The interaction between T cell immunoglobulin-and mucin-domain-containing molecule (Tim-3) expressed on T helper 1 (Th1) cells, and its ligand, galectin-9, negatively regulates Th1-mediated immune responses. However, it is poorly understood if and how the Tim-3/ galectin-9 signaling pathway is involved in immune escape in patients with hepatocellular carcinoma (HCC). Here we studied the expression, function, and regulation of the Tim-3/ galectin-9 pathway in patients with hepatitis B virus (HBV)-associated HCC. We detected different levels of galectin-9 expression on antigen-presenting cell (APC) subsets including Kupffer cells (KCs), myeloid dendritic cells (DCs), and plasmacytoid DCs in HCC. The highest galectin-9 expression was on KCs in HCC islets, not in the adjacent tissues. Furthermore, Tim-3 expression was increased on CD4 1 and CD8 1 T cells in HCC as compared to the adjacent tissues, and Tim-3 1 T cells were replicative senescent and expressed surface and genetic markers for senescence. Interestingly, tumor-infiltrating T-cell-derived interferon (IFN)-c stimulated the expression of galectin-9 on APCs in the HCC microenvironment. Immunofluorescence staining revealed a colocalization of Tim-3 1 T cells and galectin-91 KCs in HCC. Functional studies demonstrated that blockade of the Tim-3/ galectin-9 signaling pathway importantly increased the functionality of tumor-infiltrating Tim-3 1 T cells as shown by increased T-cell proliferation and effector cytokine production. Finally, we show that the numbers of Tim-3 1 tumor-infiltrating cells were negatively associated with patient survival. Conclusion: Our work demonstrates that the Tim-3/galectin-9 signaling pathway mediates T-cell senescence in HBV-associated HCC. The data suggest that this pathway could be an immunotherapeutic target in patients with HBV-associated HCC. (HEPATOLOGY 2012;56:1342-1351 H epatocellular carcinoma (HCC) is one of the most common cancers. More than 80% of patients are not candidates for curative treatments with the final diagnosis, and are linked to chronic infection with the hepatitis B (HBV) or hepatitis C (HCV) viruses based on different regions.
The carcinogenic role of Hepatitis B X (HBX) in hepatocellular carcinoma (HCC) remains largely unknown. Histone H3 lysine 4 methyltransferase SMYD3 was found to be over-expressed and have a pro-carcinogenic effect in HCC. The role of HBX in regulating SMYD3 activity and the corresponding C-MYC gene in HCC carcinogenesis was investigated. SMYD3 and C-MYC expression in HBV-negative HepG2 and HBV-positive HepG2.2.15 were detected by real time PCR and Western blot. After transfection of HBX into HepG2, SMYD3 and C-MYC protein expression was detected and the apoptosis and proliferation of hepatoma cells were assayed. After SMYD3 expression in HepG2 with HBX transfection downregulated by siRNA, the corresponding C-MYC expression, cellular apoptosis, and proliferation were assayed by FACS. SMYD3 mRNA and protein and C-MYC protein were significantly higher in HepG2.2.15 than in HepG2. HBX transfection resulted in enhanced SMYD3 and C-MYC expressions, decreased cell apoptosis, and increased cell proliferation in HepG2 cells. Knocking down of SMYD3 in HepG2 with HBX transfection inhibited C-MYC expression and promoted apoptosis. These results suggest that HBX upregulates SMYD3 expression in HepG2, which may promote hepatoma development and progress. C-MYC may act as a down-stream gene in HBX-SMYD3-related hepatocarcinogenesis.
Abstract. microRNAs (miRNAs) have been proven to play key regulatory roles in hepatocarcinogenesis. In the present study, the possible role of microRNA-450a (miR-450a) in hepatocarcinogenesis was investigated. Our study revealed that miR-450a was significantly down-regulated in hepatocellular carcinoma (HCC) tissues compared with that in normal liver (NL) and para-tumorous (PT) tissues, and miR-450a expression in HepG2 cells was significantly lower than that in L02 cells. Both the mRNA and protein levels of the miR-450a potential target gene, DNA methyltransferase 3a (DNMT3a), were obviously higher in HCC compared with levels in the NL and PT tissues. We further identified DNMT3a as the direct target gene for miR-450a, and ectopic miR-450a expression in HepG2 cells caused the down-regulation of DNMT3a and an inhibition of cell proliferation. Taken together, these findings suggest that miR-450a plays an important regulatory role in hepatocarcinogenesis through inhibition of DNMT3a expression, and miR-450a may be a potential target for the treatment of HCC. IntroductionHepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, especially in East Asia, including China. However, the pathogenesis of hepatocarcinogenesis is far from clear. microRNAs (miRNAs) are a type of highly conserved non-coding small RNAs which regulate gene expression at the post-transcriptional level. It is now clear that miRNAs can potentially regulate every aspect of cellular activity, including differentiation and development, metabolism, proliferation, and apoptotic and viral infection. Recently, miRNAs have been found to play pivotal roles in many malignancies including HCC development (1-9). The presence of a molecular prognostic miRNA signature in primary HCC clinical specimens has also been confirmed by several recent studies (4,6,7,(10)(11)(12), and many miRNAs have been found to play important regulatory roles in hepatocarcinogenesis.Our previous study found that miRNA-602 has an important regulatory activity in HBV-mediated hepatocarcinogenesis by inhibiting the tumor-suppressive gene RASSF1A from very early stages of chronic HBV hepatitis to HBV-positive cirrhosis to HCC (13). In this study, the expression of miRNA-450a and its potential target, DNA methyltransferase 3a (DNMT3a), was investigated in HCC. Materials and methodsPatients and cell lines. Histologically normal liver samples were obtained by biopsy during surgery from eight patients with gallbladder stones. Thirty-four HCC and corresponding non-malignant para-tumorous specimens were collected by radical hepatectomy. All tissues were obtained with informed consent from the patients, and were verified by biochemistry and pathological examination. The study was approved by the Institutional Review Board of Tongji Medical College, Huazhong University of Science and Technology (China). The cell lines, HepG2 and L02, were cultured in RMPI-1640 with 10% fetal bovine serum.microRNA arrays. miR-450a from 8 normal livers, 34 HCC, and corresponding non-tumor...
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