| Background and ObjectiveHepatocellular carcinoma (HCC) is the fifth most common malignancy in the world, and it is estimated to cause approximately half a million deaths annually. Although the underlying mechanisms of hepatocellular carcinogenesis remain to be investigated, accumulating evidence has indicated that signal transducer and activator of transcription (STAT) proteins, in particular STAT3, may be directly or indirectly involved in HCC development. Constitutively activated STAT3, through its phosphorylation at tyrosine residue705. has been reported in many human cancers, including carcinomas of the breast, lung, prostate, ovary, and melanoma. In general, activated STAT3participates in carcinogenesis by promoting angiogenesis or stimulating cell proliferation. Recently, activated STAT3has been shown in the progression of viral hepatitis and oncogenesis associated with hepatitis B virus (HBV) and hepatitis C virus (HCV) in the liver. In addition, STAT3activity is also increased in chemically-induced HCC. Taken together, these findings suggest that constitutively activated STAT3may play a crucial role in HCC development. However, the consequences of constitutive phosphorylation of STAT3and the mechanisms that trigger this constitutive activation require further investigation.In recent years, several studies have evaluated the gene associated with retinoid-interferon-induced mortality-19(GRIM-19). GRIM-19is a novel gene product involved in interferon-β/retinoic acid (IFN-β/RA)-induced apoptosis, and it suppresses STAT3-induced gene expression. In addition, GRIM-19plays a major role in the control of cell growth through regulation of STAT3. Lufei and others have reported that GRIM-19binds to STAT3and inhibits its transcriptional activation function. Inhibition of STAT3activation by GRIM-19appears to be an important step in oncogenesic suppression. Furthermore, constitutive activation of STATS promotes anti-apoptotic gene expression to support tumor survival. Low expression levels of GRIM-19have been shown in renal, prostate, esophageal, and colonic tumors. However. GRIM-19expression levels and the associated pathologies have not been reported for liver tumors. In the current study, we demonstrate a significant downregulation of GRIM-19protein levels in HCC that was associated with increased expression of p-STAT3and downstream target genes. In addition, we present novel findings that downregulation of GRIM-19and hyperactivation of p-STAT3expression in HCC lesions were closely correlated with an increased histological grading in HCC,Part I Expression and Clinical value of GRIM-19and P-STAT3in Hepatocellular CarcinomaObjectivel.To detect the location and the expression levels of GRIM-19and p-STAT3in hepatocellular carcinoma and adjacent nontumorous liver tissues.2. To detect the expression level of GRIM-19. STAT3, p-STAT3and VEGF in hepatocellular carcinoma and adjacent nontumorous liver tissues.3. To evaluated the correlation between expression level of the GRIM-19mRNA in HCC tissues between different patient subgroups according to various clinicopathological parameters.Methods1. Detect the location of GRIM-19and p-STAT3in hepatocellular carcinoma and adjacent nontumorous liver tissues by immunohistochemistry.2. Detect the expression level of GRIM-19and p-STAT3in hepatocellular carcinoma and adjacent nontumorous liver tissues by immunohistochemistry. western blot analysis, and RT-PCR.3. Detect the expression level of GRIM-19, STAT3, p-STAT3and VEGF in hepatocellular carcinoma and adjacent nontumorous liver tissues by western blot analvsis. Results1. The percentage of cells positively stained were categorizedThe percentage of cells positively stained in each section were categorized as follows: negative (samples with≤5%positive cells), low (5-25%positive cells), moderate (25-50%positive cells), and strong (50-100%positive) were indicated as-,+,++, and+++, respectively.2. Cellular Localization of GRIM-19in Liver TissueWe first analyzed the localization of GRIM-19in liver tissues using a purified GRIM-19antibody and an immunohistochemistry assay. GRIM-19protein expression was predominantly located in the cytoplasm with weak staining in the nucleus in ANLT, but only located in the cytoplasm in HCC tissues. To confirm this observation, pure nuclear protein was extracted separately from HCC tissues and ANLT, and GRIM-19protein expression was determined by western blot analysis. Consistent with the immunohistochemistry results, GRIM-19expression levels in the nucleus was significantly higher in ANLT compared to HCC tissues,(p<0.001).3. Lower Expression Levels of GRIM-19in HCC Compared to Adjacent Nontumorous Liver TissueGRIM-19-positive staining was found in3/4(75%) of the normal liver tissues,40/55(72.7%) of the adjacent nontumorous liver tissues, and25/55(45.5%) of the HCC tissues. GRIM-19expression in the ANLT tissues was not significantly different between the nontumorous, noncirrhotic specimens (12/16,75%) and the nontumorous but cirrhotic specimens (28/39,71.8%). In HCC tissues, however, GRIM-19expression was extremely reduced, and in some cases, even absent. In the cases with positive GRIM-19expression,23/65(35.4%) were determined to be++to+++(by the ranking system described in the methods section); and among them,20/23(87%) were ANLT. Thus, GRIM-19protein expression was extremely lower in HCC tissues than in the ANLT. To verify this finding, we examined the expression of GRIM-19mRNA using RT-PCR. Notably, the GRIM-19mRNA expression levels were significantly lower in HCC tissues compared to the ANLT (p<0.001).3. Downregulation of GRIM-19was Associated with Increased p-STAT3in HCC TissueGRIM-19is known to play an important role in the control of cell growth exerted through STAT3, which is a transcription factor known to be inhibited by GRIM-19binding. To explore the relationship between GRIM-19and p-STAT3expression in HCC, we evaluated the expression status of p-STAT3in HCC samples. In HCC tissues, immunopositive p-STAT3was confined to the nuclei of cancerous cells. Expression of p-STAT3in nonneoplastic. noncirrhotic liver was absent, while positive staining was found in14/39(35.9%) of the cases with liver cirrhosis and in47/55(85.5%) of the HCC samples. In the positively stained cases,40/61(65.6%) were determined to be++to+++, and among them,35/40(87.5%) cases were HCC tissues. These findings indicate that p-STAT3protein expression is highly elevated in HCC tissues compared to the ANLT. These results suggest that an inverse correlation existed between the expression of GRIM-19and p-STAT3in HCC tissues and the ANLT.To investigate this relationship further, we examined the expression of GRIM-19and p-STAT proteins in HCC tissues (n=55) and the ANLT (n=55) by western blot analysis. The expression of GRIM-19protein was significantly decreased in all grades of HCC tissues (p<0.05), and the expression of p-STAT3protein was significantly increased in all grades of HCC tissues (p<0.05) compared to the β-actin control. Next, we examined the expression of total STAT3and the p-STAT3target protein VEGF in HCC tissues (n=20) and the ANLT (n=20). Consistent with the reduced GRIM-19expression and the increased p-STAT3expression that were found, total STAT3expression (p=0.008) and VEGF expression (p=0.012) were significantly upregulated in the HCC tissues compared to the ANLT. Together, these results suggest that downregulation of GRIM-19may be associated with increased p-STAT3activity in HCC tissues.4. Correlation of GRIM-19Expression Levels with Clinicopathological Parameters We evaluated possible correlations of GRIM-19mRNA expression in HCC tissues between different patient subgroups according to various clinicopathological parameters (Table1). Two significant correlations of GRIM-19expression were observed for vascular invasion (p=0.032) and histological grade (p=0.001). No significant correlation of GRIM-19mRNA expression was found for age, gender. HBV infection, presence of cirrhosis, tumor size, serum a-fetoprotein levels, or local lymph node metastasis.Conclusion1、GRIM-19protein expression was predominantly located in the cytoplasm with weak staining in the nucleus in ANLT, but only located in the cytoplasm in HCC tissues.2、HCC samples exhibited low levels of GRIM-19and moderate to high levels of p-STAT3expression. In contrast,ANLT was characterized by high levels of GRIM-19and low levels of p-STAT3expression.3、Downregulation of GRIM-19was closely correlated with increased histological grade in HCC. Part Ⅱ The Affection of Retinoic Acid combined with Beta-Interferon Synergize in Inducing Growth Inhibition, Apoptosis and GRIM-19Expression in Human Hepatoma Carcinoma Cell in VitroObjective1. To detect the location and expression level of the GRIM-19in different hepatocellular carcinoma cell lines.2. To research growth inhibition and apoptosis of hepatocellular carcinoma cell (HepG2) intervened by RA combined with IFN-β.3. To investigate the changes in the expression level of GRIM-19and proto-oncogene STAT3、p-STAT3、VEGF intervened by RA combined with IFN-β.Methods1. Immunofluorescence、western blot and RT-PCR were used to detect the location and the expression of GRIM-19in LO2cell line and HepG2、MHCC-97H cell lines, respectively.2. MTT and flow cytometry were used to research growth inhibition and apoptosis of hepatocellular carcinoma cell (HepG2) intervened by RA combined with IFN-β 3. Immunofluorescence、western blot and RT-PCR were used to detect the expression level of the GR1M-19in HepG2intervened by RA combined with IFN-β.4. Western blot was used to detect the expression level of the GRIM-19、STAT3、p-STAT3and VEGF in HepG2intervened by RA combined with IFN-β.Results1. Cellular Localization of GRIM-19in hepatocellular carcinoma cell linesIn our previous studies, we first analyzed the localization of GRIM-19in liver tissues using a purified GRIM-19antibody and an immunohistochemistry assay. GRIM-19protein expression was predominantly located in the cytoplasm with weak staining in the nucleus in ANLT, but only located in the cytoplasm in HCC tissues. To confirm this observation, we analyzed the cellular localization of endogenous GRIM-19in LO2cells (a human hepatic cell line), HepG2cells and MHCC-97H cells (a human hepatocellular carcinoma cell line) using a purified GRIM-19antibody and an immunofluorescence assay. Positive staining for GRIM-19was observed in the cytoplasm of all cells, which was intensified near the peri-nuclear region. In addition, weak staining was also noticeable in the nucleus of the LO2cells but not in the nucleus of the HepG2cells and MHCC-97H.2. Expression levels of GRIM-19in different hepatocellular carcinoma cell linesSince GRIM-19was identified as a potential tumor suppressor in recent studies, we evaluated the expression status of GRIM-19in primary liver cancers using immunohistochemistry. Briefly, GRIM-19protein expression was extremely lower in HCC tissues than in the ANLT. To explore the relationship between human hepatocellular carcinoma cell lines and hepatic cell lines, we evaluated the expression status of endogenous GRIM-19expression levels in LO2cells, HepG2cells and MHCC-97H cells by immunofluorescence experiments at equal pace. Endogenous GRIM-19expression in HepG2cells was significantly lower than in LO2cells, but it was higher than in the MHCC-97H cells. These findings were confirmed by determination of the endogenous GRIM-19protein expression levels by western blot analysis and endogenous GRIM-19mRNA levels were determined by QRT-PCR. Consistent with the immunofluorescence data, endogenous GRIM-19expression in HepG2cells was higher than in MHCC-97H cells but lower than in LO2cells. In addition, endogenous STATS、p-STAT3and VEGF expression in HepG2cells was significantly higher than in LO2cells, but it was lower than in the MHCC-97H cells.3. The growth inhibition of HepG2intervened by RA combined with IFN-βThe combination of RA and IFN-βhas been shown to inhibit cell growth in other cell lines. To determine if IFN/RA exerts similar growth-suppressive effects in the hepatocellular carcinoma cells, human hepatocellular carcinoma HepG2cells grown in culture dishes were treated either with either IFN or RA or a combination of IFN/RA. MTT assays were performed. As shown in Table2, exposure to RA or IFN-β alone can inhibit the growth of HepG2cells, but RA combined with IFN-β can greatly increase it, and this is associated with concentration and time. We found that too much RA/IFN-β or too long time can induce cell death in HepG2cell lines. Exposure to RA or IFN-β alone did not cause cell death. Similarly, combined exposure to IFN/RA for24h and48h did not cause significant cell death, where as combined exposure to IFN/RA for72h caused significant cell death. So, we chose48h as best exposure time to study the biology change of HepG2cells combined exposure to different concentration of IFN-β and RA in following.4. The apoptosis of HepG2intervened by RA combined with IFN-βHuman hepatocellular carcinoma HepG2cells at a low density and then exposing to different concentration of IFN/RA for48h. To examine the effect of RA combined with IFN-P on the apoptosis of HepG2cells, flow cytometry were used. The result showed that IFN/RA exhibits a dual effect on cell growth:inhibition of cell growth and an induction of cell apoptosis. RA combined with IFN-P can promote the apoptosis of HepG2cells, and this is associated with concentration.5. GRIM-19expression increased and appear nuclear expression in HepG2after exposed to RA/IFN-pTo determine if the cell apoptosis could be correlated with the expression of GRIM-19, we analyzed the cellular localization of GRIM-19in HepG2cells after intervened by different concentration of IFN/RA for48h using the purified GRIM-19antibody by immunofluorescence. Positive staining of GRIM-19was observed in the cytoplasm of untreated HepG2cells which was intensified at the peri-nuclear region. In addition, a positive staining was also noticeable in the nucleus after the IFN-β/RA treatment, which was significantly increased following the increased of the concentration. To verify these results further, pure nuclear protein was extracted in HepG2cells which absence or presence of IFN-β/RA treatment, and GRIM-19expression was determined. Consistent with the immunofluorescence results, the expression of GRIM-19in the nucleus was increased slightly after IFN-β/RA treatment.6. STAT3, p-STAT3and VEGF expression decreased after exposed to RA/IFN-β We detected the expression of GRIM-19in HepG2cells after exposed to different concentration of IFN/RA for48h using the purified GRIM-19antibody by Western blot. The result indicated that GRIM-19protein expression increased than control group(p <0.05). More importantly, the lower expression of STAT3、p-STAT3、VEGF were found in treatment groups than control cells.Conclusion1. The location of GRIM-19in human hepatic cell lines was different with the hepatocellular carcinoma cell lines. And the expression of GRIM-19in human hepatic cell lines was higher than hepatocellular carcinoma cell lines.2. RA combination with IFN-β can inhibit the growth and promote the apoptosis of HepG2cells.3. RA combination with IFN-β can up-regulate of GRIM-19protein expression and down-regulate of STAT3、p-STAT3、VEGF expression.4. GRIM-19may be a new target spot in liver cancer gene therapy. |
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