Guohua Lou

The p53/miR-34a/SIRT1 Positive Feedback Loop in Quercetin-Induced Apoptosis.

Background: The anti-tumor effects of quercetin have been reported, but the underlying molecular mechanisms remain to be elucidated. The aim of present study was to explore the role of miRNA in the anticancer effects of quercetin. Methods: The differential miRNAs expression between the HepG2 and Huh7 cells treated by quercetin were detected by microarray. The xCELLigence, Flow cytometry, RT-PCR and Western blot were used to analyze the cell proliferation, cell apoptosis, cell cycle arrest, anti-tumor genes, and protein expression. Results: miR-34a was up-regulated in HepG2 cells treated by quercetin exhibiting wild-type p53. When inhibiting the miR-34a, the sensitivity of the cells to quercetin decreased and the expression of the SIRT1 was up-regulated, but the acetylation of p53 and the expression of some genes related to p53 down-regulated. Conclusion: miR-34a plays an important role in the anti-tumor effects of querctin in HCC, miR-34a may be a tiemolecule between the p53 and SIRT1 and is composed of a p53/miR-34a/SIRT1 signal feedback loop, which could enhance apoptosis signal and significantly promote cell apoptosis.

Mesenchymal stem cell-derived exosomes as a new therapeutic strategy for liver diseases

The administration of mesenchymal stem cells (MSCs) as a therapy for liver disease holds great promise. MSCs can differentiate into hepatocytes, reduce liver inflammation, promote hepatic regeneration and secrete protective cytokines. However, the risks of iatrogenic tumor formation, cellular rejection and infusional toxicity in MSC transplantation remain unresolved. Accumulating evidence now suggests that a novel cell-free therapy, MSC-secreted exosomes, might constitute a compelling alternative because of their advantages over the corresponding MSCs. They are smaller and less complex than their parent cells and, thus, easier to produce and store, they are devoid of viable cells, and they present no risk of tumor formation. Moreover, they are less immunogenic than their parent cells because of their lower content in membrane-bound proteins. This paper reviews the biogenesis of MSC exosomes and their physiological functions, and highlights the specific biochemical potential of MSC-derived exosomes in restoring tissue homeostasis. In addition, we summarize the recent advances in the role of exosomes in MSC therapy for various liver diseases, including liver fibrosis, acute liver injury and hepatocellular carcinoma. This paper also discusses the potential challenges and strategies in the use of exosome-based therapies for liver disease in the future.

Involvement of the NF-κB pathway in multidrug resistance induced by HBx in a hepatoma cell line

Summary.  It is widely believed that hepatocellular cancer (HCC), especially HBV associated HCC, is highly resistant to chemotherapy. To investigate the molecular influence of HBx protein on multidrug resistance (MDR) in HCC and the potential role of the NF‐κB pathway in this process. We established HBx‐expressing cells by liposome‐mediated transfection of the HBx into the HepG2 cell line. We found that HBx expression in HCC cells induces drug resistance against multiple drugs, a significantly lower apoptosis ratio in HepG2‐HBx and HepG2.2.15 cells, compared with HepG2 and HepG2‐3.1 cells (P < 0.05) after treating with 5‐FU or adriamycin. And compared with the control group, the HBx‐transfected cells showed a higher expression of MDR‐associated and anti‐apoptotic genes. Furthermore, we found that the NF‐κB activity was remarkably high in the HBx‐expressing cells as measured by p65 nuclear localization. In addition, the upregulated anti‐apoptotic genes, Gadd45b and Survivin, in HBx‐expressing HCC cells were downregulated by IMD‐0354 treatment, which is the NF‐κB pathway inhibitor. Taken together, these results suggest that HBx protein might be one of the causes for the occurrence of MDR in HCC, and the NF‐κB pathway might be involved in this change.

Isoliquiritigenin Inhibits Interferon-γ-Inducible Genes Expression in Hepatocytes through Down-Regulating Activation of JAK1/STAT1, IRF3/MyD88, ERK/MAPK, JNK/MAPK and PI3K/Akt Signaling Pathways

Background & Aims: The high expression levels of interferon-γ (IFN-γ)-inducible genes correlate positively with liver diseases. The present study aimed to explore the effect of isoliquiritigenin (ISL) on the expression of genes induced by IFN-γ in vitro, and to elucidate the underlying molecular mechanisms. Methods: HepG2 and L02 cells were divided into control, ISL, IFN-γ, and IFN-γ plus ISL groups. The cytotoxicity of compounds to cells was evaluated by Cell Counting Kit 8 (CCK8) assay; the expression levels of chemokine (C-X-C motif) ligand 9 (CXCL9), CXCL10, CXCL11, and interleukin-6 (IL-6) in cells and supernatant were measured by quantitative real time polymerase chain reaction (qRT-PCR) and ELISA, respectively. Moreover, western blot was used to examine the phosphorylated levels of janus kinase (JAK)/signal transducer and activator of transcription 1 (STAT1), nuclear factor (NF)-γB, interferon regulatory factor 3 (IRF3)/myeloid differentiation factor 88 (MyD88), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/Protein Kinase B (Akt) in HepG2 and L02 cells exposed to ISL, IFN-γ and IFN-γ plus ISL. Results: The results showed that IFN-γ treatment induced the expression of CXCL9, CXCL10, CXCL11, and IL-6 in HepG2 and LO2 cells, which could be significantly and dose-dependently inhibited by ISL treatment (P < 0.05 or P < 0.01), but the inhibitory effect of ISL on IL-6 expression was not so good as on CXCL9, CXCL10, and CXCL11 expression. Furthermore, ISL treatment dose-dependently inhibited the activation of JAK1/STAT1, IRF3/MyD88, extracellular signal-regulated kinase (ERK)/MAPK, c-Jun N-terminal kinase (JNK)/MAPK, and PI3K/Akt signaling pathways (P < 0.05), but had no effect on the activation of JAK2/STAT1, NF-γB and p38/MAPK signaling pathways. Conclusion: We demonstrate that ISL inhibits IFN-γ-induced inflammation in hepatocytes via influencing the activation of JAK1/STAT1, IRF3/MyD88, ERK/MAPK, JNK/MAPK, and PI3K/Akt signaling pathways.

Interferon-α sensitizes HBx-expressing hepatocarcinoma cells to chemotherapeutic drugs through inhibition of HBx-mediated NF-κB activation.

BackgroundHepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) is characterized by high chemotherapy resistance; however, the underlying mechanism has not been fully clarified. In addition, HBx protein has been reported to play a key role in virus-mediated hepatocarcinogenesis. Therefore, the present study aims to investigate the role of HBx in the drug-resistance of HBV-related HCC and examine whether such drug-resistance can be reversed by IFN-α treatment.MethodsWe established HBx-expressing cells by liposome-mediated transfection of HBx into the Huh7 cell line. MTT, Annexin V/PI, and cell cycle assay were used for determining the cellular growth inhibition, apoptosis, and growth arrest, respectively, after treatment with chemical drug. We further used tumor-bearing mice model to compare the tumor growth inhibition efficacy of ADM and 5-FU between the Huh7-HBx group and the control group, as well as the ADM + IFN-α or ADM + IMD treated group and the ADM treated group. SQ-Real time-PCR was performed to analyze the expression of MDR-associated genes and anti-apoptotic genes. Moreover, immunofluorescence and Western blotting were used to determine the subcellular localization of p65 and the phosphorylation of IκBα.ResultsThe IC50 values of Huh7-HBx cells against ADM and Amn were 2.317 and 1.828-folds higher than those of Huh7-3.1 cells, respectively. The apoptosis ratio and growth arrest was significantly lower in Huh7-HBx cells after treatment with ADM. The in vivo experiment also confirmed that the Huh7-HBx group was much more resistant to ADM or 5-FU than the control. Furthermore, the expression of MDR-associated genes, such as MDR1, MRP1, LRP1, and ABCG2, were significantly up-regulated in Huh7-HBx cells, and the NF-κB pathway was activated after HBx gene transfection in Huh7 cells. However, combined with IFN-α in ADM treatment, the HBx induced drug-resistance in Huh7-HBx cells can be partly abolished in in vitro and in vivo models. Moreover, we found that the NF-κB canonical pathway was affected by IFN-α treatment, and the expression of anti-apoptotic genes, such as Gadd45β, Survivin, and c-IAP-1 was down-regulated by IFN-α treatment in a dose-dependent manner.ConclusionsHBx protein can induce MDR of HBV-related HCC by activating the NF-κB pathway, which can be partly abolished by IFN-α treatment.

Direct targeting sperm-associated antigen 9 by miR-141 influences hepatocellular carcinoma cell growth and metastasis via JNK pathway

BackgroundThe aberrant expression of sperm-associated antigen 9 (SPAG9) is associated with numerous cancers, including hepatocellular carcinoma (HCC). The exploration of molecules and mechanisms regulating SPAG9 expression may provide new options for HCC therapy.MethodsMiRNA target prediction programs were used to explore SPAG9-targeted miRNAs. SPAG9 and miR-141 expression were detected in HCC tissues and cell lines by Western blot and real-time PCR. Dual-luciferase reporter assay was utilized to validate SPAG9 as a direct target gene of miR-141. Cell proliferation, invasion, and migration assays were used to determine whether miR-141-mediated regulation of SPAG9 could affect HCC progression.ResultsAn inverse correlation was observed between SPAG9 and miR-141 expression in HCC tissues and cell lines. Dual-luciferase reporter assay further showed that SPAG9 was a direct target gene of miR-141. The ectopic expression of miR-141 could markedly suppress SPAG9 expression in HCC cells. MiR-141 overexpression also resulted in significantly reduced cell proliferation, invasion, and migration, and imitation of the SPAG9 knockdown effects on HCC cells. Furthermore, SPAG9 restoration in miR-141-expressing cells sufficiently attenuated the tumor-suppressive effects of miR-141. Finally, JNK activity was found to be reduced by miR-141 overexpression the same way as by SPAG9 silencing. The overexpression of SPAG9 lacking its 3′-UTR significantly restored JNK activity and its downstream genes in miR-141-transfected HCC cells.ConclusionMiR-141 suppression may cause aberrant expression of SPAG9 and promote HCC tumorigenesis via JNK pathway.

The Sterile Inflammation in the Exacerbation of HBV-Associated Liver Injury

Exacerbation of hepatitis B virus-associated liver injury is characterized by abnormal immune response which not only mobilizes specific antiviral effects but also poses a potentially lethal nonspecific sterile inflammation to the host. How nonspecific sterile inflammation is triggered after the preexisting injury caused by specific immune injury remains elusive. In the setting of sterile inflammation, endogenous damage-associated molecular patterns are released by stressed and dying hepatocytes, which alarm the immune system through their potential pattern recognition receptors and related signaling pathways, orchestrate the influx of diverse cytokines, and ultimately amplify liver destruction. This review highlights current knowledge about the sterile hepatic inflammation in the exacerbation of chronic hepatitis B.

MiR‐122 modification enhances the therapeutic efficacy of adipose tissue‐derived mesenchymal stem cells against liver fibrosis

Mesenchymal stem cell (MSC) transplantation alone may be insufficient for treatment of liver fibrosis because of complicated histopathological changes in the liver. Given that miR‐122 plays an essential role in liver fibrosis by negatively regulating the proliferation and transactivation of hepatic stellate cells (HSCs), this study investigated whether miR‐122 modification can improve the therapeutic efficacy of adipose tissue‐derived MSCs in treating liver fibrosis. MiR‐122‐modified AMSCs (AMSC‐122) were constructed through lentivirus‐mediated transfer of pre‐miR‐122. MiR‐122‐modified AMSCs expressed high level of miR‐122, while they retained their phenotype and differentiation potential as naïve AMSCs. AMSC‐122 more effectively suppressed the proliferation of and collagen maturation in HSCs than scramble miRNA‐modified AMSCs. In addition, AMSC‐derived exosomes mediated the miR‐122 communication between AMSCs and HSCs, further affecting the expression levels of miR‐122 target genes, such as insulin‐like growth factor receptor 1 (IGF1R), Cyclin G(1) (CCNG1) and prolyl‐4‐hydroxylase α1 (P4HA1), which are involved in proliferation of and collagen maturation in HSCs. Moreover, miR‐122 modification enhanced the therapeutic efficacy of AMSCs in the treatment of carbon tetrachloride (CCl4)‐induced liver fibrosis by suppressing the activation of HSCs and alleviating collagen deposition. Results demonstrate that miR‐122 modification improves the therapeutic efficacy of AMSCs through exosome‐mediated miR‐122 communication; thus, miR‐122 modification is a new potential strategy for treatment of liver fibrosis.

MicroRNA-223 Acts as an Important Regulator to Kupffer Cells Activation at the Early Stage of Con A-Induced Acute Liver Failure via AIM2 Signaling Pathway

Background: Acute liver failure (ALF), known as a rapid and severe clinical syndrome, can induce multiple organ dysfunction and failure. It was noticed that Kupffer cells activation at the initial phase was involved in some intense inflammatory responses in the pathogenesis of ALF. However, detailed regulation mechanism of Kupffer cells activation during ALF is still obscured. Present study aimed to discover the potential regulator and explore deeper information of Kupffer cells activation at the early stage of ALF. Methods: The mouse model of ALF was established by Concanavalin A injection. Dynamic immunological statuses of Kupffer cells at the early stage of ALF were exhibited by detecting typical cytokines. The expression of inflammasome AIM2 was measured in both RNA and protein level. Its role of affecting Kupffer cells activation during ALF by inducing IL-1β production was identified by RNA interference in vitro. Moreover, the expression of miR-223 in vivo was measured by q-PCR and its role in regulating Kupffer cells activation during Con A induced ALF was determined by RNAs transfection. Results: Present study showed that mass production of IL-1β from isolated Kupffer cells in Con A treated mice might be the main driving force of Kupffer cells pro-inflammatory activation during ALF. The role of AIM2 in affecting pro-inflammatory activation of Kupffer cells by inducing IL-1β production was crucial to ALF. Further study found that miR-223 acted as a regulator in Kupffer cells activation at the early stage of ALF by influencing IL-1β production via AIM2 pathway. Conclusion: For the first time, this paper demonstrated that miR-223 acted to inhibit IL-1β production via AIM2 pathway, suppressing Kupffer cells pro-inflammatory activation at the early stage of ALF. Thus, it played an important role in the pathogenesis of ALF.

New Point Mutations in Surface and Core Genes of Hepatitis B Virus Associated with Acute on Chronic Liver Failure Identified by Complete Genomic Sequencing

The objective of this study was to identify new viral biomarkers associated with acute on chronic liver failure (ACLF) by complete genomic sequencing of HBV. Hepatitis B virus mutations associated with ACLF were screened by Illumina high-throughput sequencing in twelve ACLF cases and twelve age-matched mild chronic hepatitis B patients, which were validated in 438 chronic hepatitis B patients (80 asymptomatic carriers, 152 mild chronic hepatitis B patients, 102 severe chronic hepatitis B patients and 104 ACLF patients) by direct sequencing. The results of Illumina sequencing showed that the mutations at 7 sites (T216C, G285A, A1846T, G1896A, C1913A/G, A2159G, and A2189C) of 12 ACLF patients were significantly higher than those of 12 controls. In the validation cohorts, a significantly higher ratio of genotype B to C was found in patients with ACLF than in patients with non-ACLF. Multivariate analysis showed that T216C, G1896A, C1913A/G and A2159G/C were independent risk factors for ACLF. C216 in any combination, A/G1913 in any combination, and G/C2159 in any combination had high specificity for ACLF. In summary, T216C and A2159G/C mutations were novel factors independently associated with ACLF. Combined mutations in hepatitis B cases could play important roles in ACLF development.