Molecular Mechanism Of Heat Shock Protein 27 In Hepatocellular Carcinoma Metastasis

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 LinesFor 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 LinesThe 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 LinesMetastasis 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.Conclusion1．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 project1．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 wordshuman hepatocellular carcinoma; tumor metastasis; gene microarray; RNA-mediated interference; signal transduction pathway; Heat Shock Protein 27; MAPK pathway; apoptosisDiscipline Category numberR735．7; Q51 ; Q71...