Qi-Man Sun

Macrophage-secreted IL-8 induces epithelial-mesenchymal transition in hepatocellular carcinoma cells by activating the JAK2/STAT3/Snail pathway.

Macrophages are a major component of the leukocyte infiltrate of tumors and play a pivotal role in the progression of hepatocellular carcinoma (HCC). However, the molecular mechanisms by which macrophages promote HCC invasion are poorly understood. The present study was undertaken to investigate the relationship between macrophages and epithelial-mesenchymal transition (EMT) of HCC. Double-staining immunohistochemistry was used to observe the association between macrophages and EMT markers in clinical HCC samples and it showed that EMT primarily occurred at the edge of the tumor nest, in which infiltrating macrophages were always observed. This indicated that CD68 which is a marker of macrophages, was correlated with EMT marker levels. In addition, after being cultured with macrophages for 24 h, the ability of HCC cells to migrate and invade increased, Snail and N-Cadherin expression was upregulated, and E-Cadherin was downregulated. An antibody array assay was applied to analyze the supernatant of these cultures and it demonstrated IL-8 increased significantly in the macrophage co-culture system. Finally, the role of macrophage-derived IL-8 in the invasion of HCC cells was assayed, and downstream signaling pathways were also investigated. We found that IL-8: i) may induce EMT and promote HCC cell migration and invasion and ii) is associated with the JAK2/STAT3/Snail signaling pathway. Taking together, these findings revealed that macrophages that have infiltrated tumors may induce epithelial-mesenchymal transition of HCC cells via the IL-8 activated JAK2/STAT3/Snail pathway. Thus, this may offer a potential target for developing new HCC therapies.

Plasma microRNA, a potential biomarker for acute rejection after liver transplantation.

Background Acute rejection (AR) of an organ transplant is a life-threatening complication. Currently, there are few diagnostic biomarkers suitable for clinical application. We aim to determine the potential of plasma microRNAs as biomarkers for AR. Methods Using rat orthotopic liver transplantation model and microarrays, we compared the difference in the spectrum and levels of microRNAs in both plasma and grafts between AR rats and control. AR-related plasma microRNAs were selected and validated using real-time quantification polymerase chain reaction. Plasma from AR rats with or without tacrolimus treatment was used for microRNA dynamic monitoring. To clarify the origin of AR-related plasma microRNAs, drug-induced liver damage rat model were performed and in situ hybridization was used to detect and localize the specific microRNA in allografts. Results We found that plasma miR-122, miR-192, and miR-146a was significantly up-regulated when AR occur (fold change>2; P<0.05) and the elevation could be repressed by immunosuppression. In liver injury rat model, up-regulated plasma miR-122 (fold change=22.126; P=0.002) and miR-192 (fold change=8.833; P<0.001) rather than miR-146a (fold change=1.181; P=0.594) were observed. Further study demonstrated that miR-146a was up-regulated by sixfold in microvesicles isolated from AR plasma, whereas miR-122 and miR-192 showed no distinct change. In situ hybridization revealed that the portal areas of the AR graft were brimming with lymphocytes, which showed highly intense staining for miR-146a. Conclusions Our study provides the global fingerprint of plasma microRNAs in AR rats and suggests that plasma miR-122 and miR-192 reflect liver injury, whereas miR-146a may associate with cellular rejection.

New-onset diabetes after liver transplantation and its impact on complications and patient survival.

The aim of the present study was to investigate the incidence and risk factors of new‐onset diabetes after transplantation (NODAT) in liver transplant recipients and the influence of NODAT on complications and long‐term patient survival.

Circumventing intratumoral heterogeneity to identify potential therapeutic targets in hepatocellular carcinoma

BACKGROUND & AIMS Identifying target genetic mutations in hepatocellular carcinoma (HCC) for therapy is made challenging by intratumoral heterogeneity. Circulating cell-free DNAs (cfDNA) may contain a more complete mutational spectrum compared to a single tumor sample. This study aimed to identify the most efficient strategy to identify all the mutations within heterogeneous HCCs. METHODS Whole exome sequencing (WES) and targeted deep sequencing (TDS) were carried out in 32 multi-regional tumor samples from five patients. Matched preoperative cfDNAs were sequenced accordingly. Intratumoral heterogeneity was measured using the average percentage of non-ubiquitous mutations (present in parts of tumor regions). Profiling efficiencies of single tumor specimen and cfDNA were compared. The strategy with the highest performance was used to screen for actionable mutations. RESULTS Variable levels of heterogeneity with branched and parallel evolution patterns were observed. The heterogeneity decreased at higher sequencing depth of TDS compared to measurements by WES (28.1% vs. 34.9%, p<0.01) but remained unchanged when additional samples were analyzed. TDS of single tumor specimen identified an average of 70% of the total mutations from multi-regional tissues. Although genome profiling efficiency of cfDNA increased with sequencing depth, an average of 47.2% total mutations were identified using TDS, suggesting that tissue samples outperformed it. TDS of single tumor specimen in 66 patients and cfDNAs in four unresectable HCCs showed that 38.6% (26/66 and 1/4) of patients carried mutations that were potential therapeutic targets. CONCLUSIONS TDS of single tumor specimen could identify actionable mutations targets for therapy in HCC. cfDNA may serve as secondary alternative in profiling HCC genome. LAY SUMMARY Targeted deep sequencing of single tumor specimen is a more efficient method to identify mutations in hepatocellular carcinoma made from mixed subtypes compared to circulating cell-free DNA in blood. cfDNA may serve as secondary alternative in profiling HCC genome. Identifying mutations may help clinicians choose targeted therapy for better individual treatments.

Long non-coding RNA00364 represses hepatocellular carcinoma cell proliferation via modulating p-STAT3-IFIT2 signaling axis

The effects of long non-coding RNAs (lncRNAs) on hepatocellular carcinoma (HCC) remain largely unclear. In this study, we identified an interferon (IFN)-γ-induced LncRNA, LncRNA00364, in HCC by microarray. LncRNA00364 displays lower expression in HCC tumor samples compared to paired normal controls. Overexpression of LncRNA00364 inhibits cell proliferation, G1/S cell cycle progression and promotes apoptosis in HCC cell lines. Consistently, LncRNA00364 overexpression leads to decreased HCC tumor formation in vivo. Mechanistically, LncRNA00364 specifically binds with STAT3, resulting in inhibition of STAT3 phosphorylation and therefore leads to upregulation of IFIT2. In a clinical setting, LncRNA00364 shows an independent prognostic indicator for overall survival and cumulative recurrence in HCC patients, and correlates with IFIT2. Therefore, our study provides new insights into a novel therapeutic avenue targeting the LncRNA00364 signaling axis in HCC.

Tumour-suppressive role of PTPN13 in hepatocellular carcinoma and its clinical significance.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer mortality and carries a dismal prognosis. The present study aimed to identify the tumour-suppressive role and clinical implications of PTPN13 in HCC progression. We tested the effects of PTPN13 expression in proliferation, invasion, epithelial–mesenchymal transition and associated pathways in HCC cell lines in vitro. Furthermore, its clinical relevance was evaluated in a tissue microarray analysis of samples from 282 HCC patients. Various HCC cell lines expressed relatively low PTPN13 protein levels in vitro. PTPN13 overexpression significantly inhibited the progression of HCC cells, possibly by inhibiting epithelial–mesenchymal transition through inactivation of the EGFR/ERK signalling pathway. Tissue microarray analysis revealed that high PTPN13 expression was correlated with a favourable prognosis in postoperative HCC patients. This study demonstrated the tumour suppressor, PTPN13, as an alternative therapeutic target for HCC.

Tacrolimus promotes hepatocellular carcinoma and enhances CXCR4/SDF‑1α expression in vivo

The aim of our study was to elucidate the effect of tacrolimus (FK506) and of C-X-C chemokine receptor type 4 (CXCR4), which is a receptor specific to the stromal cell-derived factor-1α (SDF-1α), on growth and metastasis of hepatocellular carcinoma (HCC). Following treatment with different concentrations of FK506, AMD3100 or normal saline (NS), the proliferation of Morris rat hepatoma 3924A (MH3924A) cells was measured by the MTT assay, the expression of CXCR4 was analyzed with immunohistochemistry, and the morphological changes and the invasiveness of cells were studied with a transwell assay and under a scanning electron microscope, respectively. In addition, August Copenhagen Irish rat models implanted with tumor were used to examine the pathological changes and invasiveness of tumor in vivo, the expression of CXCR4 in tumor tissues and the expression of SDF-1α in the adjacent tissues to the HCC ones, using immunohistochemistry. In vitro, FK506 (100–1,000 μg/l) significantly promoted the proliferation of MH3924A cells (P<0.01), and increased the expression of CXCR4 in MH3924A cells, albeit with no significance (P>0.05). By contrast, AMD3100 had no effect on the proliferation of MH3924A cells, but significantly reduced the expression of CXCR4 (P<0.05). The invasiveness of MH3924A cells was significantly (P<0.01) enhanced following treatment with FK506, SDF-1α, FK506 + AMD3100, FK506 + SDF-1α or FK506 + AMD3100 + SDF-1α. In vivo, tumor weight (P=0.041), lymph node metastasis (P=0.002), the number of pulmonary nodules (P=0.012), the expression of CXCR4 in tumor tissues (P=0.048) and that of SDF-1α in adjacent tissues (P=0.026) were significantly different between the FK506-treated and the NS group. Our results suggest that FK506 promotes the proliferation of MH3924A cells and the expression of CXCR4 and SDF-1α in vivo. Therefore, inhibiting the formation of the CXCR4/SDF-1α complex may partly reduce the promoting effect of FK506 on HCC.

Donor liver steatosis: A risk factor for early new‐onset diabetes after liver transplantation

To investigate whether donor liver steatosis increases the incidence of new‐onset diabetes after transplantation (NODAT) in liver transplant recipients.

Whole-Exome Sequencing-Based Mutational Profiling of Hepatitis B Virus-Related Early-Stage Hepatocellular Carcinoma

Background Hepatocellular carcinoma (HCC) ranks as the third leading cause of cancer-related mortality in China with increasing incidence. This study is designed to explore early genetic changes implicated in HCC tumorigenesis and progression by whole-exome sequencing. Methods We firstly sequenced the whole exomes of 5 paired hepatitis B virus-related early-stage HCC and peripheral blood samples, followed by gene ontological analysis and pathway analysis of the single-nucleotide variants discovered. Then, the mutations of high frequency were further confirmed by Sanger sequencing. Results We identified a mutational signature of dominant T:A>A:T transversion in early HCC and significantly enriched pathways including ECM-receptor interaction, axon guidance, and focal adhesion and enriched biological processes containing cell adhesion, axon guidance, and regulation of pH. Eight genes, including MUC16, UNC79, USH2A, DNAH17, PTPN13, TENM4, PCLO, and PDE1C, were frequently mutated. Conclusions This study reveals a mutational profile and a distinct mutation signature of T:A>A:T transversion in early-stage HCC with HBV infection, which will enrich our understanding of genetic characteristics of the early-stage HCC.

Effect of interleukin-2 receptor antagonists on new-onset diabetes after liver transplantation: A retrospective cohort study.

The aim of the present retrospective observational study was to examine the effect of interleukin‐2 receptor antagonists (IL‐2Ra) on new‐onset diabetes after transplantation (NODAT) in liver transplant recipients.