RNA-Sequencing Analysis and Expression of Lymphocyte-Specific Protein Tyrosine Kinase, Linker for Activation of T Cells, and 70-kDa T-Cell Receptor Zeta-Chain Associated Protein Kinase in Rat Liver Transplant Rejection.

OBJECTIVES: The prevention and treatment of liver transplant rejection remain challenging. We investigated the pathophysiological mechanisms of liver transplant rejection in rats and screened candidate genes to determine their degree of rejection response for possible development of potential therapeutic targets. MATERIALS AND METHODS: Brown Norway-Brown Norway transplant tolerant models and Lewis-Brown Norway transplant rejection models were established. We collected liver tissue and venous blood at 7 days posttransplant for hematoxylin and eosin staining and RNA sequencing analysis, respectively. We conducted differential expression gene analysis, KEGG and GO enrichment analysis. We performed immunohistochemistry to detect highly expressed immunerelated proteins, including lymphocyte-specific protein tyrosine kinase, linker for activation of T cells, and 70-kDa T-cell receptor zeta-chain-associated protein kinase. RESULTS: Significant differences were found in liver function and Banff scores between rejection and tolerant groups, indicating the successful establishment of liver transplant models. RNA-sequencing screened 7521 differentially expressed genes, with 3355 upregulated and 3058 downregulated. KEGG analysis of upregulated genes showed that 8 of the top 20 enrichment pathways were associated with immune system processes and 5 were related to immune system diseases. Among these immune pathways, 289 genes were upregulated; of these, 147 genes were removed after comparison with the IMMPORT database, of which 97 genes were significantly changed. Our GO analysis showed upregulated genes mainly participating in immune response processes, with downregulated genes mainly participating in metabolic processes. Real-time polymerase chain reaction and immunohistochemistry verified expression of the immune-related proteins, consistent with RNAsequencing results, which were mainly expressed in inflammatory cells in sinus and portal vein. CONCLUSIONS: Immune-related genes were found to be associated with liver transplant rejection. The 3 immune-related genes that we analyzed may play a role in liver transplant rejection and can possibly serve as candidate markers for monitoring the degree of liver transplant rejection.

LncRNA ST8SIA6-AS1 Promotes Cholangiocarcinoma Progression by Suppressing the miR-145-5p/MAL2 Axis.

BACKGROUND: The tumor-promoting roles of ST8SIA6-AS1 and miR-145-5p have been found in several cancers, but their function in cholangiocarcinoma (CHOL) remains speculative. The purpose of this study was to examine the regulatory functions of the ST8SIA6-AS1/MAL2/miR-145-5p pathway in CHOL progression. METHODS: RT-qPCR assay was used to detect ST8SIA6-AS1 expression in CHOL tissues and cell lines. Cell migration, apoptosis, invasion, and proliferation abilities were assessed by RIP, RNA pull-down, and luciferase assays. CCK-8, BrdU, transwell, and FITC assays to investigate the regulatory functions of ST8SIA6-AS1, miR-145-5p, and MAL2 function in CHOL cells. RESULTS: Findings revealed the enrichment of ST8SIA6-AS1 in CHOL tissues and cell lines. It was also found that ST8SIA6-AS1 facilitated cell growth and migration, but it reduced the apoptosis level of the CHOL cells. The results of experiments showed that ST8SIA6-AS1 sponged miR-145-5p, thereby allowing MAL2 to exert its biological function on CHOL cells. CONCLUSION: This research suggested that the ST8SIA6-AS1/miR-145-5p/MAL2 axis could enhance CHOL progression, which might be useful to improve the clinical outcomes of CHOL patients.

Drainage procedure for pancreatolithiasis: re-examination of the pancreatic duct diameter standard.

PURPOSE: Pancreatic duct decompression relieves pancreatic duct stone (PDS)-associated abdominal pain, though a consensus indication for the drainage procedure of the main pancreatic duct (MPD) is lacking. Moreover, major prognostic factors for postsurgical long-term pain relief and recurrence are largely unknown. METHODS: The clinical outcomes of 65 consecutive PDS patients undergoing surgery from 2008-2012 with 3+ years of follow-up were assessed. RESULTS: At postsurgical follow-up (median, 4.5 years; range, 3-7 years; procedure: Partington, n = 32; Frey, n = 27; pancreatoduodenectomy, n = 3; distal pancreatectomy, n = 3), the early complication and complete stone clearance rates were 29.2% and 97%, respectively. Long-term, complete and partial pain relief were 93.9%, 83.1%, and 10.8%, respectively. The risk of pancreatic fistula was higher in the <8 mm group than in the >8 mm group (P < 0.05), and 80% of the pancreatic fistula cases occurred in the <8 mm group. A shorter pain duration (P = 0.007), smaller MPD diameter (P = 0.04), and lower Izbicki pain score (P < 0.001) predicted long-term pain relief. Pain recurrence after initial remission occurred in 5 patients and was only related to pain duration (P = 0.02). Stone recurrence and pancreatic exocrine functional and endocrine functional deterioration occurred in 2, 5, and 11 patients, respectively. CONCLUSION: Surgery provides excellent stone clearance, long-term pain relief, and acceptable postoperative morbidity. Using 8 mm as the criterion for drainage surgery can minimize the postoperative pancreatic fistula risk. Individualized and timely surgical treatment may improve the effect of surgery.

Long non-coding RNA BZRAP1-AS1 silencing suppresses tumor angiogenesis in hepatocellular carcinoma by mediating THBS1 methylation.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer associated with a high mortality. Long non-coding RNAs (lncRNAs) have recently emerged as regulators in the development and progression of several cancers, and therefore represent an opportunity to uncover new targets for therapy. In the present study, we aimed to investigate the potential effect of lncRNA BZRAP1-AS1 on the angiogenesis of HCC. METHODS: Microarray-based data analysis was initially employed to screen genes and lncRNAs that are differentially expressed in HCC and the candidate BZRAP1-AS1 was identified as a hit. The expression of BZRAP1-AS1 and thrombospondin-1 (THBS1) in HCC tissues and cells were then determined using RT-qPCR. The gene methylation level was measured by methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) assays. Next, the interactions between BZRAP1-AS1, DNA methyltransferase 3B (DNMT3b), and THBS1 were assessed by RIP, RNA pull-down and ChIP assays. Finally, the roles of BZRAP1-AS1, DNMT3b and THBS1 in angiogenesis in vitro as well as tumorigenesis in vivo were evaluated by a battery of the gain- and loss-of function experiments. RESULTS: BZRAP1-AS1 was identified as a highly expressed lncRNA in HCC tissues and cells. Down-regulation of BZRAP1-AS1 in HCC cells inhibited HUVEC proliferation, migration and angiogenesis. By interacting with DNMT3b, BZRAP1-AS1 induced methylation of the THBS1 promoter and inhibited the transcription of THBS1, resulting in promoted angiogenesis of HUVECs. Moreover, silencing of BZRAP1-AS1 repressed the angiogenesis as well as the tumor growth of HCC in vivo via up-regulating THBS1. CONCLUSION: This study provides evidence that angiogenesis in HCC is hindered by silencing of BZRAP1-AS1. Thus, BZRAP1-AS1 may be a promising marker for the treatment of HCC.

Mechanism of exosomal microRNA-224 in development of hepatocellular carcinoma and its diagnostic and prognostic value.

BACKGROUND: Exosomes contain proteins, lipids, and biological molecules such as DNA and RNA. Nucleic acids in exosomes are a group of molecules that can act as biomarkers. Currently, there are many reports on exosomal microRNAs, which are ideal biomarkers for the early diagnosis of cancer. However, there are few reports on the role of exosomal microRNAs in the diagnosis and prognosis of hepatocellular carcinoma (HCC). AIM: To understand the mechanism of exosomal microRNA-224 (miR-224) in the development of HCC and evaluate its diagnostic and prognostic value. METHODS: Cell culture and transfection of exosomal miRNA-224, real-time quantitative PCR, luciferase reporter assay, and other methods were used to find new biomarkers related to the development of HCC that can be used to diagnose HCC and predict HCC prognosis. RESULTS: By targeting glycine N-methyltransferase, incubating exosomes with miR-224 mimic resulted in a significant increase in cell proliferation compared to that of the control group, while incubation with the miR-224 inhibitor significantly reduced cell proliferation. The same results were obtained for the cell invasion assay. Serum exosomal miR-224 did have some ability to differentiate patients with HCC from healthy controls, with an area under the curve of 0.910, and HCC patients with higher serum exosomal miR-224 expression had lower overall survival. CONCLUSION: Exosomal miR-224 is a tumor promotor and can be a marker of diagnosis and prognosis of HCC patients, however, its ability to distinguish liver diseases needs further verification.

The Prognostic Value of Tumor-infiltrating Lymphocytes in Hepatocellular Carcinoma: a Systematic Review and Meta-analysis.

Previous clinical studies have found that the levels of tumor-infiltrating lymphocytes (TILs) significantly correlated with prognosis in hepatocellular carcinoma (HCC). However, these conclusions and data remain controversial. We performed a systematic review and meta-analysis to assess the prognostic value and clinical utilization of TILs in patients with HCC. A total of 23 relevant studies of 3173 patients were included into our meta-analysis. The results demonstrated that high levels of CD8 + and CD3 + TILs had a better prognostic value on overall survival (OS), with HRs of 0.71 (P = 0.04) and 0.63 (P = 0.03), respectively, compared to low levels, as did high levels of CD8 + , CD3 + and CD4 + TILs on disease/recurrence-free survival (DFS/RFS), with HRs of 0.66 (P = 0.01), 0.60 (P = 0.01) and 0.79 (P = 0.04), respectively. In contrast, high levels of FoxP3 + TILs had a worse prognostic value on OS and DFS/RFS, with HRs of 2.06 (P < 0.00001) and 1.77 (P < 0.00001), respectively. The FoxP3+/CD4+ and FoxP3+/CD8+ ratios negatively correlated with OS and DFS/RFS. These findings suggest that TILs may serve as a prognostic biomarker in HCC. However, further research should be performed to clarify the clinical value of TILs in HCC.

TACC3 overexpression in cholangiocarcinoma correlates with poor prognosis and is a potential anti-cancer molecular drug target for HDAC inhibitors.

Histone deacetylases (HDACs) have been implicated in multiple malignant tumors, and HDAC inhibitors (HDACIs) exert anti-cancer effects. However, the expression of HDACs and the anti-tumor mechanism of HDACIs in cholangiocarcinoma (CCA) have not yet been elucidated. In this study, we found that expression of HDACs 2, 3, and 8 were up-regulated in CCA tissues and those patients with high expression of HDAC2 and/or HDAC3 had a worse prognosis. In CCA cells, two HDACIs, trichostatin (TSA) and vorinostat (SAHA), suppressed proliferation and induced apoptosis and G2/M cycle arrest. Microarray analysis revealed that TACC3 mRNA was down-regulated in CCA cells treated with TSA. TACC3 was highly expressed in CCA tissues and predicted a poor prognosis in CCA patients. TACC3 knockdown induced G2/M cycle arrest and suppressed the invasion, metastasis, and proliferation of CCA cells, both in vitro and in vivo. TACC3 overexpression reversed the effects of its knockdown. These findings suggest TACC3 may be a useful prognostic biomarker for CCA and is a potential therapeutic target for HDACIs.

14-3-3ζ and aPKC-ι synergistically facilitate epithelial-mesenchymal transition of cholangiocarcinoma via GSK-3ß/Snail signaling pathway.

Cholangiocarcinoma (CCA) invasion and metastasis are the primary causes of poor survival rates in patients. The epithelial-mesenchymal transition (EMT) is a crucial step in cancer invasion and metastasis. However, it is still unclear of the molecular mechanism. In this study, the expression of 14-3-3ζ and atypical protein kinase C-ι (aPKC-ι) was further detected in CCA tissues and cell lines. Meanwhile, we established the EMT model of CCA cells and investigated 14-3-3ζ and aPKC-ι co-regulatory effect on the EMT in vitro and in vivo. Further, we identified the downstream molecular glycogen synthase kinase 3 beta (GSK-3ß)/Snail signalling pathway that contribute to regulating the EMT. Our data showed that the expression of 14-3-3ζ and aPKC-ι was synergistically increased in CCA tissues compared with adjacent noncancerous tissues and was intimately associated with differentiation and the tumour-node-metastasis (TNM) stage. Multivariate Cox regression analysis indicated that high 14-3-3ζ and aPKC-ι expression separately predicted a poor prognosis and were independent prognostic indicators in patients with CCA. The CO-IP experiment confirmed that the mutual binding relationship between 14-3-3ζ and aPKC-ι. Small interfering RNAs and siRNA rescue experiment demonstrated that 14-3-3ζ and aPKC-ι regulated each other. In addition, 14-3-3ζ and aPKC-ι pretreatment by si-RNA inhibit the phosphorylated GSK-3ß and Snail expression during EMT. Meanwhile, silence of 14-3-3ζ or aPKC-ι suppressed CCA cells migration, metastasis and proliferation in vitro and in vivo. Our study demonstrates that 14-3-3ζ and aPKC-ι synergistically facilitate EMT of CCA via GSK-3ß/Snail signalling pathway, and may be potential therapeutic target for CCA.