ABSTRACT
Purpose: As a major structural component of the outer membrane of Gram-negative bacteria, lipopolysaccharide (LPS) has been detected in the blood circulation and tissues in patients with chronic diseases and cancers, which plays a critical role in the tumor formation and progression. However, the biological role of LPS in human intrahepatic cholangiocarcinoma remains unclear. The aims of this study were to investigate the role of LPS in the malignant progression of intrahepatic cholangiocarcinoma. Methods: The cell migration and invasion capacities of cholangiocarcinoma cell lines were evaluated by Boyden chamber assays. Expression levels of the key molecules involved in the PI3K/AKT signaling and METTL3 were detected by qPCR and western blot. The molecular mechanism by which LPS promotes the malignant behaviors was investigated by using siRNAs, plasmids and small molecule inhibitors. Results: In vitro experiments showed that exogenous LPS treatment promoted cell migration and invasion capacities in both QBC939 and HUCCT1 cell lines, while did not affect cell proliferation and apoptosis. Mechanistically, exogenous LPS treatment had been proved to induce the increased expression of METTL3 and activate the downstream PI3K/AKTsignaling pathway. In addition, suppression of METTL3 expression reduced cell proliferation, migration and invasion capacities in both cell lines. Furthermore, inhibition of METTL3 expression or inhibition of PI3K/AKT signaling decreased LPS-induced cell migration and invasion capacities. Moreover, knockdown of METTL3 or inhibition of METTL3 significantly inhibited LPS-induced activation of the PI3K/AKT signaling. Conclusion: In general, these results suggest that the LPS-METTL3-PI3K/AKT signal axis promotes cell migration and invasion in ICC, which contributes to a reduced overall survival in patients with ICC. It may broaden the horizon of cancer therapy with potential therapeutic targets.
ABSTRACT
BACKGROUND: Liver transplantation (LT) offers a good survival chance for both the patient in short or long term, but still faces many challenges in the treatment of LT, such as the side effects associated with long-term immunosuppression, which is one of the side effects that occurs in most patients. However, the dynamics of the cellular immune system composition over time during immune tolerance to LT after immunosuppressive therapy are not known. METHODS: Using single-cell transcriptome sequencing, we analyzed five peripheral blood samples (one normal individual and four patients who underwent LT and received immunosuppressive therapy for 2 months, 1 year, 3 years, and 7 years, respectively) for immune cell composition and gene expression. RESULTS: A total of 17,462 peripheral blood mononuclear cells were acquired from a normal individual without LT and patients who underwent LT and received immunosuppressive therapy for 2 months, 1 year, 3 years, and 7 years, respectively. A total of 24 cell clusters were obtained and categorized into four different cell types based on gene expression characteristics as follows: eight clusters of T cells, two clusters of B cells, two clusters of neutrophils, two clusters of monocytes, natural killer cells, and natural killer T (NKT) cells (n = 4), and six other cell clusters. Cell subset analysis, pseudotime analysis, and intercellular communication analysis revealed that the CD8+ NKT cells specifically expressed NKG2A (KLRC1, CD159A), which may be an important cell group for CD8+ NKG2A+ NKT cells in LT, thereby highlighting the heterogeneity and functional diversity in patients who undergo LT. CONCLUSIONS: We comprehensively analyzed single-cell RNA sequencing data from a normal individual and patients who underwent LT and elucidated the mechanism underlying the development of immune tolerance in LT. CD8+ NKT cells specifically expressing KLRC1 play a crucial role in LT, and dynamic monitoring of these cells may provide novel avenues for the diagnosis and treatment of LT-related immune rejection.
ABSTRACT
Biliary complications remain a major source of morbidity in liver transplant patients. Among these complications, nonanastomotic biliary strictures (NAS) are especially common and they are frequently therapy resistant in part because biliary epithelial cells are more sensitive to warm ischemic injury than hepatocytes. It has been a challenge to maintain the physiological function of biliary epithelial cells during liver transplantation. In this work, we have examined the effect of oxygen on proliferation of biliary epithelial cells in the rat livers obtained from donation after circulatory death (DCD). Twelve rat livers from DCD were divided into two groups. Livers in the control group were isolated following a standard procedure without oxygen supply. Livers in the experimental group were isolated with a constant supply of oxygen. All livers were then connected to an ex situ liver culture system in the presence of bromodeoxyuridine (BrdU), a thymidine analogue and a marker for cell proliferation. After 6 hours of normothermic ex situ liver culture, morphology and DNA replication in hepatocytes and biliary epithelial cells were assessed and compared between the two groups. We found that about 4.5% of the biliary epithelial cells in the experimental group proliferated compared with only 0.4% of cells in the control based on BrdU staining. No significant change in cell morphology was observed in those cells between the two groups. Thus, our results indicate that oxygen supply is required for maintenance of the physiological function of biliary epithelial cells during liver transplant and suggest that a constant oxygen supply during liver isolation along with ex situ liver organ culture can enhance the repair of biliary epithelial cell injury during liver transplantation.
ABSTRACT
UNLABELLED: Liver transplantation is an effective approach to end-stage liver disease. Shortage of donor liver and increased waiting time for liver transplantation necessitate the development of an organ culture system by which livers can be cultured and maintained ex situ for a prolonged period of time. The aim of this work is to test whether cell culture condition in vitro could be used to culture whole livers ex situ without the use of erythrocytes. Twelve castrated male land race/farm young porcine livers were exposed to 30 min warm ischemia and 30 min cold perfusion. Livers were isolated and connected to an Ex situ liver culture system using a standard culture medium RPMI1640 supplied with 10% of fetal bovine serum and sufficient dissolved oxygen under a normothermic condition for 6 hours. Metabolic biomarkers, bile and urea production, hepatic cell viability and histology analysis of biopsies were examined and newly proliferated hepatic cells labeled by BrdU were analyzed after 6 hours ex situ culture. The results from biochemical assays and histology analysis indicate that livers after the organ culture still maintain the full function. CONCLUSIONS: Our data demonstrate that the liver culture system established in this work can be used to culture whole livers ex situ in the absence of erythrocytes.
