热休克蛋白27促进人肝细胞肝癌侵袭转移的分子机制研究
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摘要
在我国，肝细胞肝癌(human hepatocellular carcinoma，HCC)是居恶性肿瘤死亡率第二位的一种疾病，其复发和转移一直是影响肝癌治疗效果的重要因素。肝癌细胞要获得侵袭转移能力，必然导致细胞内外信号转导异常，揭示其浸润转移的信号转导分子机制将是肝细胞肝癌防治的关键所在。近年来，小热休克蛋白27(HSP27)作为肿瘤预后评价的可信指标之一，已经在多种肿瘤中得到证实。HSP27可通过调节一系列信号转导，参与肿瘤的侵袭转移过程。本研究通过对转移潜能不同的人肝细胞肝癌细胞系信号转导相关基因表达谱的差异分析，发现了丝裂原激活的蛋白激酶途径(MAPK pathway)和凋亡途径(apoptosis pathway)与肝癌细胞转移潜能的形成密切相关，并进一步解析了HSP27在这些通路中的调控机制及其与肝癌侵袭转移的关系。
第一部分转移潜能不同肝癌细胞系中HSP27的表达及转移相关信号转导通路的基因组学分析
本部分的研究是应用细胞免疫荧光检测技术结合反转录PCR(RT-PCR)，实时荧光定量PCR，蛋白印迹(western blot)及免疫酶联吸附实验(ELISA)等技术，对转移潜能不同的人肝细胞肝癌细胞系Hep3B、MHCC97L、MHCC97H、HCCLM6中HSP27的定位及表达进行分析。运用RNA干扰(RNAi)技术，观察HSP27与肝癌细胞侵袭迁移能力的关系。另外，利用基因芯片检测技术，比较MHCC97L、MHCC97H、HCCLM6细胞系信号转导相关基因表达谱的差异，运用KOBAS软件分析系统对差异基因所涉及的信号通路进行统计分析，筛查在肝癌细胞转移潜能形成过程中发挥重要作用的信号转导通路。
应用细胞免疫荧光技术检测HSP27在体外培养肝癌细胞系中的定位，结果显示HSP27主要定位在肝癌细胞细胞浆内，亦见于细胞核。通过RT-PCR、实时荧光定量PCR技术检测HSP27的基因表达水平，发现HSP27在有转移潜能人肝癌细胞系MHCC97L、MHCC97H、HCCLM6中mRNA表达水平明显高于无转移潜能的Hep3B，其中HCCLM6细胞系最高。利用western blot和HSP27蛋白ELISA方法，证实在四种肝癌细胞系中HSP27蛋白表达水平与上述mRNA水平一致。此外，应用RNA干扰技术，设置三个平行实验组：对照组(HCCLM6细胞)、MOCK组(转染一小段非特异性dsRNA，但该dsRNA不针对任何基因，经验证转染后不抑制基因表达)和RNA干扰组(转染HSP27特异性dsRNA)。在转染100nM HSP27特异性siRNA 36、48小时后，HCCLM6细胞内HSP27mRNA均下降了80％以上而蛋白水平分别与70％、92％；与MOCK组相比，RNA干扰组侵袭和迁移的HCCLM6细胞数分别下降了53％与64％。这些结果提示HSP27参与了肝癌细胞侵袭迁移过程，它的高表达可能与肝癌细胞转移潜能的增高有关。
选取遗传背景相同而转移潜能不同的肝癌细胞系MHCC97L、MHCC97H、HCCLM6，提取总RNA，逆转录成cDNA，cy3和cy5标记后，进行人类信号转导寡核苷酸微阵列检测，结果显示三种肝癌细胞系中连续变化的差异基因有62个，差异倍数.1.5倍且连续上调的基因17个。通过KOBAS软件分析，发现芯片中的897个信号转导相关基因涉及了142条KEGG通路，其中62个连续变化的差异基因涉及66条通路，连续上调、差异倍数.1.5倍的17个差异基因涉及28条通路。这28条信号通路中通过FDR校正后仅有6条通路的P值＜0.05，其中MAPK通路(0.0189)、凋亡通路(0.0365)排在前两位，推测这些通路的持续活化可能与肝癌侵袭转移有关。
第二部分HSP27经由PKC.-ERK／p38 MAPK信号通路促进肝癌转移的分子机制研究
本部分的研究目的是应用RNA干扰技术结合激酶特异性抑制剂和激动剂、蛋白免疫印迹技术、体外侵袭迁移实验，探讨肝细胞肝癌细胞内MAPK通路与HSP27的关系，证实通路中关键靶标分子的功能效应，揭示其在肝癌细胞转移潜能形成中的作用，以获得潜在的肝癌诊断标记及治疗靶标。
本研究应用蛋白免疫印迹技术检测MHCC97L、MHCC97H、HCCLM6肝癌细胞中HSP27和MAPK通路三个关键活性分子ERK1／2、p38MAPK、JNK及显著差异表达基因PKC.的蛋白磷酸化水平，结果显示在三种转移性肝癌细胞内磷酸化HSP27、磷酸化PKC.和磷酸化ERK1／2、磷酸化p38 MAPK逐渐增高，而磷酸化JNK没有明显变化，这提示HSP27、PKC.的持续活化及ERK1／2和p38MAPK通路的激活可能与肝癌细胞转移能力增强有关。
在转移潜能最高的HCCLM6肝癌细胞内，分别利用MAPK特异性抑制剂SB203580(5.M，p38MAPK特异性抑制剂)、U0126(10.M，ERK1／2特异性抑制剂)和SP600125(10.M，JNK特异性抑制剂)处理2h后，发现前两者能有效抑制HSP27的磷酸化，而SP600125对HSP27的磷酸化没有明显抑制，说明在肝癌细胞内HSP27的激活受p38 MAPK和ERK1／2所调节。利用RNA干扰技术、PKC.特异性抑制剂LY317615结合PKC激动剂PMA，发现LY317615能有效抑制肝癌细胞内p38MAPK、ERK1／2、和HSP27的磷酸化，而对JNK的磷酸化没有明显抑制，这说明PKC.的活性缺失能有效抑制ERK1／2-HSP27和p38MAPK-HSP27的活化；还发现与PMA处理组相比，PKC.RNAi+PMA处理组磷酸化ERK1／2、p38 MAPK和HSP27减少。这些结果证实了肝癌细胞内HSP27是PKC.-ERK1／2和PKC.-p38MAPK下游的活性分子。
为了探究PKC.-ERK1／2／p38MAPK-HSP27通路与肝癌细胞转移表型的关系，我们利用RNA干扰技术和PKC.特异性抑制剂LY317615结合体外细胞侵袭迁移实验观察HCCLM6肝癌细胞侵袭迁移能力的变化，发现与MOCK组相比，PKC.RNAi(70nM)处理组侵袭和迁移的细胞数分别下降了56％和51％；LY317615(0.025.M)处理组与其对照组相比则分别下降了36％和38％。此外，还发现PMA(PKC激动剂)处理组与未处理组(HCCLM6细胞)相比，侵袭、迁移的细胞数分别增加了26％和23％，而PKC.RNAi处理组与PKC.RNAi+PMA处理组侵袭、迁移的肝癌细胞数没有统计学差别。以上结果表明PKC.蛋白的缺失及其活性抑制降低了肝癌细胞的侵袭迁移能力；PMA所致肝癌细胞侵袭迁移能力的增强是PKC.依赖性的。
进一步试验发现，与PMA处理组相比，SB203580+PMA处理组与U0126+PMA处理组侵袭、迁移的细胞分别降低了29％、31％和34％、37％。这些结果说明p38 MAPK和ERK1／2的抑制能降低PMA激活的、PKC.介导的肝癌细胞侵袭迁移的增加。同时发现，与PMA处理组相比，SB203580+PMA处理组、U0126+PMA处理组中HSP27的磷酸化水平降低，这提示SB203580、U0126降低PMA激活的PKC.介导的肝癌细胞侵袭、迁移的增加可能是由HSP27活性降低所致。为了进一步证实这个结果，采用RNA干扰技术阻断了HCCLM6细胞内HSP27的表达，再用PMA处理后，观察肝癌细胞侵袭、迁移能力的变化。结果显示，与PMA处理组相比，HSP27 RNAi+PMA处理组HCCLM6肝癌细胞侵袭、迁移细胞数目明显减少，说明HSP27的缺失能有效逆转PMA激活的、PKC.-ERK1／21 PKC.-p38 MAPK介导的肝癌细胞侵袭迁移的增强。
综合以上结果，表明HSP27作为PKC.-ERK／p38 MAPK下游的活性分子在肝癌细胞的侵袭转移过程发挥着重要作用。
第三部分HSP27在转移潜能不同肝癌细胞内参与核因子-.B通路激活的分子机制研究
转移是肿瘤最重要的恶性表型之一，是多分子参与高度复杂有序的过程。越来越多的证据显示细胞凋亡与肿瘤转移存在相关性。大量证据表明，HSP27可以作用于凋亡通路上的多个环节，抑制细胞凋亡，使损伤刺激下的细胞存活率显著提高，它在不同信号通路中所起的作用与细胞内的其他凋亡信号分子组成了一个复杂的调节网络。本部分的研究目的就是探讨在人类肝细胞肝癌细胞系中HSP27与细胞凋亡的关系，并进一步分析其参与肝癌细胞凋亡的分子机制及其与肝癌侵袭转移的内在联系。
应用RNA干扰技术，抑制高转移潜能人肝癌细胞系MHCC97H的HSP27的表达。瞬时转染化学合成的双链HSP27小干扰RNA(siRNA，100nM)后，检测到MHCC97H细胞中HSP27的mRNA和蛋白表达均受到明显抑制。以MHCC97H细胞作为试验的细胞模型，设置对照组(MHCC97H细胞)、MOCK组(转染非特异性的阴性对照dsRNA)、RNAi组(转染HSP27 siRNA)3个试验平行组，应用流式细胞仪分析(JC-1标记)和脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)，观察HSP27对MHCC97H细胞凋亡比例的影响。应用流式细胞仪分析显示，与MOCK组相比，RNAi组中MHCC97H细胞凋亡比例增加了20.07％，HSP27 RNA干扰后MHCC97H细胞凋亡比例明显增加。TUNEL法标记细胞核也显示HSP27 RNA干扰后MHCC97H细胞凋亡比例增加。
取对照组、MOCK组和RNAi组细胞进行人类信号转导通路发现者基因芯片的检测，结果显示RNAi组中IL2(0.12±0.019)、LTA(0.38±0.029)、NFKB1(NF.B 0.03±0.027)、NFKBIA(I.B. 0.27±0.033)、PECAM1(0.43±0.026)均出现明显下调，并经实时定量PCR证实。通过pathway miner软件分析，这些基因均位于NF-.B通路，提示HSP27与细胞内NF-.B通路的激活相关。应用蛋白印迹与免疫共沉淀技术发现，HSP27干扰实验后，MHCC97H细胞核内活化的NF-.Bp65减少，细胞内磷酸化的I.B.也减少，说明HSP27影响了肝癌细胞内NF-.B通路的激活；对不同转移潜能肝癌细胞细胞核内活化的NF-.B p65检测发现，Hep3B、MHCC97L及MHCC97H细胞核内活化的NF-.B p65蛋白随转移潜能的增加而增高；在三种肝癌细胞中HSP27可同IKK.、I.B.共沉淀，而不能与活化的NF-.B p65共沉淀；同时IKK.免疫共沉淀结果显示，HSP27干扰实验后，IKK.与IKK.的结合减少，说明HSP27的缺失降低了IKK复合物的稳定性。以上结果表明HSP27的抗凋亡作用可能是通过参与肝癌细胞内NF-.B通路的活化所致。
结论
1．HSP27的高表达可能与肝细胞肝癌的转移潜能正相关。
2．转移潜能不同肝细胞肝癌细胞的基因表达谱存在明显的差异性，涉及多条信号转导通路的激活，表明肝癌细胞转移潜能与多种信号转导通路的活化相关。其中MAPK通路、凋亡通路发挥着重要作用。
3．PKC.-ERK1／2／p38 MAPK-HSP27通路的活化在肝癌转移中发挥着重要的作用。
4．HSP27可通过与NF-.B通路中IKK.及I.B.成分的相互作用及影响IKK复合物的稳定性来参与肝癌细胞内NF-.B途径的激活，抑制肝癌细胞的凋亡，参与肝癌的发生发展和侵袭转移过程。
潜在应用价值
1．筛检出的差异信号转导通路有可能成为今后研究肝癌转移复发的靶标。
2．探讨了HSP27参与的信号转导机制及其在肝癌侵袭转移中的作用，有助于肝癌病理机制的阐明，为肝癌的干预治疗研究提供新的靶点。
3．HSP27可以作为一种潜在的、有应用价值的测定肝癌及其转移的标志物。
创新点
1．运用高通量的基因组学技术首次比较了转移潜能不同的肝细胞肝癌细胞的信号转导相关的基因表达谱，揭示了信号转导通路与肝癌细胞转移的关系。
2．首次发现了PKC.-ERK1／2／p38 MAPK-HSP27通路，初步阐明了其与肝癌细胞侵袭转移的关系。
3．首次在肝癌细胞中揭示HSP27与NF-.B通路激活的关系，分析了其抑制凋亡及与肝癌侵袭转移的潜在关系。
Hepatocellular carcinoma (HCC) is the 2rd most common cause of death from cancer in China. Metastasis and recurrence is the major cause for lower post-operation survival rate of HCC patients. Like other cancers, the development and migration of HCC is a multifactorial and multistage pathogenesis. For HCC cells, the ability to form metastasis phenotype may depend on the alteration of a series signal transduction network that enables them to complete all the steps of metastatic cascade. Therefore, understanding the molecular mechanisms of specific signal transduction pathways by which tumors could progress to metastatic state and id entifying roles of key regulators of signal transduction pathways that take place in malignant cells would provide insight into development of new intervention strategies. For recent decades, as a powerful prognostic indicator, aberrant expression of small heat shock protein 27(HSP27) has been proved to be associated with some cancers on base of cumulative data. HSP27 appears to modulate the polymerization of actin and is proposed to play a role in cytoskeleton dynamics through the broader relevance of MAPK pathway and HSP27 in some cancers. Moreover, recent evidence has shown that Hsp27 regulates apoptosis through an ability to interact with key components of the apoptotic signalling pathway. These characters have HSP27 involved in some cancer cells metastasis. In our study, we used signal transduction gene microarray to identify the genes with different expressing level among a series human HCC cell lines with different metastatic potentials which have been established at the authors' institute. Analysis of the microarray data revealed prominent roles of MAPK pathway and apoptosis pathway during human HCC metastasis, which was confirmed by further biochemical and functional investigation. We also addressed some molecular mechanism of HSP27 involved in regulation of above pathways through RNA interference technique and some following experiments related to cell properties ofapoptosis, migration and invasion. This study elucidated at some extent interior mechanisms of HCC metastasis.Part One Expression of HSP27 and Exploration of Prominent Signal Pathways in Different Metastatic Human HepatocellularCarcinoma Cell Lines
For the sake of evaluating relationship of expression of HSP27 and HCC metastasis in different metastatic hepatocellular carcinoma (HCC) cell lines, immunofluoscence, RT-PCR, real time-PCR, western blot, HSP27 protein ELISA kit and RNA interference were integratedly used. In order to screen key and prominent altered signal pathways related to HCC metastasis, a human signal transduction gene microarray was performed and followed by KOBAS (KEGG Orthology-Based Annotation System) analysis.
Immunofluorescence staining showed HSP27 immunoreactivity was prominently in the cytoplasm of cells and occasionally in nuclei. Using Semi-quantitive RT-PCR and real time-PCR, mRNA level of HSP27 was evaluated and showed consistence with HCC metastatic potential. The analysis of Western blot images and HSP27 ELISA data confirmed that level of HSP27 protein increased constitutively from Hep3B, MHCC97L, MHCC97H to HCCLM6．Using RNA interference technique, after HSP27 mRNA and protein were inhibited efficiently, HCCLM6 cell invasive and migratory ability stepped down obviously up to 50%, compared with control group by in vitro invasion and migration assays.
MHCC97L, MHCC97H and HCCLM6 cells with a similar genetic background and yet stepwise increasing metastatic potential were chose for further signal transduction relative gene microarray analysis. Total RNA were extracted by Trizol method, converted into cDNA, labeled with cy3 or cy5 and hybridized. The result showed a total of 62 statistically and consecutively changed genes passed statistic t-test, if combined with ratio values 1．5-fold difference, among which 17 genes showed stepwise up-regulation. Using KOBAS software, it mapped 897 genes to 142 KEGG pathways, including 62 consecutive changed genes to 66 pathways and 17 up-regulated genes to 28 pathways. Fourteen of latter 28 pathways were significantly up-regulated (P < 0．05) associated with HCC cell metastasis if judged by P value based on hypergeometric distribution. Only six of these pathways had P values < 0．05 after FDR correction. MAPK pathway and apoptosis pathway ranked top two with an FDR-corrected P value of 0．0189 and 0．0365 respectively. The results implied that various distinct different signal pathways may join together in HCC metastasis.Part TwoConstruction and Functional Exploration of PKC.-ERK1/2/ p38 MAPK-HSP27 in Metastatic Human HepatocellularCarcinoma Cell Lines
The object of this part is to elucidate internal relationship of MAPK pathway and HSP27 molecule, explore functional effect of key molecules of constructed pathway related to HCC metastasis by using RNA interference, western blot, specific kinase inhibitors or activators and in vitro migration and invasion assay, which may result in discovery of possible and potential diagnostic biomarkers or therapeutic targets.
Current evidence suggests mammalian cells express at least three groups of MAPKs: extracellular signal-regulating kinases (ERK), p38 MAPK and c-Jun N-terminal kinases (JNK), which are pivotal and representative signal molecules involved in different MAPK pathway. It was showed that function of HSP27 is phosphorylation-dependent, it can be activated by kinases other than p38 MAPK. To verify the possible sequential activation of MAPK pathway and further determine which kinases activated HSP27 in the HCC cell lines, we observed protein levels of HSP27 and three kinases mentioned above and their phosphorylation through Western blot analysis. The results showed that both of HSP27, ERK1/2 and their phosphorylation were consecutively over-expressed stepwise from MHCC97L to MHCC97H to HCCLM6 cells. Although total p38 MAPK were not obviously changed, but its phosphorylation level showed significantly elevated in these cell lines. For JNK and phosphor-JNK, protein levels hardly changed in three cell lines. Furthermore, phosphorylated HSP27 reduced after U0126, SB203580 treatment, but not SP600125．It is believable that ERK/MAPK and p38 MAPK were up-stream of HSP27 activation and consecutive activation of ERK1/2-HSP27 and p38 MAPK-HSP27 could potentially contribute to the up-regulation of HCC cells metastatic potentials. Since PKC. transcription level was significantly and markedly up-regulated from MHCC97L, MHCC97H to HCCLM6 cells, we evaluated levels of PKC. protein and its phosphorylated status in HCC cell lines and found the protein level was in agreement with microarray data. To address this relationship of PKC. and consecutive activation of ERK1/2-HSP27 and p38 MAPK-HSP27 in three HCC cell lines, we firstly observed phosphorylated levels of HSP27, ERK, JNK and p38 MAPK after treating HCCLM6 cells with PKC. special inhibitor LY317615．It showed that phosphor-HSP27, phosphor-ERK1/2 and phosphor-p38 were decreased in HCCLM6 cells after treated with LY317615, while phosphor-JNK was not difference in such treated cell samples. Reciprocally HCCLM6 cells were pretreated with PKC. RNAi followed by PMA(or not). It showed that although PMA enhan ced independently phosphorylated levels of HSP27, p38 MAPK or ERK1/2, phosphorylated HSP27, ERK1/2 and p38 reduced after absence of PKC. protein, compared with PMA-treatment group. These findings above revealed HSP27 was downstream signal molecule of PKC.-ERK1/2 and PKC.-p38 MAPK and effectively activated by PKC.-ERK1/2 and PKC.-p38 MAPK.
To demonstrate whether the absence of PKC. or inhibition of its activation was directly related to HCC cells metastatic phenotype, We chose the HCCLM6 cell line with the highest metastatic potential among the three cell samples and treated HCCLM6 with PKC. special siRNA or inhibitor LY317615 respectively. As evidenced by in vitro migration assay and invasion assay, motility and invasion of HCCLM6 cells stepped greatly down when absence of PKC. protein after PKC. RNAi and the same result was reconfirmed after HCCLM6 cells treated with 0．025．M LY317615, which inhibited obviously phosphor-PKC. and hardly inhibit phosphorylation of other PKC isoforms. To further investigate whet her a prominent role of PKC. among PKC isozymes in HCC metastasis, HCCLM6 cells were transfected with PKC. special siRNA, then treated by PKC activator PMA. Compared with increasing of migrated and invasive HCCLM6 cells by PMA stimulation, PKC. RNAi-followed-by-PMA-treated HCCLM6 cells motility and invasion significantly reduced, While there was no statistical difference between PKC. RNAi-followed-by-PMA-treated HCCLM6 cells and independently PKC. RNAi-treated HCCLM6 cells. This indicated a prominent role of PKC. in HCC cells motility and invasion, compared with other PKC isozymes and increasing of PMA-stimulated HCCLM6 cells motility and invasion was mainly through PKC.. Altogether, these results above indicate that absence/deficiency of PKC. protein and inhibition of its activation decrease metastatic HCC cells motility and invasion.
In addition, respective treatment of SB203580 and U0126 significantly and specifically reduced the level of phosphor-ERK and phosphor-p38 and the number of migrated and invasive cells treated with PMA, it was furthermore found that SB203580 or U0126 hardly decrease phosphorylation of PMA-stimulated PKC.. These data manifested that inhibition of p38 MAPK or ERK/MAPK pathways could negate increasing of PMA-activated PKC.-mediated HCC motility and invasion. Moreover, we inhibited HSP27 expression in HCCLM6 cells through HSP27 RNAi and then treated these cells by PMA and analyzed their migrated and invasive ability. These results showed that HSP27 RNAi-pretreated-PMA-stimulated HCCLM6 cells invasion and motility were significantly decreased. These findings above indicated that HSP27 was involved in PMA activated PKC.-ERK1/2/p38MAPK mediated HCC cells motility and invasion.Part ThreeVerification and Mechanism Analysis of HSP27 Involved inActivation of NF-kB Pathway in Metastatic HumanHepatocellular Carcinoma Cell Lines
Metastasis is a complex process that requires the sequential completion of multiple steps in which various molecules were involved. During the process of metastasis cells are subjected to various apoptotic stimuli. Heat shock protein 27 (Hsp27) has been shown to interact with and inhibit components of both stress and death-receptor induced apoptotic pathways. Over-expression of Hsp27 in transformed cell lines enhanced tumorigenic potential and contributed to tumor metastasis by blocking apoptosis. Aim of this part is to elucidate mechanism of HSP27 involved in metastatic HCC cell apoptosis.
Using RNA interference technique, mRNA and protein level of HSP27 in MHCC97H cell line were specifically reduce by 87% and 85%. Compared with control group, MHCC97H cells in RNAi group apoptosis ration increased 20．07% by flow cytometry analysis, which was reconfirmed by TUNEL (terminal-deoxynucleotidyl transferase mediated nick end labeling) morphological assessment. The significant changes in apoptosis of MHCC97H implied HSP27 was involved in HCC cell apoptosis. Furthermore a Human Q Series Signal Transduction in Cancer Gene Array analysis showed NFKB1 (NF. B 0．03±0．027) , IL2(0．12±0．019) , NFKBIA(I. Ba 0．27±0．033) , LTA (0．38±0．029) , PECAM1 (0．43±0．026) genes significantly downregulated in HSP27 RNAi group and were proved to be related to NF-. B pathway through pathway miner software analysis. This revealed that NF-. B pathway activation was inhibited after absence of HSP27 protein. Immunobloting analysis showed that the decreasing of nuclear activated NF-. B p65 in MHCC97H cells after HSP27 RNAi, and co-immunoprecipitation assay showed interaction of IKK.、I. B. with HSP27 in three HCC cell lines, while decreasing of phosphorylated I. B. and reducing of the association between IKK. and IKK. were found in MHCC97H cells after HSP27 RNAi. Together, these findings revealed roles of HSP27 in being of HCC cell lines metastatic potentials through involving in cellular NF-. B pathway activation.
Conclusion
1．Over-expression of heat shock protein 27(HSP27) presents a positive correlationwith enhancement of human hepatocellular carcinoma (HCC) cells metastatic potentials.
2．The signal transduction relative genes profile of different metastatic HCC celllines displayed obviously difference. The results implied that various distinct up-regulated signal pathways may lead to HCC metastasis together. Among these pathways, MAPK pathway and apoptosis pathway may play prominent roles.
3．The first time constructed PKC.-ERK/MAPK/ p38 MAPK-HSP27 pathway wasinvolved in HCC metastsis through modulating HCC cells motility and invasion.
4．HSP27 can promote HCC cells metastatic potentials and inhibit cell apoptosisthrough be involved in activation of NF-. B pathway through HSP27 association with IKK. and I. B. and its effect on stabilization of IKK complexes.
The potential application of this work
1．Aberrant signal pathways may be used as potential study targets for HCCmetastasis and recurrence.
2．Molecular functional exploration of HSP27 involved in some signal pathwaysrelated to HCC metastasis help to elucidate internal pathological mechanism of HCC tumorigenesis and progress and provide a therapeutic target.
3．HSP27 may be used as a potential and valuable biomarker for HCC prognosis andprediction of metastasis.
Novelty of this project
1．First comparation of signal transduction related genes expression profile in different metastatic HCC cell lines by high throughout gene microarray, which revealed relationship of signal transduction and HCC cell metastatic potentials.
2．The PKC.-ERK/MAPK/ p38 MAPK-HSP27 pathway was the first time constructed. The broader relevance of this pathway to HCC was supported by the fact that we demonstrated effects of various molecular targets on metastatic HCC cells.
3．Our works firstly discovered the possible molecular mechanism of HSP27involved in HCC cell apoptosis through participating in activation of NF-. B pathway.
Key words
human hepatocellular carcinoma; tumor metastasis; gene microarray; RNA-mediated interference; signal transduction pathway; Heat Shock Protein 27; MAPK pathway; apoptosis
Discipline Category number
R735．7; Q51 ; Q71
第一部分转移潜能不同肝癌细胞系中HSP27的表达及转移相关信号转导通路的基因组学分析
本部分的研究是应用细胞免疫荧光检测技术结合反转录PCR(RT-PCR)，实时荧光定量PCR，蛋白印迹(western blot)及免疫酶联吸附实验(ELISA)等技术，对转移潜能不同的人肝细胞肝癌细胞系Hep3B、MHCC97L、MHCC97H、HCCLM6中HSP27的定位及表达进行分析。运用RNA干扰(RNAi)技术，观察HSP27与肝癌细胞侵袭迁移能力的关系。另外，利用基因芯片检测技术，比较MHCC97L、MHCC97H、HCCLM6细胞系信号转导相关基因表达谱的差异，运用KOBAS软件分析系统对差异基因所涉及的信号通路进行统计分析，筛查在肝癌细胞转移潜能形成过程中发挥重要作用的信号转导通路。
应用细胞免疫荧光技术检测HSP27在体外培养肝癌细胞系中的定位，结果显示HSP27主要定位在肝癌细胞细胞浆内，亦见于细胞核。通过RT-PCR、实时荧光定量PCR技术检测HSP27的基因表达水平，发现HSP27在有转移潜能人肝癌细胞系MHCC97L、MHCC97H、HCCLM6中mRNA表达水平明显高于无转移潜能的Hep3B，其中HCCLM6细胞系最高。利用western blot和HSP27蛋白ELISA方法，证实在四种肝癌细胞系中HSP27蛋白表达水平与上述mRNA水平一致。此外，应用RNA干扰技术，设置三个平行实验组：对照组(HCCLM6细胞)、MOCK组(转染一小段非特异性dsRNA，但该dsRNA不针对任何基因，经验证转染后不抑制基因表达)和RNA干扰组(转染HSP27特异性dsRNA)。在转染100nM HSP27特异性siRNA 36、48小时后，HCCLM6细胞内HSP27mRNA均下降了80％以上而蛋白水平分别与70％、92％；与MOCK组相比，RNA干扰组侵袭和迁移的HCCLM6细胞数分别下降了53％与64％。这些结果提示HSP27参与了肝癌细胞侵袭迁移过程，它的高表达可能与肝癌细胞转移潜能的增高有关。
选取遗传背景相同而转移潜能不同的肝癌细胞系MHCC97L、MHCC97H、HCCLM6，提取总RNA，逆转录成cDNA，cy3和cy5标记后，进行人类信号转导寡核苷酸微阵列检测，结果显示三种肝癌细胞系中连续变化的差异基因有62个，差异倍数.1.5倍且连续上调的基因17个。通过KOBAS软件分析，发现芯片中的897个信号转导相关基因涉及了142条KEGG通路，其中62个连续变化的差异基因涉及66条通路，连续上调、差异倍数.1.5倍的17个差异基因涉及28条通路。这28条信号通路中通过FDR校正后仅有6条通路的P值＜0.05，其中MAPK通路(0.0189)、凋亡通路(0.0365)排在前两位，推测这些通路的持续活化可能与肝癌侵袭转移有关。
第二部分HSP27经由PKC.-ERK／p38 MAPK信号通路促进肝癌转移的分子机制研究
本部分的研究目的是应用RNA干扰技术结合激酶特异性抑制剂和激动剂、蛋白免疫印迹技术、体外侵袭迁移实验，探讨肝细胞肝癌细胞内MAPK通路与HSP27的关系，证实通路中关键靶标分子的功能效应，揭示其在肝癌细胞转移潜能形成中的作用，以获得潜在的肝癌诊断标记及治疗靶标。
本研究应用蛋白免疫印迹技术检测MHCC97L、MHCC97H、HCCLM6肝癌细胞中HSP27和MAPK通路三个关键活性分子ERK1／2、p38MAPK、JNK及显著差异表达基因PKC.的蛋白磷酸化水平，结果显示在三种转移性肝癌细胞内磷酸化HSP27、磷酸化PKC.和磷酸化ERK1／2、磷酸化p38 MAPK逐渐增高，而磷酸化JNK没有明显变化，这提示HSP27、PKC.的持续活化及ERK1／2和p38MAPK通路的激活可能与肝癌细胞转移能力增强有关。
在转移潜能最高的HCCLM6肝癌细胞内，分别利用MAPK特异性抑制剂SB203580(5.M，p38MAPK特异性抑制剂)、U0126(10.M，ERK1／2特异性抑制剂)和SP600125(10.M，JNK特异性抑制剂)处理2h后，发现前两者能有效抑制HSP27的磷酸化，而SP600125对HSP27的磷酸化没有明显抑制，说明在肝癌细胞内HSP27的激活受p38 MAPK和ERK1／2所调节。利用RNA干扰技术、PKC.特异性抑制剂LY317615结合PKC激动剂PMA，发现LY317615能有效抑制肝癌细胞内p38MAPK、ERK1／2、和HSP27的磷酸化，而对JNK的磷酸化没有明显抑制，这说明PKC.的活性缺失能有效抑制ERK1／2-HSP27和p38MAPK-HSP27的活化；还发现与PMA处理组相比，PKC.RNAi+PMA处理组磷酸化ERK1／2、p38 MAPK和HSP27减少。这些结果证实了肝癌细胞内HSP27是PKC.-ERK1／2和PKC.-p38MAPK下游的活性分子。
为了探究PKC.-ERK1／2／p38MAPK-HSP27通路与肝癌细胞转移表型的关系，我们利用RNA干扰技术和PKC.特异性抑制剂LY317615结合体外细胞侵袭迁移实验观察HCCLM6肝癌细胞侵袭迁移能力的变化，发现与MOCK组相比，PKC.RNAi(70nM)处理组侵袭和迁移的细胞数分别下降了56％和51％；LY317615(0.025.M)处理组与其对照组相比则分别下降了36％和38％。此外，还发现PMA(PKC激动剂)处理组与未处理组(HCCLM6细胞)相比，侵袭、迁移的细胞数分别增加了26％和23％，而PKC.RNAi处理组与PKC.RNAi+PMA处理组侵袭、迁移的肝癌细胞数没有统计学差别。以上结果表明PKC.蛋白的缺失及其活性抑制降低了肝癌细胞的侵袭迁移能力；PMA所致肝癌细胞侵袭迁移能力的增强是PKC.依赖性的。
进一步试验发现，与PMA处理组相比，SB203580+PMA处理组与U0126+PMA处理组侵袭、迁移的细胞分别降低了29％、31％和34％、37％。这些结果说明p38 MAPK和ERK1／2的抑制能降低PMA激活的、PKC.介导的肝癌细胞侵袭迁移的增加。同时发现，与PMA处理组相比，SB203580+PMA处理组、U0126+PMA处理组中HSP27的磷酸化水平降低，这提示SB203580、U0126降低PMA激活的PKC.介导的肝癌细胞侵袭、迁移的增加可能是由HSP27活性降低所致。为了进一步证实这个结果，采用RNA干扰技术阻断了HCCLM6细胞内HSP27的表达，再用PMA处理后，观察肝癌细胞侵袭、迁移能力的变化。结果显示，与PMA处理组相比，HSP27 RNAi+PMA处理组HCCLM6肝癌细胞侵袭、迁移细胞数目明显减少，说明HSP27的缺失能有效逆转PMA激活的、PKC.-ERK1／21 PKC.-p38 MAPK介导的肝癌细胞侵袭迁移的增强。
综合以上结果，表明HSP27作为PKC.-ERK／p38 MAPK下游的活性分子在肝癌细胞的侵袭转移过程发挥着重要作用。
第三部分HSP27在转移潜能不同肝癌细胞内参与核因子-.B通路激活的分子机制研究
转移是肿瘤最重要的恶性表型之一，是多分子参与高度复杂有序的过程。越来越多的证据显示细胞凋亡与肿瘤转移存在相关性。大量证据表明，HSP27可以作用于凋亡通路上的多个环节，抑制细胞凋亡，使损伤刺激下的细胞存活率显著提高，它在不同信号通路中所起的作用与细胞内的其他凋亡信号分子组成了一个复杂的调节网络。本部分的研究目的就是探讨在人类肝细胞肝癌细胞系中HSP27与细胞凋亡的关系，并进一步分析其参与肝癌细胞凋亡的分子机制及其与肝癌侵袭转移的内在联系。
应用RNA干扰技术，抑制高转移潜能人肝癌细胞系MHCC97H的HSP27的表达。瞬时转染化学合成的双链HSP27小干扰RNA(siRNA，100nM)后，检测到MHCC97H细胞中HSP27的mRNA和蛋白表达均受到明显抑制。以MHCC97H细胞作为试验的细胞模型，设置对照组(MHCC97H细胞)、MOCK组(转染非特异性的阴性对照dsRNA)、RNAi组(转染HSP27 siRNA)3个试验平行组，应用流式细胞仪分析(JC-1标记)和脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)，观察HSP27对MHCC97H细胞凋亡比例的影响。应用流式细胞仪分析显示，与MOCK组相比，RNAi组中MHCC97H细胞凋亡比例增加了20.07％，HSP27 RNA干扰后MHCC97H细胞凋亡比例明显增加。TUNEL法标记细胞核也显示HSP27 RNA干扰后MHCC97H细胞凋亡比例增加。
取对照组、MOCK组和RNAi组细胞进行人类信号转导通路发现者基因芯片的检测，结果显示RNAi组中IL2(0.12±0.019)、LTA(0.38±0.029)、NFKB1(NF.B 0.03±0.027)、NFKBIA(I.B. 0.27±0.033)、PECAM1(0.43±0.026)均出现明显下调，并经实时定量PCR证实。通过pathway miner软件分析，这些基因均位于NF-.B通路，提示HSP27与细胞内NF-.B通路的激活相关。应用蛋白印迹与免疫共沉淀技术发现，HSP27干扰实验后，MHCC97H细胞核内活化的NF-.Bp65减少，细胞内磷酸化的I.B.也减少，说明HSP27影响了肝癌细胞内NF-.B通路的激活；对不同转移潜能肝癌细胞细胞核内活化的NF-.B p65检测发现，Hep3B、MHCC97L及MHCC97H细胞核内活化的NF-.B p65蛋白随转移潜能的增加而增高；在三种肝癌细胞中HSP27可同IKK.、I.B.共沉淀，而不能与活化的NF-.B p65共沉淀；同时IKK.免疫共沉淀结果显示，HSP27干扰实验后，IKK.与IKK.的结合减少，说明HSP27的缺失降低了IKK复合物的稳定性。以上结果表明HSP27的抗凋亡作用可能是通过参与肝癌细胞内NF-.B通路的活化所致。
结论
1．HSP27的高表达可能与肝细胞肝癌的转移潜能正相关。
2．转移潜能不同肝细胞肝癌细胞的基因表达谱存在明显的差异性，涉及多条信号转导通路的激活，表明肝癌细胞转移潜能与多种信号转导通路的活化相关。其中MAPK通路、凋亡通路发挥着重要作用。
3．PKC.-ERK1／2／p38 MAPK-HSP27通路的活化在肝癌转移中发挥着重要的作用。
4．HSP27可通过与NF-.B通路中IKK.及I.B.成分的相互作用及影响IKK复合物的稳定性来参与肝癌细胞内NF-.B途径的激活，抑制肝癌细胞的凋亡，参与肝癌的发生发展和侵袭转移过程。
潜在应用价值
1．筛检出的差异信号转导通路有可能成为今后研究肝癌转移复发的靶标。
2．探讨了HSP27参与的信号转导机制及其在肝癌侵袭转移中的作用，有助于肝癌病理机制的阐明，为肝癌的干预治疗研究提供新的靶点。
3．HSP27可以作为一种潜在的、有应用价值的测定肝癌及其转移的标志物。
创新点
1．运用高通量的基因组学技术首次比较了转移潜能不同的肝细胞肝癌细胞的信号转导相关的基因表达谱，揭示了信号转导通路与肝癌细胞转移的关系。
2．首次发现了PKC.-ERK1／2／p38 MAPK-HSP27通路，初步阐明了其与肝癌细胞侵袭转移的关系。
3．首次在肝癌细胞中揭示HSP27与NF-.B通路激活的关系，分析了其抑制凋亡及与肝癌侵袭转移的潜在关系。
Hepatocellular carcinoma (HCC) is the 2rd most common cause of death from cancer in China. Metastasis and recurrence is the major cause for lower post-operation survival rate of HCC patients. Like other cancers, the development and migration of HCC is a multifactorial and multistage pathogenesis. For HCC cells, the ability to form metastasis phenotype may depend on the alteration of a series signal transduction network that enables them to complete all the steps of metastatic cascade. Therefore, understanding the molecular mechanisms of specific signal transduction pathways by which tumors could progress to metastatic state and id entifying roles of key regulators of signal transduction pathways that take place in malignant cells would provide insight into development of new intervention strategies. For recent decades, as a powerful prognostic indicator, aberrant expression of small heat shock protein 27(HSP27) has been proved to be associated with some cancers on base of cumulative data. HSP27 appears to modulate the polymerization of actin and is proposed to play a role in cytoskeleton dynamics through the broader relevance of MAPK pathway and HSP27 in some cancers. Moreover, recent evidence has shown that Hsp27 regulates apoptosis through an ability to interact with key components of the apoptotic signalling pathway. These characters have HSP27 involved in some cancer cells metastasis. In our study, we used signal transduction gene microarray to identify the genes with different expressing level among a series human HCC cell lines with different metastatic potentials which have been established at the authors' institute. Analysis of the microarray data revealed prominent roles of MAPK pathway and apoptosis pathway during human HCC metastasis, which was confirmed by further biochemical and functional investigation. We also addressed some molecular mechanism of HSP27 involved in regulation of above pathways through RNA interference technique and some following experiments related to cell properties ofapoptosis, migration and invasion. This study elucidated at some extent interior mechanisms of HCC metastasis.Part One Expression of HSP27 and Exploration of Prominent Signal Pathways in Different Metastatic Human HepatocellularCarcinoma Cell Lines
For the sake of evaluating relationship of expression of HSP27 and HCC metastasis in different metastatic hepatocellular carcinoma (HCC) cell lines, immunofluoscence, RT-PCR, real time-PCR, western blot, HSP27 protein ELISA kit and RNA interference were integratedly used. In order to screen key and prominent altered signal pathways related to HCC metastasis, a human signal transduction gene microarray was performed and followed by KOBAS (KEGG Orthology-Based Annotation System) analysis.
Immunofluorescence staining showed HSP27 immunoreactivity was prominently in the cytoplasm of cells and occasionally in nuclei. Using Semi-quantitive RT-PCR and real time-PCR, mRNA level of HSP27 was evaluated and showed consistence with HCC metastatic potential. The analysis of Western blot images and HSP27 ELISA data confirmed that level of HSP27 protein increased constitutively from Hep3B, MHCC97L, MHCC97H to HCCLM6．Using RNA interference technique, after HSP27 mRNA and protein were inhibited efficiently, HCCLM6 cell invasive and migratory ability stepped down obviously up to 50%, compared with control group by in vitro invasion and migration assays.
MHCC97L, MHCC97H and HCCLM6 cells with a similar genetic background and yet stepwise increasing metastatic potential were chose for further signal transduction relative gene microarray analysis. Total RNA were extracted by Trizol method, converted into cDNA, labeled with cy3 or cy5 and hybridized. The result showed a total of 62 statistically and consecutively changed genes passed statistic t-test, if combined with ratio values 1．5-fold difference, among which 17 genes showed stepwise up-regulation. Using KOBAS software, it mapped 897 genes to 142 KEGG pathways, including 62 consecutive changed genes to 66 pathways and 17 up-regulated genes to 28 pathways. Fourteen of latter 28 pathways were significantly up-regulated (P < 0．05) associated with HCC cell metastasis if judged by P value based on hypergeometric distribution. Only six of these pathways had P values < 0．05 after FDR correction. MAPK pathway and apoptosis pathway ranked top two with an FDR-corrected P value of 0．0189 and 0．0365 respectively. The results implied that various distinct different signal pathways may join together in HCC metastasis.Part TwoConstruction and Functional Exploration of PKC.-ERK1/2/ p38 MAPK-HSP27 in Metastatic Human HepatocellularCarcinoma Cell Lines
The object of this part is to elucidate internal relationship of MAPK pathway and HSP27 molecule, explore functional effect of key molecules of constructed pathway related to HCC metastasis by using RNA interference, western blot, specific kinase inhibitors or activators and in vitro migration and invasion assay, which may result in discovery of possible and potential diagnostic biomarkers or therapeutic targets.
Current evidence suggests mammalian cells express at least three groups of MAPKs: extracellular signal-regulating kinases (ERK), p38 MAPK and c-Jun N-terminal kinases (JNK), which are pivotal and representative signal molecules involved in different MAPK pathway. It was showed that function of HSP27 is phosphorylation-dependent, it can be activated by kinases other than p38 MAPK. To verify the possible sequential activation of MAPK pathway and further determine which kinases activated HSP27 in the HCC cell lines, we observed protein levels of HSP27 and three kinases mentioned above and their phosphorylation through Western blot analysis. The results showed that both of HSP27, ERK1/2 and their phosphorylation were consecutively over-expressed stepwise from MHCC97L to MHCC97H to HCCLM6 cells. Although total p38 MAPK were not obviously changed, but its phosphorylation level showed significantly elevated in these cell lines. For JNK and phosphor-JNK, protein levels hardly changed in three cell lines. Furthermore, phosphorylated HSP27 reduced after U0126, SB203580 treatment, but not SP600125．It is believable that ERK/MAPK and p38 MAPK were up-stream of HSP27 activation and consecutive activation of ERK1/2-HSP27 and p38 MAPK-HSP27 could potentially contribute to the up-regulation of HCC cells metastatic potentials. Since PKC. transcription level was significantly and markedly up-regulated from MHCC97L, MHCC97H to HCCLM6 cells, we evaluated levels of PKC. protein and its phosphorylated status in HCC cell lines and found the protein level was in agreement with microarray data. To address this relationship of PKC. and consecutive activation of ERK1/2-HSP27 and p38 MAPK-HSP27 in three HCC cell lines, we firstly observed phosphorylated levels of HSP27, ERK, JNK and p38 MAPK after treating HCCLM6 cells with PKC. special inhibitor LY317615．It showed that phosphor-HSP27, phosphor-ERK1/2 and phosphor-p38 were decreased in HCCLM6 cells after treated with LY317615, while phosphor-JNK was not difference in such treated cell samples. Reciprocally HCCLM6 cells were pretreated with PKC. RNAi followed by PMA(or not). It showed that although PMA enhan ced independently phosphorylated levels of HSP27, p38 MAPK or ERK1/2, phosphorylated HSP27, ERK1/2 and p38 reduced after absence of PKC. protein, compared with PMA-treatment group. These findings above revealed HSP27 was downstream signal molecule of PKC.-ERK1/2 and PKC.-p38 MAPK and effectively activated by PKC.-ERK1/2 and PKC.-p38 MAPK.
To demonstrate whether the absence of PKC. or inhibition of its activation was directly related to HCC cells metastatic phenotype, We chose the HCCLM6 cell line with the highest metastatic potential among the three cell samples and treated HCCLM6 with PKC. special siRNA or inhibitor LY317615 respectively. As evidenced by in vitro migration assay and invasion assay, motility and invasion of HCCLM6 cells stepped greatly down when absence of PKC. protein after PKC. RNAi and the same result was reconfirmed after HCCLM6 cells treated with 0．025．M LY317615, which inhibited obviously phosphor-PKC. and hardly inhibit phosphorylation of other PKC isoforms. To further investigate whet her a prominent role of PKC. among PKC isozymes in HCC metastasis, HCCLM6 cells were transfected with PKC. special siRNA, then treated by PKC activator PMA. Compared with increasing of migrated and invasive HCCLM6 cells by PMA stimulation, PKC. RNAi-followed-by-PMA-treated HCCLM6 cells motility and invasion significantly reduced, While there was no statistical difference between PKC. RNAi-followed-by-PMA-treated HCCLM6 cells and independently PKC. RNAi-treated HCCLM6 cells. This indicated a prominent role of PKC. in HCC cells motility and invasion, compared with other PKC isozymes and increasing of PMA-stimulated HCCLM6 cells motility and invasion was mainly through PKC.. Altogether, these results above indicate that absence/deficiency of PKC. protein and inhibition of its activation decrease metastatic HCC cells motility and invasion.
In addition, respective treatment of SB203580 and U0126 significantly and specifically reduced the level of phosphor-ERK and phosphor-p38 and the number of migrated and invasive cells treated with PMA, it was furthermore found that SB203580 or U0126 hardly decrease phosphorylation of PMA-stimulated PKC.. These data manifested that inhibition of p38 MAPK or ERK/MAPK pathways could negate increasing of PMA-activated PKC.-mediated HCC motility and invasion. Moreover, we inhibited HSP27 expression in HCCLM6 cells through HSP27 RNAi and then treated these cells by PMA and analyzed their migrated and invasive ability. These results showed that HSP27 RNAi-pretreated-PMA-stimulated HCCLM6 cells invasion and motility were significantly decreased. These findings above indicated that HSP27 was involved in PMA activated PKC.-ERK1/2/p38MAPK mediated HCC cells motility and invasion.Part ThreeVerification and Mechanism Analysis of HSP27 Involved inActivation of NF-kB Pathway in Metastatic HumanHepatocellular Carcinoma Cell Lines
Metastasis is a complex process that requires the sequential completion of multiple steps in which various molecules were involved. During the process of metastasis cells are subjected to various apoptotic stimuli. Heat shock protein 27 (Hsp27) has been shown to interact with and inhibit components of both stress and death-receptor induced apoptotic pathways. Over-expression of Hsp27 in transformed cell lines enhanced tumorigenic potential and contributed to tumor metastasis by blocking apoptosis. Aim of this part is to elucidate mechanism of HSP27 involved in metastatic HCC cell apoptosis.
Using RNA interference technique, mRNA and protein level of HSP27 in MHCC97H cell line were specifically reduce by 87% and 85%. Compared with control group, MHCC97H cells in RNAi group apoptosis ration increased 20．07% by flow cytometry analysis, which was reconfirmed by TUNEL (terminal-deoxynucleotidyl transferase mediated nick end labeling) morphological assessment. The significant changes in apoptosis of MHCC97H implied HSP27 was involved in HCC cell apoptosis. Furthermore a Human Q Series Signal Transduction in Cancer Gene Array analysis showed NFKB1 (NF. B 0．03±0．027) , IL2(0．12±0．019) , NFKBIA(I. Ba 0．27±0．033) , LTA (0．38±0．029) , PECAM1 (0．43±0．026) genes significantly downregulated in HSP27 RNAi group and were proved to be related to NF-. B pathway through pathway miner software analysis. This revealed that NF-. B pathway activation was inhibited after absence of HSP27 protein. Immunobloting analysis showed that the decreasing of nuclear activated NF-. B p65 in MHCC97H cells after HSP27 RNAi, and co-immunoprecipitation assay showed interaction of IKK.、I. B. with HSP27 in three HCC cell lines, while decreasing of phosphorylated I. B. and reducing of the association between IKK. and IKK. were found in MHCC97H cells after HSP27 RNAi. Together, these findings revealed roles of HSP27 in being of HCC cell lines metastatic potentials through involving in cellular NF-. B pathway activation.
Conclusion
1．Over-expression of heat shock protein 27(HSP27) presents a positive correlationwith enhancement of human hepatocellular carcinoma (HCC) cells metastatic potentials.
2．The signal transduction relative genes profile of different metastatic HCC celllines displayed obviously difference. The results implied that various distinct up-regulated signal pathways may lead to HCC metastasis together. Among these pathways, MAPK pathway and apoptosis pathway may play prominent roles.
3．The first time constructed PKC.-ERK/MAPK/ p38 MAPK-HSP27 pathway wasinvolved in HCC metastsis through modulating HCC cells motility and invasion.
4．HSP27 can promote HCC cells metastatic potentials and inhibit cell apoptosisthrough be involved in activation of NF-. B pathway through HSP27 association with IKK. and I. B. and its effect on stabilization of IKK complexes.
The potential application of this work
1．Aberrant signal pathways may be used as potential study targets for HCCmetastasis and recurrence.
2．Molecular functional exploration of HSP27 involved in some signal pathwaysrelated to HCC metastasis help to elucidate internal pathological mechanism of HCC tumorigenesis and progress and provide a therapeutic target.
3．HSP27 may be used as a potential and valuable biomarker for HCC prognosis andprediction of metastasis.
Novelty of this project
1．First comparation of signal transduction related genes expression profile in different metastatic HCC cell lines by high throughout gene microarray, which revealed relationship of signal transduction and HCC cell metastatic potentials.
2．The PKC.-ERK/MAPK/ p38 MAPK-HSP27 pathway was the first time constructed. The broader relevance of this pathway to HCC was supported by the fact that we demonstrated effects of various molecular targets on metastatic HCC cells.
3．Our works firstly discovered the possible molecular mechanism of HSP27involved in HCC cell apoptosis through participating in activation of NF-. B pathway.
Key words
human hepatocellular carcinoma; tumor metastasis; gene microarray; RNA-mediated interference; signal transduction pathway; Heat Shock Protein 27; MAPK pathway; apoptosis
Discipline Category number
R735．7; Q51 ; Q71
引文
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