【作者】 付华；

【导师】 文继舫；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 前言:胃癌是一种常见的恶性肿瘤,据有关文献报道,在我国其死亡率居各种恶性肿瘤之首。胃癌患者的主要死因为肿瘤病灶的侵袭及转移。因此探讨其侵袭转移机制显得尤为重要。在众多的促转移分子中,转化生长因子β(transforming growthfactor beta,TGFβ)是一重要分子,它参与了多种肿瘤的侵袭和转移过程。用基因转染的方法诱导肿瘤细胞中TGF-β1表达可增加肿瘤细胞的转移能力。同时有研究表明,阻止TGF-β信号通路可以抑制胃癌的转移。我们的早期研究发现TGF-β1可以促进胃癌细胞SGC7901和BGC823的浸润和转移。一些临床研究也显示:TGF-β1的表达与淋巴结的转移和预后不良呈正相关。目前虽然已有许多研究证实了TGF-β1在肿瘤侵袭和转移中的作用,但其具体机制尚不明确。研究表明TGF-β促浸润转移作用可由smad依赖通路及smad非依赖通路介导。TGF-β与细胞膜Ⅱ型受体(TβRⅡ)结合后,激活Ⅰ型受体,活化的Ⅰ型受体激活下游分子smad2和smad3,随后smad2/smad3与smad4结合形成smad复合物并转移到核内,调节TGF-β介导的靶基因转录。而在smad非依赖通路中,JNK,Erk和P38通路则被认为是关键性的通路。基于TGF-β在肿瘤发生与转移中的重要性,所以深入探讨TGF-β促转移机制具有非常重要的理论及临床意义。人类fascin 1基因属于fascin家族,其蛋白质编码产物是一种分子量为55 kDa的细胞骨架蛋白,可与F肌动蛋白结合,定位于细胞质张力纤维和细胞膜皱褶(ruffles)边缘与细胞的运动、癌细胞的转移有密切关系的公状伪足、微棘(microspikes)的核心肌动蛋白束中。细胞内fascin 1基因表达上调引起细胞间连接发生解离,同时细胞的运动也相应增加。而将fascin 1基因转染至fascin 1基因低表达的结直肠癌细胞系SW 1222中,结果细胞膜表面突起增多,细胞分化程度降低,细胞运动性增强。Fascin蛋白在胃癌中表达明显增高,它的高表达与胃癌的浸润深度、淋巴结转移、TNM分期和复发密切相关。有研究表明在肺癌中TGF-β1可诱导fascin1基因表达上调,且TGF-β1和fascin1蛋白都与肺癌的侵袭与转移有关,但是目前TGF-β与fascin1在胃癌侵袭与转移中是否存在调控关系尚未见文献报道。RNA干扰(RNA interference,RNAi)是近年来发展起来的一种新技术,由于它能特异而有效地阻断靶基因的表达,目前被广泛应用于各种肿瘤的研究。我们构建了针对Fascin1的短发夹RNA(shortharpin RNA,shRNA)真核表达载体并首次转染人胃癌细胞MKN45,观察抑制fascin1表达前后,TGF-β1作用对胃癌侵袭和转移的影响,并对其机制作初步探讨。目的:研究TGF-β1对fascin1基因表达的影响,以及fascin1在TGF-β促胃癌侵袭与转移中的作用。探讨以fascin1为靶的胃癌基因治疗以及进一步揭示TGF-β1促胃癌侵袭和转移机制提供理论和实验依据。方法:1.通过细菌转化、质粒提取、酶切、凝胶电泳、基因测序鉴定和基因重组技术等方法构建针对Fascin1的RNAi质粒pGen-1-FSCN1和阴性对照质粒pGen-1-con。2.利用脂质体介导转染技术转染人胃癌细胞MKN45,经G418筛选得到稳定抑制fascin1表达的胃癌细胞模型(pGen-1-FSCN1细胞);分别采用逆转录聚合酶链反应(RT-PCR)和Western blotting检测RNAi组(siFascin1)、对照组(CON)及未转染组(NT)三组细胞中Fascin1的表达。3.以10ng/ml的TGFβ1处理siFascin1,CON,NT三组胃癌细胞,观察处理前后各组细胞生物学行为的变化,采用MTT比色法测定细胞的黏附能力,Boyden小室测定法测定转染细胞的迁移能力和侵袭能力;腹腔移植法建立裸鼠胃癌细胞转移模型。4.分别瞬时转染smad2,smad4的siRNA(small interfering RNA),检测smad依赖通路在TGF-β诱导fascin1促胃癌浸润与转移中的作用;应用JNK,Erk和P38通路的特异性抑制剂检测smad非依赖通路在TGF-β诱导fascin1促胃癌浸润与转移中的作用。结果:1.TGF-β1(10ng/ml)在作用于MKN45细胞24小时后,fascin1基因表达约为0小时的2.2倍。2.限制性酶切及基因测序证实成功构建了针对fascin1基因的RNAi载体pGen-1-FSCN1。经G418筛选,RT-PCR及Western blotting鉴定,成功建立了稳定抑制fascin1基因表达的胃癌细胞模型。3.与CON和NT比较,siFascin1细胞的黏附能力、迁移能力、侵袭能力和转移能力明显降低(p＜0.05);三组细胞经TGF-β1(10ng/ml)预处理后,其中CON与NT细胞黏附能力、迁移能力、侵袭能力和转移能力明显增高(p＜0.05);而在siFascin1组,TGF-β1处理前后细胞黏附能力、迁移能力、侵袭能力和转移能力无明显变化(p＞0.05)。4.分别瞬时转染smad2,smad4的siRNA于MKN45细胞中,予TGF-β1处理后,经western blotting检测发现,MKN45中smad2,smad4的表达水平较未转染前减少约70%,而在干扰smad2,smad4后,MKN45细胞中的fascin1表达无明显变化;选用JNK,Erk和P38通路的特异性抑制剂,SP600125,PD98095和SB203580分别预孵育MKN45细胞1h,结果发现:SP600125和PD98095组予TGF-β1处理24小时后,fascin1的表达明显降低(p＜0.05),而P38通路的特异性抑制剂SB203580对fascin1的表达无明显影响。结论:1.首次证实,在胃癌细胞MKN45中fascin1基因表达受外源性TGF-β1(10ng/ml)调控。2.成功构建了针对fascin1基因的RNAi质粒pGen-1-FSCN1,建立了稳定抑制fascin1基因表达的胃癌细胞模型。3.稳定抑制fascin1基因表达后,TGF-β1促胃癌细胞侵袭和转移能力下降。4.TGF-β1主要通过JNK和Erk通路诱导fascin1基因促进胃癌的侵袭和转移。更多还原

【Abstract】 Gastric carcinoma(GC) is a frequent malignant neoplasm.In China, the mortality of patients with GC is quite high.Invasion and metastasis is the major always responsible for the death of patients with GC. Therefore,further investigations on the mechanism of invasion and metastasis are of great importance.Transforming growth factor beta(TGF-β) has been reported to be expressed in many human tumors and is believed to play an important role in tumor invasion as well as tumor metastasis.Cancer cells overexpressing active TGF-β1 by gene transfection showed increased metastatic ability.Targeting of TGF-βsignaling can prevent metastasis in GC.Our previous study showed that TGF-β1 significantly promoted the invasiveness and metastasis of GC cell lines SGC7901 and BGC823. Similarly,clinical studies showed the positive correlation of TGF-β1 expression with lymph node metastasis and poor prognosis in gastric carcinoma.Although many efforts were made to clarify the role of TGF-βin the invasion and metastasis of neoplasms,the mechanism is not fully elucidated.Study indicated that TGFβ1 actives Smad-dependent and Smadindependent pathways.TGFβ1 combines with TGFβtypeⅡreceptor (TβR-Ⅱ) and then actives TβR-Ⅰ.Smad2 and Smad3 are phosphorylated by TβR-Ⅰand then form the heteromeric complexes by associating with Smad4.These Smad complexes move into the nucleus and regulate expression of their target genes.In Smad-independent pathway,JNK,Erk and P38 have been identified as key signaling processes in response to TGF-β.Fascin1,a 55-kDa globular protein,belongs to a unique family of actin-binding proteins.Fascin1 locates in structures containing actin bundles including filopodia,stress fibers,cell membrane ruffles and microspikes.Studies reported cells expressing high levels of fascin showed an increasing activity of membrane extensions,cell motility and loss of cell-cell adhesion.In human colorectal carcinoma cell SW1222, up-regulation of fascin gene expression contributed to the formation of cell-motility structure and the increased migration ability.In normal gastric mucosa,fascin expression is usually undetectable but is often dramatically up-regulated in gastric carcinoma.Moreover increased expression of fascin was found positively correlated with invasion, metastasis and prognosis in GC.Recently,study showed that TGF-β1 up-regulated fascin1 expression in lung cancer.So,we postulate that TGF-β1 can induce the expression of fascin1 and then promote the invasion and metastasis of GC cells.In recent years,RNA interference(RNAi) has emerged as a powerful method of gene therapy,and has been widely used for silencing malignant cellular and viral genes.In this study,we employed RNAi-mediated suppression of Fascin1 gene to evaluate its effects on invasion and metastasis of GC cells and investigate its preliminary mechanisms.ObjectiveOur current investigation attempts to study the effects and mechanisms of TGF-β1-induced fascin1 expression on invasion and metastasis of GC cells,and to provide theoretical and experimental evidences for the gene therapy of GC through specifically turning off Fascin1 gene.MethodsA single strand DNA was synthesized according to the hair loop RNA sequence,and subcloned into eukaryotic expression vector pGenesil-1 to construct a shRNA-expression pDNAs driven by human U6 promoter of fascin1(pGen-1-FSCN1),then transformed into DH5αcompetent cells.One additional construct of random siRNA (pGen-1-con) not homologous to any human genes was made in the same way as control.Positive clones were identified and verified by using restrictive cleavage and sequence.pGen-1-FSCN1 and pGen-1-con were transfected into gastric carcinoma cell line MKN45 with liposome,respectively.Positive colonies were selected with G418.Expression of fascin1 protein and mRNA in the transfected and non-transfected MKN45 cells(NT) was examined by Western blotting and RT-PCR,respectively.Adherent ability of transfected cells was detected by MTT method; The ability of migration and invasion of transfected cells was examined by Bodern method;Nude mice metastasis models were established by abdominal cavity transfer method.To examine the signaling pathway by which the TGF-βincreases the fascin1 expression and then promotes the ability of invasion and migration,Smad2 siRNA and Smad4 siRNA were transiently transfcted into MKN45 cells,and the the expression of fascin1 were measured. Specific protein kinase inhibitors were used to determine that whether the Smad-independent signaling involved in the changes of fascin1 production and promoting the ability of invasion and migration induced by TGF-β.ResultsFascin1 expression levels were found to markedly decrease in MKN45 cells transfected with pGen-1-FSCN1(siFascin1).Transfection of pGen-1-con plasmid(CON) showed no obvious effect on fascin1 expression in MKN45.Down-regulation of fascin1 was accompanied with decreased adhesiveness,invasiveness and migratory abilities.To investigate whether TGF-β1-mediated fascin1 expression is involved in the invasive and metastatic potential of MKN45 cells,NT, CON and siFascin1 cells with or without TGF-β1 treatment were used in our study.Compared with untreated NT and CON cells,the siFascin1 cells showed lower adherent,invasive and metastatic abilities(p＜0.05). TGF-β1 significantly promoted the adhesiveness,invasiveness and migration of NT and CON cells.However,TGF-β1 showed no effect on the adherent,invasive and metastatic abilities in siFascin1 cells.In vivo, the migration rate of the siFascin1 cells was decreased than NT and CON cells(p＜0.05).In vivo,TGF-β1 pretreatment could increase liver metastasis in NT and CON cells.However,TGF-β1 treatment had no effect on the metastatic potential in siFascin1 cells.To address whether smad2 and smad4 were involved in TGF-β1-mediated fascin1 expression,the levels of fascin1 expression in the cells were detected by western blotting after silencing smad2 and smad4.We found both smad2 and smad4 expressions were suppressed successsfuly(p＜0.05),but fascin1 expression was not altered significantly.The specific inhibitors of P38,JNK and Erk,SB203580,SP600125, and PD98059 were used in our study,PD98059 and SP600125 decreased the TGF-β1-induced fascin1 production(by 78%and 75%,respectively), but SB203580 showed no effect on fascin1 expression.Conclusion1.It is the first time to address that TGF-β1 can induce the fascin1 expression in gastric carcinomacells MKN45.2.siRNA plasmid targeted against fascin1,pGen-1-FSCN1,was successfully constructed and transfected into MKN45 cells,and cell model steadily suppressing fascin1 was obtained.3.We for the first time revealed that RNAi plasmid targeted against fascin1 could obviously suppress the invasion and metastasis of GC cells MKN45 mediated by TGF-β1,in vitro and in vivo.4.TGF-β1 promoted the invasion and metastasis of MKN45 cells by increasing fascin1 expression through JNK and ErK signaling mediated.更多还原