【作者】 覃丽；

【导师】 廖端芳；

【作者基本信息】 中南大学 ， 分子药理学， 2009， 博士

【摘要】 第一部分WNT5A/PCK介导上皮间质转化与鼻咽癌转移[研究背景]鼻咽癌（Nasopharyngeal carcinoma,NPC）是我国华南地区及东南亚常见的一种恶性肿瘤。鼻咽癌的治疗主要以放疗为主,对放化疗都比较敏感,早期疗效好,而晚期鼻咽癌的疗效甚差,并且有30%以上的鼻咽癌患者会产生远处转移,所以转移是导致鼻咽癌患者死亡的原因之一。但迄今为止,导致鼻咽癌转移的具体机制尚未明了。因此,探讨鼻咽癌转移的特征性基因及其分子机制,对于找到鼻咽癌转移治疗的新靶点及提高转移患者的生存率具有重大的意义。WNT家族是一类含丝氨酸丰富的分泌性糖蛋白,包括19种成员,主要通过细胞表面受体调节特异性转录因子参与细胞生命调控、迁移以及细胞极化等。WNT信号通路能抑制凋亡,促进肿瘤细胞的增殖,同时WNT家族及其成员也参与了多种肿瘤的发生,包括鼻咽癌。WNT5A被归于WNT家族中无转化能力的成员之一,主要参与了非经典的WNT信号通路。有研究表明WNT5A的表达增加与肿瘤发生发展密切相关。但是WNT5A在鼻咽癌转移中的作用及机制尚不清楚。因此,本研究旨在探讨WNT5A在鼻咽癌侵袭、转移过程中的作用及其相关的分子机制。1)WNT5A是鼻咽癌转移的特征性分子之一前期的研究已经从人鼻咽癌细胞株CNE2中分离出不同转移能力的克隆细胞株Clone-18,Clone-22和Clone-26。其中Clone-18具有显著的侵袭转移能力,而亲代鼻咽癌细胞株CNE2及其克隆Clone-22,Clone-26的侵袭转移能力较弱。对上述细胞我们通过全基因组表达基因的高通量分析,分别筛选出多个在Clone-18上调和下调的基因。对裸鼠移植瘤进行全基因组表达基因的高通量分析,得出类似的结果,其中WNT5A在高转移细胞株Clone-18中的表达明显上调。2)高转移细胞株Clone-18发生EMT现象光镜观察发现人鼻咽癌细胞株CNE2及其低转移能力的克隆Clone-22和Clone-26呈现典型的上皮细胞形状,而高转移细胞克隆Clone-18的形态则类似于成纤维细胞的形态。由于这种细胞形态的改变类似于细胞发生上皮-间质转变时的典型特征,于是检测了相关的分子标志物。在Clone-18中E-cadherin的表达明显降低,而Snail的表达明显增加。3)抑制WNT5A的表达能降低高转移细胞株Clone-18的体外迁移和侵袭能力为了阐明WNT5A对鼻咽癌细胞运动能力的影响,设计了三条针对WNT5A mRNA的干扰序列,并筛选出最有效的一条用于后续的实验。通过划痕实验和侵袭实验,证实了干扰WNT5A能有效降低Clone-18细胞株的迁移和侵袭能力。4)抑制PKC的活性能降低高转移细胞克隆Clone-18的运动能力有研究报道在黑色素瘤细胞中PKC能介导WNT5A通过非经典的WNT/Ca²⁺通路参与细胞的侵袭转移。另外在乳腺癌细胞中PKC的活化能反馈性的上调WNT5A的表达。因此我们研究PKC的抑制剂是否能阻碍高转移细胞克隆Clone-18的运动。GF-10923X是PKC的经典抑制剂,能有效抑制Clone-18中PKC的磷酸化,导致PKC的失活,下调WNT5A的表达。同时影响到EMT标志性分子的改变,即导致Snail表达的下降和增加E-cadherin的表达,最终降低Clone-18的运动能力。采用另一种PKC的抑制剂G（O|¨） 6983能产生类似的效果。因此,PKC参与了鼻咽癌细胞的EMT,调节细胞的运动。5)WNT5A通过PKC途径调节鼻咽癌细胞EMT由于WNT5A能增加PKC的磷酸化,而PKC参与了细胞的运动和上皮间质转化,我们因此进一步探究WNT5A是否能直接诱导细胞的EMT。在WNT5A被干扰之后,Clone-18中磷酸化PKC的表达显著降低,而EMT的标志性分子Snail表达下调,E-cadherin表达增加。而在低转移细胞克隆Clone-22中加入外源性重组WNT5A蛋白后,Snail的表达增加,E-cadherin的表达下调,提示促进了细胞发生EMT改变。6)PKC的活化通过调节Snail和E-cadherin的表达促进低转移细胞克隆的运动为了进一步研究PKC的激活是否能增强低转移鼻咽癌细胞克隆的运动能力,我们采用PKC的激活剂佛波酯PMA来处理细胞。结果显示当采用低浓度的PMA（200nM）处理后,磷酸化PKC的表达在30分钟左右达到顶峰,随后缓慢地衰减,12小时至24小时左右表达最低。有趣的是,WNT5A和Snail的表达则随着时间的延长缓慢增加,12小时至24小时达最高,而E-cadherin的表达却是缓慢地减弱。正如所预测的,当加入PMA或重组WNT5A因子之后,划痕实验显示细胞的运动能力增强,但是这一运动能力可以被PKC抑制剂G（O|¨） 6983阻断。7)WNT5A的表达上调与鼻咽癌转移的临床相关性我们也关心WNT5A促进鼻咽癌细胞在体外的迁移侵袭能力是否具有临床相关性。我们收集了49例临床新鲜活检组织样本,包括20例非肿瘤的鼻咽黏膜组织样本,19例鼻咽原位肿瘤样本,5例鼻咽癌淋巴结转移灶的穿刺活检样本（局部转移）,4例鼻咽癌肝转移灶的穿刺活检样本（远处转移）,进行荧光实时定量PCR检测WNT5A的mRNA表达水平。结果显示WNT5A在鼻咽癌淋巴结转移灶的的mRNA表达水平显著高于鼻咽原位肿瘤样本,在鼻咽癌肝转移样本的表达显著高于淋巴结转移样本。但是WNT5A的mRNA在非肿瘤鼻咽黏膜组织和鼻咽原位肿瘤的表达没有显著差异性。[结论]1)WNT5A通过激活PKC通路,调节Snail和E-cadherin的表达,促进鼻咽癌细胞发生上皮间质转化,从而导致肿瘤的转移;2)PKC的激活剂PMA以及抑制剂GF.10923X和G（O|¨） 6983也能通过调节Snail和E-cadherin的表达,影响细胞EMT和鼻咽癌细胞的迁徙能力;3)WNT5A与PKC之间存在反馈调控作用:第二部分BMI-1对鼻咽癌细胞药敏性的影响[研究背景]鼻咽癌的发生是多因素导致的一种恶性肿瘤,包括遗传易感性、EB（Epstein-Barr）病毒的感染以及其他环境因素等。目前,5-氟脲嘧啶是治疗鼻咽癌的一线化疗药物。然而,有部分鼻咽癌患者在进行5-氟脲嘧啶治疗后出现复发甚至恶化,其主要原因是体内的肿瘤细胞出现了抗药性。因此,如何增加肿瘤细胞对药物的敏感性越来越受到重视。BMI-1（B-cell-specific moloney leukemia virus insert site 1）属于多梳基因家族抑制复合体PRC1（Polycomb Repressive Complexl）的成员,被公认为是一个癌基因。它在多种恶性肿瘤中高表达,其表达与恶性肿瘤的进程相关,并且与患者的生存预后密切相关。研究表明,BMI-1具有诱导细胞永生化,促进肿瘤的早期转化,维持及促进干细胞的自我更新等重要功能。此外,有文献报道,BMI-1在鼻咽癌中高表达,且其表达水平与鼻咽癌原发灶的浸润范围以及患者生存预后密切相关;而BMI-1的表达下调能促进肿瘤细胞的凋亡。因此我们假设BMI-1的下调能通过促进鼻咽癌细胞的凋亡而增加化疗药物的敏感性,从而进一步阐明其分子机制,旨在为解决肿瘤细胞的抗药性提供新的药物靶点。1)BMI-1的干扰能增加鼻咽癌细胞对5-氟脲嘧啶的敏感性在人鼻咽癌细胞株CNE2和HONE1中,通过逆转录病毒感染系统建立BMI-1稳定干扰的细胞株即CNE2-BMI-1/RNAi,CNE2-vector和HONE1-BMI-1/RNAi,HONE1-vector。以上述细胞作为研究模型,加入不同浓度的5-氟脲嘧啶处理72小时,MTT法检测药物的IC50值。结果显示:CNE2-vector,CNE2-BMI-1/RNAi中5-氟脲嘧啶的IC50值分别为9.1504±0.6997mg/L,3.1151±0.8073mg/L（P<0.05）:HONE1-vector,HONE1-BMI-1/RNAi中5-氟脲嘧啶的IC50值分别为3.9023±0.752mg/L,1.5815±0.433mg/L（P<0.05）。2)BMI-1的干扰能增加5-氟脲嘧啶诱导的鼻咽癌细胞的凋亡为了探讨BMI-1干扰后对细胞凋亡的影响,采用5mg/L 5-氟脲嘧啶分别处理CNE2-vector,CNE2-BMI-1/RNAi细胞株72小时,Hoechst 33258染色观察细胞凋亡的形态学改变,流式细胞仪检测细胞凋亡率。结果显示,CNE2-BMI-1/RNAi细胞株出现明显的凋亡形态如细胞染色质浓缩、核固缩,凋亡小体形成等。而CNE2-vector的凋亡形态改变相对不明显。同时CNE2-BMI-1/RNAi的细胞凋亡率为45.4%,而CNE2-vector的细胞凋亡率仅为32.1%（P<0.05）。3)BMI-1干扰后通过P13K/AKT途径增加5-氟脲嘧啶诱导的鼻咽癌细胞的凋亡为了进一步探讨5-氟脲嘧啶诱导鼻咽癌细胞株凋亡的分子机制,我们检测了AKT、磷酸化AKT、BAX和BCL-2的蛋白表达水平。结果显示,当BMI-1稳定干扰后,磷酸化AKT的水平显著降低,BCL-2的蛋白表达明显减少;而BAX的表达上调;加入5-氟脲嘧啶处理后上述变化更明显。随后,采用P13K的抑制剂Wortmanin预处理细胞1小时后,再加入不同浓度的5-氟脲嘧啶分别处理CNE2-vector,CNE2-BMI-1/RNAi细胞,MTT检测细胞的存活率。结果显示,Wortmanin和5.氟脲嘧啶联合处理后,细胞的存活率明显降低,但两株细胞的存活率没有显著的差异,提示BMI-1干扰后可能通过P13K/AKT途径增加5-氟脲嘧啶诱导的鼻咽癌细胞的凋亡。[结论]1)BMI-1干扰能增加肿瘤细胞对化疗药物5.氟脲嘧啶的敏感性,该效应与促进5-氟脲嘧啶诱导的细胞凋亡有关。2)BMI-1干扰能抑制AKT的活性,上调BAX的表达,下调BCL-2的表达。3)BMI-1干扰可能通过P13K/AKT途径增加5-氟脲嘧啶诱导的鼻咽癌细胞的凋亡。更多还原

【Abstract】 PartⅠWNT5A/PKC Induces Epithelial to MesenchymalTransition and Nasopharyngeal Carcinoma Metastasis[BACKGROUD]Nasopharyngeal carcinoma（NPC） is a prevalent malignancy in South China and Southeast Asia.It is sensitive to radiotherapy and/or radiochemotherapy.The therapeutic effect of NPC patients in early stage is good while it is extremely poor in late stage,and more than 30%of NPCs will develop distant metastases.The underlying mechanism（s） of NPC have been poorly explored.Therefore,it is of great importance to investigate specific moleculars and its mechanism for the guidance of new theraputic targets as well as increasing the 5-year survival rate.WNTs belong to a large family of cysteine-rich secreted glycoproteins consisting of at least 19 members in humans.WNT proteins control cell fate,migration and cellular polarity through cell surface receptors that modulate the transcription of specific target genes. WNT signaling can suppress apoptosis and promote invasive growth of cancer cells.The importance of aberrant WNT signaling has been reported in many human cancers,including NPC.WNT5A signaling has been classified as a noncanonical and nontransforming pathway.There is many evidences that increased WNT5A expression is associated with cancer progression.But little is known about the role and mechanism of WNT5A in the metastasis of NPC.This study aims to explore whether and how WNT5A induces invasion and metastasis of NPC.1) WNT5A is one of the most specific gene in NPC metastasisAmong the cellular clones isolated from CNE-2,Clone-18 has the highest metastatic ability;Clone-22,Clone-26,and the parental CNE-2 line possess limited metastatic potential.Through whole-genome expression profiling of the cultured cells in vitro,we identified 25 up-regulated and 24 down-regulated genes in the high-metastasis Clone-18.Using the same methodology,parallel gene sets in Clone-18 were also identified from xenograft tumors.WNT5A was found to be up-regulated in Clone-18 in both the in vitro and in vivo scenarios.2) Characteristics of epithelial-to-mesenehymal transition（EMT） in Clone-18 cellsMorphologically,the parental line CNE2,as well as Clone-22 and Clone-26,displayed a tightly packed cobblestone appearance.Clone-18 cells,however,displayed a fibroblast-like morphology,suggesting an epithelial-to-mesenchymal transition（EMT）.In the Clone-18 cells,the expression of Snail was up-regulated and E-cadherin was down-regulated relative to their expressions in CNE2,Clone-22,and Clone-26 cells, confirming the existence of EMT in the highly metastatic Clone-18 cells.3) Inhibition of Wn5a expression reduced the movement and invasive ability of Clone-18 cellsTo elucidate the role of WNT5A in the motility of NPC cells, three different siRNA sequences targeting WNT5A were designed and tested,siRNA-3 was the most efficient sequence and was chosen for subsequent experiments.Knocking down WNT5A using siRNA-3 dramatically impaired the process of wound healing in Clone-18 cells. Moreover,suppression of WNT5A could significantly reduce the invasive ability of Clone-18 cells（P＜0.05）.These results indicate that WNT5A plays a key role in NPC cell migration and invasion.4) Inhibition of PKC activity decreased the motility of Clone-18 cellsThe role of WNT5A in the invasiveness of melanoma cells depends on the Wnt/Ca²⁺ pathway mediated by PKC.It has been reported that PKC activation can up-regulate WNT5A expression in a positive feedback loop in breast cancer cells.We therefore tested whether inhibition of PKC would impede the motility of Clone-18 cells. GF-10923X,a widely used PKC inhibitor,efficiently inhibited the phosphorylation of PKC in Clone-18 cells,producing an inactivated form of PKC.The inhibition of PKC activity by GF-10923X reduced the expression of WNT5A in the Clone-18 cells.The same concentration of GF-10923X also down-regulated the expression of Snail,which is the most prominent EMT-inducing regulator and E-cadherin-specific transcriptional repressor,resulting in the up-regulation of E-cadherin. Consequently,the motility of Clone-18 cells was reduced after GF-10923X treatment.These results were consistently repeated using another conventional PKC inhibitor,G（o|¨） 6983.Therefore,PKC is involved in the EMT of NPC cells,regulating the motility of the cells.5) WNTSA regulates EMT through PKC activityGiven that WNT5A is able to increase the phosphorylation of PKC and that PKC is involved in cell motility and in EMT,we tested whether WNT5A could directly induce EMT.First,we found that knocking down WNT5A mRNA using siRNA-3 in the highly metastatic Clone-18 cells significantly diminished PKC phosphorylation,and also EMT,as indicated by down-regulation of Snail protein and accumulation of E-cadherin in the cells.Second,treatment with recombinant WNT5A protein in the low-metastatic Clone-22 cells resulted in the overexpression of Snail and the reduction of E-cadherin protein.6) Activation of PKC induces motility in low-metastasis cells through Snail and E-cadherin expressionTo determine if activation of PKC can increase cellular motility in the low-metastasis Clone-22 and Clone-26 cells,which possess low levels of WNT5A protein,the clones were treated with the PKC activator PMA.The level of phospho-PKC peaked 30 min after treatment with a low concentration of PMA（200 nM）,then gradually decreased to very low levels by 12-24h.Interestingly,both WNT5A and Snail protein levels gradually increased and reached their peaks at 12-24 h,whereas E-cadherin gradually decreased during this period.As expected,cellular motility increased in Clone-22 and Clone-26 cells after treatment with PMA or recombinant WNT5A for 12 h as assessed by the wound healing assay.More importantly,the increase of cellular motility induced by recombinant WNT5A could be abolished by pretreatment with the PKC inhibitor G（o|¨） 6983.7) Up-regulation of WNT5A is associated with NPC metastases in clinical scenariosFinally,we sought to test if the critical role of WNT5A in the migration and invasion of NPC cells revealed in the present study is clinically relevant.Quantitative real-time PCR was performed to evaluate the level of WNT5A mRNA in the tissues of 20 noncancerous nasopharyngeal mucosa,19 primary NPCs,5 metastatic NPC in the cervical lymph nodes（regional metastasis）,and 4 metastatic NPC in the liver（distant metastasis）.The expression level（relative to GAPDH） of WNT5A mRNA in lymph node metastatic NPC was significantly higher than that in the primary tumor.Further,the expression was much higher in the distant（liver） metastatic NPCs than in regional metastases.There was no significant difference in WNT5A mRNA levels between the noncancerous NP mucosa and primary NPC tissues. [CONCLUSION]1) WNT5A activates the PKC pathway,regulating the expressions of Snail and E-cadherin and subsequently inducing EMT and metastasis in NPC;2) Changes in PKC activity caused by PMA,GF-10923X,or G（o|¨） 6983 can modulate the expressions of Snail and E-cadherin and subsequently affect EMT and metastasis in NPC;3) A positive feedback loop between WNTSA and phosphorylated PKC exists in NPC metastasis; PartⅡthe Effects of BMI-1 on Chemosensitivity ofNasopharyngeal Carcinoma Cells[BACKGROUD]Nasopharyngeal carcinoma（NPC） is a common malignancy in southern China with uncertain etiologic factors.The development and progression of NPC are believed to result from the interplay of several factors,including genetic susceptibility,Epstein-Barr virus（EBV） infection,and other environmental factors.Currently,the administration of 5-fluorouracil（5-FU）—the most widely used anticancer agent—is one of the standard chemoradio-therapy regimens for NPC.However,a sizable proportion of NPC patients show tumor recurrence after 5-FU treatment that is mainly caused by drug-resistant cancer cells.Therefore, during the past 2 decades,new strategies for enhancing the sensitivity of cancer cells to drug-induced apoptosis for cancer therapy have been intensively explored.The B-cell-specific moloney leukemia virus insert site 1 is one of the components of Polycomb Group protein repression complex 1 （PRC1）.It was originally isolated as an oncogene cooperatingwith c-Myc in lymphomagenesis in a murine model.BMI-1 is closely associated with the development and progression of malignant tumors.It is upregulated in a number of cancers and the expression correlates with the invasion and metastasis phenotype,as well as the prognosis of patients.BMI-1 plays critical roles in immortalization of normal epithelial cells and early transformation of malignants,as well as maintenance of the self-renewal of stem cell.Forthermore,some reports showed that BMI-1 is upregulated in nasopharyngeal carcinoma and its expression level is strongly associated with the invasion phenotype of NPC and poor survival of patients.Recently,it has been reported that the downregulation of BMI-1 can result in the apoptosis of cancer cells. Therefore,we hypothesize that the abrogation of BMI-1 expression may be an effective strategy for sensitizing human cancer cells,including NPC cells,to cancer chemotherapy.1) BMI-1 knock down made the ceils more sensitive to 5-FUTo examine the effect of 5-FU on the survival of BMI-1 knock-down cells,an MTT assay was performed after the CNE2-BMI-1/ RNAi,HONE1-BMI-1/RNAi,CNE2-vector,and HONE1-vector cells were treated with 5-FU for 72h.After treatment with various concentrations of 5-FU,we found that both the BMI-1 shRNA-transfected cells,i.e.,the CNE2-BMI-1/RNAi and HONE1-BMI-1/ RNAi cells,showed lower cell viabilities than the empty vector-transfected cells.The IC50 of 5-FU in the CNE2-vector and CNE2-BMI-1/RNAi cells were 9.1504±0.6997 mg/L and 3.1151±0.8073 mg/L（P＜0.05）,respectively;the IC50 values of 5-FU in the HONE1-vector and HONE1-BMI-1/RNAi cells were 3.9023±0.752 mg/L and 1.5815±0.433 mg/L（P＜0.05）,respectively.These results indicated that BMI-1 knockdown made the cells more sensitive to 5-FU.2) Depletion of BMI-1 enhanced 5-FU-induced apoptosisIn order to evaluate the effect of BMI-1 knockdown on the induction of apoptosis,the CNE2-BMI-1/RNAi and CNE2-vector cells that were treated with 5 mg/mL of 5-FU for 72h were subjected to Hoechst 33258 staining.The cells were examined under a fluorescence microscope.Typical apoptotic morphological changes,such as condensed chromatin,shrunken nuclei,and loss of cell volume,were frequently observed in the CNE2-BMI-1/RNAi cells.Incontrast,only few apoptotic CNE2-vector cells were observed with the same apoptotic morphological changes.In order to further confirm the aforementioned results,we examined the apoptotic rate in the CNE2-BMI-1/RNAi and CNE2-vector cells after treating them with 5 mg/L of 5-FU for 72h.The flow cytometry data showed that the apoptotic rate among the CNE2-BMI-1/RNAi cells was 45.4%as compared to 32.1%among the CNE2-vector cells（P＜0.05）.3) PI3K/AKT pathway was essential for the sensitization effect of BMI-1 to 5-FU treatmentTo further explore the mechanism underlying the enhancement of 5-FU-induced apoptosis by the silencing of BMI-1,we examined the expression levels of total-AKT,phospho-AKT,BAX,and BCL-2 in the CNE2-BMI-1/RNAi and CNE2-vector ceils.Our results showed that the knockdown of endogenous BMI-1 led to substantial reduction in the levels of phospho-AKT,while the total-AKT levels remained uninfluenced.Consistent with this reduction in the phospho-AKT level, Westernblot analysis showed significantly decreased expression of BCL-2 in BMI-1-knocked down cells exposed to 5-FU.The accumulation of BAX in the BMI-1-knocked down ceils was more prominent after the cells were exposed to 5-FU.To investigate whether the depletion of BMI-1 enhances 5-FU-induced apoptosis through the PI3K/AKT pathway,the CNE2-BMI-1/RNAi cells were treated with a PI3K inhibitor.The cells were pretreated with 1μM of the PI3K inhibitor wortmarmin for 1 h and then treated with various concentrations of 5-FU; this was followed by the measurement of cell viability determined by the MTT assay.The inhibition rates of the CNE2-BMI-1/RNAi and CNE2-vector cells by 5-FU treatment were not significantly different after application of the PI3K inhibitor.However,the rate of inhibition of the CNE2-vector cells by 5-FU increased after pretreatment of the cells with wortmannin;this was in contrast to the low rate of inhibition of the CNE2-vector cells by 5-FU without influencing PI3K activity.Taken together,the abrogation of the PI3K/AKT pathway could not further increase the sensitivity of the CNE2-BMI-1/RNAi cells to 5-FU treatment.This suggested that PI3K/AKT pathway was essential for the sensitization effect of BMI-1 to 5-FU treatment.[CONCLUSION]1) Knockdown of BMI-1 makes cancer cells more sensitive to 5-FU as well as enhances 5-FU-induced apoptosis.2) Knockdown of BMI-1 inhibits AKT activation and upregulates the expression level of BAX as well as downregulats expression of BCL-23) Knockdown of BMI-1 enhances apoptosis of NPC cells via AKT/PI3K pathway.更多还原