Aiqun Zhang

Long non-coding RNA HNF1A-AS1 functioned as an oncogene and autophagy promoter in hepatocellular carcinoma through sponging hsa-miR-30b-5p

Long non-coding RNAs (lncRNAs) had been proved to be pivotal regulators in carcinogenesis. On the basis of competitive endogenous RNAs (ceRNAs) system, lncRNAs significantly expanded their regulating networks. In our research, we aimed to figure out the exact role of lncRNA HNF1A-AS1 in the pathogenesis of hepatocellular carcinoma (HCC), in a ceRNA-dependent way. First, we revealed: HNF1A-AS1 was frequently overexpressed in HCC tissues and cell lines and its relative high expression was closely related to larger tumor size, multiple tumor lesions, poor differentiation and advanced TNM stage. Then we found: HNF1A-AS1 functioned as an oncogene in tumor growth and apoptosis through sponging tumor-suppressive hsa-miR-30b-5p (miR-30b) and de-repressing Bcl-2. Further experiments identified: HNF1A-AS1-miR-30b axis significantly promoted autophagy under starvation and ATG5 was first proved to be a target of miR-30b. In summary, we identified HNF1A-AS1-miR-30b axis as a key regulator in hepatocarcinogenesis, which may be promising biomarkers and therapeutic targets in the future.

STAT3 regulates the migration and invasion of a stem‑like subpopulation through microRNA‑21 and multiple targets in hepatocellular carcinoma.

Despite advances in the detection and treatment of hepatocellular carcinoma (HCC), the prognosis remains poor partly due to recurrence or extra/intrahepatic metastasis. Stem‑like cancer cells are considered the source of malignant phenotypes including metastasis in various types of cancer. HCC side population (SP), considered as stem‑like cancer cells, plays an important role in the migration and invasion in HCC, while the mechanisms involved remain unknown. In the present study, high levels of STAT3 and phospho‑STAT3 were observed in MHCC97H SP cells compared with the main population (MP) cells. Inhibition of phospho‑STAT3 led to a reduction of miR‑21 expression, an increase of PTEN, RECK, and programmed cell death 4 (PDCD4) expression as well as the migration and invasion of SP cells. A set of rescue experiments was performed using different combinations of STAT3 inhibitor, miR‑21 mimics and siRNAs to observe the expression of miR‑21 targets, cell migration and invasion alterations. Data indicated that the alterations induced by STAT3 inhibition were partly reversed by the upregulation of miR‑21. Additionally, the cells migration and invasion when silencing the targets of miR‑21 were also reversed by STAT3 inhibition. In conclusion, the present study revealed the aberrant expression of STAT3 and miR‑21 in HCC SP cells. Targeting STAT3 may limit HCC migration and invasion, which is likely to involve the regulation of miR‑21 and its targets PTEN, RECK and PDCD4. Strategies directed towards STAT3 may therefore be a novel approach for the treatment of HCC.

Establishment of a Rat Model of Portal Vein Ligation Combined with In Situ Splitting

Background Portal vein ligation (PVL) combined with in situ splitting (ISS) has been shown to induce remarkable liver regeneration in patients. The purpose of this study was to establish a model of PVL+ISS in rats for exploring the possible mechanisms of liver regeneration using these techniques. Materials and Methods Rats were randomly assigned to three experimental groups: selective PVL, selective PVL+ISS and sham operation. The hepatic regeneration rate (HRR), Ki-67, liver biochemical determinations and histopathology were assessed at 24, 48, and 72 h and 7 days after the operation. The microcirculation of the median lobes before and after ISS was examined by laser speckle contrast imaging. Meanwhile, cytokines such as TNF-α, IL-6, HGF and HSP70 in regenerating liver lobes at 24 h was investigated by RT-PCR and ELISA. Results The HRR of PVL+ISS was much higher than that of the PVL at 72 h and 7 days after surgery (p<0.01). The expression of Ki-67 in hepatocytes in the regenerating liver lobe was stronger in the PVL+ISS group than in the PVL group at 48 and 72 h (p<0.01). There was a significant reduction in microcirculation blood perfusion of the left median lobe before and after ISS. Liver biochemical determinations and histopathology demonstrated more severe hepatocyte injury in the PVL+ISS group. Both the mRNA levels of TNF-α and IL-6 and the protein levels of TNF-α, IL-6 and HGF in regenerating liver lobes were higher in the PVL+ISS than the PVL alone. Conclusions The higher HRR in the PVL+ISS compared with the PVL confirmed that we had successfully established a PVL+ISS model in rats. The possible mechanisms included the reduced microcirculation blood perfusion of the left median lobe and up-regulation of cytokines in the regenerating lobes after ISS.

The monitoring of microvascular liver blood flow changes during ischemia and reperfusion using laser speckle contrast imaging

OBJECTIVE The recovery of microvascular liver blood flow (LBF) after ischemia is an important determinant of the degree of hepatocellular injury. Laser speckle contrast imaging (LSCI) was recently suggested to be a suitable instrument for monitoring the LBF. This study was designed to evaluate LSCI in monitoring the LBF changes during liver ischemia and reperfusion (IR). METHODS A rat model with 120-min ischemia and 60-min reperfusion to 90% of the liver (entire liver except the caudate lobe, which was kept as portal blood bypass) was used. The LBF of the sham operation (SO) group and the IR group was measured with LSCI at the following time points: before ischemia (Baseline), 5 min after the start of ischemia (I-5 min), 5 min before the end of ischemia (I-115 min) and 5 and 60 min after the start of reperfusion (R-5 min and R-60 min). The reproducibility among different rats or repeated measurements, the liver histopathology, the liver biological zero (BZ) and the influence of liver movement on the LSCI measurements were investigated. RESULTS The entire exposed liver surface after laparotomy was suitable for full-view LSCI imaging. Establishing many circular or oval regions of interest (ROIs) on the LSCI flux image was a simple and convenient method for calculating and comparing the LBF of different ROIs and different liver lobes. There was good-to-moderate intra-individual and inter-individual reproducibility for the LSCI measurements of the LBF in the rats of the SO group. In the IR group, the total blood inflow occlusion resulted in a notable drop of the LBF from the baseline (P<0.05) that remained for the 120 min of ischemia. The LBF decreased further after the reperfusion (P<0.05), reflecting the IR-induced liver microcirculation dysfunction. The histopathological examination revealed severe hepatic sinus congestion and damaged hepatocytes in the IR group. The no flow BZ and liver movement contributed to the LBF values. CONCLUSIONS LSCI technology is a simple, convenient and accurate method for the real-time monitoring of microvascular LBF changes during ischemia and reperfusion, regardless of the contribution of biological zero and liver movement. This finding suggests the possible application of LSCI for monitoring the microvascular LBF changes intraoperatively.

A new view of the roles of blood flow dynamics and Kupffer cell in intra-hepatic metastasis of hepatocellular carcinoma

Viral cirrhotic primary liver cancer (VCPLC) is characterized by intra-hepatic metastasis (IHM) at early stage. By contrast, simple primary liver cancer without cirrhosis (SPLC) has high extra-hepatic metastasis (EHM) rate. Historically, studies on mechanism of metastasis of PLC mainly focused on biological behaviors of cancer cells, however tumor microenvironment is always neglected, especially blood flow dynamics and Kupffer cells. In SPLC, few damages of hepatic lobules occur, to the contrary, VCPLC is characterized by obvious hepatic fibrosis which results in varieties of blood flow disturbances, such as central lobular vein obstruction, hepatic artery-portal vein fistula (HAPVF), and portal vein countercurrent (PVC). Traditionally, Kupffer cells in liver are regarded as powerful defenses against PLC and metastasis lesions in liver, however, recent studies show under long term ischemic-hypoxic stress, Kupffer cells can secrete varieties of pro-inflammatory mediators to promote fibrosis or even cancerogenesis. Tumor associated macrophages (TAMs) are a group of macrophages living in ischemic tumor lesions which can promote tumor progression and metastasis. Based on the totally different metastasis routes and significantly different tumor microenvironment of VCPLC and SPLC, we hypothesize: (1) unlike the normal Kupffer cells in non-cirrhosis liver, Kupffer cells of VCPLC, a group of dominant hepatic resident macrophages living in extremely cirrhotic ischemic-hypoxic sinusoids, might not act as fighters against cancer cells, what is worse, ischemic-hypoxic situation might incite Kupffer cells to secrete cytokines and chemokines, just like TAMs, to promote tumor growth and metastasis; (2) the blood flow disturbances, not the behaviors of cancer cells, are the main force to determine the different metastasis preferences. In VCPLC, central lobular vein is mechanically compressed by cirrhosis nodes, so cancer cells cannot go extra-hepatically, instead, the hepatic artery-portal vein fistula (HAPVF) and portal vein countercurrent (PVC) load cancer cells intra-hepatically. In SPLC, cancer cells can run directly extra-hepatically through central lobular vein fluently. In short, the different blood flow dynamics between these two PLC is the predominant factor to determine the preferences of metastasis route. In VCPLC, the Kupffer cells should not be the powerful defenses against tumor and metastasis lesions, instead, they should be tamed to be some TAMs by long term ischemic-hypoxic stress and tumor derived factors to play important roles in tumorigenesis or even metastasis, so Kupffer cells can be a new target for preventing hepatocancerogenesis and metastasis.

Prognostic Significance of Neutrophil-to-Lymphocyte Ratio in Colorectal Liver Metastasis: A Systematic Review and Meta-Analysis.

Background and Objective Inflammation is deemed to play critical roles in tumor progression and metastasis, and an increased neutrophil-lymphocyte ratio (NLR) has been reported to correlate with poor survivals in various malignancies. However, association between NLR elevation and survival outcome in patients with colorectal liver metastasis (CRLM) remains controversial. The aim of this study was to investigate the prognostic significance of elevated NLR in CRLM. Methods The meta-analysis was conducted in adherence to the MOOSE guidelines. PubMed, Embase, Cochrane Library, Web of Science and the Chinese SinoMed were systematically searched to identify eligible studies from the initiation of the databases to May, 2016. Overall survival (OS) and recurrence free survival (RFS) were pooled by using hazard ratio (HR) with corresponding 95% confidence interval (CI). Correlation between NLR values and clinicopathological features was synthesized by using odds ratio (OR) with corresponding 95% CI. Results A total of 1685 patients from 8 studies (9 cohorts) were analyzed, consisting 347 (20.59%) in high pretreatment NLR value group and 1338 (79.41%) in low pretreatment NLR value one. The results demonstrated that elevated pretreatment NLR was significantly related to poor OS (HR 2.17, 95% CI 1.82–2.58) and RFS (HR 1.96, 95% CI 1.64–2.35) in patients with CRLM. Conclusion The result of this systematic review and meta-analysis indicated that an elevated pretreatment NLR was closely correlated with poor long-term survival (OS and RFS) in CRLM patients. NLR can be routinely monitored and serve as a useful and cost-effective marker with strong prognostic significance in patients with CRLM.

Selective bowel decontamination improves the survival of 90% hepatectomy in rats

BACKGROUND Clinically, hepatectomy is a clean procedure performed without routine antimicrobial prophylaxis, regardless of the extent of liver loss. Translocation of endotoxin has been recognized as a fatal complication leading to liver failure. After extended hepatectomy, the portal hypertension, mucosal damage, intrahepatic bile acid retention, inhibited enterokinesia, and so forth are likely to contribute to enhanced endotoxin absorption. The effect of selective bowel decontamination (SBD) on the prognosis of hepatectomy were investigated. METHODS We adopted rat models of partial hepatectomy (70%, PHx) and subtotal hepatectomy (90%, SHx), gentamicin or saline of the same amount was administrated preoperatively. Liver damage makers, portal and systemic lipopolysaccharide, mucosal damage, signaling pathways, liver regeneration, and bile canalicular networks reconstruction were investigated. RESULTS We found that SHx but not PHx resulted in significantly enhanced portal and systemic endotoxin. Inhibition of gastrointestinal gram-negative bacteria by gentamicin significantly reduced lipopolysaccharide levels and improved survival after SHx (56% with gentamicin, 24% with saline, P < 0.05). We also found SBD with gentamicin protected intestinal mucosa barrier, alleviated liver parenchymal damage, and promoted liver regeneration and bile canalicular networks reconstruction after extended liver resection. CONCLUSIONS We conclude that SBD is beneficial and necessary for extended hepatectomy.

Genome‐wide Inference of Transcription Factor–DNA Binding Specificity in Cell Regeneration Using a Combination Strategy

The cell growth, development, and regeneration of tissue and organ are associated with a large number of gene regulation events, which are mediated in part by transcription factors (TFs) binding to cis‐regulatory elements involved in the genome. Predicting the binding affinity and inferring the binding specificity of TF–DNA interactions at the genomic level would be fundamentally helpful for our understanding of the molecular mechanism and biological implication underlying sequence‐specific TF–DNA recognition. In this study, we report the development of a combination method to characterize the interaction behavior of a 11‐mer oligonucleotide segment and its mutations with the Gcn4p protein, a homodimeric, basic leucine zipper TF, and to predict the binding affinity and specificity of potential Gcn4p binders in the genome‐wide scale. In this procedure, a position‐mutated energy matrix is created based on molecular modeling analysis of native and mutated Gcn4p–DNA complex structures to describe the position‐independent interaction energy profile of Gcn4p with different nucleotide types at each position of the oligonucleotide, and the energy terms extracted from the matrix and their interactives are then correlated with experimentally measured affinities of 19 268 distinct oligonucleotides using statistical modeling methodology. Subsequently, the best one of built regression models is successfully applied to screen those of potential high‐affinity Gcn4p binders from the complete genome. The findings arising from this study are briefly listed below: (i) The 11 positions of oligonucleotides are highly interactive and non‐additive in contribution to Gcn4p–DNA binding affinity; (ii) Indirect conformational effects upon nucleotide mutations as well as associated subtle changes in interfacial atomic contacts, but not the direct nonbonded interactions, are primarily responsible for the sequence‐specific recognition; (iii) The intrinsic synergistic effects among the sequence positions of oligonucleotides determine Gcn4p–DNA binding affinity and specificity; (iv) Linear regression models in conjunction with variable selection seem to perform fairly well in capturing the internal dependences hidden in the Gcn4p–DNA system, albeit ignoring nonlinear factors may lead the models to systematically underestimate and overestimate high‐ and low‐affinity samples, respectively.

Laser speckle contrast imaging and Oxygen to See for assessing microcirculatory liver blood flow changes following different volumes of hepatectomy

OBJECTIVE Portal hyperperfusion after extended hepatectomy or small-for-size liver transplantation may induce organ dysfunction and failure. This study was designed to monitor and characterize the hepatic microcirculatory perfusion following different volumes of hepatectomy in rats by using laser speckle contrast image (LSCI) and Oxygen to See (O2C), a spectrometric device. METHODS The microcirculatory liver blood flow of the rats that underwent 68%, 85% and 90% hepatectomy (68PH, 85PH and 90PH) was monitored with LSCI and O2C before and following the hepatectomy. The portal venous flow (PVF) and hepatic arterial flow (HAF) were measured with an ultrasonic flowmeter. Liver regeneration, liver injury, histologic evaluation and gene expression were also assessed at 12h, 24h, 3d and 7d post hepatectomy. RESULTS All the 68PH and 85PH rats survived, and 57% of the 90PH rats survived. After hepatectomy, both PVF and HAF decreased transiently, with the PVF of the 85PH and 90PH rats significantly lower than that of the 68PH rats. In contrast, the PVF and HAF per gram of liver weight were greatly increased after liver resection and were proportional to the volume of resected liver. Correspondingly, the microcirculatory liver blood flow of the 68PH, 85PH and 90PH rats, as assessed by both LSCI and O2C, were increased after hepatectomy, and the 90PH group was significantly higher than the 68PH and 85PH groups. The hyperperfusion continued for approximately 3days and returned to baseline following the completion of liver regeneration. The liver venous oxygen saturation of the three groups decreased immediately after hepatectomy and returned to baseline from 24h after hepatectomy. The 90PH rats also showed delayed liver regeneration and the most severe liver injury, as reflected by increased serum ALT, AST and TBIL levels, hepatocellular vacuolization, and inflammatory and endothelial constriction gene expressions (TNF-α, IL-1β, MIP-1α, ET-1 and TM-1). CONCLUSION Hepatic microcirculation hyperperfusion resulting from major and extended liver resection could be assessed by LSCI and O2C methods. The 90PH in rats led to extraordinary sinusoidal hyperperfusion, severe endothelial injury and liver failure. Monitoring the changes of hepatic microcirculation perfusion following extended hepatectomy or small-for-size liver transplantation may help to analyze the extent of hyperperfusion.

Effects of Combined Anisodamine and Neostigmine Treatment on the Inflammatory Response and Liver Regeneration of Obstructive Jaundice Rats after Hepatectomy

Background. Cholestasis is associated with high rates of morbidity and mortality in patients undergoing major liver resection. This study aimed to evaluate the effects of a combined anisodamine and neostigmine (Ani+Neo) treatment on the inflammatory response and liver regeneration in rats with obstructive jaundice (OJ) after partial hepatectomy. Materials and Methods. OJ was induced in the rats by bile duct ligation. After 7 days biliary drainage and partial hepatectomy were performed. These rats were assigned to a saline group or an Ani+Neo treatment group. The expressions of inflammatory mediators, liver regeneration, and liver damage were assessed at 48 h after hepatectomy. Results. The mRNA levels of TNF-α, IL-1β, IL-6, MCP-1, and MIP-1α, in the remnant livers, and the serum levels of TNF-α and IL-1β were substantially reduced in the Ani+Neo group compared with saline group (P < 0.05). The Ani+Neo treatment obviously promoted liver regeneration as indicated by the liver weights and Ki-67 labeling index (P < 0.05). The serum albumin and γ-GT levels and liver neutrophil infiltration also significantly improved in the Ani+Neo group (P < 0.05) compared with the saline group. Conclusions. These results demonstrate that the combined anisodamine and neostigmine treatment is able to improve the liver regeneration in rats with OJ by substantially alleviating the inflammatory response.