【作者】 陈琦；

【导师】 高国兰； 罗时文；

【作者基本信息】 南昌大学医学院 ， 外科学， 2013， 博士

【摘要】 背景与目的：卵巢癌是世界范围内最致命的妇科恶性疾病之一,由于缺乏典型的临床症状、特异的体征及肿瘤标志物等有效的筛查手段,绝大多数卵巢癌病人在得到诊断时已处于晚期,使其死亡率高居妇科恶性肿瘤首位。肿瘤的转移能力有赖于细胞的迁移与侵袭,然而决定肿瘤转移能力的分子机制目前仍然不十分清楚。因此,努力探索调控卵巢癌细胞转移潜能的分子靶标将至关重要。Hedgehog (Hh)信号通路在细胞命运决定以及细胞增殖过程中发挥重要作用。该通路由Hh配体,膜受体Ptch和跨膜信号转导蛋白Smo以及参与Hh信号转导的胞浆蛋白复合体组成,其级联传导的末点被公认为锌指转录因子Gli (Glioma-associated oncogene transcription factors),是该信号通路的关键节点,在信号转导中起枢纽作用。Hh靶基因涉及细胞增殖、细胞存活、细胞分化、细胞周期以及干细胞形成和细胞侵袭等多方面。有研究发现：卵巢癌中Hedgehog (Hh)信号通路异常活化,但信号通路异常活化与卵巢癌侵袭、转移的关系有待进一步明晰。本研究试图发现受Hh信号通路调控的靶基因,从临床标本、细胞及分子水平、动物模型等多个层次,采用细胞学、生物化学、分子生物学、生物信息学等多学科手段阐明受Hh信号通路调控的靶基因的作用及分子机制,进一步揭示Hh信号通路与卵巢癌侵袭与转移的关系,为卵巢癌诊疗策略的选择及抗癌新药研发提供理论依据。方法：第一部分人上皮性卵巢癌组织中Hedgehog信号通路异常活化与临床病理特征的关系(1)为了证实在卵巢癌中存在Hh信号通路的异常活化,我们采用免疫组化技术检测了65例卵巢癌病人卵巢肿瘤组织石蜡切片中Hh信号通路重要组分蛋白Gli2的表达情况,并且在此基础上将Gli2的表达情况与卵巢癌的临床病理特征进行了相关性分析。(2)同时,通过Western-blot及real-time PCR等方法检测卵巢癌及正常卵巢组织中Hh信号通路重要组分蛋白Shh、Gli1、Gli2等的蛋白水平及mRNA水平的表达情况。第二部分抑制Hedgehog信号通路降低卵巢癌细胞增殖与迁移能力(1)为了探索Hh信号通路与卵巢癌细胞增殖与迁移能力的关系,我们以卵巢癌细胞系(SKO-V3和ES-2)为研究对象,采用转录因子Gli特异性小分子抑制剂GANT61处理SKO-V3和ES-2细胞,对加药处理(实验组加30μM GANT61,对照组加等体积的DMSO)的SKO-V3和ES-2细胞通过Western-blot、 real-time PCR及免疫细胞化学方法在蛋白水平以及mRNA水平检测了Hh信号通路中相关组分,如跨膜受体Smo,终末转录因子Gli1和Gli2的表达情况。(2)加药处理(实验组加30μM GANT61,对照组加等体积的DMSO)后的SKO-V3和ES-2细胞,分别通过MTT比色法及BrdU增殖实验检测其细胞活力及增殖能力的变化。(3)加药处理(实验组加30μM GANT61,对照组加等体积的DMSO)后的ES-2细胞,通过细胞划痕实验及软琼脂克隆形成实验检测其细胞迁移及克隆形成能力的变化。第三部分抑制Hedgehog信号通路改变卵巢癌细胞基因表达谱(1)为了研究抑制Hh信号通路对卵巢癌细胞基因表达谱的影响,采用转录因子Gli特异性小分子抑制剂GANT61(实验组加30μM GANT61,对照组加等体积的DMSO),处理SKO-V3和ES-2细胞60hr,采用基因表达谱芯片技术分析其受Gli调控的差异表达基因,并对差异表达的基因进行GO (gene ontology) pathway分析和KEGG (Kyoto encyclopedia of genes and genomes) pathway分析,发现受Hh信号通路调控的靶基因。并采用real-time PCR和Western blot分别对芯片发现的富集于某些信号通路的差异基因以及相关基因(如ITGB4、FAK、 CD24、MMP7等)在mRNA水平和蛋白水平进行验证,以确认芯片结果的可靠性。(2)用Gli特异性小分子抑制剂GANT61,转录因子Gli的siRNA(miR-Gli1)和含Hh配体的条件培养液(Shh-Med)分别抑制或激活Hh信号通路,采用real-time PCR、Western blot以及细胞迁移实验研究Hh信号通路调控卵巢癌细胞的侵袭与转移的分子机制。第四部分抑制Hedgehog信号通路减慢卵巢癌生长为了在体内水平研究Hh信号通路对卵巢癌生长的影响,我们采用卵巢癌组织块皮下移植法构建荷人卵巢癌裸鼠皮下移植瘤模型；并利用此动物模型,给予GANT61(浓度10mg/ml,0.2m1/次)和同体积的溶剂对照处理,观测卵巢癌体积的变化,比较瘤体重量的改变；同时采用Western blot技术检测瘤组织中Gli1和Gli2的表达水平的变化,免疫组化技术检测瘤体中整合素integrinβ4(ITGB4)和磷酸化的粘着斑激酶(p-FAK)的表达情况。结果：第一部分人上皮性卵巢癌组织中Hedgehog信号通路异常活化与临床病理特征的关系(1)人卵巢癌组织标本行Western blot检测及real-time PCR检测,发现其Shh, Gli1和Gli2蛋白表达水平明显高于正常卵巢组织；Shh, Gli1和Gli2mRNA水平明显高于正常卵巢组织,差异有极显著统计学意义(p<0.01)提示：卵巢癌组织中Hh信号通路异常活化。(2)人卵巢癌组织标本免疫组织化学染色提示：Gli2的蛋白表达水平与卵巢癌患者的临床病理分期相关,其中晚期(Ⅲ-Ⅳ期)的卵巢癌患者的Gli2蛋白的高评分发生率明显高于早期(Ⅰ-Ⅱ期)的卵巢癌患者,差异有统计学意义(p<0.05)；与卵巢癌患者的年龄、病理组织学类型、分化程度及是否存在淋巴结转移无明显相关,差异无统计学意义(p>0.05)。第二部分抑制Hedgehog信号通路降低卵巢癌细胞增殖与迁移能力(1)Gli特异性小分子抑制剂GANT61降低ES-2和SKO-V3细胞的Gli1及Gli2蛋白表达水平；GANT61降低ES-2和SKO-V3细胞的Gli1和Gli2的mRNA表达水平,差异有极显著统计学意义(p<0.01)；经GANT61处理后细胞免疫荧光显示：Gli1蛋白表达水平显著降低。(2)Gli表达下调可直接抑制卵巢癌细胞活力,(MTT法；p<0.01)；降低其增殖活性,(BrdU法；p<0.01)。(3) GANT61抑制卵巢癌细胞(ES-2)的迁移能力,(划痕实验；p<0.05)；GANT61抑制卵巢癌细胞(ES-2)的克隆形成能力,(软琼脂法；p<0.01)。第三部分抑制Hedgehog信号通路改变卵巢癌细胞基因表达谱(1)基因芯片表达谱及通路网络分析结果显示：SKO-V3细胞经GANT61处理60hr后的差异表达基因的通路网络中,主要影响细胞粘附相关信号通路为主,既有部分表现为上调,同时也有部分表现为下调,其中以整合素蛋白integrins与骨架蛋白lamin为主。同时,MAPK信号通路也有明显的差异基因的富集,而该通路中差异表达基因绝大多数表现为上调,仅少数几个表现为下调。(2)采用real-time PCR和Western blot在mRNA以及蛋白水平验证芯片结果,发现经GANT61处理的SKO-V3细胞,ITGB4, COL1A1, COL5A1等基因mRNA水平表达明显下降,(p<0.01)；而LAMC2, ITGA5, LAMA3等基因mRNA水平表达明显升高,(p<0.01),与Microarray所获结果一致。此外,CD24,CD70, S100A2及MMP7等mRNA表达水平下降,(p<0.01);而S100A4和FAK mRNA水平无显著差异,(p>0.05),与Microarray所获结果基本一致；Western blot结果显示：GANT61处理的SKO-V3细胞,ITGB4, CD24, MMP7等蛋白表达水平明显下降,(p<0.01)；而FAK蛋白表达水平改变不明显,(p>0.05),与real-time PCR结果基本一致。(3)采用Shh配体条件培养液(Shh-Med)激活Hh信号通路,观察上述差异基因的表达变化,结果提示：Shh配体条件培养液(Shh-Med)处理SKO-V3细胞后,ITGB4的蛋白表达水平以及mRNA表达水平明显增加,(p<0.01)。此外,细胞侵袭实验提示：Shh-Med刺激Hh信号通路所引起的SKO-V3细胞侵袭能力的增加可以被抗ITGB4抗体(anti-ITGB4)所阻断(p<0.01)。(4)为了证实GANT61对转录因子Gli作用的特异性,我们采用Gli1特异的siRNA (miR-Gli1-720)干扰Glil的表达,Western blot结果显示：miR-Glil-720转染的SKO-V3细胞中Gli1及ITGB4的蛋白表达水平明显下降,与GANT61的抑制效果一致。(5)有文献报道ITGB4可调控FAK信号,为了阐明Hh信号通路影响ITGB4表达的生物学意义,我们采用Shh-Med和GANT61分别处理SKO-V3细胞,结果提示：Shh-Med处理SKO-V3细胞后,FAK的蛋白表达水平未明显改变,而磷酸化的FAK (p-FAK)水平明显增加,(p<0.01); GANT61处理SKO-V3细胞后,FAK的蛋白表达水平未见明显改变,而p-FAK水平明显下降,(p<0.01),另一方面,Western blot结果显示：Hh信号通路激活所引起的SKO-V3细胞p-FAK水平的增加可以被anti-ITGB4所阻断。提示Hh信号通路激活可通过ITGB4介导促进FAK蛋白磷酸化。(6)此外,我们采用免疫细胞化学方法观察抑制Hh信号通路对FAK磷酸化及细胞骨架的影响,结果显示：经GANT61处的SKO-V3细胞p-FAK-Y397的表达水平明显下降,提示：抑制Hh信号通路可影响FAK的活化；同时,经GANT61处理后,SKO-V3细胞骨架发生破坏,细胞伪足明显减少,提示：抑制Hh信号通路可影响SKO-V3细胞的侵袭能力。第四部分抑制Hedgehog信号通路减慢卵巢癌生长(1)为了在体内水平研究Hh信号通路对卵巢癌细胞生长的影响,我们成功采用瘤组织块皮下移植法构建荷人卵巢癌裸鼠皮下移植瘤模型。(2)实验组(GANT61组)比对照组(溶剂组)裸鼠肿瘤生长速度明显下降,尤其是在给药4次后,对照组肿瘤体积增长迅速,而实验组肿瘤体积缓慢增长。实验结束时将两组裸鼠皮下移植瘤离体后称重,实验组瘤体重量明显低于对照组,差异有极显著统计学意义(p<0.01)。(3) Western blot结果显示：实验组裸鼠皮下移植瘤中Gli1和Gli2蛋白表达水平明显低于对照组裸鼠,相对定量结果提示差异有极显著统计学意义,(p<0.01)；免疫组织化学法检测实验组裸鼠及对照组裸鼠皮下移植瘤中ITGB4及p-FAK的蛋白表达水平,结果显示实验组相比对照组瘤体中ITGB4及p-FAK的蛋白表达水平明显下降。结论：1、卵巢癌组织中Hh信号通路异常活化,Hh信号通路重要组分蛋白Gli2的表达水平与卵巢癌的临床病理分期密切相关。2、GANT61降低卵巢癌细胞(ES-2和SKO-V3)的Gli1和Gli2表达水平,同时,抑制ES-2和SKO-V3细胞的活力、增殖、迁移及克隆形成能力。3、采用Gli特异性小分子抑制剂GANT61处理SKO-V3细胞,通过基因芯片表达谱分析筛选出差异表达基因,并且选择部分与细胞粘附及迁移密切相关的基因进行蛋白水平及mRNA水平的鉴定,鉴定结果与芯片分析结果一致,转录调节因子Gli的下调降低ITGB4的表达,通过抗ITGB4抗体阻断ITGB4/FAK信号途径可以抑制Shh介导的卵巢癌细胞的迁移和侵袭,提示Hh信号通路很可能是通过ITGB4/FAK信号途径来调控卵巢癌的侵袭、转移。4、GANT61缩小荷人卵巢癌裸鼠皮下移植瘤模型中肿瘤的体积及重量,且实验组移植瘤中Gli1、Gli2、ITGB4及p-FAK的蛋白表达水平均下降,提示：Hh信号通路的终末转录因子Gli有望成为卵巢癌诊疗策略的新靶点,为抗癌新药研发提供理论依据。更多还原

【Abstract】 Background and PurposeOvarian cancer is one of the most lethal gynecologic malignancies worldwide. Due to the non-specific symptoms, most of ovarian cancer cases are presented with advanced stage disease and associate with the highest mortality rate.Both cell migration and invasion contribute the metastatic ability of the tumor cells and the genetic mechanism that regulates metastasis remains largely unknown. Therefore, it is of paramount importance to identify molecular mediators conferring the metastatic potential to ovarian cancer cells that may be used as tumor markers in predicting risk of ovarian cancer progression. Hedgehog (Hh) signaling pathway plays an important role in the process of cell fate decisions and cell proliferation. The pathway by Hh ligands, membrane receptors PTCH and transmembrane signal transduction protein Smo and the cytoplasmic protein involved in the Hh signal transduction complex, the last point of the cascade conduction is recognized as the zinc finger transcription factor Gli (glioma-associated oncogene transcription factors Gli), is a key node of the signaling pathway plays a pivotal role in signal transduction. Hh target genes are involved in cell proliferation, survival, differentiation, cell cycle, stem cells and invasion. Several reports indicate that the aberrant activation of Hh signaling is implicated in ovarian cancer, however, the exact role and regulatory mechanisms of Hh signaling for invasion and metastasis in ovarian cancer are not well understood. This project will discover new genes regulated by Hh signaling pathway. Using clinical research, cellular biological method, DNA tip technique, animal experiment, bioinformatics and other regular assays, we will illustrate the role and requlatory mechanisms of the new genes and further reveal the exact influence of Hh signaling pathway on tumorigenesis, invasion and matestasis of ovarian cancer. Our final objective is to offer theoretical guidance of new treatment strategies and drug development targeting to ovarian cancer. MethodsPart Ⅰ. The relation between the aberrant activation of Hh signaling pathway and clinicopathological parameters in ovarian cancer(1) To detect Gli2protein in65ovarian cancer samples by using two steps immunohistochemistry; then observed the relationship between the expression change of the key protein in Hedgehog signaling pathway and the clinicopathological paramenters.(2) Simultaneously, the expression of Shh, Gli1, Gli2(critical components of Hedgehog signaling pathway) mRNA and protein was assayed by real-time PCR and western-blot, respectively.Part Ⅱ. Blocking Hh signaling pathway decreases cell proliferation and migration in ovarian cancer(1) In order to explore the relationship of Hedgehog signaling pathway and cell proliferation and migration in ovarian cancer, Ovarian cancer cell lines (SKO-V3and ES-2) for the object of study, the transcription factor Gli-specific small molecule inhibitors GANT61processing the SKO-V3and ES-2cells, we detected Smo, Gli1, Gli2(critical components of Hedgehog signaling pathway) mRNA and protein in ovarian cancer cells (SKO-V3and ES-2) that were treated with DMSO or GANT61(30μim) by real-time PCR, western-blot and immunocytochemistry, respectively.(2) To detect cell viability and proliferation in ovarian cancer cells (SKO-V3and ES-2) that treated with DMSO or GANT61(30μm) by MTT assays and BrdU assays respectively;(3) To detect cell migration and cell colony formation in ovarian cancer cells (SKO-V3and ES-2) that treated with DMSO or GANT61(30μm) by wound-healing assays and soft agar assays, respectively. Part III. Blocking Hh signaling pathway changes cDNA microarrays genesexpression profiling in ovarian cancer cells(1) The transcription factor Gli-specific small molecule inhibitors GANT61processing the SKO-V3and ES-2cells, we detected the differentially expressed genes (DEGs) that regulated by Hedgehog signaling pathway in ovarian cancer cells (SKO-V3) that were treated with DMSO or GANT61(30μm) by cDNA microarrays gene expression profiling, and these differentially expressed genes were uploaded for GO pathway and KEGG pathway analysis by bioinformatics. we found that some genes are regulated by Hedgehog signaling pathway. Then, we validated the DEGs (such as ITGB4, FAK, CD24, MMP7, etc.) by real-time PCR and western-blot,respectively.(2) In order to explore the molecular mechanisms by which Hh signaling pathway resulates cell invasion and metastasis in ovarian cancer, Gli-specific small molecule inhibitors GANT61, siRNA of Gli1and Hh signaling pathway ligand conditional medium (Shh-Med), were used to inhibit or activate the Hh signaling, The real-time PCR,western blot and cell invasion assay were performed to test cell migration and invasion of ovarian of cancer.Part IV. Blocking Hh signaling pathway reduces tumor growth in vivo(1) In order to test potential tumor growth inhibitory effects of Gli inhibitor GANT61in vivo, We establish the nude mice subcutaneous xenograft models employed human ovarian epithelial carcinoma tissue masses.(2) The nude mice subcutaneous xenograft models were treated with GANT61(50mg/5ml) or control. Tumor sizes were measured every other day. The volume and weight of tumor were compared each other.(3) Gli1and Gli2protein in xenografts were detected by western-blot. Simultaneously, ITGB4and p-FAK protein in xenografts were detected by immunohistochemistry. ResultsPart Ⅰ. The relation between the aberrant activation of Hedgehog signaling pathway and clinicopathological parameters in ovarian cancer(1) The expression of Shh, Gli1and Gli2protein in ovarian carcinoma was significantly higher than that in the adjacent tissues by western-blot, and the expression of Shh, Gli1and Gli2mRNA in ovarian carcinoma was significantly higher than that in the adjacent tissues by real-time PCR, the difference was statistically significant (p<0.01), The above data suggest that abnormal activation of the Hh signaling pathway was occured in ovarian cancer tissue.(2) Gli2protein expression was related with tumor TNM stage, the difference was statistically significant (p<0.05); and the expression of Gli2protein with patients age, histological type of tumor, the degree of tumor differentiation and lymph node matestasis were not significantly related (p>0.05).Part Ⅱ. Blocking Hedgehog signaling pathway decreases cell proliferation and migration in ovarian cancer(1) Gli-specific small molecule inhibitors GANT61induced down-regulation of transcription factor Gli and its downstream targets Ptch in protein level in ovarian cancer cells; GANT61induced down-regulation of transcription factor Gli1and Gli2in mRNA level in ovarian cancer cells, the difference was statistically significant (p<0.01); Gli2protein expression was significantly reduced in the cells treated with GANT61.(2) GANT61inhibited the viability of ovarian cancer cells, the difference was statistically significant (MTT assays; p<0.01); Also, GANT61inhibited the proliferation of ovarian cancer cells, the difference was statistically significant (BrdU assays; p<0.01);(3) GANT61inhibited cell migration of ovarian cancer cells, the difference was statistically significant (wound-healing assays; p<0.01); and GANT61inhibited the cell colony formation of ovarian cancer cells, the difference was statistically significant (soft agar assays; p<0.01).Part III. Blocking Hedgehog signaling pathway changes cDNA microarrays genes expression profiling in ovarian cancer cells(1) cDNA microarrays gene expression profiling and pathway network analysis indicated that the mapped DEGs in SKO-V3following GANT61treatment for60hr chiefly involved the focal adhesion pathway, including both up-and down-regulated genes. Of above genes, integrin and lamin are the main DEGs. Simultaneously, the mapped DEGs in SKO-V3are also accumulated in MAPK signaling pathway and most of those DEGs were up-regulated, several of those DEGs were down-regulated.(2) Real-time PCR and Western blot verified the microarray results at the mRNA and protein levels, the results indicated that the expression of ITGB4, COL1A1, COL5A1mRNA was significantly declined following GANT61treatment compared with DMSO treatment, the difference was statistically significant (p<0.01); However, the expression of LAMC2, ITGA5, LAMA3mRNA was significantly higher, the difference was statistically significant (p<0.01), consistent with cDNA microarrays. The expression of CD24, CD70, S100A2, MMP7mRNA were all declined,(p<0.01); The results were also consistent with cDNA microarrays; In addition, the expression of ITGB4, CD24, MMP7protein were all significantly declined following GANT61treatment compared with DMSO treatment, There consistent with real-time PCR assays.(3) In order to observe the differences in gene expression changes. Shh ligand conditioned medium (Shh-Med) is used to activate the Hh signaling pathway, we found that the expression of ITGB4protein and mRNA in SKO-V3was significantly higher following Shh-Med treatment compared with control treatment, the difference was statistically significant (p<0.01). In addition, cell invasion assay indicated that the motility and invasiveness of ovarian cancer cells caused by Shh-Med can be blocked by the anti-ITGB4,(p<0.01).(4) In order to confirm the specific role of GANT61to the transcription factor Gli, Gli1siRNA(miR-Gli1-720) was used to interfere the expression of Gli, Western blot results indicated that Gli1and ITGB4protein expression levels of SKO-V3cells transfected with miR-Gli1-720were significantly decreased, consistent with the inhibitory effect of GANT61.(5) In order to clarify the biological significance of Hh signaling pathway affect1TGB4expression, eithor Shh-Med or GANT61was used to treat SKO-V3cells, the results suggested that compared with control treatment, the expression of p-FAK protein in SKO-V3was significantly higher following Shh-Med treatment, the difference was statistically significant (p<0.01); But the difference in the expression of FAK protein was not statistically significant (p>0.05); However the expression of p-FAK levels were significantly decreased following GANT61treatment (p<0.01). Western blot results suggested that increase in p-FAK levels of SKO-V3cell caused by stimulation of Shh-Med can be blocked by the anti-ITGB4,(p<0.01). the above data confirmed that the Hh signaling pathway can promote FAK phosphorylation.(6) Next, we employed immunofluorescence technique to examine the effects of GANT-61on the expression of p-FAK. SKO-V3cells were treated with GANT61, and the expression of p-FAK-Y397was observed. GANT61inhibited the expression of p-FAK-Y397. In addition, we found that cytoskeleton of ovarian cancer cell was destroyed following GANT61treatment for48hr, and cell pseudopodia were significantly lessen.Part Ⅳ. Blocking Hedgehog signaling pathway reduces tumor growth(1) The nude mice subcutaneous xenograft models employed human ovarian epithelial carcinoma tissue masses were successfully established.(2) The volumes of the nude mice subcutaneous xenografts were significantly reduced following GANT61treatment compared with control treatment, especially at the ninth day. The tumor weight of the nude mice was also significantly reduced following GANT61treatment compared with control treatment, the difference was statistically significant (p<0.01).(3) Western-blot assays indicated that the expression of Glil and Gli2protein in xenografts that treated with GANT61was significantly lower than that treated with vehicle, the difference was statistically significant (p<0.01); While immunohisto chemistry assays indicated that the expression of ITGB4and p-FAK protein in xenografts that treated with GANT61was significantly lower than that treated with vehicle.Conclusion1. The aberrant activation of Hh signaling pathway was implicated in ovarian cancer. Gli2as the important component of Hh signaling, its protein expression was related with tumor stage of ovarian cancer.2. GANT61decreased the expression of Gli1, Gli2and Ptch protein, Simultaneously, it inhibited cell viability, proliferation, migration and colony formation.3. Some DEGs were screened by cDNA micrearrays following GANT61treatment, and of these genes, several ones related to cell adhesion and migration were verified in protein and mRNA level. The results were consistent with cDNA micrearrays. Down-regulation of Gli decreased the expression of ITGB4protein and mRNA, and anti-ITGB4inhibited Shh mediated cell migration and invasion of ovarian cancer by destroying ITGB4/FAK pathway. We found that Hh pathway regulated cell migration and invasion of ovarian cancer, which may was mediated by ITGB4/FAK signaling.4. GANT61reduced tumor volume and weight of xenografts in the nude mice, and the expression of Gli1,Gli2, ITGB4and p-FAK protein all declined following GANT61treatment, Gli as the final transcription factor of Hh pathway, it may become the new promising target of ovarian cancer, and it will offer theoretical guidance of treatment strategies and drug development targeting to ovarian cancer.更多还原