Browsing by Subject "hepatocellular carcinoma"
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Item Identification of long non-coding RNA-related and -coexpressed mRNA biomarkers for hepatocellular carcinoma(BioMed Central Ltd., 2019-01-31) Zhang, Fan; Ding, Linda; Cui, Li; Barber, Robert C.; Deng, BinBackground: While changes in mRNA expression during tumorigenesis have been used widely as molecular biomarkers for the diagnosis of a number of cancers, the approach has limitations. For example, traditional methods do not consider the regulatory and positional relationship between mRNA and lncRNA. The latter has been largely shown to possess tumor suppressive or oncogenic properties. The combined analysis of mRNA and lncRNA is likely to facilitate the identification of biomarkers with higher confidence. Results: Therefore, we have developed an lncRNA-related method to identify traditional mRNA biomarkers. First we identified mRNAs that are differentially expressed in Hepatocellular Carcinoma (HCC) by comparing cancer and matched adjacent non-tumorous liver tissues. Then, we performed mRNA-lncRNA relationship and coexpression analysis and obtained 41 lncRNA-related and -coexpressed mRNA biomarkers. Next, we performed network analysis, gene ontology analysis and pathway analysis to unravel the functional roles and molecular mechanisms of these lncRNA-related and -coexpressed mRNA biomarkers. Finally, we validated the prediction and performance of the 41 lncRNA-related and -coexpressed mRNA biomarkers using Support Vector Machine model with five-fold cross-validation in an independent HCC dataset from RNA-seq. Conclusions: Our results suggested that mRNAs expression profiles coexpressed with positionally related lncRNAs can provide important insights into early diagnosis and specific targeted gene therapy of HCC.Item Inhibition of the Glutaredoxin System Increases Doxorubicin Sensitivity in Hepatocellular Carcinoma By Impairing the Nrf2-Dependent Antioxidant Response(2015-05-01) Xavier, Christy; Wu, Hongli; Di Pasqua, Anthony J.; Yan, Liang-JunPurpose: Hepatocellular carcinoma (HepG2) is the most common type of liver cancer, causing approximately 1.25 million deaths annually. Even with premier anti-cancer drugs like doxorubicin, the lethality of hepatocellular carcinoma has increased and is mainly attributed to growing drug resistance. Specifically, overexpression of key antioxidant enzymes such as the glutaredoxin system (Grxs) may enable drug resistance. Glutaredoxin is a powerful protective thiol repair enzyme that increases cancer cell survival. In this study, we explored a new anti-cancer strategy, the inhibition of Grxs, as a way to both increase apoptosis and doxorubicin sensitivity in HepG2 by impairing the Nrf2-dependent antioxidant response. Methods: Hepatocellular carcinoma tissue and neighboring healthy liver tissue was obtained from five patients from Sun-Yat-Sen Hospital and tested for Grx1 and Grx2 expression levels using western blot. HepG2 cells were transfected with Grxs or scramble shRNA vector. Scramble sh-RNA, Grx1 shRNA, and Grx2shRNA HepG2 cells were treated with 1 uM and 10 uM doxorubicin, and cell viability was measured by the WST-8 colorimetric assay. Western blot was performed to test expression levels of pro- and anti-apoptotic proteins like Bax, Bcl2, and cleaved caspase-3. The level of protein glutathionylation (PSSG) in whole cell lysates and mitochondrial fractions were measured by immunoblotting using anti-PSSG antibody. Western blot was used to also examine the expression levels of Nrf2 and its downstream proteins in Grxs-inhibited cells before and after doxorubicin treatment. Nrf2 translocation assay and co-immunoprecipitation with Grxs and PSSG was also performed. Immunostaining was done to confirm Grx2’s presence in the nucleus and Grx2-Nrf2 binding. NucBlue live cell staining was performed to analyze Grx2’s possible function in the nucleus. Antioxidant gene screening for 48 Nrf2-pathway related genes for scramble and Grxs shRNA after doxorubicin treatment was analyzed. Results: All five patients in our clinical study showed much higher Grx1 and Grx2 levels in cancerous tissue than in normal liver tissue. shRNA transfection gave a 50-70% Grxs knockdown. Grxs inhibition caused increased doxorubicin sensitivity with approximately a 20% lower cell viability at 1 and 10 uM doxorubicin treatment, higher pro-apoptotic protein expression levels, and increased glutathionylation than control. Moreover, actin glutathionylation increased in Grxs shRNA cells. Grxs inhibition also decreased the expression of antioxidant enzyme transcription factor regulator, Nrf2, and its downstream antioxidant proteins like HO-1, catalase, thioredoxin, and NQO1 especially after doxorubicin treatment. Nrf2’s presence in the nucleus and cytoplasm decreased with glutaredoxin knockdown. Doxorubicin treatment enhanced Grx1 and Grx2 binding with Nrf2 in scramble shRNA HepG2 cells. Glutaredoxin inhibition also significantly increased Nrf2 glutathionylation, indicating lower Nrf2 activation. Immunostaining showed abundant amount of Grx2 in the nucleus and that Grx2 specifically interacts and repairs Nrf2 in the nucleus. Grx2 may also be involved in DNA repair and synthesis in the nucleus. Gene screening also showed significant decrease in mRNA levels of Bcl2, GSTs, and Prdx6 with Grxs inhibition after doxorubicin treatment. Conclusions: Grx1 is vital to repair Nrf2 in the cytoplasm, whereas Grx2 repairs Nrf2 in the nucleus, and if inhibited, can cause an increase in Nrf2 glutathionylation and inactivation. Grxs inhibition causes increased doxorubicin sensitivity and apoptosis of hepatocellular carcinoma by attenuating Nrf2 and its downstream antioxidant genes’ activation, making it an ideal pharmacological target for future anti-liver cancer treatment.Item ROLE OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 NEF IN ACCELERATION OF HEPATITIS C VIRUS-MEDIATED LIVER DISEASE.(2013-04-12) Park, In-WooPurpose: HIV-1 infection has profound, adverse consequences for every stage of the natural history of HCV infection via significantly elevating HCV viral load and expediting HCV-mediated liver disease progression in the co-infected host. However, molecular details for how HIV-1 accelerates this pathogenesis are largely unknown. According to recent publications, HIV-1 Nef can be transferred from HIV-1 susceptible cells to other uninfected susceptible cells and even to non-susceptible target cells by formation of conduits or by exosomes, suggesting that Nef is a leading candidate molecule for explaining the occurrence of HIV-1-mediated opportunistic diseases in non-susceptible target tissues. Accordingly, we have investigated the role of HIV-1 and viral protein Nef in HCV-infected hepatocytes to better understand the pathobiology of HIV-1 and HCV co-infection. Methods: Infectivity of HIV-1 in human hepatocytes was monitored at the indicated time point by measuring reverse transcriptase (RT) activity in the clarified culture supernatants, and effect of Nef on the expression of HCV replicon was examined by measuring reporter gene, Luciferase (Luc), expression in the replicon cells. Transfer of Nef to target hepatocytes and subcellular distribution of lipid droplets (LD) were studied by immunofluoscence and confocal microscopic analyses as well as by flow cytometric analyses. Results: Infectious HIV-1 failed to replicate in human hepatocytic cell lines. No discernible virus replication was observed, even when the hepatocytes transfected with HIV-1 proviral DNA were co-cultured with Jurkat T cells, indicating that the problem of liver deterioration in the co-infected patient is not due to the replication of HIV-1 in the hepatocytes of the HCV infected host. Instead, HIV-1 Nef protein was found to be transferred from expressing T cells to hepatocytes through conduits, wherein up to 16% (average 10%) of hepatocytes harbored the transferred Nef, when the hepatocytes were co-cultured with nef-expressing Jurkat cells for 24 h. Further, Nef altered the size and numbers of LD, and consistently up-regulated HCV replication by 1.5~2.5 fold in the target hepatocytes, which is remarkable in relation to the initially indolent viral replication. Conclusions: HIV-1 Nef is a critical element in accelerating HCV-mediated liver pathogenesis through modulation of lipid molecules and changes in HCV replication.