Elevation of plasma tRNA fragments as a promising biomarker for liver fibrosis in nonalcoholic fatty liver disease.

Fibrotic tissue remodelling in nonalcoholic fatty liver disease (NAFLD) will probably emerge as the leading cause of end-stage liver disease in the coming decades, but the ability to diagnose liver fibrosis in NAFLD patients noninvasively is limited. The abnormal expression of tRNA-derived small RNA (tsRNA) in plasma provides a novel idea for noninvasive diagnosis of various diseases, however, the relationship between tsRNAs and NAFLD is still unknown. Here, we took advantage of small RNA-Seq technology to profile tsRNAs in NAFLD patients and found the ubiquitous presence of hepatic tsRNAs secreted into circulating blood. Verification in a cohort of 114 patients with NAFLD and 42 patients without NAFLD revealed that three tsRNAs (tRF-Val-CAC-005, tiRNA-His-GTG-001, and tRF-Ala-CGC-006) were significantly elevated in the plasma of NAFLD patients, and the expression level are associated with NAFLD activity score (calculated from 0 to 8) and fibrosis stage (scored from 0 to 4). In mouse models, we further found that increased plasma levels of these three tsRNAs were positively correlated with the degree of liver fibrosis. Our study potentially identifies a new class of NAFLD biomarkers and reveal the possible existence of tsRNAs in the blood that can be used to predict fibrogenesis risk in patients diagnosed with NAFLD.

MicroRNA-137 inhibits autophagy and chemosensitizes pancreatic cancer cells by targeting ATG5.

PURPOSE: Autophagy play an important role in tumor chemotherapy resistance. It has been reported that miR-137 expression was reducedand involved in the regulation of sensitivity of PC cells to chemotherapy. However, little is known about the underlying molecular mechanisms. In this study, we hypothesized that miR-137 might sensitize PC cells to chemotherapy thought regulating cell autophagy. METHODS: Cell survival was determined with MTT assay. Apoptotic cells were assessed with flow cytometric analysis. Fluorescence intensity of GFP-LC3 and RFP-GFP-LC3 were examined with immunofluorescence analysis to determine the autophagy and autophagic flux level. Western blotting assay was used to determine protein expression levels of LC3II/LC3I, P62, FUNDC1 and ATG5. mRNA expression level of miR-137 was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Dual-luciferase reporter assay was used to evaluate the directly binding of miR-137 with its targets. Xenograft model was setup to evaluate tumor growth. RESULTS: The results showed that doxorubicin (Dox) induced autophagy but downregulated the expression level of miR-137 in pancreatic cancer (PC) cells. In turn, overexpression of miR-137 enhanced the effect of Dox on decreasing cell survival, inducing cell apoptosis and inhibiting autophagy rather than influencing autophagic flux in PC cells. Further mechanistic study identified that ATG5 was a direct target of miR-137. Moreover, overexpression of ATG5 dramatically reversed the promotion of apoptosis and inhibition of autophagy mediated by higher expression level of miR-137. We also demonstrated that miR-137 sensitized PANC-1 cells to Dox through inhibiting ATG5 and autophagy in vivo. CONCLUSIONS: Our findings demonstrated for the first time that miR-137 was able to promote sensitivity of PC cells to chemotherapy via inhibition of autophagy mediated by ATG5. Therefore, miR-137 may act as a potential therapeutic target for pancreatic cancer.

LncRNA MEG3 functions as a ceRNA in regulating hepatic lipogenesis by competitively binding to miR-21 with LRP6.

BACKGROUND: Hepatic lipogenesis dysregulation is essential for the development of non-alcoholic fatty liver disease (NAFLD). Emerging evidence indicates the importance of the involvement of long non-coding RNAs (LncRNAs) in lipogenesis. However, the specific mechanism underlying this process is not clear. OBJECTIVE: This study aimed to investigate the functional implication of LncRNA MEG3 (MEG3) in fatty degeneration of hepatocytes and in the pathogenesis of NAFLD. METHODS: The expression of MEG3 was analysed in in vitro and in vivo models of NAFLD, which were established by free fatty acid (FFA)-challenged HepG2 cells and high-fat diet-fed mice, respectively. Endogenous MEG3 was over-expressed by a specific pcDNA3.1-MEG3 to evaluate the regulatory function of MEG3 on triglyceride (TG)- and lipogenesis-related genes. Bioinformatic analysis was used to predict the target genes and binding sites, and the targeted regulatory relationship was verified with a dual luciferase assay. Finally, the possible pathway that regulates MEG3 was also evaluated. RESULTS: We found that the downregulation of MEG3 in vitro and in vivo models of NAFLD was negatively correlated with lipogenesis-related genes and that overexpression of MEG3 reversed FFA-induced lipid accumulation in HepG2 cells. miR-21 was upregulated in the FFA-challenged HepG2 cells and was physically associated with MEG3 in the process of lipogenesis. Our mechanistic studies demonstrated that MEG3 competitively binds to miR-21 with LRP6, followed by the inhibition of the mTOR pathway, which induces intracellular lipid accumulation. CONCLUSION: Our data are the first to document the working model of MEG3 functions as a potential hepatocyte lipid degeneration suppressor. MEG3 helps to alleviate lipid over-deposition, probably by binding to miR-21 to regulate the expression of LRP6. Our results suggest the potency of MEG3 as a biomarker for NAFLD and as a therapeutic target for treatment.

RELA/NEAT1/miR-302a-3p/RELA feedback loop modulates pancreatic ductal adenocarcinoma cell proliferation and migration.

Pancreatic ductal adenocarcinoma (PDAC) remains a challenging malignancy due to distant metastasis. RELA, a major component of the NF-κB pathway, could serve as an oncogene through activating proliferation or migration-related gene expression, including NEAT1, a well-known oncogenic long noncoding RNA. In the current study, the expression and function of RELA and NEAT1 in PDAC were examined. The potential upstream regulatory microRNAs of RELA were screened and verified for their correlation with RELA and NEAT1. The expression and function of the selected miR-302a-3p were evaluated. RELA and NEAT1 expression were upregulated in PDAC tissues, particularly in PDAC tissues with lymph node metastasis, and their expression correlated with clinical parameters. RELA overexpression promoted PDAC cell proliferation and migration, which could be partially attenuated by the NEAT1 knockdown. By binding to RELA, miR-302a-3p inhibited RELA expression, as well as PDAC cell proliferation and migration. RELA downstream NEAT1 expression was negatively regulated by miR-302a-3p; the suppressive effect of NEAT1 knockdown on PDAC cell proliferation and migration was partially attenuated by miR-302a-3p inhibition. Moreover, through direct binding, the expression of miR-302a-3p was also negatively regulated by NEAT1. The expression of miR-302a-3p was downregulated and negatively correlated with RELA or NEAT1 in tissue samples, indicating that rescuing miR-302a-3p expression may inhibit PDAC cell proliferation and migration through RELA/NEAT1. In summary, RELA, NEAT1, and miR-302a-3p form a feedback loop in PDAC to modulate PDAC cell proliferation and migration.

Liver X Receptor Inverse Agonist SR9243 Suppresses Nonalcoholic Steatohepatitis Intrahepatic Inflammation and Fibrosis.

Abnormal metabolism of cholesterol may be a contributing factor in nonalcoholic steatohepatitis (NASH) pathogenesis. Accumulating evidence has shown that liver X receptor (LXR) is closely related to intrahepatic inflammation and fibrosis. In this study, we evaluated the effects of a novel liver-specific LXR inverse agonist, SR9243, on antifibrosis in NASH mice. A high-cholesterol diet was employed to induce NASH in BALB/c mice by either carbon tetrachloride (CCL4) administration or bile-duct ligation (BDL). Once NASH was induced, mice were treated with SR9243 for one month by intraperitoneal (i.p.) injection. Liver tissues were collected to determine the degree of fibrosis and intrahepatic inflammation via pathological examination and QPCR; serum was collected to analyze the plasma lipid levels and liver function by clinical biochemistry. The mice developed hepatic steatosis, severe hepatic inflammation, and fibrosis by BDL or CCL4. Treatment with SR9243 significantly reduced the severity of hepatic inflammation and ameliorated hepatic fibrosis; simultaneously, body weight, serum glucose, and plasma lipid levels were controlled effectively. Our data demonstrate that SR9243 exerts an antifibrotic and anti-inflammatory effect in NASH mice; hence these findings highly suggest that LXR inverse agonist could be therapeutically important in NASH treatment.

Glutathione-s-transferase A 4 (GSTA4) suppresses tumor growth and metastasis of human hepatocellular carcinoma by targeting AKT pathway.

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies of cancers and its prognosis remains dismal due to the paucity of effective therapeutic targets. Up-regulation of glutathione-s-transferase A 4 (GSTA4) is associated with poor prognosis of HCC, but its functional mechanism in HCC remains unclear. In this study, we investigated the roles of GSTA4 in tumor growth and metastasis of HCC and found that GSTA4 was frequently up-regulated in HCC tissues. Through gain- and loss-of-function studies, GSTA4 was demonstrated to significantly regulate cell proliferation, migration, and invasion in vitro. Furthermore, GSTA4 overexpressing significantly promoted the tumorigenicity and metastasis of HCC cells in nude mice models bearing human HCC, whereas silencing endogenous GSTA4 caused an opposite outcome. Moreover, we demonstrated that GSTA4 enhanced HCC aggressiveness by activating protein kinase B (AKT) signaling. In multivariate analysis, our results GSTA4 overexpression promotes the progression of hepatocellular carcinoma and might represent a novel therapeutic target for its treatment.

Association between reversal of multidrug resistance by methyl jasmonate and P-glycoprotein ATPase activity in hepatocellular carcinoma.

OBJECTIVE: To study the effects of methyl jasmonate on multidrug resistance in a mouse model of hepatocellular carcinoma. METHODS: Multidrug resistant H22 (H22/FAP) hepatocellular carcinoma cells were produced in vitro by continuous exposure to increasing doses of doxorubicin, cisplatin and 5-fluorouracil (FAP regimen). Cell toxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolum bromide (MTT) assay. Survival time was calculated for BALB/c mice that received intraperitoneal injections of H22/FAP cells followed by treatment with methyl jasmonate or verapamil in combination with FAP for 7 days. Adenosine triphosphate (ATP) hydrolysis was used to measure the activity of permeability-glycoprotein (P-gp) ATPase activity in plasma membranes. RESULTS: The MTT assay showed that methyl jasmonate significantly enhanced the cytotoxicity of the FAP regimen in multidrug resistant H22/FAP cells. Methyl jasmonate (10 mg/kg and 5 mg/kg) combined with FAP significantly increased survival time in BALB/c mice by 44.25% and 48.01%, respectively, compared with FAP. Methyl jasmonate increased P-gp ATPase activity. CONCLUSION: The combined use of methyl jasmonate and the FAP regimen might be a novel strategy for overcoming the multidrug resistance often observed in hepatocellular carcinoma.

Amelioration of carbon tetrachloride-induced cirrhosis and portal hypertension in rat using adenoviral gene transfer of Akt.

AIM: To investigate whether a virus constitutively expressing active Akt is useful to prevent cirrhosis induced by carbon tetrachloride (CCl4). METHODS: Using cre-loxp technique, we created an Ad-myr-HA-Akt virus, in which Akt is labeled by a HA tag and its expression is driven by myr promoter. Further, through measuring enzyme levels and histological structure, we determined the efficacy of this Ad-myr-HA-Akt virus in inhibiting the development of cirrhosis induced by CCl4 in rats. Lastly, using western blotting, we examined the expression levels and/or phosphorylation status of Akt, apoptotic mediators, endothelial nitric oxide synthase (eNOS), and markers for hepatic stellate cells activation to understand the underlying mechanisms of protective role of this virus. RESULTS: The Ad-myr-HA-Akt virus was confirmed using polymerase chain reaction amplification of inserted Akt gene and sequencing for full length of inserted fragment, which was consistent with the sequence reported in the GenBank. The concentrations of Ad-myr-HA-Akt and adenoviral enhanced green fluorescent protein (Ad-EGFP) virus used in the current study were 5.5 × 10(11) vp/mL. The portal vein diameter, peak velocity of blood flow, portal blood flow and congestion index were significantly increased in untreated, saline and Ad-EGFP cirrhosis groups when compared to normal control after the virus was introduced to animal through tail veil injection. In contrast, these parameters in the Akt cirrhosis group were comparable to normal control group. Compared to the normal control, the liver function (Alanine aminotransferase, Aspartate aminotransferase and Albumin) was significantly impaired in the untreated, saline and Ad-EGFP cirrhosis groups. The Akt cirrhosis group showed significant improvement of liver function when compared to the untreated, saline and Ad-EGFP cirrhosis groups. The Hyp level and portal vein pressure in Akt cirrhosis groups were also significantly lower than other cirrhosis groups. The results of HE and Van Gieson staining indicated that Akt group has better preservation of histological structure and less fibrosis than other cirrhosis groups. The percentage of apoptotic cell was greatly less in Akt cirrhosis group than in other cirrhosis groups. Akt group showed positive HA tag and an increased level of phosphorylated Akt as well as decreased levels of Fas. In contrast, Caspase-3 and Caspase-9 levels in Akt group were significantly lower than other cirrhosis groups. Noticeable decrease of DR5 and α-SMA and increase of phosphorylated eNOS were observed in the Akt group when compared to other cirrhosis groups. The NO level in liver was significantly higher in Akt group than other cirrhosis groups, which was consistent with the level of phosphorylated eNOS in these groups. CONCLUSION: This study suggest that Ad-myr-HA-Akt virus is a useful tool to prevent CCl4-induced cirrhosis in rat model and Akt pathway may be a therapeutic target for human cirrhosis.

[Risk factors for anastomotic leakage after anterior resection for rectal cancer].

OBJECTIVE: To identify the risk factors associated with anastomotic leakage following an anterior resection for rectal cancer. METHODS: Between June, 1999 and June, 2009, 628 patients underwent anterior resection for rectal cancer. A retrospective study of the cases was performed to identify the risk factors for anastomotic leakage following the resection. RESULTS: The overall incidence rate of anatomic leak was 8.6% (54/628) in these patients. A low albumin level (less than 35 g/L), diabetes, absence of a protective stoma, a distance less than 7 cm from the tumor to the anal edge, and a tumor diameter over 5 cm were identified as the risk factors for anastomotic leakage after anterior resection. CONCLUSION: For patients at a high risk for anastomotic leakage, a protective stoma can significantly decrease the rate of clinical leaks and subsequent reoperation after low anterior resection for rectal cancer.

[Therapeutic effect of subtotal gastrectomy and Roux-en-Y gastrojejunostomy on type 2 diabetes mellitus in non-obese patients].

OBJECTIVE: To evaluate the therapeutic effect of Roux-en-Y anastomosis following subtotal gastrectomy on type 2 diabetes mellitus (T2DM) in non-obese patients. METHODS: We performed a retrospective analysis of 16 non-obese patients with T2DM undergoing Roux-en-Y anastomosis following subtotal gastrectomy for stomach cancer and upper gastrointestinal tract ulcer. RESULTS: All the patients were followed up for 6 months after the surgery. Roux-en-Y gastrojejunostomy significantly lowered the levels of fasting plasma glucose (FPG), 2 h postprandial plasma glucose (2hPG), and glycated hemoglobin (HbA1c)(P<0.05). Of these patients, 8 (50%) achieved adequate glycemic control without antidiabetic medication and 5 (31.25%) showed obvious improvement. The total effectiveness rate of the surgery was 81.25%. CONCLUSION: Roux-en-Y gastrectomy can effectively ameliorate the diabetic symptoms and might serve as a new treatment option for T2DM in non-obese patients.

Labeling and tracking human amniotic epithelial cells with green fluorescent protein in an adeno-associated virus vector.

Human amniotic epithelial cells (hAECs) are a recently identified type of stem cell. Thanks to their ready availability and the lower risk of teratoma formation, hAECs have been studied and tested for a variety of human disease treatments and tissue reconstruction efforts. This aim of this study was to establish a stable tracking system to further monitor hAECs in vivo after transplantation. hAECs were isolated from the placentas of patients who visited the Hunan Province Maternity and Child Care Hospitals between Jan 2008 and Jan 2009. Using the classic transfection/infection technique, we successfully introduced green fluorescent protein (GFP) into cultured hAECs with an adeno-associated virus (AAV) vector. The initial preparation of the AAV-GFP virus stock was titrated using HT1081 cells, and further used for the infection of hAECs. GFP(+) hAECs preserve the capacity of differentiation into hepatocyte-like cells with the expression of cytokeratin-18 (CK18) and albumin (ALB). AAV-GFP virus-infected hAECs were transplanted through the spleen into severe combined immune deficiency (SCID) mice via hepatectomy. Four weeks later, the GFP and human albumin expressions were examined in multiple organs through immunofluorescence staining. In culture, over 50% of the hAECs were GFP-positive 3 days after infection. Following transplantation, AAV-GFP-infected hAECs survived and continued to express GFP in the host for up to 4 weeks. These cells were primarily found in the spleen and liver, expressing human albumin. This study provides a feasible and stable system to track hAECs. It may prove useful to further identify their biological characteristics after transplantation and to elucidate their beneficial roles for therapeutic purposes.

[Akt gene therapy for cirrhotic rats with portal hypertension].

OBJECTIVE: To determine whether there is an impaired Akt and eNOS activation in cirrhotic livers, and to investigate the feasibility of transferring adenovirus-mediated Akt gene to the liver for portal hypertension. METHODS: Recombinant adenovirus Ad-myr-HA-Akt and Ad-EGFP were produced by homologoas recombination in 293 cells . The Methods of compound factor, carbon tetrachloride (CCl4), corn flour, and cholesterol plus alcohol were used to construct the hepatic cirrhosis rat models. Ten normal rats were served as a normal control group, and 40 cirrhotic rats were divided into 4 groups randomly: an untreated group, an Ad-myr-HA-Akt treated group, an Ad-EGFP group, and a saline group. Ad-myr-HA-Akt, Ad-EGFP, and saline were transduced into the Ad-myr-HA-Akt treated group, Ad-EGFP group, and saline group via the tail vein respectively. Portal vein pressure, mean arterial pressure, and heart rate were measured in all rats. Protein abundance and phosphorylation status of Akt and eNOS were examined by Western blot. Spectrophotometry was used to measure the NO level. Frozen sections of the liver, heart, lung, kidney, brain, spleen, and testis were made to examine the expression of enhanced green fluorescent protein (EGFP) by fluorescence microscopy on Day 3 in the Ad-EGFP group. RESULTS: The concentration of recombinant adenovirus Ad-myr-HA-Akt after the purification was 5.5 x 10(11)vp/mL and that of Ad-EGFP was 6.0 x 10(11)vp/mL. Akt and eNOS phosphorylations in the liver of cirrhotic rats were obviously impaired. Adenoviral delivery of myr-Akt restored eNOS phosphorylation, increased the NO level and decreased the portal pressure after 3 days of adenoviral infection. In contrast, the livers infected with Ad-EGFP and saline were not changed. The EGFP expression was mainly found under the fluorescence microscopy on the frozen section of liver. Very little fluorescence was detected in the lung and kidney; and there was no detectable EGFP in other organs. CONCLUSION: There is an impaired Akt and eNOS activation in the cirrhotic livers; myr-Akt gene therapy can restore the Akt activation and NO production in the cirrhotic liver, suggesting that this therapy may be helpful in treating portal hypertension.

[Effects of different treatment complex on esophageal vascular structures in patients with portal hypertension].

OBJECTIVE: To assess the effects of different treatment complex on esophageal vascular structures in patients with portal hypertension. METHODS: Patients (142 cases) with esophageal varices received either endoscopic variceal ligation (EVL) alone (54 cases), pericardial devascularization procedure (PDP) alone (23 cases), a combination of EVL and partial splenic embolization (PSE) (34 cases), or a combination of EVL and PDP (31 cases) for variceal eradication. Esophageal vascular structures were examined with miniature ultrasonic probe. The recurrence and rebleeding of esophageal varices were investigated. RESULTS: Esophageal submucous varices were obliterated and collateral veins remained unchanged in patients treated by EVL or EVL combined with PSE; esophageal submucous varices were diminished in size and collateral veins were obliterated by PDP, and both esophageal submucous varices and collateral veins were obliterated by the combination of EVL and PDP. CONCLUSIONS: The combination of EVL and Hassab's procedure can effectively shut off the portoazygous shunt, prevent esophageal varices from bleeding and recurrence. It's a simply and less cost procedure.

[Dynamic variation of endothelin in portal hypertensive patients with hemorrhage and its clinical significance].

OBJECTIVE: To investigate variations of plasma endothelin (ET) and its clinical significance in portal hypertensive patients with esophageal variceal hemorrhage. METHODS: Sixty-six patients with portal hypertension were randomly divided into 2 groups. Group I (32 patients) received general therapy and Group II (34 patients) received general therapy and UTI after hemorrhage. The plasma ET concentration and liver function were determined at 1, 2, 4, 7, 10, and 14 d after the hemorrhage. Another 20 patients without the hemorrhage were elected as the control group. RESULTS: At 7 and 14 d after the hemorrhage, the levels of TBIL, ALT and AST were elevated at first and then decreased in Groups I and II. The decrease of TBIL, ALT and AST levels was significantly faster in Group II than in Group I (P < 0.05, P < 0.01, P < 0.05, respectively) on 14 d after the hemorrhage. At 1 d after the hemorrhage the ET concentration was markedly increased in Group I and II as compared with the control group (P < 0.01). Then it was gradually decreased on 10 d after the hemorrhage. The ET concentration in Group II was decreased more rapidly than that in Group I on 2, 4 and 7 d after the hemorrhage (P < 0.05; P < 0.01; P < 0.05, respectively). The ET concentration was positively correlated to TBIL levels in groups I and II (r = 0.734, P < 0.01). And the decreased index of ET concentration was negatively correlated to the increased index of TBIL (r = -0.486, P < 0.05). CONCLUSION: The increased plasma ET in portal hypertensive patients with hemorrhage may contribute to liver injury. UTI can protect the liver function by inhibiting ALT, AST, TBIL and ET level.

[Laser inducing mucosal fibrosis for preventing recurrence of esophageal varices].

OBJECTIVE: To investigate the prevention of esophageal varices recurrence by laser inducing esophageal mucosal fibrosis. METHODS: Our study included 42 patients after esophageal varices eradicated by endoscopic varices ligation, and they were divided into 2 groups randomly, each group included 21 patients. One group was assigned to received laser treatment, and indocyanine green solution (1 mg/ml) was injected submucosally, a diode laser (power 10 watts) was applied to the surface from the esophagogastric junction to 5 cm above it. Another group was controlling without any treatments. All patient were followed up by endoscopy every 3 months until 12 months. RESULTS: Laser irradiation was performed safely without any major complications. And lower esophageal mucosa produced fibrosis widely after laser irradiated 1 month. After 12 months follow up, the cumulative recurrence rate was significantly lower than the control group, 14% (3/21) vs 43% (9/21) (chi(2) = 4.20, P < 0.05). CONCLUSIONS: Our study indicates that laser inducing mucous fibrosis is safely and can prevent recurrence of esophageal varices.

[Treatment of portal hypertension by endoscopic esophageal ligation of varicose veins combined with partial splenic embolization].

OBJECTIVE: To evaluate the feasibility and efficacy of a new method of endoscopic esophageal variceal ligation combined with partial splenic embolization (EVL-PSE) for the patients with portal hypertension. METHODS: From May 1999 to February 2003, sixty-eight patients with portal hypertension underwent EVL-PSE, and hemodynamics of the portal trunk (PT), the left gastric vein and azygos vein, including maximum velocity, flow volume, vein diameter, were assessed using color ultrasound Doppler. RESULTS: The esophageal varices and hypersplenism were greatly ameliorated after operation in patients who had undergone EVL-PSE. Postoperative portal trunk flow volume and velocity were significantly reduced (P < 0.05), and flow volume of the left gastric vein as well as the azygos vein were also reduced after operation. During 2 - 24-month follow-up, no recurrent bleeding was found. CONCLUSIONS: EVL-PSE is less traumatic with less complications, and results in marked eradication of esophageal varices, it can be carried out safely in the clinical treatment for patients with portal hypertension.