋白与H/RS细胞和B淋巴细胞调控因子的关系
信息学分析、western blot检测验证Septin-2与调控因子nf-kappaB和c-Myb的关系。
2、Septin-2在H/RS细胞和B淋巴瘤细胞转化中初步功能验证
瞬时干扰L428细胞中Septin-2表达,通过镜下观察、荧光共聚焦检测、CCK8实验和流式细胞检测Septin-2基因干扰对L428细胞形态、细胞骨架蛋白、细胞增殖能力和免疫表型的影响。
3、Septin-2在H/RS细胞和B淋巴瘤细胞转化中可能参与的信号通路分析
采用Western blot检测H/RS细胞L428稳定过表达CD99基因前后及瞬时干扰Septin-2基因前后相关通路蛋白的表达；结合蛋白组学分析结果,通过生物信息学和文献资料分析Septin-2在CD99调控H/RS细胞向B淋巴瘤细胞转化中可能参与的信号调控通路。
结果
一、L428-CD99和LV-mCD99L2-A20细胞亚系的鉴定及分析
1.L428-CD99细胞亚系的鉴定
(1) RT-PCR,实时荧光定量RT-PCR和Western blot检测显示,与空载体组比较,L428-CD99细胞亚系CD99基因和蛋白表达均增高。
(2)培养细胞镜下形态观察、HE染色后计数,L428-CD99细胞亚系细胞体积变小,差异具有显著性(t=7.131,P=0.018)；鬼笔环肽染色发现,与对照组相比,L428-CD99细胞骨架发生重建。
(3)MTT检测发现,CD99基因过表达组较空载体组增殖减慢,差异具有显著性(F=773.374,P=0.000)；
(4)免疫细胞化学和流式细胞检测发现,CD99基因过表达后,与对照组相比,CD30、CD15和MUM1表达降低,CD10、CD19、CD79α、BCL-6、PAX5和CD38表达增高,而CD20和CD138的表达没有明显改变。
2. LV-mCD99L2-A20细胞亚系的鉴定
(1)PCR检测干扰载体稳定整合于LV-mCD99L2-A20细胞基因组,RT-PCR和实时荧光定量RT-PCR检测LV-mCD99L2-A20细胞中目的基因片段mCD99L2的nRNA表达稳定降低,mCD99L2基因的干扰效率约51%。
(2)传代培养细胞于倒置显微镜下和HE染色发现,LV-mCD99L2-A20细胞细胞体积明显增大,出现类人H/RS细胞的巨大细胞；MTT实验检测mCD99L2干扰使A20细胞增殖能力下降,差异具有显著性(F=77.452,P=0.000)。
二、CD99+/mCD99L2-调控H/RS细胞与B淋巴瘤细胞转化的蛋白质组学分析
1.人源性转化细胞组荧光差异双向凝胶电泳和质谱分析
人源性转化细胞组通过荧光差异双向凝胶电泳和质谱分析得到38个差异蛋白,与L428-CD99细胞而言正相关的蛋白21个,负相关的蛋白17个。其中RhoGDP-dissociation inhibitor2(GDIR2)和Septin-2(SEPT2)参与细胞骨架；DNAmismatch repair protein Msh2(MSH2), Hematopoietic lineage cell-specific protein(HS1)参与细胞分化；Rho GDP-dissociation inhibitor2(GDIR2)、Prostaglandin E synthase3(TEBP)、Prohibitin(PHB)、Hematopoietic lineage cell-specific protein(HS1)、Sorcin (SORCN)和GEM-interacting protein (GMIP)参与信号通路；Prohibitin (PHB)、Heat shock protein beta-1(HSPB1)和Hematopoietic lineage cell-specific protein (HS1)参与调节基因表达。
2.鼠源性转化细胞组荧光差异双向凝胶电泳和质谱分析
鼠源性转化细胞组通过荧光差异双向凝胶电泳和质谱分析得到41个差异蛋白,其中与LV-mCD99L2-A20细胞而言正相关的蛋白21个,负相关的蛋白20个。其中参与细胞骨架的蛋白有Actin, cytoplasmic1(ACTB)、Septin-2(SEPT2)、 PDZ and LIM domain protein1(PDLI1), Stathmin (STMN1);参与细胞分化的蛋白有stathmin (STMN1)、ADP-ribosylation factor-like protein6(ARL6);参与信号通路的蛋白有Ran GTPase-activating protein1(RAGP1)、 ADP-ribosylation factor-like protein6(ARL6);参与调节基因表达的蛋白有Eukaryotic translation initiation factor4E (IF4E)、Nucleophosmin (NPM)、60kDa heat shock protein, mitochondrial (CH60)、PDZ and LIM domain protein1(PDLI1)、Hypermethylated in cancer2protein (HIC2)> Poly(U)-binding-splicing factor PUF60(GCP60)等。
三、CD99+/mCD99L2-调控H/RS细胞与B淋巴瘤细胞转化差异蛋白筛选及验证
1.通过生物信息学检索和人鼠两组转化细胞差异蛋白的分析比较,选取骨架蛋白Septin-2和Stathmin作为验证的目标蛋白。
2.实时荧光定量RT-PCR.免疫细胞和细胞化学、Western blot(?)和荧光共聚焦检测显示Septin-2和Stathmin的nRNA水平、蛋白水平在人转化细胞组L428和L428-CD99中存在差异表达,蛋白水平在组织标本中也存在差异,Septin-2和CD99表达负相关,Stathmin(?)和CD99表达正相关。
3.免疫细胞化学、Western blot检测显示Septin-2和Stathmin在鼠源性转化细胞A20和LV-mCD99L2-A20中存在差异表达,与人源性转化细胞上表达一致。
四、差异蛋白Septin-2在L428细胞和B淋巴瘤细胞初步功能验证及相关信号通路分析
1. Septin-2上游调控因子NF-kappaB1、c-Myb和XBP-1也是促进H/RS细胞形成和B细胞分化的调控因子。Western blot检测显示NF-kappaB1和c-Myb参与H/RS细胞和B淋巴细胞的转化,Septin-2的表达受nf-kappaB、c-Myb调控,与nf-kappaB表达呈正相关,与c-Myb表达呈负相关；nf-kappaB和c-Myb不受Septin-2的调控。
2.倒置显微镜下连续观察和荧光共聚焦检测显示L428瞬时干扰SEPT2后细胞骨架发生重建,细胞伪足状突起消失。
3.CCK8试验显示与空载体组L428-cn细胞相比,L428-sisept2细胞株生长较缓慢,差异具有显著性(F=204.927,P=0.000)。
4.流式细胞检测结果显示L428细胞瞬时干扰Septin-2后细胞的部分抗原标记出现了小幅改变,H/RS细胞特征性抗原标记CD30和CD15表达下降,B细胞抗原标记CD19表达上升,生发中心标记CD10和早期浆细胞标记CD38表达也有上升。
5. Western blot检测发现,干扰L428中Septin-2和过表达L428中CD99表达,RhoA蛋白表达都明显降低；结合RhoGDI2、HS1、Stathmin等差异蛋白和信息学检索,提出CD99调控H/RS细胞和B淋巴瘤细胞转化过程中,可能通过上调RhoGDI2来抑制Rho Family GTPases信号通路中的Rho GTP酶,主要是抑制了RhoA活性,从而抑制细胞骨架GTP结合蛋白Septin-2的表达,促使骨架重建并抑制了细胞的增殖活性；同时Septin-2也能通过调节RhoA蛋白参与B细胞受体通路,和HS1共同平衡转化细胞的骨架重组,参与调节B细胞转录因子和B细胞抗原的形成分化。另一骨架蛋白Stathmin可能起到协同改变细胞骨架和形态作用。
结论
1.前期构建L428-CD99和LV-mCD99L2-A20细胞亚系反复传代培养并验证CD99+/mCD99L2-调控了H/RS细胞与B淋巴瘤细胞的转化；
2.人H/RS细胞株L428过表达CD99后得到38个差异蛋白,21个表达上调,17个表达下调；鼠源性B淋巴瘤细胞株A20敲低mCD99L2表达后得到41个差异蛋白,21个表达上调,20个表达下调；部分蛋白参与细胞分化、信号通路、骨架改变和基因表达调节等功能,和细胞转化密切相关；
3.骨架蛋白Septin-2和Stathmin在人鼠两组转化细胞中皆存在差异表达,Septin-2与CD99表达负相关,Stathmin与CD99表达正相关；
4.初步验证了Septin-2在人H/RS细胞转化中通过骨架重建参与细胞形态改变,降低细胞增殖活性,参与H/RS细胞向B淋巴瘤细胞转化过程中的抗原分化；分析其可能主要通过Rho Family GTPases信号通路和B细胞受体的信号通路发挥作用。
创新之处
1.本研究通过蛋白质组学技术筛选CD99+/mCD99L2-调控了H/RS细胞与B淋巴瘤细胞转化的差异蛋白,分析出一批和转化密切相关的蛋白；
2.初步验证Septin-2的功能并分析相关作用信号通路,初步提出CD99调控H/RS细胞向B淋巴瘤细胞转化的分子机制。
Backgroud
Hodgkin's lymphoma (HL) is one category of malignant lymphomas, with characteristic Hodgkin/Reed-Sternberg (H/RS) tumorous cells. But the proportion of H/RS cells is of little and less than1%of the whole bulk of the tumor, with the remanent predominant T cell background of reactive inflammation. The frequent variation of the tumors further strengthen the diagnostic dilemma. To master a series problems including the intrinsic qulity of the H/RS cells, the mechanism of the origination and development of H/RS cell, the complicated relationship between H/RS cells and the background must be elucidated. To explore these promlems from the root, a suitable animal model with similar characteristic pathologic feature of HL is required. Though a large quantity of human originated H/RS exist, their tumorigenesis of H/RS cell in animal is rarely reported. Research indicates that successful tumorigenesis of H/RS cell in animals restricted to the severe immunodeficient mice like SCID, BNX, NOG. But the more severe the immunodeficiency of the animal, the less the tumorigenesis can reveal the character of the tumor with a background of abundant immunity and inflammation. It is our a long-term vision to built animal model with H/RS cells of mouse.
Kiippers et al. revealed that H/RS cell originated from crippled B cells in the germinal center. The research speculated that some cells underwent a favored mutation selection, and accepted by T helper cells and follicular dendritic cells. With a repeating process of proliferation, mutation and selection, the positively selected cells differentiated to plasma cells and memory B cells. The other group, however underwent an unfavorable mutation, like negative mutation, automatic reactive acquisition, and became the so called functionally crippled pro-apoptotic cells, and underwent a programmatic cell death. Some crippled pro-apoptotic cells lost their superficial markers partly or completely, and presented the characteristic antigen marker CD15and CD30, as one of the biological character of H/RS cell. Because of the change of immune phenotype and receptor of H/RS cell, the cell escaped the immune surveillance and cell apoptosis, survived and proliferated in clone.
The human CD99is a glycosylated transmembrane protein. Research showed that the down-regulation of CD99was related to the forming of human H/RS. Our research group has previously transfected the CD99gene into the CD99negative HL cell line L428by lipidosome transfection, and screened out the CD99stably expressing cell suline L428-CD99. The preliminary studies indicated that L428-CD99cell subline reappeared with some of the B cell features. We further found that the mouse originated CD99(mouse CD99antigen-like2, mCD99L2) was highly homologous with that of human. Letivirus ShRNA vector was applied the transfect the endogenous mCD99L2gene positive B lymphoma cell line A20, and screened out the interfering vector stably expressing cell subline LV-mCD99L2-A20, and preliminarily proved that some of the LV-mCD99L2-A20cells owned similar character with that of human H/RS cells.
Our research group have previously constructed subseries of L428cell line with stable overexpression of CD99gene and A20cell line with low mCD99L2gene. On this basis,2D-DIGE and Mass Spectrometry will be applied to compare the protein expression difference between the two cells lines before and after their treatment. The target protein that interact with human and mouse CD99will be isolated and indentified. From the data obtained from the bioinformatics research and analysis of both the group of human and mouse, we expect to screen out the important proteins that functioning during the cell transformation, analyze and prove their functions and the possible related signal transduction pathway. Our study may provide the role and a theoretical basis for interacting protein with mCD99L2in building H/RS cell model and HL animal model.
Objective
Contents and methods
1. Identification of L428-CD99cell subline and LV-mCD99L2-A20cell subline.
(1) Identification of L428-CD99cell subline
The previously constructed L428-CD99clone cell line with stable expressiom of CD99gene was contiously subcultured, and the mRNA and protein expression of CD99was measured by real-time PCR, Western blot and confocal microscope.The effect of CD99low expression on proliferation, celluar size and cytoskeletal proteins was detected by MTT, HE staining and phalloidin staining.The diognosis markers were detected by immunocytochemistry and flow cytometry.
(2) Identification of LV-mCD99L2-A20cell subline
The previously screened out LV-mCD99L2-A20cell line with stably interfering expressiom of mCD99L2was contiously subcultured. The general primers for amplification of the vector gene were applied for PCR detection of the situation of the interfering vector that integrating into LV-mCD99L2-A20cells. The mRNA and protein expression of mCD99L2was measured by real-time PCR, Western blot.The effect of mCD99L2overexpression on morphological changes and proliferation was detected by HE staining and MTT.
2.Proteomic analysis of CD99+/mCD99L2-regulating the transformation between H/RS and B lymphoma cells
Differential proteome analysis of upregulating human CD99or downregulating mouse CD99Like2gene during translation between Hodgkin/Reed-Sternberg and B lymphocyte cell was conducted using two-dimensional differfntial in gel electrophoresis combined with matrix-assisted laser desorption/time of fight (MALDITOF)mass spectrometry. Further, cluster analysis was carried out using the ways included the software Gofact (http://www.hupo.org.cn/gofact), annotation of differentially expressed proteins with the proceeds of the involved biological processes, composition of cell components and molecular function.
3. The screening and primary identification of differentially expressed proteins in CD99+/mCD99L2-regulating the transformation between H/RS and B lymphoma cells
(1) Screening and analysis of differently expressed proteins
Bioinformatics network was employed to predict interactive proteins with CD99and mCD99L2before2-D DIGE and mass spectral analysis to screen the differentially expressed proteins in human CD99+/mice mCD99L2-regulating the transformation between H/RS and B cell lymphoma cells
(2) Expression verification of targeted proteins in human cells and tissues
mRNA and expression of targeted proteins in L428-CD99and L428-CTR were tested by RT-PCR, immunocytochemistry, Western blot and Confocal Immunofluorescence Microscopy
(3) Expression verification of targeted proteins in mice cells
Expression of targeted Proteins in LV-mCD99L2-A20with stable RNA interference of mCD99L2and LV-Gus-A20cells transfected with empty vector were examined by immunocytochemistry and Western blot.
4. Primary biofunction verification and signal transduction analysis of differently expressed Septin-2in L428cells and B lymphoma cells
(1) The interaction between Septin-2and regulating factors of H/RS cells and B lymphocytes
The interaction between Septin-2and nf-kappaB and c-Myb was examined by Bioinformatics and Western Blot
(2) The biofunction verification of Septin-2in H/RS-B cell lymphoma cells transformation
Microscopy and Fluorescent Phalloidin Staining of Septin-2interfered L428cells were performed to test the effect of Septin-2RNA interference on cellular morphology and cytoskeletal protein. CCK-8experiment and flow cytometry was conducted to test the effect of Septin-2RNA interference on cell proliferation and immunophenotype of L428cells.
(3) The analysis of the possible role of Septin-2in signal transduction of H/RS-B cell lymphoma cells transformation
Signal cascase proteins were tested by Western blot before and after stable RNA interference of CD99and transient interference of Septin-2. Combined with bioinformatics, proteomics and literature data, the role of Septin-2in the signal transduction pathway of CD99regulating H/RS to transform into B cell lymphoma was analyzed.
Results
1. Identification and analysis of cell fraction of L428-CD99and LV-mCD99L2 -A20cells
(1) Iidentification of cell fraction of L428-CD991) RT-PCR, Real-Time Reverse Transcription PCR and Western Blot showed the increase CD99gene and its expression in L428-CD99compared to empty vector
2) Significant difference was found (t=7.131, P=0.018) in cell volume of L428-CD99microscopically, phalloidin staining revealed cytoskeleton remodelling in L428-CD99compared with control group.
3) MTT Assay showed cells with CD99over expression has a decreased cell proliferation(F=773.374, P=0.000),compared with empty vector
4) Flow cytometry (FCM) and immunohistochemistry (IHC) showed that in comparison with control group, cells with CD99over expression significantly reduced the expression of CD30, CD15and MUM1, promoted the expression of CD10、CD19、CD79α、BCL-6、PAX5and CD38, but had no impact on CD20and CD138.
(2) Iidentification of cell fraction of LV-mCD99L2-A20
1) PCR confirmed the stable intergration of interference vector into LV-mCD99L2-A20genomem, RT-PCR and Real-Time RT-PCR showed the mRNA copy numbers of mCD99L2in LV-mCD99L2-A20is reduced with an efficient rate of51%.
2) Reverse microscope and HE staining demonstrated that the cell volume of the subcultureed LV-mCD99L2-A20increased significantly, presented with giant cells the resembling human H/RS cells. MTT assays revealed RNA interence of mCD99L2significantly supressed the proliferation of A20.
2.Proteomics of CD99+/mCD99L2-regulating the transformation between H/RS and B lymphoma cells.
(1)2-D DIGE and mass spectral analysis of human tumor derived cell lines harvested38differently expressed proteins,21of which was positivly correlated with L428-CD99and17had negative correlation. Rho GDP-dissociation inhibitor2(GDIR2) and Septin-2(SEPT2) are related to cytoskeleton organization; DNA mismatch repair protein Msh2(MSH2), Hematopoietic lineage cell-specific protein are related to cell differentiation; Rho GDP-dissociation inhibitor2(GDIR2)、 Prostaglandin E synthase3(TEBP)、Prohibitin (PHB)、Hematopoietic lineage cell-specific protein (HS1)、Sorcin (SORCN) and GEM-interacting protein (GMIP) are related to signal transduction; Prohibitin(PHB)、Heat shock protein beta-1(HSPB1)和Hematopoietic lineage cell-specific protein (HS1) are related to regulation of gene expression
(2)2-D DIGE and mass spectral analysis of cell lines derived from murine tumor targeted42proteins,21and20of them were positively and negatively correlated with LV-mCD99L2-A20, respectively. Actin, cytoplasmic1(ACTB)、Septin-2(SEPT2)、 PDZ and LIM domain protein1(PDLI1)、Stathmin (STMN1) are related to cytoskeleton organization; stathmin(STMN1) and ADP-ribosylation factor-like protein6(ARL6)are related to cell differentiation; Ran GTPase-activating protein1(RAGP1)、 ADP-ribosylation factor-like protein6(ARL6) are related to signal transduction; Eukaryotic translation initiation factor4E (IF4E)、Nucleophosmin (NPM)、60kDa heat shock protein, mitochondrial (CH60)、PDZ and LIM domain protein1(PDLI1)、 Hypermethylated in cancer2protein (HIC2)、Poly(U)-binding-splicing factor PUF60(GCP60) are related to regulation of gene expression.
3. Screening and identification of the differential expressing proteins in the process of CD99+/mCD99L2regulating the transformation between H/RS and B lymphoma cells
(1) Bioinformatics retrieving and differential protomics analysis of humanity and mouse transforming cell lines pinpointed Septin-2and Stathmin as the targeted protein for further investigation.
(2) Real time RT-PCR, IHC, ICC, WB and Confocol showed significant difference between L428and L428-CD99in terms of mRNA and proteins expression of Septin-2and Stathmin. Septin-2negatively correlated with CD99expression while Stathmin positively correlated with CD99expression.
(3) ICC and WB detection indicated that differential expression of Septin-2and Stathmin existed between mouse originated transforming cell A20andLV-mCD99L2-A20, while they were consistent with that of humanity transformed cells.
4. Biofunction verification and signal transduction pathways analysis of Septin-2in L428and B lymphoma cells
(1) The Septin-2upstream regulating factors NF-kappaB1, c-Myb and XBP-1were also the regulating factors in promoting H/RS formation and B lymphocyte differentiation. WB showed that NF-kappaB1and c-Myb were involved in H/RS-B cell lymphoma cells transformation. The expression of Septin-2was regulated by nf-kappaB and c-Myb. Septin-2expression was positively correlated with that of nf-kappa B and negatively correlated with that of c-Myb, while the expression nf-kappa and c-Myb were not modulated by Septin-2.
(2) Consecutive observation with inverse microscope and confocol assay showed that L428with transcient RNA interference of SEPT2presented with remodeling of cell structure and vanishing of pseudo foot process.
(3) CCK8experiment revealed the reducing of cell proliferation of L428-sisept2compared with that of L428-cn (F=204.927, P=0.000).
(4) FCM showed partial changes of antigen phenotype in L428after RNAi of Septin-2. The expression of characteristic markers CD30and CD15reduced while the B cell marker CD19increased. The expression of germinal center marker CD10and early stage marker of plasma cell CD38also increased.
(5) WB tests showed RNAi of SEPT2and over-expression of CD99in L428was related to the reduction of RhoA expression. Combined with of differential protein expression analysis and bioinformatic retrieving of RhoGDI2, HS1and Stathmin, we suggested that CD99regulating H/RS cell and B lymphoma cell transformation by up-regulating RhoGDI2for the inhibition of Rho GTPases in the Rho Family GTPases signal transtdution pathway. It mainly inhibited the activity of RhoA as the way to inhibit the expression of cell structure GTP binding protein Septin-2, promote the remodeling of cell structure and inhibit cell proliferation. Meanwhile, Septin-2also participated in the B cell receptor pathway by regulating RhoA protein, remodeling the transformed cell structure through co-balancing with HS1, participated in regulating B cell transcript factor and B cell antigen differentiation with the help of Stathmin..
Conclusion
1. The formerly constructed cell subline L428-CD99and LV-mCD99L2-A20are confirmed by consecutive subculture and assay that CD99+/mCD99L2regulates the transformation between H/RS cell and B cell lymphoma.
2.Among the38targeted proteins identified in CD99over-expressed L428,21are up-regulated and17down-regulated. Among the41proteins selected from mCD99L2dow-regulated A20,22are upregulated and20are down-regulated. Some of targeted proteins are involved in the process of cell differentiation, signal transduction and gene transcription regulation.
3. Septin-2and Stathmin are differently expressed in both human and mice derived cells. Septin-2negatively correlated with CD99while Stathmin positively correlated with CD99.
4. Primary conclusion can be drawn that Septin-2is involved in cellular morphological change by participating structure remodeling in H/RS cells transformation, reducing cell proliferation and plays an important role in antigen differentiation process in H/RS-B lymphocyte cells transformation by Rho Family GTPases signal cascade and B cell receptor signal pathway.
Discoveries and innovations
1. Based on previously established cell subline with the character that CD99+/mCD99L2-regulates H/RS-B lymphocyte cells transformation with cell models of L428-CD99and LV-mCD99L2-A20, this study further investigate the role of different expression of Septin-2and Stathmin in transfoming cells. Protomics detection reveals the related proteins in cell differentiation, signal transduction,gene transcription regulation and cell transformation.
2. This study provided Preliminary verification that in the process CD99+/mCD99L2-regulating the transformation between H/RS and B lymphoma cells, Septin-2may suppress cell proliferation by regulating cell structure remodeling and morphological change, as well as participation in antigen differentiation. While CD99regulates H/RS to lymphocyte transformation, Septin-2participates in ho Family GTPases and BCR signal transduction pathways.
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