Zhongzhou Si

Long non-coding RNA CCAT2 functions as an oncogene in hepatocellular carcinoma, regulating cellular proliferation, migration and apoptosis

An increasing number of studies have demonstrated that the dysregulation of long non-coding RNAs (lncRNAs) may serve an important role in tumor progression. Previous studies have reported that the lncRNA, colon cancer associated transcript 2 (CCAT2), was highly expressed in various tumors. However, the function of CCAT2 in hepatocellular carcinoma (HCC) has not yet been elucidated. The aim of the present study was to identify novel oncogene lncRNAs and investigate their physiological function and mechanism in HCC. Using reverse transcription-quantitative polymerase chain reaction, it was observed that CCAT2 was upregulated in HCC tissues and human HCC cell lines. Furthermore, the impacts of CCAT2 on cell proliferation, migration and apoptosis were analyzed using cell migration, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and enzyme-linked immunosorbent assay analysis respectively. The overexpression of CCAT2 using a synthesized vector significantly promoted cell migration and proliferation, and inhibited apoptosis of HCC cells in vitro. The suppression of CCAT2 expression resulted in opposing effects. To the best of our knowledge, the present study is the first to demonstrate that CCAT2 functions as a oncogene in HCC. Further investigation is required to clarify the molecular mechanisms of this lncRNA in HCC development.

Cytoprotective Effects of Human Interleukin-10 Gene Transfer Against Necrosis and Apoptosis Induced by Hepatic Cold Ischemia/Reperfusion Injury

BACKGROUND Apoptosis as well as necrosis may play an important role in hepatic ischemia/reperfusion (I/R) injury. Interleukin 10 (IL-10), a Th2 type cytokine, modulates inflammatory responses by inhibiting the production of proinflammatory cytokines. The study focused on cytoprotective and antiapoptotic pathways to assess mechanisms by which gene transduction of human IL-10 (hIL-10) may renders grafts resistant to the cold I/R injury. MATERIALS AND METHODS Adenoviruses encoding hIL-10 or beta-galactosidase (LacZ) were injected via the superior mesenteric vein into prospective donor animals. The donor liver was harvested 48h after transduction, and stored for 12h at 4 degrees C lactated Ringers solution prior to being transplanted. Graft survival, liver function, the degree of necrosis and apoptosis, and the molecules of apoptotic networks were assessed. RESULTS Ad-hIL-10 pretreatment significantly prolonged the survival of liver grafts by improving liver function, preserving hepatocyte integrity and architecture, and depressing intrahepatic apoptosis and necrosis. In addition, Ad-hIL-10 pretreatment diminished the release of cytochrome c from mitochondria into cytoplasm and caspase-3 activity, with simultaneous up-regulated of antioxidant HO-1 and anti-antiapoptotic Bcl-2 molecules. CONCLUSION Adenoviral gene transfer of hIL-10 ameliorated cold I/R injury by decreasing hepatic necrosis and apoptosis. The underlying mechanism of cytoprotective effects may at least be involved with the inhibition of caspase-3 activity and mitochondrial cytochrome c release, and the up-regulation of antiapoptotic (Bcl-2) and antioxidant (HO-1) molecules.

Alterations of axis inhibition protein 1 (AXIN1) in hepatitis B virus-related hepatocellular carcinoma and overexpression of AXIN1 induces apoptosis in hepatocellular cancer cells.

Axis inhibition protein 1 (AXIN1) is a negative regulator of Wnt/beta-catenin signaling via regulating the level of beta-catenin. However, the role of AXIN1 in the tumorigenesis and progression of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is less clear. PCR sequence analysis, immunohistochemistry, and Western blot were performed on 22 HBV-related HCC samples and corresponding nontumor liver tissues to detect variants in AXIN1 gene and the expression level of AXIN1. Human hepatoma cell lines SNU475 and SNU423 were transfected with pCDNA3.1-AXIN1-myc or AXIN1 G425S-myc mutant. The growth curve and apoptosis rate of cell lines, phosphorylation of beta-catenin, and cell cycle regulatory proteins depending on beta-catenin transcriptional activity were detected. We identified four mutations of AXIN1 in 22 primary HBV-related HCCs and demonstrated a lower expression of AXIN1 in HBV-related HCC tissues than that in paired adjacent nontumor tissues. Overexpression of AXIN1 wild-type but not AXIN1 mutant inhibited the growth of HCC cell lines, accelerated their apoptosis, and negatively regulated beta-catenin-dependent transcriptional activity. Our study revealed that alterations of AXIN1 were involved in HBV-related HCC. Overexpression of AXIN1 but not AXIN1 mutant negatively regulated beta-catenin-dependent transcriptional activity and downregulated the level of cell cycle regulatory proteins, suggesting that AXIN1 may be a potential target for gene therapy of primary HCC.

Induction of Lymphocyte Apoptosis in Rat Liver Allograft by Adenoviral Gene Transfection of Human Interleukin-10

Background/Aims: Gene therapy can provide a possible avenue in organ transplantation to treat acute allograft rejection. This study was designed to investigate the effect of adenovirus-mediated human IL-10 (hIL-10) gene transfer on the apoptosis of infiltrating lymphocytes and examine the efficacy of hIL-10 gene transfer in combination with subtherapeutic doses of cyclosporine A (CsA) in a rat liver transplantation model. Methods: Inbred male DA and LEW rats were used for liver donors and recipients, respectively. The rats were divided into saline, Ad-lacZ, CsA, Ad-hIL-10 and Ad-hIL-10 + CsA groups. Graft survival, histopathological, enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction and flow cytometry were performed in liver specimens obtained from different time points after transplantation in the 5 groups. Results: Ad-hIL-10 pretreatment inhibited allograft rejection, prolonged the survival of hepatic allografts, and downregulated the expression of IFN-γ and IL-2 mRNA, with simultaneous upregulation of IL-4 mRNA. In addition, Ad-hIL-10 pretreatment upregulated the expression of Fas mRNA in the isolated graft-infiltrating lymphocytes and induced graft-infiltrating lymphocyte apoptosis. A single subtherapeutic dose of CsA acted synergistically with it. Conclusion: hIL-10 gene therapy induced alloreactive lymphocyte apoptosis via Fas/FasL pathway. hIL-10 gene transfection in combination with subtherapeutic doses of CsA facilitates the long-term survival of liver grafts.

IL-17 in the early diagnosis of acute renal allograft rejection in mice.

OBJECTIVE To investigate the expression of T helper (Th) 17 cells and the related interleukin 17 (IL-17) in acute renal allograft rejection in mice and its significance. METHODS We established a mouse renal allograft model, in which mice were randomly divided into a renal isograft group and an acute renal allograft rejection group. Three and 7 d after the transplantation, the serum interferon (IFN)-γ and IL-17 levels in the mice were determined by enzyme-linked immunosorbent assay, the percentage of Th1 and Th17 cells in the total kidney-infiltrating lymphocytes was investigated by flow cytometry, and the transplanted kidney species were given routine pathological examination after fixation with 10% formalin. RESULTS Compared with the isograft group, the allograft mice showed a significantly higher content of IL-17 (P<0.05) but not IFN-γ in the serum 3 d after transplantation, and showed significantly higher serum IL-17 and IFN-γ contents 7 d after transplantation (P<0.05). Also, compared with the isograft group, the allograft mice exhibited significantly higher percentage of Th1 and Th17 cells on both day 3 and day 7 (P<0.05). In the allograft group, the contents of serum IFN-γ and IL-17 and the percentage of Th1 and Th17 cells were significantly higher on day 7 than on day 3 (P<0.05). Routine pathological examination indicated that, as time passed, the allograft mice showed gradually stronger rejection responses. CONCLUSION Th17 cells might play an important role in the development of acute renal allograft rejection, and IL-17 can be used as an early indicator of acute rejection.

Restoration of miR-20a expression suppresses cell proliferation, migration, and invasion in HepG2 cells

Objective To study microRNA (miR)-20a expression in hepatocellular carcinoma (HCC) and its effects on the proliferation, migration, and invasion of HepG2. Methods The real-time polymerase chain reaction was used to detect the expression of miR-20a in HCC tissue and normal tissue, as well as in HCC cell lines and normal liver cells. miR-20a mimic and miR negative control (NC) were transfected into HepG2 cells. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay was used to detect cell proliferation. Annexin fluorescein isothiocyanate/propidium iodide assay was run to examine the early apoptosis of cells. Transwell chamber assay was carried out to investigate the cell invasion and migration abilities. Results miR-20a was lowly expressed both in HCC tissues and HCC cell lines. After transfection of exogenous miR-20 mimics, miR-20a expression in HepG2 cells was significantly increased by 61.29% compared to the blank group (P<0.01). MTT assay showed that the growth of HepG2 cells in the miR-20a mimics group was significantly inhibited, and optical density values during the 36–96 hour time period were dramatically decreased compared to the blank group (P<0.01). Apoptosis rates of the miR-20a mimics group were higher than those of the blank and NC groups (both P<0.01). The number of HCC cells after transfection by miR-20a mimics in the G1 and S phases were 15.88% and 7.89%, respectively, which were lower than in the blank and NC groups (both P<0.05). Transwell assay showed that in the miR-20a mimics group the number of cell migration and invasion were 0.459 and 0.501 times that of the blank group (both P<0.01), and the migration and inhibition rates were 54.1% and 51.4%, respectively. After closing target gene CCND1 in HepG2 cells, the number of cell migration and invasion in the small interfering (si)-CCND1 group were 0.444 and 0.435 times that of the si-NC group (P<0.05); and compared to the si-NC group, the migration and inhibition rates were 55.6% and 56.5%, respectively. Conclusion miR-20a can inhibit the growth, invasion, and migration of HepG2 cells, and is therefore promising as a new molecular target for diagnosis and therapy of HCC.

Gastroduodenal Arterial Reconstruction of the Pancreaticoduodenal Allograft

Simultaneous procurement of the pancreas and liver necessitates division of vessels supplying both organs. The integrity of the pancreatic arterial supply appears to be related to surgical complications after pancreas transplantation. We have described herein three cases of gastroduodenal artery (GDA) reconstruction during pancreas transplantation, and reviewed other options for GDA reconstruction. These techniques performed safely during bench reconstruction can be applied to various clinical situations.

Wernicke encephalopathy in a patient after liver transplantation: A case report

Wernicke encephalopathy (WE) is an acute neurological disorder resulting from vitamin B1 deficiency, which is common in chronic alcoholism and is rare in acute liver failure. So far, there are 2 cases of WE reported after liver transplantation. Here, we report a case of a 45-year-old nonalcoholic male patient who developed psychiatric and neurological disturbance 15 d after receiving orthotopic liver transplantation because of hepatitis B-related cirrhosis and portal hypertension. Brain magnetic resonance imaging (MRI) showed symmetric high-signal intensities in the periaqueductal area. The patient was diagnosed with WE and given intravenous high-dose vitamin B1 immediately. His neurological disturbance resolved in 7 d after receiving the vitamin B1. Brain MRI after 5 mo showed nearly complete recovery. Most WE cases may be misdiagnosed in patients after liver transplantation, and we should pay more attention to its onset.

Splenic artery trunk embolization reduces the surgical risk of liver transplantation

BACKGROUND Portal hypertension is one of the most important clinical conditions that cause intraoperative intensive hemorrhage in cirrhotic patients undergoing liver transplantation. Pre-transplant portal decompression may reduce the intraoperative bleeding during liver transplantation. METHODS Splenic artery trunk embolization (SATE) was performed one month prior to liver transplantation. Platelet count, prealbumin, international normalized ratio, and blood flow in the portal vein and hepatic artery were monitored before and one month after SATE. The measurements above were collected on admission and before surgery in the non-SATE patients, who served as controls. We also recorded the intraoperative blood loss, operating time, required transfusion, post-transplant ascites, and complications within three months after operation in all patients. RESULTS SATE significantly reduced portal venous blood flow, increased hepatic arterial blood flow, normalized platelet count, and improved prealbumin and international normalized ratio in the patients before liver transplantation. Compared to the non-SATE patients, the pre-transplant SATE significantly decreased the operating time, intraoperative bleeding, post-transplant ascites and severe surgical complications. CONCLUSION Pre-transplant SATE decreases portal pressure, improves liver function reserve, and reduces the surgical risk of liver transplantation effectively in patients with severe portal hypertension.

[Effect of interleukin-17 and T helper cell 17 on acute heart allograft rejection in mice].

OBJECTIVE To investigate the role of T helper cell 17(Th17) cells and their cytokines IL-17 in heart allograft rejection in mice. METHODS The heart transplantation models were randomly divided into 2 groups: a allograft group (n=12) and an isograft group (n=12).On the post-operative day (POD) 3 and 7, serum IL-17 level was tested by enzyme-linked immunosorbent assay, and Th17 cells in heart grafts were measured by flow cytometry. The heart grafts were harvested and saved in 10% formalin, and then embedded in paraffin. RESULTS Compared with the iso-graft group, the allograft group had a higher serum level of IL-17 on POD3 and POD7 (P<0.05), and the level of IL-17 was higher on POD7 than that on POD3 (P<0.05). The allograft group had more Th17 cells infiltrating in grafts on POD3 and POD7 (P<0.05) and there were more Th17 cells infiltrating on POD7 than that on POD3 (P<0.05). Histological examination showed that the prolongation of post transplantation time resulted in more rejection pathological changes. CONCLUSION Th17 cells may play an important role in the development of heart transplant rejection. IL-17 may serve as a predictive parameter for allograft rejection in the future.