Raf激酶抑制蛋白介导的信号通路对肝癌侵袭转移的抑制作用
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摘要
肝细胞癌(hepatocellular carcinoma,HCC)位居世界恶性肿瘤发病率的第五位,是全球第三位的癌症死亡原因;HCC是我国最常见的恶性肿瘤之一,具有恶性程度高,易复发、转移、预后差的特点。即使施行肝癌根治性切除的肝癌患者,术后3年生存率只有40-50%,其原因与肝细胞癌易侵袭转移的生物学特性有关。至今肿瘤转移及其调控的确切机制仍不清楚,而且无法在临床上采取有效的治疗措施,因此,揭示恶性肿瘤转移及其调控的确切分子机制已成为临床提高肿瘤治疗效果的迫切需要,这一领域也成为分子肿瘤学、分子生物学等基础理论学科研究的热点。
Raf激酶抑制蛋白(Raf kinase inhibitor protein,RKIP)属于高度保守的磷脂酰乙醇胺结合蛋白(phosphatidylethanolamine binding protein,PEBP)家族,广泛存在于多种生物中。RKIP参与多条信号传导通路的调控,在细胞生长、增殖、分化、凋亡等生理过程中发挥重要作用。最近研究表明RKIP与肿瘤转移密切相关,但RKIP在肝细胞癌中的作用及其机制如何,目前国内外研究报道较少。
本研究首先检测肝细胞癌组织中RKIP及其可能相关蛋白P- RKIP、p65和P-ERK的表达,了解RKIP与肝细胞癌临床病理学特征的关系及其与P- RKIP、p65和P-ERK的相关性。其次,通过RKIP基因转染或干扰体外培养的肝癌细胞,观察上调或抑制RKIP表达后肝癌细胞增殖、凋亡、侵袭转移能力的变化,进一步明确RKIP在肝癌细胞侵袭转移过程中的作用。最后,检测RKIP表达或抑制后肝癌细胞Raf-MEK1/2-ERK1/2和NF-κB信号转导通路相关蛋白的表达,了解RKIP在肝细胞癌侵袭转移中的作用机制,最终明确肝癌的侵袭转移是否与RKIP的失活及Raf-MEK1/2 -ERK1/2和NF-κB信号转导通路的激活有关。
第一部分肝癌组织中Raf激酶抑制蛋白的表达及其临床意义
目的:观察肝细胞癌组织、癌旁肝组织、正常肝组织中RKIP及其可能相关蛋白P- RKIP、p65和P-ERK的表达情况,了解RKIP与肝细胞癌临床病理学特征的关系及其与P- RKIP、p65和P-ERK的相关性。
方法:RT-PCR方法检测肝细胞癌、癌旁及正常肝组织中RKIP mRNA的表达,免疫组织化学和western方法检测肝细胞癌、癌旁及正常肝组织中RKIP、P- RKIP、p65和P-ERK蛋白的表达,分析RKIP与肝细胞癌临床病理学特征的关系及其与P- RKIP、p65和P-ERK的相关性。
结果:1肝细胞癌和对应的癌旁组织、正常肝组织RKIP mRNA表达分别为1.357±0.569、1.512±0.623和1.550±0.426,差异无显著性(P=0.427)。
2 Western-blot结果显示肝细胞癌及对应的癌旁组织、正常肝组织中RKIP蛋白的表达量分别为0.579±0.380、1.178±0.659和1.115±0.442,P-RKIP蛋白的表达量分别为0.499±0.560、0.885±0.864和1.104±0.622,RKIP和P-RKIP蛋白在肝癌组织中的表达水平均显著低于癌旁及正常肝组织(P<0.05),但在癌旁和正常肝组织之间表达差异无显著性(P>0.05)。
3 Western-blot结果显示肝细胞癌组织、癌旁和正常肝组织中P65蛋白的表达量分别为0.83±0.376、0.63±0.337和0.466±0.345,P-ERK蛋白的表达量分别为1.023±0.478、0.605±0.367和0.461±0.293。P65和P-ERK在肝癌组织中的表达水平均显著高于癌旁及正常肝组织(P<0.05),但在癌旁和正常肝组织之间P65和P-ERK的表达差异无显著性(P>0.05)。
4免疫组织化学染色结果显示RKIP在72例肝细胞癌组织、50例癌旁和16例正常肝组织中的阳性表达率分别为22.2%、86%、93.8%;P-RKIP在肝细胞癌组织、癌旁和正常肝组织中的阳性表达率分别为29.2%、84%、93.8%。RKIP和P-RKIP在肝癌组织中的表达水平均显著低于癌旁及正常肝组织(P<0.05)。RKIP和P-RKIP蛋白表达在中高分化肝癌组高于低分化组,无肝内或淋巴转移组高于有肝内或淋巴转移组(P<0.05),但与肝癌肿瘤大小、AFP水平及有无门静脉或胆管癌栓无关(P>0.05)。
5免疫组织化学染色结果显示P65在72例肝细胞癌组织、50例癌旁和16例正常肝组织中的阳性表达率分别为73.6%、56%、37.5%;P-ERK在肝细胞癌组织、癌旁和正常肝组织中的阳性表达率为65.3%、38%、31.3%。P65和P-ERK蛋白在肝癌组织中的表达水平显著高于癌旁及正常肝组织(P<0.05),但在癌旁及正常肝组织表达差异无显著性(P>0.05)。P65蛋白表达水平与肝癌分化程度、有无门静脉或胆管癌栓有关(P<0.05),而与AFP水平、肿瘤大小及有无肝内或淋巴结转移无关;P-ERK蛋白表达水平与肝癌分化程度、有无门静脉或胆管癌栓及有无肝内或淋巴结转移有关(P<0.05),而与AFP水平和肿瘤大小无关。
6肝癌组织中RKIP蛋白表达与P-RKIP的表达呈显著正相关(P=0.000),与P-ERK的表达呈显著负相关(P=0.04),但与P65的表达无相关性(P=0.143)。
结论:1肝细胞癌、癌旁及正常肝组织中RKIP mRNA表达差异无显著性说明RKIP的表达可能是在转录后水平受到调节。
2 RKIP和P-RKIP表达的减少或缺失、P65和P-ERK的高表达与肝癌的发生发展密切相关。
3在中高分化肝癌组RKIP和P-RKIP蛋白表达显著高于低分化组,无肝内或淋巴转移组显著高于有肝内或淋巴转移组,而且肝癌组织中RKIP和P-RKIP蛋白表达呈正相关,提示RKIP和P-RKIP蛋白在抑制肝癌侵袭转移过程中共同发挥作用。
4 P65蛋白表达水平与肝癌分化程度、有无门静脉或胆管癌栓有关, P-ERK蛋白表达与肝癌分化程度、有无门静脉或胆管癌栓以及有无肝内或淋巴结转移有关,提示P65和P-ERK在肝癌侵袭转移过程中发挥作用。
5 RKIP与P-ERK的表达呈显著负相关,提示RKIP表达的减少或缺失可能通过上调P-ERK的表达促进肝细胞癌的侵袭转移。
第二部分上调Raf激酶抑制蛋白的表达对肝癌细胞生物学行为的影响
目的:RKIP表达质粒稳定转染RKIP低表达肝癌细胞株,体内外实验明确RKIP对肝癌细胞增殖、侵袭、凋亡及细胞周期的影响。
方法:RT-PCR和western-blot方法对BEL7402、HepG2、HCCLM3和SMMC7721四种人肝癌细胞株进行检测,筛选出RKIP低表达肝癌细胞株,行RKIP质粒转染,G418筛选后,经RT-PCR和western-blot方法鉴定,获得稳定转染空载体和表达RKIP蛋白的细胞克隆。通过MTT法绘制细胞生长曲线,观察肝癌细胞增殖的变化;流式细胞术(FCM)观察RKIP表达对肝癌细胞的细胞周期和凋亡率的影响;通过进行单层细胞划痕实验和Matrigel侵袭实验检测肝癌细胞迁移能力和侵袭力的变化;对照组和RKIP转染组肝癌细胞接种裸鼠,建立人肝癌细胞裸鼠皮下移植瘤模型,观察RKIP对肝癌细胞增殖和转移能力的影响。
结果:1 BEL7402、HepG2、HCCLM3和SMMC7721四种人肝癌细胞株RKIPmRNA的表达量分别为0.349±0.025、0.885±0.015、0.290±0.015和0.602±0.031,RKIP蛋白的表达量分别为0.582±0.056、1.389±0.079、0.428±0.021和0.733±0.089。HepG2肝癌细胞RKIP mRNA和蛋白表达水平均显著高于其它三株(P<0.05),而HCCLM3肝癌细胞RKIP mRNA和蛋白表达水平均显著低于其它三株(P<0.05)。
2 RKIP表达质粒和空质粒稳定转染HCCLM3人肝癌细胞, RT-PCR和western blot结果显示RKIP转染组HCCLM3细胞中RKIP mRNA和蛋白的表达显著高于对照组(P<0.05)。
3根据MTT结果绘制细胞生长曲线,结果示:从第3天开始,RKIP转染组细胞生长显著慢于对照组(P<0.05)。
4划痕实验结果显示划痕24h后,对照组的细胞迁移了52.67±5.67%,而RKIP转染组的细胞仅迁移31.67±3.79%,差异具有显著性(P=0.006);48h后,对照组细胞迁移了74.33±7.02%,而RKIP转染组细胞仅迁移43.33±8.73%,差异有显著性(P=0.009)。
5侵袭实验结果显示对照组和RKIP转染组HCCLM3细胞发生侵袭的细胞个数分别为:101±12和67±14,转染组侵袭细胞数显著低于对照组(P<0.05), RKIP对HCCLM3肝癌细胞侵袭力抑制率为33.7%。
6与对照组比较,RKIP转染组HCCLM3肝癌细胞的细胞周期和凋亡率无显著改变(P>0.05)。
7种植对照组和RKIP转染组HCCLM3肝癌细胞的裸鼠,在接种细胞后2周左右均成瘤,6周后肿瘤体积分别为(2.207±0.752)cm3和(1.197±0.406)cm3,瘤重分别为(1.580±0.625)g和(0.836±0.254)g,转染组瘤重和体积均显著小于对照组(P<0.05),两组均未见肺转移。
结论:1高转移性肝癌细胞系HCCLM3细胞RKIPmRNA和蛋白低表达,提示RKIP的低表达可能与肝癌细胞的侵袭、转移等恶性行为有关。
2 RKIP基因成功转入并在HCCLM3肝癌细胞中稳定表达。
3 RKIP基因转染后,HCCLM3肝癌细胞体外增殖率、细胞的迁移速度和体外侵袭力均明显降低,说明RKIP表达能抑制HCCLM3肝癌细胞的体外增殖能力和侵袭力。
4成功建立转染组和对照组HCCLM3人肝癌细胞裸鼠皮下移植瘤模型,结果显示RKIP不影响HCCLM3细胞成瘤性,但能够抑制HCCLM3肝癌细胞的体内增殖能力。
5 RKIP对HCCLM3肝癌细胞的细胞周期和凋亡率无影响,说明RKIP表达不能促进HCCLM3肝癌细胞凋亡,RKIP表达抑制HCCLM3肝癌细胞增殖和侵袭力与细胞周期的改变无关。
第三部分抑制Raf激酶抑制蛋白的表达对肝癌细胞生物学行为的影响
目的:观察RKIP-siRNA对肝癌细胞中RKIP mRNA及其蛋白表达的抑制作用以及对肝癌细胞生物学行为的影响;同时建立人肝癌细胞裸鼠皮下移植瘤模型,观察RKIP基因沉默对HepG2细胞体内增殖活性的影响。
方法:构建RKIP-siRNA和针对性设计的阴性对照RKIP-ncRNA质粒载体,脂质体介导转染RKIP高表达细胞株HepG2细胞,G418筛选,扩大培养的细胞分别命名为HepG2-NC和HepG2-si。HepG2-NC和HepG2-si细胞经RT-PCR和western-blot方法鉴定,观察RKIP-siRNA是否成功沉默人HepG2肝癌细胞中RKIP基因表达。通过MTT法绘制细胞生长曲线,观察肝癌细胞增殖的变化;流式细胞术观察RKIP表达对肝癌细胞的细胞周期和凋亡率的影响;通过Matrigel侵袭实验检测肝癌细胞侵袭力的变化;HepG2-NC和HepG2-si细胞接种裸鼠,建立人肝癌细胞裸鼠皮下移植瘤模型,体内实验观察RKIP对肝癌细胞增殖的影响。
结果:1 HepG2-NC和HepG2-si组细胞RKIPmRNA表达水平分别为0.873±0.035和0.421±0.028,RKIP蛋白表达水平分别为1.36±0.068和0.524±0.024。HepG2-si组RKIP基因表达水平显著低于HepG2-NC组(P<0.05)。
2 MTT结果显示,在相同的初始细胞浓度条件下,各个时间点细胞增殖差异无统计学意义(P>0.05)。细胞生长曲线示HepG2-NC组和HepG2-si组细胞增殖速度相近。
3在侵袭实验中,HepG2-NC组和HepG2-si组细胞发生侵袭的细胞个数分别为87±13和118±17,HepG2-si组侵袭细胞数显著高于HepG2-NC组(P=0.005)。
4 HepG2-NC组和HepG2-si组凋亡率和细胞周期变化无显著性(P>0.05)。
5 HepG2-si和HepG2-NC组细胞接种裸鼠,在接种细胞后10天左右均可见肿瘤结节形成。40天后,HepG2-si组和HepG2-NC组肿瘤体积分别为:(2.109±0.340)cm3和(1.948±0.539)cm3;瘤重分别为(1.308±0.27)g和(1.236±0.283)g,两组之间肿瘤体积和重量差异均无显著性(P>0.05)。
结论:1 RKIP-siRNA成功沉默人HepG2肝癌细胞中RKIP基因。
2 RKIP-siRNA不改变HepG2肝癌细胞的细胞凋亡率和细胞周期,也不影响HepG2肝癌细胞体内外的增殖能力,但能够增强HepG2细胞的体外侵袭能力,说明RKIP基因在抑制肝癌侵袭转移方面发挥重要作用。
第四部分Raf激酶抑制蛋白抑制肝癌侵袭转移的作用机制
目的:以稳定转染空质粒和RKIP基因的HCCLM3肝癌细胞、稳定转染RKIP-ncRNA质粒和RKIP-siRNA质粒的HepG2肝癌细胞为研究对象,检测肝癌细胞Raf-MEK1/2 -ERK1/2和NF-κB信号转导通路相关蛋白的表达,明确RKIP在肝癌侵袭转移中的作用机制。
方法:1 HCCLM3- C(对照组)、HCCLM3-RKIP(转染组)、HepG2-NC和HepG2-si肝癌细胞加入NF-κB特异性抑制剂PDTC, 48h后EGF处理组加入EGF,激活ERK通路,15分钟后提取细胞胞浆和胞核蛋白,考马斯亮兰法蛋白定量。Western blot检测P65、ERK和P-ERK蛋白表达,观察RKIP表达或抑制对肝癌细胞ERK信号通路的影响。
2 HCCLM3-C(对照组)、HCCLM3-RKIP(转染组)、HepG2-NC和HepG2-si组肝癌细胞加入ERK信号通路特异性抑制剂PD98059, 48h后TNF-α处理组加入TNF-α,激活NF-κB通路,30分钟后提取细胞胞浆和胞核蛋白,考马斯亮兰法蛋白定量。Western blot检测P65、ERK和P-ERK蛋白表达,观察RKIP表达或抑制对肝癌细胞NF-κB通路信号通路的影响。
结果:1 RKIP转染组HCCLM3细胞RKIP蛋白的表达显著高于对照组(P<0.05),EGF对RKIP的表达无影响;EGF(+)对照组中HCCLM3细胞胞浆和胞核P-ERK的表达明显高于EGF(-)对照组(P<0.05),而在EGF(-)转染组和EGF(+)转染组之间P-ERK的表达差异无统计学意义(P>0.05);与EGF(-)对照组比较,EGF(-)转染组HCCLM3细胞胞浆和胞核P-ERK的表达显著降低(P<0.05);EGF(+)转染组HCCLM3细胞胞浆和胞核P-ERK的表达显著低于EGF(+)对照组(P<0.05)。
2 TNF-α(+)对照组中HCCLM3细胞胞浆和胞核P65的表达明显高于TNF-α(-)对照组(P<0.05),而在TNF-α(-)转染组和TNF-α(+)转染组之间P65的表达差异无统计学意义(P>0.05);与TNF-α(-)对照组比较,TNF-α(-)转染组HCCLM3细胞胞浆和胞核P65的表达显著降低(P<0.05);TNF-α(+)转染组HCCLM3细胞胞浆和胞核P65的表达显著低于TNF-α(+)对照组(P<0.05)。
3 RKIP-siRNA组HepG2细胞RKIP蛋白表达显著低于对照组(P<0.05),EGF对两组HepG2细胞RKIP的表达无影响;EGF(+)NC组中HepG2细胞胞浆和胞核P-ERK的表达显著高于EGF(-)NC组(P<0.05);EGF(+)-si组HepG2细胞P-ERK的表达显著高于EGF(-)-si组;与EGF(-)-NC组比较,EGF(-)-si组HepG2细胞胞浆和胞核P-ERK的表达显著升高(P<0.05);在EGF(+)-si组和EGF(+)-NC组之间,HepG2细胞胞浆和胞核P-ERK的表达无显著性差异(P>0.05)。
4 TNF-α对两组HepG2细胞RKIP的表达无影响(P>0.05);TNF-α(+)-NC组中HepG2细胞胞浆和胞核P65的表达明显高于TNF-α(-)-NC;在TNF-α(+)-si组P65的表达高于TNF-α(-)-si组;与TNF-α(-)-NC比较,TNF-α(-)-si组HepG2细胞胞浆和胞核P65的表达无显著性差异(P>0.05);与TNF-α(+)-NC组比较,TNF-α(+)-si组HepG2细胞胞浆和胞核P65的表达无显著差异(P>0.05)。
结论:1 RKIP表达不仅能够抑制HCCLM3细胞ERK的磷酸化,还能够抑制EGF诱导的HCCLM3细胞ERK的磷酸化,说明RKIP抑制肝癌细胞的增殖和侵袭可能是通过抑制ERK信号通路的活化来实现的。
2 RKIP不仅能够抑制HCCLM3细胞NF-κB信号通路的活化,还能够抑制TNF-α对HCCLM3细胞NF-κB信号传导通路的激活作用,说明RKIP抑制HCCLM3肝癌细胞的增殖、侵袭、转移不仅与抑制ERK信号通路的活化有关,也与NF-κB信号传导通路抑制有关。
3 RKIP表达的下调够活化HepG2细胞ERK信号通路,而对HepG2细胞NF-κB信号传导通路无影响,说明RKIP表达下调可能通过活化ERK信号通路促进HepG2肝癌细胞的侵袭,也说明肝癌细胞信号传导通路调节的复杂性以及RKIP在不同的细胞其作用机制不同。
Hepatocellular carcinoma(HCC) is the 5th commonest malignancy worldwide and is the third most common cause of cancer-related death. HCC is one of the most common malignant tumors in our country, which is frequently associated with metastasis in early stage and has a high rate of recurrence after operation or intervention treatment. However, the long-term prognosis of patients undergoing potentially curative hepatic resection is still poor, with reported 3-year survival rates ranging from 40% to 50%. This poor prognosis mainly contributes to the high rate of intra-hepatic and distant metastasis after resection or transplantation. To date, the metastatic mechanisms and the regulated mechanisms of tumors are not known clearly, so it is very important for understanding the mechanisms of invasion and metastasis and searching effective approaches to prevent the recurrence and metastasis of HCC is of great importance.
Raf kinase inhibitor protein(RKIP) is a member of the phosphatidylethanolamine binding protein family, a ubiquitously expressed and evolutionarily conserved group of proteins. In recent years, it has been reported that RKIP influence intracellular signaling cascades, cell cycle regulation, the suppression of metastasis, neurode generative processes, the modulation of emotions, and reproduction, and RKIP has been identified as a member of a novel class of molecules that suppress the metastatic spread of tumors, but little is known about the role and the regulating mechanisms of RKIP in human hepatocarcinogenesis.
In this study, we used immunohistochemistry and western blot to analyze expressions of RKIP, P- RKIP, p65 and P-ERK in HCC tissues, paratumor tissues and normal liver tissues, and to determine the relationship between the former four factors in HCC tissues and the feature of invasion and metastasis, and between RKIP and P-RKIP, p65, P-ERK in HCC. Nextly, cell proliferation, cell cycle, apoptosis rates and migration assays were analyzed after RKIP over expression or knockdown HCC cell lines in vitro or vivo. Finally, we underlying mechanisms of RKIP were assessed with immunoblot analysis after either over expression or knockdown of RKIP expression in HCC cell lines.
Part I Expression and significance of Raf kinase inhibitor protein in hepatocellular carcinoma
Objective: To investigate the expression of RKIP, P-RKIP, p65 and P-ERK in HCC and the correlation with the invasion and metastasis of HCC. Methods: Reverse transcription polymerase chain reaction(RT-PCR) was used to detect expression of RKIP mRNA. The expression levels of RKIP, P- RKIP, p65 and P-ERK protein in HCC tumor and peritumoral tissues were determined by immunohistochemistry and Western blot analysis. Statistical analysis was used to determine the relationship between their expression and clinicopathological parameters.
Results: 1 Semi-quantity RT-PCR analysis showed there was no significant difference in RKIP mRNA expression levels among HCC tumors, corresponding peritumoral tissues and normal liver tissues(P=0.427).
2 Immunoblot analysis of 12 normal liver tisues, 36 paired HCCs and adjacent uninvolved tissue samples showed the ratio of RKIP protein expression level(RKIP/actin) was 0.579±0.380 in HCCs, 1.178±0.659 in peritumoral tissues and 1.115±0.442 in normal liver tissues; the ratio of P-RKIP protein expression level(RKIP/actin) was 0.499±0.560, 0.885±0.864 and 1.104±0.622, respectively. RKIP and P-RKIP protein expression levels were decreased in HCCs compared with adjacent peritumoral tissues and normal liver tisues(P<0.05), but there was no significant difference in RKIP or P-RKIP protein expression levels between peritumoral tissues and normal liver tissues(P>0.05).
3 Immunoblot analysis showed the ratio of P65 protein expression level(RKIP/actin) was 0.83±0.376 in HCCs, 0.63±0.337 in peritumoral tissues and 0.466±0.345 in normal liver tissues; the ratio of P-ERK protein level(RKIP/actin) was 1.023±0.478, 0.605±0.367 and 0.461±0.293, respectively. The results showed increased P65 and P-ERK protein levels in HCCs compared with adjacent peritumoral tissues and normal liver tisues(P<0.05), but there was no significant difference in P65 or P-ERK protein expression levels between peritumoral tissues and normal liver tissues(P>0.05).
4 The expression of RKIP, P-RKIP, P65 and P-ERK protein were evaluated by immunohistochemistry in 72 human HCC tumors, 50 peritumoral tissues and 16 normal live tissues. RKIP-positive stainning was detected only in sixteen of 72 tumor tissues(22.2%), P-RKIP-positive stainning in twenty one (29.2%), P65-positive stainning in fifty three(73.6%), and P-ERK-positive stainning in forty seven(65.3%). In contrast, RKIP-positive stainning was in forty seven of 50 tumor-surrounding tissues(86.0%), P-RKIP-positive stainning in forty two (84.0%), P65-positive stain in twenty eight(56.0%), and P-ERK-positive stainning in 19(38%). In normal liver tissues fifteen of 16 RKIP-positive and P-RKIP-positive stainning was observed(93.8%), P65-positive stainning only in six(37.5%) and P-ERK-positive stainning in five(31.3%). P65-positive and P-ERK-positive stainning rates were higher in tumor tissues than that in tumor-surrounding tissues and in normal liver tissues(P<0.05), but RKIP-positive and P-RKIP-positive stainning rates were lower in tumor tissues than that in tumor-surrounding tissues and in normal liver tissues(P<0.05).
6 RKIP and P-RKIP protein expression levels were significantly lower in HCCs with intrahepatic or lymphatic metastasis than that in without respectively. In moderately and well differentiated HCCs both RKIP and P-RKIP positive stain rate were significantly higher than that in poorly differentiated HCCs, but both of them did not correlated with age, sex, the tumor size, AFP level and portal vein or biliary duct tumor thrombosis (P>0.05).
7 P65-positive stain was significantly higher in HCC with portal vein or biliary duct tumor thrombosis than that in without respectively. In poorly differentiated HCC P-ERK positive stain expression was significantly higher than that in moderately and well differentiated HCC (P<0.05). P65 protein expression has nothing to do with age, sex, the tumor size, intrahepatic or lymphatic metastasis and AFP level (P>0.05),
8 P-ERK-positive stain was significantly higher in HCC with intrahepatic or lymphatic metastasis, and portal vein or biliary duct tumor thrombosis than that in without respectively. In poorly differentiated HCC P-ERK positive stain expression is significantly higher than that in moderately and well differentiated HCC (P<0.05). P-ERK protein expression has nothing to do with age, sex, the tumor size and AFP level (P>0.05).
9 There was a positive relationship between RKIP and P-RKIP expression and a negative relationship between RKIP and P-ERK expression, but RKIP expression didn’t correlated with P65 expression in HCCs. Conclusions: 1 Differences observed in RKIP protein expression in HCC are probably due to posttranscriptional mechanisms.
2 P65 and P-ERK-positive stain rate were higher, but RKIP and P-RKIP-positive stain rates were lower in tumor tissues than that in tumor-surrounding tissues and in normal liver tissues, which indicated that down-regulation of RKIP and P-RKIP protein expression and up-regulation of P65 and P-ERK protein expression are associated with occurrence and development of HCC.
3 RKIP and P-RKIP protein expressions related to portal vein or biliary duct tumor thrombosis and the level of tumor differentiation in HCCs. P65 protein expression correlated with portal vein or biliary duct tumor thrombosis and the level of tumor differentiation in HCCs. P-ERK protein expression correlated with portal vein or biliary duct tumor thrombosis, intrahepatic or lymphatic metastasis and the level of tumor differentiation. These suggested that high expression of P65 and P-ERK and low expression of RKIP and P-RKIP are closely correlated with invasion and metastatis of HCC and they will be valuable indicators to prognosis, monitoring recurrence and metastasis, assessing therapeutic effect in HCC.
4 Negative correlation between RKIP and P-ERK protein expression in HCCs revealed that down-regulation of RKIP expression might serve as a risk marker for HCC development, progression and metastasis, which may contribute to elevated ERK activity.
Part II Effects of RKIP restoration on biological characters of HCC cells
Objective: To investigate the effect of RKIP restoration in HCC cells on cell proliferation, cell cycle, apoptosis, migration and invasiveness in vitro and vivo.
Methods: RKIP protein and mRNA expression levels in 4 HCC cell lines: BEL-7402, HCCLM3, SMMC-7721, and HepG2 were evaluated by RT-PCR and western-blot methods respectively. HCC cells which express lowest level of RKIP protein were transfected with pCMV5-HA-RKIP plasmid or empty vector plasmid (control) using Lipofectamine 2000 reagent. To obtain cell line transfected stably with empty vector or RKIP plasmid, selection was initiated using G418. Cell proliferation was determined using MTT methods; changes of cell cycle and apoptotic rates were measured with flow cytometry(FCM); cell motility was observed using wound Healing assay; cell invasiveness was evaluated by Matrigel invasion assay and expression of RKIP protein were detected with western blot after transfected stably with either empty vector or RKIP plasmid. Ten nude mice were inoculated subcutaneously with cells transfected stably with either empty vector or RKIP plasmid. Six weeks later, mice were executed and the length and short-diameter and weight of the transplantation tumor were measured.
Results: 1 RT-PCR and Western blot analysis showed the lowest level of RKIP mRNA and protein in HCCLM3 cells, whereas HepG2 cells expressed the highest level of RKIP.
2 The RKIP gene and protein expression in HCCLM3 cells transfected stably with RKIP plasmid increased significantly compared with those in control group (P<0.05).
3 Compared with control group, ectopic expression of RKIP decreased HCCLM3 cell proliferation significantly (P<0.05).
4 To further confirm the effect of RKIP on cell migration, control and RKIP-over expressing HCCLM3 cells were plated under confluence conditions and the monolayers were scrape wounded. Control cells migrated rapidly and closed the wound by (52.67±5.67)% after 24h and (74.33±7.02)% after 48h. In contrast, RKIP over expression only closed the wound by (31.67±3.79)% and (43.33±8.73)% in the same time interval. The wound closure rate decreased significantly in RKIP transfected group compared with that in control group (P<0.05).
5 The invasive cells of HCCLM3 were (67±14) in RKIP transfected group and were (101±12) in control group. The invasive cells decreased significantly in RKIP transfected group compared with that in control group (P<0.05). The inhibitory rate of ectopic expression of RKIP was 33.7%.
6 Flow cytometry analysis showed no difference was found in apoptotic rate and cell cycle between control and RKIP-over expressing HCCLM3 cells.
7 Six weeks after inoculated with cells transfected stably with either empty vector or RKIP plasmid average tumor volume was (2.207±0.752)cm3 versus (1.197±0.406)cm3 and average tumor weight was (1.580±0.625)g versus (0.836±0.254)g. Tumor volume and tumor weight were lower in RKIP plasmid transfected group than that in control group, respecively(P<0.05).
Conclusions: 1 HCCLM3 cell as a high metastatic human HCC cell line display decreased expression of RKIP mRNA and protein, which indicated that down-regulation of RKIP expression is associated with invasion and metastatis of HCC.
2 Restoration of RKIP can reduce cell proliferation, migration and invasiveness in HCCLM3 cells in vitro.
3 The experiment revealed that restoration of RKIP can suppress HCCLM3 cell tumor growth in vivo.
4 RKIP inhibits HCCLM3 cell proliferation, migration and invasiveness, which have nothing to do with altering the cell cycle and promoting apoptosis.
Part III Effects of down-regulation of RKIP expression on biological characters of HCC cells
Objective: To analyse whether RKIPsiRNA can inhibit RKIP expression and to investigate whether impaired RKIP may contribute to enhance the ability of cell proliferation and invasiveness in HepG2 cells in vitro and vivo.
Methods: Control small interfering RNA(C-siRNA) and siRNA specific for human RKIP(RKIP-siRNA) were synthesized and cloned into the pGenesil-2.1 plasmid vector, respectively. HepG2 cells were transfected with RKIP-siRNA plasmid or C-siRNA plasmid with LiporfectamineTM 2000 reagent. To obtain cell line transfected stably with RKIP-siRNA plasmid named HepG2-si or C-siRNA plasmid named HepG2-NC, selection was initiated using G418. Cell proliferation was determined using MTT methods; changes of cell cycle and apoptosis rates were measured by flow cytometry; cell invasiveness was evaluated by Matrigel invasion assay and expression of RKIP mRNA and protein were detected with RT-PCR and western blot, respectively. Ten nude mice were inoculated subcutaneously with cells transfected stably with either RKIP-siRNA plasmid or C-siRNA plasmid. Forty days later, mice were executed and the length and short-diameter and weight of the transplantation tumor were measured.
Results: 1 The RKIP gene and protein expression in HepG2-si group decreased significantly compared with those in HepG2-NC group (P<0.05).
2 Compared with HepG2-NC group, cell proliferation didn’t changed significantly in HepG2-si group (P>0.05).
3 The invasive cells were (87±13) in HepG2-NC group and were (118±17) in HepG2-si group. The invasive cells increased significantly in HepG2-si group compared with that in HepG2-NC group (P<0.05).
4 Flow cytometry analysis showed no difference was found in apoptotic rates and cell cycle between HepG2-NC and HepG2-si cells.
5 Forty days after inoculated, average tumor volume was (2.109±0.340)cm3 in HepG2-si group and (1.948±0.539)cm3 in HepG2-NC group. Average tumor weight was (1.308±0.27)g versus (1.236±0.283)g. Statistical analysis showed no difference was found in tumor volume and tumor weight between HepG2-NC and HepG2-si group(P>0.05).
Conclusions: 1 RKIP-siRNA can inhibit RKIP expression in mRNA and protein levels in HepG2 cells.
2 Absence of RKIP don’t alter the cell cycle and apoptosis and don’t affect cell proliferation of HepG2 cells in vitro and vivo, but can promote HepG2 cell invasion in vitro. The results indicated that RKIP plays an important role in suppressing HCC cell invasion.
PartⅣMechanisms of RKIP inhibiting invasion and metastasis in HCC
Objective: To investigate the molecular mechanisms by which RKIP inhibits HCC cell proliferation or metastasis and whether restoration or impaired RKIP may contribute to enhance or suppress activity of the Ras/Raf/MEK/ERK and /or NF-κB signaling pathway.
Methods: 1 HCCLM3 cells were transfected stably with either RKIP or vector plasmid. HepG2 cells were transfected stably with either control siRNA plasmid(C-siRNA) or RKIP siRNA plasmid(RKIP-siRNA). RKIP or vector transfected HCCLM3 cells, HepG2-NC and HepG2-RKIP-siRNA cells were stimulated with(+) or without(-) 100 nmol/L of EGF for 15 minutes after treated with 0.4μΜPDTC for 24 hours. Cytosolic and nuclear fractions were immunoblotted for RKIP, P65, phospho-ERK(PERK), and total ERK expression; actin and LaminB1 served as cytosolic and nuclear loading control, respectively.
2 RKIP or vector transfected HCCLM3 cells, HepG2-NC and HepG2-si cells were stimulated with(+) or without(-) 20 ng/ml of TNF(+) for 30 minutes after 24 hours of treated with 50μmol/L PD98059. Cytosolic and nuclear fractions were immunoblotted for RKIP, P65, phospho-ERK, and total ERK expression; actin and LaminB1 served as cytosolic and nuclear loading control, respectively.
Results: 1 Examination of ERK1/2 and phospho-ERK1/2 levels by Western blot in control and RKIP transfected HCCLM3 cells revealed downregulation of ERK activity in RKIP over expressing cells in comparison to control cells. EGF stimulation of HCCLM3 cells resulted in accumulation of phospho-ERK in the cytoplasm, which was abolished by restoration of RKIP(P<0.05). In addition, EGF stimulation resulted in increased nuclear accumulation of phospho-ERK, which was inhibited by restoration of RKIP(P<0.05). The RKIP had no effect on the total amount of ERK present in the HCCLM3 cell(P>0.05).
2 Restoration of RKIP can decrease P65 expression in the HCCLM3 cells. TNF-αstimulation of HCCLM3 cells resulted in accumulation of P65 in the cytoplasm, which was abolished by restoration of RKIP(P<0.05). In addition, TNF-αstimulation resulted in increased nuclear accumulation of P65, which was inhibited by restoration of RKIP (P<0.05).
3 Depletion of RKIP expression levels in HepG2 cells by transfection of antisense RKIP construct yielded an increase in ERK1/2 activity. In addition, EGF stimulation of HepG2 cells resulted in accumulation of phospho-ERK in the cytoplasm and nuclear (P<0.05), which was not enhanced by depletion of RKIP. The impaired RKIP had no effect on the total amount of ERK present in HepG2 cells (P>0.05).
4 The impaired RKIP had no effect on P65 expression in HepG2 cells(P>0.05). TNF-αstimulation of HepG2 cells resulted in accumulation of P65 in the cytoplasm and nuclear(P<0.05), which was not enhanced by depletion of RKIP.
Conclusions: 1 Both EGF-stimulated activation of the ERK/MAPK pathway and TNF-α-induced NF-κB signaling pathway activation can be blocked by restoration of RKIP levels, which indicated that the inhibitory effects of RKIP on proliferative and invasive potential of a human hepatocellular carcinoma cell line, HCCLM3, not only via inhibition of ERK1/2 cascade directly through inhibition of ERK1/2 phosphorylation, but also via inhibition of NF-κB signaling pathway.
2 Down-regulation of RKIP expression can promote HepG2 cell invasive potential through activation of ERK1/2 cascade but not through activation of NF-κB signaling pathway, which suggests that the mechanisms of signaling pathway regulating are complex in different HCC cells and RKIP may modulate different signaling pathway in different cell type.
Raf激酶抑制蛋白(Raf kinase inhibitor protein,RKIP)属于高度保守的磷脂酰乙醇胺结合蛋白(phosphatidylethanolamine binding protein,PEBP)家族,广泛存在于多种生物中。RKIP参与多条信号传导通路的调控,在细胞生长、增殖、分化、凋亡等生理过程中发挥重要作用。最近研究表明RKIP与肿瘤转移密切相关,但RKIP在肝细胞癌中的作用及其机制如何,目前国内外研究报道较少。
本研究首先检测肝细胞癌组织中RKIP及其可能相关蛋白P- RKIP、p65和P-ERK的表达,了解RKIP与肝细胞癌临床病理学特征的关系及其与P- RKIP、p65和P-ERK的相关性。其次,通过RKIP基因转染或干扰体外培养的肝癌细胞,观察上调或抑制RKIP表达后肝癌细胞增殖、凋亡、侵袭转移能力的变化,进一步明确RKIP在肝癌细胞侵袭转移过程中的作用。最后,检测RKIP表达或抑制后肝癌细胞Raf-MEK1/2-ERK1/2和NF-κB信号转导通路相关蛋白的表达,了解RKIP在肝细胞癌侵袭转移中的作用机制,最终明确肝癌的侵袭转移是否与RKIP的失活及Raf-MEK1/2 -ERK1/2和NF-κB信号转导通路的激活有关。
第一部分肝癌组织中Raf激酶抑制蛋白的表达及其临床意义
目的:观察肝细胞癌组织、癌旁肝组织、正常肝组织中RKIP及其可能相关蛋白P- RKIP、p65和P-ERK的表达情况,了解RKIP与肝细胞癌临床病理学特征的关系及其与P- RKIP、p65和P-ERK的相关性。
方法:RT-PCR方法检测肝细胞癌、癌旁及正常肝组织中RKIP mRNA的表达,免疫组织化学和western方法检测肝细胞癌、癌旁及正常肝组织中RKIP、P- RKIP、p65和P-ERK蛋白的表达,分析RKIP与肝细胞癌临床病理学特征的关系及其与P- RKIP、p65和P-ERK的相关性。
结果:1肝细胞癌和对应的癌旁组织、正常肝组织RKIP mRNA表达分别为1.357±0.569、1.512±0.623和1.550±0.426,差异无显著性(P=0.427)。
2 Western-blot结果显示肝细胞癌及对应的癌旁组织、正常肝组织中RKIP蛋白的表达量分别为0.579±0.380、1.178±0.659和1.115±0.442,P-RKIP蛋白的表达量分别为0.499±0.560、0.885±0.864和1.104±0.622,RKIP和P-RKIP蛋白在肝癌组织中的表达水平均显著低于癌旁及正常肝组织(P<0.05),但在癌旁和正常肝组织之间表达差异无显著性(P>0.05)。
3 Western-blot结果显示肝细胞癌组织、癌旁和正常肝组织中P65蛋白的表达量分别为0.83±0.376、0.63±0.337和0.466±0.345,P-ERK蛋白的表达量分别为1.023±0.478、0.605±0.367和0.461±0.293。P65和P-ERK在肝癌组织中的表达水平均显著高于癌旁及正常肝组织(P<0.05),但在癌旁和正常肝组织之间P65和P-ERK的表达差异无显著性(P>0.05)。
4免疫组织化学染色结果显示RKIP在72例肝细胞癌组织、50例癌旁和16例正常肝组织中的阳性表达率分别为22.2%、86%、93.8%;P-RKIP在肝细胞癌组织、癌旁和正常肝组织中的阳性表达率分别为29.2%、84%、93.8%。RKIP和P-RKIP在肝癌组织中的表达水平均显著低于癌旁及正常肝组织(P<0.05)。RKIP和P-RKIP蛋白表达在中高分化肝癌组高于低分化组,无肝内或淋巴转移组高于有肝内或淋巴转移组(P<0.05),但与肝癌肿瘤大小、AFP水平及有无门静脉或胆管癌栓无关(P>0.05)。
5免疫组织化学染色结果显示P65在72例肝细胞癌组织、50例癌旁和16例正常肝组织中的阳性表达率分别为73.6%、56%、37.5%;P-ERK在肝细胞癌组织、癌旁和正常肝组织中的阳性表达率为65.3%、38%、31.3%。P65和P-ERK蛋白在肝癌组织中的表达水平显著高于癌旁及正常肝组织(P<0.05),但在癌旁及正常肝组织表达差异无显著性(P>0.05)。P65蛋白表达水平与肝癌分化程度、有无门静脉或胆管癌栓有关(P<0.05),而与AFP水平、肿瘤大小及有无肝内或淋巴结转移无关;P-ERK蛋白表达水平与肝癌分化程度、有无门静脉或胆管癌栓及有无肝内或淋巴结转移有关(P<0.05),而与AFP水平和肿瘤大小无关。
6肝癌组织中RKIP蛋白表达与P-RKIP的表达呈显著正相关(P=0.000),与P-ERK的表达呈显著负相关(P=0.04),但与P65的表达无相关性(P=0.143)。
结论:1肝细胞癌、癌旁及正常肝组织中RKIP mRNA表达差异无显著性说明RKIP的表达可能是在转录后水平受到调节。
2 RKIP和P-RKIP表达的减少或缺失、P65和P-ERK的高表达与肝癌的发生发展密切相关。
3在中高分化肝癌组RKIP和P-RKIP蛋白表达显著高于低分化组,无肝内或淋巴转移组显著高于有肝内或淋巴转移组,而且肝癌组织中RKIP和P-RKIP蛋白表达呈正相关,提示RKIP和P-RKIP蛋白在抑制肝癌侵袭转移过程中共同发挥作用。
4 P65蛋白表达水平与肝癌分化程度、有无门静脉或胆管癌栓有关, P-ERK蛋白表达与肝癌分化程度、有无门静脉或胆管癌栓以及有无肝内或淋巴结转移有关,提示P65和P-ERK在肝癌侵袭转移过程中发挥作用。
5 RKIP与P-ERK的表达呈显著负相关,提示RKIP表达的减少或缺失可能通过上调P-ERK的表达促进肝细胞癌的侵袭转移。
第二部分上调Raf激酶抑制蛋白的表达对肝癌细胞生物学行为的影响
目的:RKIP表达质粒稳定转染RKIP低表达肝癌细胞株,体内外实验明确RKIP对肝癌细胞增殖、侵袭、凋亡及细胞周期的影响。
方法:RT-PCR和western-blot方法对BEL7402、HepG2、HCCLM3和SMMC7721四种人肝癌细胞株进行检测,筛选出RKIP低表达肝癌细胞株,行RKIP质粒转染,G418筛选后,经RT-PCR和western-blot方法鉴定,获得稳定转染空载体和表达RKIP蛋白的细胞克隆。通过MTT法绘制细胞生长曲线,观察肝癌细胞增殖的变化;流式细胞术(FCM)观察RKIP表达对肝癌细胞的细胞周期和凋亡率的影响;通过进行单层细胞划痕实验和Matrigel侵袭实验检测肝癌细胞迁移能力和侵袭力的变化;对照组和RKIP转染组肝癌细胞接种裸鼠,建立人肝癌细胞裸鼠皮下移植瘤模型,观察RKIP对肝癌细胞增殖和转移能力的影响。
结果:1 BEL7402、HepG2、HCCLM3和SMMC7721四种人肝癌细胞株RKIPmRNA的表达量分别为0.349±0.025、0.885±0.015、0.290±0.015和0.602±0.031,RKIP蛋白的表达量分别为0.582±0.056、1.389±0.079、0.428±0.021和0.733±0.089。HepG2肝癌细胞RKIP mRNA和蛋白表达水平均显著高于其它三株(P<0.05),而HCCLM3肝癌细胞RKIP mRNA和蛋白表达水平均显著低于其它三株(P<0.05)。
2 RKIP表达质粒和空质粒稳定转染HCCLM3人肝癌细胞, RT-PCR和western blot结果显示RKIP转染组HCCLM3细胞中RKIP mRNA和蛋白的表达显著高于对照组(P<0.05)。
3根据MTT结果绘制细胞生长曲线,结果示:从第3天开始,RKIP转染组细胞生长显著慢于对照组(P<0.05)。
4划痕实验结果显示划痕24h后,对照组的细胞迁移了52.67±5.67%,而RKIP转染组的细胞仅迁移31.67±3.79%,差异具有显著性(P=0.006);48h后,对照组细胞迁移了74.33±7.02%,而RKIP转染组细胞仅迁移43.33±8.73%,差异有显著性(P=0.009)。
5侵袭实验结果显示对照组和RKIP转染组HCCLM3细胞发生侵袭的细胞个数分别为:101±12和67±14,转染组侵袭细胞数显著低于对照组(P<0.05), RKIP对HCCLM3肝癌细胞侵袭力抑制率为33.7%。
6与对照组比较,RKIP转染组HCCLM3肝癌细胞的细胞周期和凋亡率无显著改变(P>0.05)。
7种植对照组和RKIP转染组HCCLM3肝癌细胞的裸鼠,在接种细胞后2周左右均成瘤,6周后肿瘤体积分别为(2.207±0.752)cm3和(1.197±0.406)cm3,瘤重分别为(1.580±0.625)g和(0.836±0.254)g,转染组瘤重和体积均显著小于对照组(P<0.05),两组均未见肺转移。
结论:1高转移性肝癌细胞系HCCLM3细胞RKIPmRNA和蛋白低表达,提示RKIP的低表达可能与肝癌细胞的侵袭、转移等恶性行为有关。
2 RKIP基因成功转入并在HCCLM3肝癌细胞中稳定表达。
3 RKIP基因转染后,HCCLM3肝癌细胞体外增殖率、细胞的迁移速度和体外侵袭力均明显降低,说明RKIP表达能抑制HCCLM3肝癌细胞的体外增殖能力和侵袭力。
4成功建立转染组和对照组HCCLM3人肝癌细胞裸鼠皮下移植瘤模型,结果显示RKIP不影响HCCLM3细胞成瘤性,但能够抑制HCCLM3肝癌细胞的体内增殖能力。
5 RKIP对HCCLM3肝癌细胞的细胞周期和凋亡率无影响,说明RKIP表达不能促进HCCLM3肝癌细胞凋亡,RKIP表达抑制HCCLM3肝癌细胞增殖和侵袭力与细胞周期的改变无关。
第三部分抑制Raf激酶抑制蛋白的表达对肝癌细胞生物学行为的影响
目的:观察RKIP-siRNA对肝癌细胞中RKIP mRNA及其蛋白表达的抑制作用以及对肝癌细胞生物学行为的影响;同时建立人肝癌细胞裸鼠皮下移植瘤模型,观察RKIP基因沉默对HepG2细胞体内增殖活性的影响。
方法:构建RKIP-siRNA和针对性设计的阴性对照RKIP-ncRNA质粒载体,脂质体介导转染RKIP高表达细胞株HepG2细胞,G418筛选,扩大培养的细胞分别命名为HepG2-NC和HepG2-si。HepG2-NC和HepG2-si细胞经RT-PCR和western-blot方法鉴定,观察RKIP-siRNA是否成功沉默人HepG2肝癌细胞中RKIP基因表达。通过MTT法绘制细胞生长曲线,观察肝癌细胞增殖的变化;流式细胞术观察RKIP表达对肝癌细胞的细胞周期和凋亡率的影响;通过Matrigel侵袭实验检测肝癌细胞侵袭力的变化;HepG2-NC和HepG2-si细胞接种裸鼠,建立人肝癌细胞裸鼠皮下移植瘤模型,体内实验观察RKIP对肝癌细胞增殖的影响。
结果:1 HepG2-NC和HepG2-si组细胞RKIPmRNA表达水平分别为0.873±0.035和0.421±0.028,RKIP蛋白表达水平分别为1.36±0.068和0.524±0.024。HepG2-si组RKIP基因表达水平显著低于HepG2-NC组(P<0.05)。
2 MTT结果显示,在相同的初始细胞浓度条件下,各个时间点细胞增殖差异无统计学意义(P>0.05)。细胞生长曲线示HepG2-NC组和HepG2-si组细胞增殖速度相近。
3在侵袭实验中,HepG2-NC组和HepG2-si组细胞发生侵袭的细胞个数分别为87±13和118±17,HepG2-si组侵袭细胞数显著高于HepG2-NC组(P=0.005)。
4 HepG2-NC组和HepG2-si组凋亡率和细胞周期变化无显著性(P>0.05)。
5 HepG2-si和HepG2-NC组细胞接种裸鼠,在接种细胞后10天左右均可见肿瘤结节形成。40天后,HepG2-si组和HepG2-NC组肿瘤体积分别为:(2.109±0.340)cm3和(1.948±0.539)cm3;瘤重分别为(1.308±0.27)g和(1.236±0.283)g,两组之间肿瘤体积和重量差异均无显著性(P>0.05)。
结论:1 RKIP-siRNA成功沉默人HepG2肝癌细胞中RKIP基因。
2 RKIP-siRNA不改变HepG2肝癌细胞的细胞凋亡率和细胞周期,也不影响HepG2肝癌细胞体内外的增殖能力,但能够增强HepG2细胞的体外侵袭能力,说明RKIP基因在抑制肝癌侵袭转移方面发挥重要作用。
第四部分Raf激酶抑制蛋白抑制肝癌侵袭转移的作用机制
目的:以稳定转染空质粒和RKIP基因的HCCLM3肝癌细胞、稳定转染RKIP-ncRNA质粒和RKIP-siRNA质粒的HepG2肝癌细胞为研究对象,检测肝癌细胞Raf-MEK1/2 -ERK1/2和NF-κB信号转导通路相关蛋白的表达,明确RKIP在肝癌侵袭转移中的作用机制。
方法:1 HCCLM3- C(对照组)、HCCLM3-RKIP(转染组)、HepG2-NC和HepG2-si肝癌细胞加入NF-κB特异性抑制剂PDTC, 48h后EGF处理组加入EGF,激活ERK通路,15分钟后提取细胞胞浆和胞核蛋白,考马斯亮兰法蛋白定量。Western blot检测P65、ERK和P-ERK蛋白表达,观察RKIP表达或抑制对肝癌细胞ERK信号通路的影响。
2 HCCLM3-C(对照组)、HCCLM3-RKIP(转染组)、HepG2-NC和HepG2-si组肝癌细胞加入ERK信号通路特异性抑制剂PD98059, 48h后TNF-α处理组加入TNF-α,激活NF-κB通路,30分钟后提取细胞胞浆和胞核蛋白,考马斯亮兰法蛋白定量。Western blot检测P65、ERK和P-ERK蛋白表达,观察RKIP表达或抑制对肝癌细胞NF-κB通路信号通路的影响。
结果:1 RKIP转染组HCCLM3细胞RKIP蛋白的表达显著高于对照组(P<0.05),EGF对RKIP的表达无影响;EGF(+)对照组中HCCLM3细胞胞浆和胞核P-ERK的表达明显高于EGF(-)对照组(P<0.05),而在EGF(-)转染组和EGF(+)转染组之间P-ERK的表达差异无统计学意义(P>0.05);与EGF(-)对照组比较,EGF(-)转染组HCCLM3细胞胞浆和胞核P-ERK的表达显著降低(P<0.05);EGF(+)转染组HCCLM3细胞胞浆和胞核P-ERK的表达显著低于EGF(+)对照组(P<0.05)。
2 TNF-α(+)对照组中HCCLM3细胞胞浆和胞核P65的表达明显高于TNF-α(-)对照组(P<0.05),而在TNF-α(-)转染组和TNF-α(+)转染组之间P65的表达差异无统计学意义(P>0.05);与TNF-α(-)对照组比较,TNF-α(-)转染组HCCLM3细胞胞浆和胞核P65的表达显著降低(P<0.05);TNF-α(+)转染组HCCLM3细胞胞浆和胞核P65的表达显著低于TNF-α(+)对照组(P<0.05)。
3 RKIP-siRNA组HepG2细胞RKIP蛋白表达显著低于对照组(P<0.05),EGF对两组HepG2细胞RKIP的表达无影响;EGF(+)NC组中HepG2细胞胞浆和胞核P-ERK的表达显著高于EGF(-)NC组(P<0.05);EGF(+)-si组HepG2细胞P-ERK的表达显著高于EGF(-)-si组;与EGF(-)-NC组比较,EGF(-)-si组HepG2细胞胞浆和胞核P-ERK的表达显著升高(P<0.05);在EGF(+)-si组和EGF(+)-NC组之间,HepG2细胞胞浆和胞核P-ERK的表达无显著性差异(P>0.05)。
4 TNF-α对两组HepG2细胞RKIP的表达无影响(P>0.05);TNF-α(+)-NC组中HepG2细胞胞浆和胞核P65的表达明显高于TNF-α(-)-NC;在TNF-α(+)-si组P65的表达高于TNF-α(-)-si组;与TNF-α(-)-NC比较,TNF-α(-)-si组HepG2细胞胞浆和胞核P65的表达无显著性差异(P>0.05);与TNF-α(+)-NC组比较,TNF-α(+)-si组HepG2细胞胞浆和胞核P65的表达无显著差异(P>0.05)。
结论:1 RKIP表达不仅能够抑制HCCLM3细胞ERK的磷酸化,还能够抑制EGF诱导的HCCLM3细胞ERK的磷酸化,说明RKIP抑制肝癌细胞的增殖和侵袭可能是通过抑制ERK信号通路的活化来实现的。
2 RKIP不仅能够抑制HCCLM3细胞NF-κB信号通路的活化,还能够抑制TNF-α对HCCLM3细胞NF-κB信号传导通路的激活作用,说明RKIP抑制HCCLM3肝癌细胞的增殖、侵袭、转移不仅与抑制ERK信号通路的活化有关,也与NF-κB信号传导通路抑制有关。
3 RKIP表达的下调够活化HepG2细胞ERK信号通路,而对HepG2细胞NF-κB信号传导通路无影响,说明RKIP表达下调可能通过活化ERK信号通路促进HepG2肝癌细胞的侵袭,也说明肝癌细胞信号传导通路调节的复杂性以及RKIP在不同的细胞其作用机制不同。
Hepatocellular carcinoma(HCC) is the 5th commonest malignancy worldwide and is the third most common cause of cancer-related death. HCC is one of the most common malignant tumors in our country, which is frequently associated with metastasis in early stage and has a high rate of recurrence after operation or intervention treatment. However, the long-term prognosis of patients undergoing potentially curative hepatic resection is still poor, with reported 3-year survival rates ranging from 40% to 50%. This poor prognosis mainly contributes to the high rate of intra-hepatic and distant metastasis after resection or transplantation. To date, the metastatic mechanisms and the regulated mechanisms of tumors are not known clearly, so it is very important for understanding the mechanisms of invasion and metastasis and searching effective approaches to prevent the recurrence and metastasis of HCC is of great importance.
Raf kinase inhibitor protein(RKIP) is a member of the phosphatidylethanolamine binding protein family, a ubiquitously expressed and evolutionarily conserved group of proteins. In recent years, it has been reported that RKIP influence intracellular signaling cascades, cell cycle regulation, the suppression of metastasis, neurode generative processes, the modulation of emotions, and reproduction, and RKIP has been identified as a member of a novel class of molecules that suppress the metastatic spread of tumors, but little is known about the role and the regulating mechanisms of RKIP in human hepatocarcinogenesis.
In this study, we used immunohistochemistry and western blot to analyze expressions of RKIP, P- RKIP, p65 and P-ERK in HCC tissues, paratumor tissues and normal liver tissues, and to determine the relationship between the former four factors in HCC tissues and the feature of invasion and metastasis, and between RKIP and P-RKIP, p65, P-ERK in HCC. Nextly, cell proliferation, cell cycle, apoptosis rates and migration assays were analyzed after RKIP over expression or knockdown HCC cell lines in vitro or vivo. Finally, we underlying mechanisms of RKIP were assessed with immunoblot analysis after either over expression or knockdown of RKIP expression in HCC cell lines.
Part I Expression and significance of Raf kinase inhibitor protein in hepatocellular carcinoma
Objective: To investigate the expression of RKIP, P-RKIP, p65 and P-ERK in HCC and the correlation with the invasion and metastasis of HCC. Methods: Reverse transcription polymerase chain reaction(RT-PCR) was used to detect expression of RKIP mRNA. The expression levels of RKIP, P- RKIP, p65 and P-ERK protein in HCC tumor and peritumoral tissues were determined by immunohistochemistry and Western blot analysis. Statistical analysis was used to determine the relationship between their expression and clinicopathological parameters.
Results: 1 Semi-quantity RT-PCR analysis showed there was no significant difference in RKIP mRNA expression levels among HCC tumors, corresponding peritumoral tissues and normal liver tissues(P=0.427).
2 Immunoblot analysis of 12 normal liver tisues, 36 paired HCCs and adjacent uninvolved tissue samples showed the ratio of RKIP protein expression level(RKIP/actin) was 0.579±0.380 in HCCs, 1.178±0.659 in peritumoral tissues and 1.115±0.442 in normal liver tissues; the ratio of P-RKIP protein expression level(RKIP/actin) was 0.499±0.560, 0.885±0.864 and 1.104±0.622, respectively. RKIP and P-RKIP protein expression levels were decreased in HCCs compared with adjacent peritumoral tissues and normal liver tisues(P<0.05), but there was no significant difference in RKIP or P-RKIP protein expression levels between peritumoral tissues and normal liver tissues(P>0.05).
3 Immunoblot analysis showed the ratio of P65 protein expression level(RKIP/actin) was 0.83±0.376 in HCCs, 0.63±0.337 in peritumoral tissues and 0.466±0.345 in normal liver tissues; the ratio of P-ERK protein level(RKIP/actin) was 1.023±0.478, 0.605±0.367 and 0.461±0.293, respectively. The results showed increased P65 and P-ERK protein levels in HCCs compared with adjacent peritumoral tissues and normal liver tisues(P<0.05), but there was no significant difference in P65 or P-ERK protein expression levels between peritumoral tissues and normal liver tissues(P>0.05).
4 The expression of RKIP, P-RKIP, P65 and P-ERK protein were evaluated by immunohistochemistry in 72 human HCC tumors, 50 peritumoral tissues and 16 normal live tissues. RKIP-positive stainning was detected only in sixteen of 72 tumor tissues(22.2%), P-RKIP-positive stainning in twenty one (29.2%), P65-positive stainning in fifty three(73.6%), and P-ERK-positive stainning in forty seven(65.3%). In contrast, RKIP-positive stainning was in forty seven of 50 tumor-surrounding tissues(86.0%), P-RKIP-positive stainning in forty two (84.0%), P65-positive stain in twenty eight(56.0%), and P-ERK-positive stainning in 19(38%). In normal liver tissues fifteen of 16 RKIP-positive and P-RKIP-positive stainning was observed(93.8%), P65-positive stainning only in six(37.5%) and P-ERK-positive stainning in five(31.3%). P65-positive and P-ERK-positive stainning rates were higher in tumor tissues than that in tumor-surrounding tissues and in normal liver tissues(P<0.05), but RKIP-positive and P-RKIP-positive stainning rates were lower in tumor tissues than that in tumor-surrounding tissues and in normal liver tissues(P<0.05).
6 RKIP and P-RKIP protein expression levels were significantly lower in HCCs with intrahepatic or lymphatic metastasis than that in without respectively. In moderately and well differentiated HCCs both RKIP and P-RKIP positive stain rate were significantly higher than that in poorly differentiated HCCs, but both of them did not correlated with age, sex, the tumor size, AFP level and portal vein or biliary duct tumor thrombosis (P>0.05).
7 P65-positive stain was significantly higher in HCC with portal vein or biliary duct tumor thrombosis than that in without respectively. In poorly differentiated HCC P-ERK positive stain expression was significantly higher than that in moderately and well differentiated HCC (P<0.05). P65 protein expression has nothing to do with age, sex, the tumor size, intrahepatic or lymphatic metastasis and AFP level (P>0.05),
8 P-ERK-positive stain was significantly higher in HCC with intrahepatic or lymphatic metastasis, and portal vein or biliary duct tumor thrombosis than that in without respectively. In poorly differentiated HCC P-ERK positive stain expression is significantly higher than that in moderately and well differentiated HCC (P<0.05). P-ERK protein expression has nothing to do with age, sex, the tumor size and AFP level (P>0.05).
9 There was a positive relationship between RKIP and P-RKIP expression and a negative relationship between RKIP and P-ERK expression, but RKIP expression didn’t correlated with P65 expression in HCCs. Conclusions: 1 Differences observed in RKIP protein expression in HCC are probably due to posttranscriptional mechanisms.
2 P65 and P-ERK-positive stain rate were higher, but RKIP and P-RKIP-positive stain rates were lower in tumor tissues than that in tumor-surrounding tissues and in normal liver tissues, which indicated that down-regulation of RKIP and P-RKIP protein expression and up-regulation of P65 and P-ERK protein expression are associated with occurrence and development of HCC.
3 RKIP and P-RKIP protein expressions related to portal vein or biliary duct tumor thrombosis and the level of tumor differentiation in HCCs. P65 protein expression correlated with portal vein or biliary duct tumor thrombosis and the level of tumor differentiation in HCCs. P-ERK protein expression correlated with portal vein or biliary duct tumor thrombosis, intrahepatic or lymphatic metastasis and the level of tumor differentiation. These suggested that high expression of P65 and P-ERK and low expression of RKIP and P-RKIP are closely correlated with invasion and metastatis of HCC and they will be valuable indicators to prognosis, monitoring recurrence and metastasis, assessing therapeutic effect in HCC.
4 Negative correlation between RKIP and P-ERK protein expression in HCCs revealed that down-regulation of RKIP expression might serve as a risk marker for HCC development, progression and metastasis, which may contribute to elevated ERK activity.
Part II Effects of RKIP restoration on biological characters of HCC cells
Objective: To investigate the effect of RKIP restoration in HCC cells on cell proliferation, cell cycle, apoptosis, migration and invasiveness in vitro and vivo.
Methods: RKIP protein and mRNA expression levels in 4 HCC cell lines: BEL-7402, HCCLM3, SMMC-7721, and HepG2 were evaluated by RT-PCR and western-blot methods respectively. HCC cells which express lowest level of RKIP protein were transfected with pCMV5-HA-RKIP plasmid or empty vector plasmid (control) using Lipofectamine 2000 reagent. To obtain cell line transfected stably with empty vector or RKIP plasmid, selection was initiated using G418. Cell proliferation was determined using MTT methods; changes of cell cycle and apoptotic rates were measured with flow cytometry(FCM); cell motility was observed using wound Healing assay; cell invasiveness was evaluated by Matrigel invasion assay and expression of RKIP protein were detected with western blot after transfected stably with either empty vector or RKIP plasmid. Ten nude mice were inoculated subcutaneously with cells transfected stably with either empty vector or RKIP plasmid. Six weeks later, mice were executed and the length and short-diameter and weight of the transplantation tumor were measured.
Results: 1 RT-PCR and Western blot analysis showed the lowest level of RKIP mRNA and protein in HCCLM3 cells, whereas HepG2 cells expressed the highest level of RKIP.
2 The RKIP gene and protein expression in HCCLM3 cells transfected stably with RKIP plasmid increased significantly compared with those in control group (P<0.05).
3 Compared with control group, ectopic expression of RKIP decreased HCCLM3 cell proliferation significantly (P<0.05).
4 To further confirm the effect of RKIP on cell migration, control and RKIP-over expressing HCCLM3 cells were plated under confluence conditions and the monolayers were scrape wounded. Control cells migrated rapidly and closed the wound by (52.67±5.67)% after 24h and (74.33±7.02)% after 48h. In contrast, RKIP over expression only closed the wound by (31.67±3.79)% and (43.33±8.73)% in the same time interval. The wound closure rate decreased significantly in RKIP transfected group compared with that in control group (P<0.05).
5 The invasive cells of HCCLM3 were (67±14) in RKIP transfected group and were (101±12) in control group. The invasive cells decreased significantly in RKIP transfected group compared with that in control group (P<0.05). The inhibitory rate of ectopic expression of RKIP was 33.7%.
6 Flow cytometry analysis showed no difference was found in apoptotic rate and cell cycle between control and RKIP-over expressing HCCLM3 cells.
7 Six weeks after inoculated with cells transfected stably with either empty vector or RKIP plasmid average tumor volume was (2.207±0.752)cm3 versus (1.197±0.406)cm3 and average tumor weight was (1.580±0.625)g versus (0.836±0.254)g. Tumor volume and tumor weight were lower in RKIP plasmid transfected group than that in control group, respecively(P<0.05).
Conclusions: 1 HCCLM3 cell as a high metastatic human HCC cell line display decreased expression of RKIP mRNA and protein, which indicated that down-regulation of RKIP expression is associated with invasion and metastatis of HCC.
2 Restoration of RKIP can reduce cell proliferation, migration and invasiveness in HCCLM3 cells in vitro.
3 The experiment revealed that restoration of RKIP can suppress HCCLM3 cell tumor growth in vivo.
4 RKIP inhibits HCCLM3 cell proliferation, migration and invasiveness, which have nothing to do with altering the cell cycle and promoting apoptosis.
Part III Effects of down-regulation of RKIP expression on biological characters of HCC cells
Objective: To analyse whether RKIPsiRNA can inhibit RKIP expression and to investigate whether impaired RKIP may contribute to enhance the ability of cell proliferation and invasiveness in HepG2 cells in vitro and vivo.
Methods: Control small interfering RNA(C-siRNA) and siRNA specific for human RKIP(RKIP-siRNA) were synthesized and cloned into the pGenesil-2.1 plasmid vector, respectively. HepG2 cells were transfected with RKIP-siRNA plasmid or C-siRNA plasmid with LiporfectamineTM 2000 reagent. To obtain cell line transfected stably with RKIP-siRNA plasmid named HepG2-si or C-siRNA plasmid named HepG2-NC, selection was initiated using G418. Cell proliferation was determined using MTT methods; changes of cell cycle and apoptosis rates were measured by flow cytometry; cell invasiveness was evaluated by Matrigel invasion assay and expression of RKIP mRNA and protein were detected with RT-PCR and western blot, respectively. Ten nude mice were inoculated subcutaneously with cells transfected stably with either RKIP-siRNA plasmid or C-siRNA plasmid. Forty days later, mice were executed and the length and short-diameter and weight of the transplantation tumor were measured.
Results: 1 The RKIP gene and protein expression in HepG2-si group decreased significantly compared with those in HepG2-NC group (P<0.05).
2 Compared with HepG2-NC group, cell proliferation didn’t changed significantly in HepG2-si group (P>0.05).
3 The invasive cells were (87±13) in HepG2-NC group and were (118±17) in HepG2-si group. The invasive cells increased significantly in HepG2-si group compared with that in HepG2-NC group (P<0.05).
4 Flow cytometry analysis showed no difference was found in apoptotic rates and cell cycle between HepG2-NC and HepG2-si cells.
5 Forty days after inoculated, average tumor volume was (2.109±0.340)cm3 in HepG2-si group and (1.948±0.539)cm3 in HepG2-NC group. Average tumor weight was (1.308±0.27)g versus (1.236±0.283)g. Statistical analysis showed no difference was found in tumor volume and tumor weight between HepG2-NC and HepG2-si group(P>0.05).
Conclusions: 1 RKIP-siRNA can inhibit RKIP expression in mRNA and protein levels in HepG2 cells.
2 Absence of RKIP don’t alter the cell cycle and apoptosis and don’t affect cell proliferation of HepG2 cells in vitro and vivo, but can promote HepG2 cell invasion in vitro. The results indicated that RKIP plays an important role in suppressing HCC cell invasion.
PartⅣMechanisms of RKIP inhibiting invasion and metastasis in HCC
Objective: To investigate the molecular mechanisms by which RKIP inhibits HCC cell proliferation or metastasis and whether restoration or impaired RKIP may contribute to enhance or suppress activity of the Ras/Raf/MEK/ERK and /or NF-κB signaling pathway.
Methods: 1 HCCLM3 cells were transfected stably with either RKIP or vector plasmid. HepG2 cells were transfected stably with either control siRNA plasmid(C-siRNA) or RKIP siRNA plasmid(RKIP-siRNA). RKIP or vector transfected HCCLM3 cells, HepG2-NC and HepG2-RKIP-siRNA cells were stimulated with(+) or without(-) 100 nmol/L of EGF for 15 minutes after treated with 0.4μΜPDTC for 24 hours. Cytosolic and nuclear fractions were immunoblotted for RKIP, P65, phospho-ERK(PERK), and total ERK expression; actin and LaminB1 served as cytosolic and nuclear loading control, respectively.
2 RKIP or vector transfected HCCLM3 cells, HepG2-NC and HepG2-si cells were stimulated with(+) or without(-) 20 ng/ml of TNF(+) for 30 minutes after 24 hours of treated with 50μmol/L PD98059. Cytosolic and nuclear fractions were immunoblotted for RKIP, P65, phospho-ERK, and total ERK expression; actin and LaminB1 served as cytosolic and nuclear loading control, respectively.
Results: 1 Examination of ERK1/2 and phospho-ERK1/2 levels by Western blot in control and RKIP transfected HCCLM3 cells revealed downregulation of ERK activity in RKIP over expressing cells in comparison to control cells. EGF stimulation of HCCLM3 cells resulted in accumulation of phospho-ERK in the cytoplasm, which was abolished by restoration of RKIP(P<0.05). In addition, EGF stimulation resulted in increased nuclear accumulation of phospho-ERK, which was inhibited by restoration of RKIP(P<0.05). The RKIP had no effect on the total amount of ERK present in the HCCLM3 cell(P>0.05).
2 Restoration of RKIP can decrease P65 expression in the HCCLM3 cells. TNF-αstimulation of HCCLM3 cells resulted in accumulation of P65 in the cytoplasm, which was abolished by restoration of RKIP(P<0.05). In addition, TNF-αstimulation resulted in increased nuclear accumulation of P65, which was inhibited by restoration of RKIP (P<0.05).
3 Depletion of RKIP expression levels in HepG2 cells by transfection of antisense RKIP construct yielded an increase in ERK1/2 activity. In addition, EGF stimulation of HepG2 cells resulted in accumulation of phospho-ERK in the cytoplasm and nuclear (P<0.05), which was not enhanced by depletion of RKIP. The impaired RKIP had no effect on the total amount of ERK present in HepG2 cells (P>0.05).
4 The impaired RKIP had no effect on P65 expression in HepG2 cells(P>0.05). TNF-αstimulation of HepG2 cells resulted in accumulation of P65 in the cytoplasm and nuclear(P<0.05), which was not enhanced by depletion of RKIP.
Conclusions: 1 Both EGF-stimulated activation of the ERK/MAPK pathway and TNF-α-induced NF-κB signaling pathway activation can be blocked by restoration of RKIP levels, which indicated that the inhibitory effects of RKIP on proliferative and invasive potential of a human hepatocellular carcinoma cell line, HCCLM3, not only via inhibition of ERK1/2 cascade directly through inhibition of ERK1/2 phosphorylation, but also via inhibition of NF-κB signaling pathway.
2 Down-regulation of RKIP expression can promote HepG2 cell invasive potential through activation of ERK1/2 cascade but not through activation of NF-κB signaling pathway, which suggests that the mechanisms of signaling pathway regulating are complex in different HCC cells and RKIP may modulate different signaling pathway in different cell type.
引文
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