【作者】 张传海；

【导师】 许戈良；

【作者基本信息】 天津医科大学 ， 外科学， 2011， 博士

【摘要】 目的：1、观察肝细胞癌(hepatocellular carcinoma, HCC,简称肝癌)组织中活化形式(磷酸化)的信号转导及转录活化子3 (phosphorylated signal transducer and activator of transcription 3, p-STAT3)、上皮间质转化(epithelial-mesenchymal transition, EMT)主要标志物的表达情况及其临床意义；2、细胞学实验体外探讨调控STAT3活化水平对肝癌细胞EMT的影响及其可能的分子机制；3、动物实验体内探讨抑制STAT3活化水平对肝癌细胞EMT的影响及其可能的分子机制；4、荟萃(meta)分析评价临床使用的具有STAT3活化抑制作用的干扰素-a(interferon-a, IFN-a)在预防肝癌根治性治疗术后复发及改善病人生存方面的疗效。方法：1、采用免疫组织化学方法检测了100例肝癌及10例正常肝组织标本中p-STAT3、EMT标志物(上皮标志物E-cadherin、间质标志物vimentin)表达,分析所检测指标之间及其与肝癌侵袭转移的相关性;2、采用不同转移潜能的肝癌细胞株(SMMC7721、HepG2、HCC97L、MHCC97H、HCCLM3), transwell侵袭实验检测肝癌细胞体外侵袭力、RT-PCR及Western-blot检测其STAT3、p-STAT3表达,筛选出低侵袭、低P-STAT3表达及高侵袭、高p-STAT3表达水平的2株具有代表性的肝癌细胞,利用表皮生长因子(epidermal growth factor, EGF)诱导肝癌细胞STAT3(?)舌化及STAT3磷酸化抑制剂JSI-124抑制STAT3活化水平；观察调控STAT3活化对肝癌细胞形态学的影响,共聚焦显微镜观察FITC-Phalloidin(鬼笔环肽)标记的F-actin细胞骨架变化,RT-PCR、Western-blot及免疫荧光染色检测EMT分子标志物(E-cadherin、vimentin)及转录因子Twist表达变化,transwell侵袭、细胞伤口愈合实验检测细胞体外侵袭迁移力的变化；3、建立裸鼠皮下种植性肝癌自发性肺转移模型,采用JSI-124腹腔注射,观察抑制STAT3活化对肝癌皮下移植瘤生长、转移的影响,RT-PCR、Western-blot及免疫组化染色检测EMT分子标志物(E-cadherin、vimentin)及Twist表达变化；4、采用meta分析方法对临床使用的具有STAT3活化抑制作用的IFN-a在预防肝癌治愈性切除或消融治疗术后复发及改善病人生存方面的作用进行系统评价。结果：1、免疫组化检测发现肝癌组织中P-STAT3、vimentin表达较正常肝组织明显升高而E-cadherin表达降低,E-cadherin、vimentin表达呈负相关,P-STAT3与vimentin表达呈正相关、而与E-cadherin表达呈负相关：此外,p-STAT3、E-cadherin及vimentin表达均与肝癌侵袭转移有关；这提示肝癌中存在EMT现象,而STAT3活化可能通过介导EMT、促进肝癌侵袭转移。2、transwell侵袭实验、RT-PCR及Western-blot检测结果提示SMMC7721细胞为具有低侵袭力、低p-STAT3表达水平的代表性细胞株,HCCLM3细胞为具有高侵袭力、高p-STAT3表达水平的代表性细胞株；故本研究中采用SMMC7721、HCCLM3作为实验对象。研究结果如下：(1)EGF处理SMMC7721细胞24小时后,细胞呈梭形、纺锤样形态,细胞间隙增宽,F-actin细胞骨架发生重排而对照组细胞却呈典型的上皮样表型,在合并使用JSI-124的情况下,部分细胞仍维持明显的上皮样形态；这初步提示EGF诱导的STAT3活化可能介导了肝癌细胞EMT。(2)通过RT-PCR、Western-blot和免疫荧光染色方法进一步检测了改变STAT3活化水平后肝癌细胞EMT分子标志物表达的变化,结果发现EGF处理SMMC7721细胞后,E-cadherin表达降低而vimentin表达升高,合并使用JSI-124却能够部分阻断上述效应的发生,JSI-124处理HCCLM3细胞后其E-cadherin表达升高而vimentin表达降低。(3)细胞侵袭和伤口愈合实验结果显示,EGF处理SMMC7721后,细胞的侵袭迁移能力明显高于对照组,而在联合应用JSI-124的情况下,这种变化却能够部分被抑制,JSI-124处理HCCLM3细胞后其侵袭迁移能力亦明显下降。(4)进一步通过RT-PCR、Western-blot和免疫荧光染色方法检测了调节STAT3活化水平对肝癌细胞Twist表达的变化,结果表明EGF处理SMMC7721细胞后,Twist表达显著升高,合并使用JSI-124却能够部分阻断上述变化,JSI-124处理HCCLM3细胞后其Twist表达明显降低。综合以上结果,表明：STAT3活化介导了肝癌细胞EMT发生,而Twist可能在此过程中起重要作用。3、裸鼠体内实验表明：与对照组相比,JSI-124能显著抑制HCCLM3肝癌皮下移植瘤生长并抑制其肺转移；采用RT-PCR、Western-blot及免疫组化检测发现JSI-124治疗组肿瘤组织P-STAT3降低E-cadherin表达升高而vimentin表达降低,此外,JSI-124处理后Twist表达明显降低；提示：抑制STAT3活化能抑制或部分抑制EMT而降低肝癌转移,这同时也验证了体外实验结果。4、meta分析结果提示IFN-a能显著的降低肝癌治愈性切除或消融术后早期复发、提高肝癌病人存活率；这预示着STAT3抑制剂用于肝癌的临床干预治疗具有潜在的价值。结论：1、人肝癌组织中存在STAT3活化及EMT现象,且与肝癌侵袭转移相关;p-STAT3/ E-cadherin信号轴可作为肝癌侵袭转移潜能预测指标：2.STAT3活化能通过介导肝癌细胞EMT、促进肝癌侵袭转移,其分子机制可能涉及转录因子Twist；3.IFN-a能有效降低肝癌治愈性治疗术后早期复发并改善病人预后。更多还原

【Abstract】 Objective:1. To examine the expression of phosphorylated STAT3 (p-STAT3), the major markers of epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) tissues; evaluate their correlations and clinical significance; 2. In vitro, to investigate the effect of altered STAT3 activation on EMT in HCC cells and its possible molecular mechanisms; 3. In vivo, to investigate the effect of altered STAT3 activation on EMT in HCC cells and its possible molecular mechanisms; 4. Meta analysis:to assess the efficacy of interferon-a (IFN-a), which has the potential ability to inhibit STAT3 activation, on the recurrence and survival in HCC after curative treatment.Methods:1. Immunohistochemistry was used to detect the expression of p-STAT3, EMT markers (E-cadherin and vimentin) in 100 cases of HCC and 10 cases of normal liver tissue; then analyze the correlation between each protein expression and HCC invasion、metastasis; 2. HCC cell lines with different metastatic potential (SMMC7721、HepG2、MHCC97L、MHCC97H、HCCLM3) were used. The invasion ability of HCC cells were investigated by transwell cell invasion assay, the expression of STAT3 and p-STAT3 was detected by RT-PCR and Western-blot; to identify two representative HCC cell lines which exhibited low invasion ability-. low p-STAT3 expression and high invasion ability、high p-STAT3 expression respectively. By using epidermal growth factor (EGF) to induce STAT3 activation and applying p-STAT3 inhibitor JSI-124 to inhibit STAT3 activation, to examine the effect of altered STATS activation on HCC cell morphology, observe the change of F-actin cell skeleton labeled by FITC-Phalloidin under laser confocal microscope, analyze the expression of EMT molecular markers (E-cadherin and vimentin) and Twist by RT-PCR、Western-blot and immunofluorescent staining, detect the invasion and migration abilities of HCC cells by transwell invasion and wound-healing assays; 3. By establishing the HCC nude mouse model of spontaneous lung metastasis, injecting JSI-124intraperitoneally; to observe the efficacy of decreased STAT3 activation on HCC growth and metastasis, detect the expression of EMT molecular markers (E-cadherin and vimentin) and Twist by RT-PCR、Western-blot and immunohistochemical staining; 4. By systematic review and meta-analysis, to investigate the effect of IFN-a on the recurrence and survival of HCC patients who received curative resection or ablation.Results:1. Immunohistochemistry revealed that HCC tissues exhibited increased p-STAT3 and vimentin expression, decreased E-cadherin expression when compared with normal liver tissues. E-cadherin and vimentin expression were negatively correlated, p-STAT3 and vimentin expression were positively correlated while the correlation between p-STAT3 and E-cadherin was negative; besides, p-STAT3、E-cadherin and vimentin expression were all associated with HCC invasion and metastasis. It signified that EMT existed in HCC, STAT3 activation might mediate EMT and promote HCC invasion and metastasis.2. The results of invasion assay^ RT-PCR and Western-blot analyses indicated that SMMC7721 was a representative cell line that had low invasion ability、low p-STAT3 expression, while HCCLM3 was a representative cell line which owned a high invasion ability、high p-STAT3 expression; Thus, SMMC7721 and HCCLM3 cells were selected as our experimental subjects in this study finally. Study results were as follows:(1) After EGF treatment of SMMC7721 cells for 24 hours, we observed differences in the gross appearance of EGF-treated cells as compared with the control cells. The morphologic changes included loss of cell polarity causing a spindle-like shape, increased intercellular separation, the rearrangement of F-actin cell skeleton; while the control cells displayed the classical epithelial morphology; in the presence of JSI-124, part of the cells still kept the epithelial phenotype like the control cells; which preliminarily suggested that activation of STAT3 induced by EGF could mediate EMT in HCC cells. (2) In order to further know the effect of altered STAT3 activation on the expression of EMT molecular markers, RT-PCR、Western-blot and immunofluorescent staining were performed. The results indicated that after EGF treatment, SMMC7721 cells displayed decreased E-cadherin and increased vimentin expression; in the presence of JSI-124, these changes were partly blocked. Moreover, JSI-124 treatment increased E-cadherin and decreased vimentin expression in HCCLM3 cells. (3) Cell invasion and wound-healing assays demonstrated that EGF-treated SMMC7721 cells exhibited higher invasion and migration abilities than the control cells, while in the presence of JSI-124, these changes were partly inhibited. JSI-124 treatment also decreased invasion and migration abilities in HCCLM3 cells. (4) By using RT-PCR^ Western-blot and immunofluorescent staining, we wanted to further know the effect of altered STAT3 activation on Twist expression. The results suggested that upon EGF treatment, SMMC7721 cells exhibited increased Twist expression; while this change was in part blocked in the presence of JSI-124. JSI-124 treatment could reduce Twist expression significantly in HCCLM3 cells. Taken together, our study results demonstrated that activation of STAT3 could mediate EMT in HCC cells, and Twist might play an important role in this process.3. In vivo nude mice experiments showed:as compared with the control group, JSI-124 could significantly suppress the growth and metastasis of HCC xenografts; the results of RT-PCR、Western-blot and immunohistochemical staining indicated that after JSI-124 treatment, the p-STAT3、vimentin expression in HCC were reduced but the E-cadherin expression was increased. Moreover, the Twist expression was significantly decreased upon JSI-124 treatment. All these suggested that inhibition of STAT3 activation could block or partly block EMT. and prevent HCC metastasis; which also confirmed our in vitro experiment results.4. Meta-analysis results suggested that IFN-a could significantly reduce recurrence and increase overall survival in patients with HCC after curative resection or ablation, signifying that STAT3 inhibitors might have the potential value to be used in the clinical management of HCC.Cone I us ions:1. HCC tissues display significant STAT3 activation and EMT phenomenon, as were all correlated significantly with HCC invasion and metastasis; p-STAT3/E-cadherin signal axis can be used as predictive marker for HCC invasion and metastasis potential; 2. STAT3 activation can mediate HCC cell EMT、promote HCC invasion and metastasis, Twist may be involved in this process; 3. IFN-a can efficiently reduce postsurgical or ablative early HCC recurrence and improve the overall prognosis of HCC patients.更多还原