| Increased DJ-1 and its prognostic significance in hepatocellular carcinomaObjective:To investigate the expression profile of DJ-1 gene and its clinical relevance and prognostic value in hepatocellular carcinoma (HCC).Methods:Specimens from 149 HCC patients were applied for DJ-1 expression through immunohistochemistry. The correlation of DJ-1 levels with clinicopathologic variables and prognosis was analyzed.32 paired HCC and para-carcinomatous liver tissue (PCLT) specimens from 149 HCC patients plus 10 hepatic cirrhosis specimens and 10 normal liver specimens were detected by reverse transcription quantitative polymerase chain reaction (RT-PCR) and Western Blot.Results:DJ-1 was up-regulated significantly in HCC by semi-quantitative/Real-Time PCR and Western blot. DJ-1 expression closely correlated with preoperative AFP, liver cirrhosis, vein invasion, differentiation and Edmondson grade in HCCs by Pearson Chi-square test. Both of tumor-free survival time and overall survival time in the DJ-1 high expression group were shorter than those in the low expression group. DJ-1 was adopted as an independent prognostic factor for overall survival of HCC patients through multivariate Cox proportional hazard model analysis (HR,2.568; P=0.003). Additionally, immunohistochemistry analysis revealed that expression of DJ-1 negatively correlated with expression of tumor suppressor gene phosphatase and tensin homolog deleted on chromosome ten (PTEN) in HCC (r=-0.836; P<0.001).Conclusion:DJ-1 expression is significantly up-regulated in HCC, and its expression level correlates with clinicopathological variables and prognosis of HCC patients, which suggests that DJ-1 maybe a candidate prognostic biomarker of HCC. Prognosis.The expression of oncogene DJ-1 in human hepatoma cell lines and DJ-1 small hairpin RNA stably transfected HepG2 cellsObjective:To detect the expression of oncogene DJ-1 in human hepatoma cell lines, construct the DJ-1 small hairpin RNA (short hair RNA, shRNA) eukaryotic expression vector, obtain RNA interference DJ-1 expression plasmid and the stably transfected HepG2 cell lines.Methods:Adopt Western Blot and immunocytochemistry for exploring the protein expression level and subcellular localization of oncogene DJ-1 in different human hepatoma cell lines, respectively. Design interference sequence structures targeted oncogene DJ-1, and inserted into RNA interference (RNAi) eukaryotic expression vector plasmid pGenesil-1.1, digested with restriction enzymes and DNA sequencing. The recombinant RNAi vectors were transfected into human hepatoma HepG2 cells with Lipofectamine 2000. The transfected cells were persistently screened under G418, and isolated with a limited dilution. The mRNA expression and protein expression of DJ-1 in the selected clones were evaluated by semi-RT PCR and Western Blot, respectively.Results:DJ-1 was highly expressed in human hepatoma cell lines, including HepG2, MHCC-97L, MHCC-97H, SMMC-7721 and Huh-7, in which the expressions have evidently differences (P<0.05). DJ-1 expression was mainly localized in the cytoplasm of human hepatoma cells detected by Confocal. However, the expression of DJ-1 was negative in human fetal liver cell line L02 (control). Synthesis and contruct recombinant DJ-1 shRNA plasmid vectors, and DNA sequencing indicated that RNAi eukaryotic expression vectors targeting DJ-1 with correct reading frame and nucleotide sequence. Green fluorescence of the stably transfected HepG2 cell lines were observed under the fluorescence microscope. semi-RT PCR revealed that DJ-1 shRNA effectively silenced mRNA expression level of DJ-1. Western Blot discovered that DJ-1 shRNA obviously suppressed protein expression level of DJ-1, of which the inhibition rate was 98.78% (P< 0.05).Conclusion:DJ-1 was highly expression in the cytoplasm of human hepatoma cell lines, of which the expression levels could correlate with the malignant biological characteristics in different cell lines. DJ-1 shRNA eukaryotic expression vectors were constructed successfully and the establishment of stably transfected HepG2 cell lines provided an original route for exploring the mechanism of DJ-1 in human hepatocellular carcinoma cells further.Role of DJ-1 down regulation by shRNA on the Akt/NF-kappaB signaling pathway in human hepatocellular carcinoma HepG2 cellsObjective:To explore the expression profile and potential downstream effectors of the oncogene DJ-1 in hepatocellular carcinoma (HCC) cell lines and its role in the Akt/nuclear factor (NF)-kappaB signaling pathway.Methods:Expressions of DJ-1 and PTEN proteins were analyzed by Western Blot in several human HCC cell lines, including HepG2, SMMC-7721, Huh-7, Hep3B, MHCC-97L and MHCC-97H. Knock-down of DJ-1 was achieved by transfecting DJ-1-specific small hairpin RNAs into HepG2 cells. Potential targets of DJ-1, including Akt, phosphorylated Akt, nuclear factor kappaB (NF-κB) and inhibitor of NF-κB (IκB), were observed in vitro and in vivo. Cell cycle, proliferation, adhesion and invasion were analyzed via flow cytometry, MTT assay, adhesion and invasion assays in vitro, respectively. Tumor growth was evaluated in vivo.Results:DJ-1 was profoundly up-regulated in different HCC cell lines with different hepatoma cell metastasis potential including HepG2, SMMC-7721, Huh-7, Hep3B, MHCC-97L and MHCC-97H, in which the expressions have evidently differences (P< 0.05). PTEN was constitutively low expression in HCC cell lines. A DJ-1 shRNA eukaryotic expression vector was successfully obtained and transfected stably into HepG2 cells. Tumor formation in the nude mouse model was successfully established, the tumor growth velocity of silencing DJ-1 expression was significantly slow (P<0.05). The down-regulation of DJ-1 in HepG2 cells increased PTEN expression, decreased phosphorylation of Akt and inhibited activation of NF-κB both in hepatoma cells (in vitro) and in nude mouse tumor samples (in vivo). Furthermore, suppression of DJ-1 expression resulted in decreased proliferation, adhesion and invasion of HepG2 cells in vitro, inhibited tumor formation in vivo and blocked the G1/S transition in the cell cycle.Conclusion:DJ-1 shRNA expression vector-mediated RNAi could downregulate DJ-1 expression, phosphorylation of Akt and activation of NF-κB, and upregulate PTEN expression in human HCC HepG2 cells. Meanwhile, DJ-1 downregulation suppressed the proliferation, adhesion and invasion of HepG2 cells. Our findings provide a clue that DJ-1 plays a crucial role in the oncogenesis of HCC and regulates the activation of the Akt/NF-κB signaling pathway.
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