ABSTRACT
Objective To explore the mechanism of microRNA (miRNA, miR)-155 in the rejection after liver transplantation in rats. Methods The rats were divided into two groups. In the xenograft model group (rejection group, n=10),the donors were male Lewis rats and the recipients were male BN rats.In the allograft model group(control group, n=10),both the donors and recipients were male Lewis rats.The rat models with orthotopic liver transplantation were established by two-cuff technique in two groups. At postoperative 7 d, the animals were sacrificed for the collection of blood and liver tissue samples. The serum levels of alanine aminotransferase (ALT), total bilirubin (TB), and cytokines of interleukin (IL)-2, IL-4, interferon (IFN)-γ were quantitatively measured. The pathological changes of liver tissues were observed under light microscope. In each group, three liver tissue samples were prepared and subject to high-throughput sequencing. The miRNAs related to rejection were identified for bioinformatics analysis to predict and analyze relevant signaling pathways and genes. Results In the rejection group, the serum levels of ALT and TB were significantly higher than those in the control group (both P<0.01). Compared with the control group, the levels of IL-2 and IFN-γ were considerably up-regulated (both P<0.01), whereas the level of IL-4 was dramatically down-regulated (P<0.01). Pathological examination demonstrated that more evident rejections were observed in the rejection group than the control group. High-throughput sequencing revealed that the expression level of miR-155 was significantly up-regulated in the rejection group, which was 5.89 times of that in the control group. Bioinformatics analysis demonstrated that up-regulation of miR-155 was associated with the mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK) and T cell receptor signaling pathways. The genes which were probably responsible for regulation included the yeast autophagy related gene 1(ATG1) and its homologous gene ULK2, insulin-like growth factor-1 (Igf-1) and G protein-coupled receptor regulatory gene(Arrb1),etc.Conclusions miR-155 might promote the incidence and progression of rejection after liver transplantation in rats. The involved signaling pathways probably include the mTOR, MAPK signaling pathway and T cell receptor signaling pathway.ATG1,ULK2,Igf-1,and Arrb1 genes may participate in this process.
ABSTRACT
Objective To investigate the expression of zeste white 10 interactor (Zwint) in primary hepatocellular carcinoma (HCC) and its effect on the prognosis of liver transplantation for HCC. Methods HCC tissues, paracancerous tissues and clinical data of 50 liver transplant recipients for HCC were collected. The expression levels of Zwint messenger RNA (mRNA) and Zwint protein in 20 pairs of HCC tissues and paracancerous tissues of 20 liver transplant recipients for HCC were compared using real-time fluorescence quantitative polymerase chain reaction (PCR), Western Blot and immunohistochemistry (IHC). Two HCC cell lines HepG-2 which interfered with the expression of Zwint successfully were selected as si-Zwint-1 group and si-Zwint-2 group, and the blank control was taken as si-NC group. The cell proliferation and cell cycle of various groups were compared using cell counting kit (CCK) -8 experiment, flat-cloning assay and cell cycle experiment. The consistency of the expression of Zwint and cyclin D1 in HCC tissues and cells was analyzed using Western Blot and IHC. The enrolled patients were divided into high expression group (22 cases) and low expression group (28 cases) based on the median of Zwint protein expression level, and the relationship of the expression level of Zwint protein and clinical characteristics, overall survival rate and disease free survival rate of liver transplant recipients for HCC was analyzed. Results The results of real-time fluorescence quantitative PCR showed that the expression level of Zwint mRNA in HCC tissues was higher than that of paracancerous tissues (P=0.03). The results of Western Blot and IHC showed that the expression level of Zwint protein in HCC tissues was higher than that of paracancerous tissues(both P<0.05).After the Zwint gene of HCC cell line HepG-2 was interfered,CCK-8 and flat-cloning assay showed that the cell proliferation potential was significantly weakened (all P<0.01), and the cell cycle arrested at stage G1(all P<0.05). The expression level of Zwint protein was closely related to tumor diameter and tumor, node, metastasis (TNM) staging (all P<0.05). The overall survival rate of liver transplant recipients for HCC in the high Zwint expression group was lower than that of the low expression group (P=0.02). Conclusions Zwint is highly expressed in HCC tissues, and it can promote the proliferation of HCC cells through regulating cell cycle. The expression level of Zwint is negatively correlated with the prognosis of liver transplantation for HCC.
ABSTRACT
Objective To establish male rat models for fertility following liver transplantation. Methods Male Sprague-Dawley (SD) rats were used as the donors and recipients of liver transplantation. The donor liver was transplanted with two-cuff technique. Liver transplantation was performed in 15 male SD rats. At 3 weeks after liver transplantation, 5 rats were randomly sacrificed for detection of sperm deformity rate. The remaining male rats were mixed bred and mated with healthy female SD rats at a ratio of 1︰2. General conditions of the rats undergoing liver transplantation were recorded. Liver function parameters were detected after liver transplantation. Postoperative sperm deformity rate was observed. The pregnant status of female rats and health situation of their offsprings was monitored. Results All 15 rats (100%) underwent liver transplantation successfully. Nine rats (9/10) survived longer than 8 weeks. Liver function parameters were normal in male rats following liver transplantation. The sperm deformity rate was ranged from 0.5% to 1.3%. Ten male rats undergoing liver transplantation were mixed bred with female rats at a ratio of 1︰2 for 1 week. All female rats were successfully mated and delivered their offsprings after 3 weeks. The offsprings had no evident physiological deformity. Conclusions Male rat models for fertility are successfully established after liver transplantation, which serve as an animal model to evaluate the fertility performance in male patients undergoing liver transplantation.