| 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 carcinomaObjective: 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 cellsObjective: 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 cellsObjective: 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 HCCObjective: 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. |
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