Upregulation of microRNA­300 induces the proliferation of liver cancer by downregulating transcription factor FOXO1.

In the present study, we investigated whether miRNA­300 (miR­300) is an oncogene in human liver cancer and sought to determine the mechanism underlying its activity. We also investigated the effect of miRNA­300 on the growth in liver cancer. To identify its target molecule, we performed luciferase assays. The downstream signaling pathway was detected by immunohistochemical (IHC) analysis in human HCC tissues, and the protein levels of AKT, 4E­BP1, S6K1, SNAIL and MMP2 were determined using western blotting. miR­300 levels were higher in patients with high­stage HCC, and miR­300 promoted cell growth both in vitro and in vivo. miRNA­300 inhibited the luciferase activity of FOXO1 by targeting its 3'­untranslated region (UTR), and overexpression of miR­300 upregulated the protein levels of phospho­AKT, phospho­4E­BP1, phospho­S6K1, SNAIL and MMP2. These data revealed that miRNA­300 functions as an oncogene in liver cancer by inhibiting FOXO1 and interacting with the AKT/mTOR signaling pathway.

Long non-coding RNA FOXD2-AS1 plays an oncogenic role in hepatocellular carcinoma by targeting miR­206.

Recently, long non-coding RNA (lncRNA) FOXD2 adjacent opposite strand RNA 1 (FOXD2-AS1) has been recognized to function as an oncogene in several human tumors, and FOXD2­AS1 dysregulation has been closely associated with carcinogenesis and tumor progression. Nevertheless, the correlation between the aberrant expression of FOXD2­AS1 and the prognosis of hepatocellular carcinoma (HCC) has not yet been elucidated. In the present study, FOXD2­AS1 was found to be overexpressed in HCC tissues, and FOXD2­AS1 overexpression resulted in significantly shortened patient survival. FOXD2­AS1 overexpression enhanced the viability and metastasis of HCC cells in vitro and in vivo, as revealed by MTT, wound healing and cell migration assays. In addition, mechanistic studies revealed that FOXD2­AS1 upregulated the expression of the miR­206 target gene annexin A2 (ANXA2) by acting as a miR­206 sponge. In summary, FOXD2­AS1 was concluded to function as an oncogene in HCC and to upregulate ANXA2 expression in part by 'sponging' miR­206.

Activation of corticotropin-releasing factor receptor 1 aggravates dextran sodium sulphate-induced colitis in mice by promoting M1 macrophage polarization.

The corticotropin-releasing factor (CRF) family is involved in modulating gastrointestinal motility, sensitivity and inflammation. CRF signalling exerts an important role in inflammatory bowel disease (IBD), predominantly by activating CRF receptors. The aim of the present study was to investigate the function of CRF receptor 1 (CRF­R1) in the development of mucosal inflammation induced by dextran sulphate sodium (DSS) and the underlying mechanism. Consecutive administration of CRF or CP154526 was used to activate or block the CRF­R1 in DSS­treated mice. Colonic inflammation was evaluated by determining the Disease Activity Index (DAI) and histology score. CRF­R1 expression was proportional to the DAI, the histology score and the number of macrophages. Activation of CRF­R1 aggravated mucosal inflammation by activating nuclear factor (NF)­κB and subsequently increasing the expression levels of tumour necrosis factor (TNF)­α and interleukin (IL)­6. Inhibition of CRF­R1 decreased the expression level of CRF­R1, macrophage infiltration, NF­κB activation, and TNF­α and IL­6 expression levels, ultimately alleviating the mucosal inflammation. Thus, CRF­R1 expression was proportional to the severity of DSS­induced colitis. Activation of CRF­R1 increased the DAI and histological scores of the colons from DSS­treated mice by promoting M1 macrophage polarization, demonstrated as increased NF­κB activation, and TNF­α and IL­6 release. These results provide evidence of the pro­inflammatory role of CRF­R1 in a DSS­induced ulcerative colitis (UC) model and a possible underlying mechanism, which may facilitate the elucidation of potential treatment approaches for UC.

Dynamic remodeling of subcellular chemical gradients using a multi-directional flow device.

Elucidation of the mechanisms by which external chemical cues regulate polarized cellular behaviors requires tools that can rapidly recast chemical landscapes with subcellular resolution. Here, we describe an approach for creating steep microscopic gradients of cellular effectors at any desired position in culture that can be reoriented rapidly to evaluate dynamic responses. In this approach, micrometre pores are ablated in a membrane that supports cell adherence, allowing dosing reagent from an underlying reservoir to enter the cell-culture flow chamber as sharp streams that are directed at subcellular targets by using a system of paired sources and drains to specify flow direction. This tool substantially extends capabilities for chemical interaction with cultured cells, enabling investigations of chemotaxis via precise placement and reorientation of peptide gradients formed at the boundaries of dosing streams. These studies demonstrate that neutrophil precursor cells can repolarize and redirect their migration paths using morphological responses that depend on the subcellular localization of chemoattractant gradients.

GG genotype of cyclin D1 G870A polymorphism is associated with non-cardiac gastric cancer in a high-risk region of China.

OBJECTIVE: Cyclin D1 (CCND1) is a regulatory protein involved in the cell cycle of both normal and neoplastic cells. Polymorphism of this gene at codon 242 in exon 4 (A870G) has an impact on the risk of several human cancers. The purpose of this study was to study the relation between the CCND1 A870G gene polymorphism and the risk of non-cardiac gastric cancer in a Chinese population. MATERIAL AND METHODS: The study population consisted of 159 patients with non-cardiac gastric cancer and 162 cancer-free controls. CCND1 870A/G polymorphism was genotyped by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay and sequencing. RESULTS: CCND1 genotype distribution among the patients was significantly different from that among controls; AA (odds ratio (OR)=0.348, 95% CI: 0.163-0.742) and GA (OR=0.715, 95% CI: 0.506-1.012) genotypes were significantly lower in the gastric cancer patients than in the controls when subjects with the GG genotype served as the reference category. In other words, the risk of gastric cancer for subjects with the GG genotype was 2.8 times that of subjects with the AA genotype, and 1.4 times that of subjects with the GA genotype. Furthermore, in the stratification analyses, the risk of GG genotype was more evident in subjects >or=60 years of age and those positive for Helicobacter pylori (H. pylori) infection. CONCLUSIONS: The CCND1870 GG genotype is associated with an increased risk for non-cardiac gastric cancer in patients in a high-risk area of China. Larger studies with multiple polymorphisms are needed to verify this finding and the function of this polymorphism needs to be further investigated.

Effect of Mexican tea herb and pilular adina herb on concrescence of gastric mucosa in experimental gastric ulcer rats.

OBJECTIVE: To study the effect and mechanism of mexican tea herb and pilular adina herb (abbreviated to MP) on concrescence of gastric mucosa in experimental gastric ulcer rats by observing the changes of epidermal growth factor (EGF), nitrogen monoxidum (NO) and expression of epidermal growth factor receptor (EGFR). METHODS: The rat ulcer model was established by 100% glacial acetic injection into the subserosa. The ulcer index (UI) was measured by sliding caliper. The levels of NO and EGF in tissue and serum were measured by the nitrate reductase method and enzyme-linked immunosorbent assay, respectively. The expression of EGFR in the mucosa around the ulcer was detected by the immunohistochemical assay and microimage analysis system. RESULTS: (1) Compared with the model group, UI of MP groups (10, 15 and 20 mg.kg(-1).d(-1)) and ranitidine group was lower (P<0.05 or P<0.01), the levels of NO and EGF in the tissue and serum were higher (P<0.05), the thickness of regenerated mucous membrane increased, and the width loss of lamina muscularis mucosa decreased (all P<0.05). (2) The expression of EGFR is weakly positive in gastric mucosa cells in the normal group, mainly in the cytoplasm and cytomembrane. In the model group, the expression of EGFR was mainly in epithelial cells in cervical part and basilar part of gastric gland around the ulcer margin, and the number of cells with EGFR weakly positive expression was more than that in the normal group. Compared with that in the normal and model groups, the number of cells with EGFR positive in MP groups and ranitidine group increased (all P<0.05), with weakly positive expression. CONCLUSION: MP can protect gastric mucosa, cure gastric ulcer, restrain the secretion of gastric acid, and boost multiplication, differentiation, migration and repair of the endothelial cell by promoting the secretion of NO and EGF, and increasing the expression of EGFR of gastric mucosa epithelial cells.

[Preparation of tetrandrine solid lipid nanoparticles].

OBJECTIVE: To prepare solid lipid nanoparticles (SLN) loaded tetrandrine (TET) extracted from traditional Chinese medicine with ultrasonication and high pressure homogenization, and to compare the physicochemical characteristics of the particles produced by the two methods. METHODS: TET was incorporated into SLN by ultrasonication and high pressure homogenization separately. Transmission electron microscopy was employed to study the shape. Particle characterization system and Zeta potential analyzer were used to study the diameter and Zeta potential of SLN in suspension. The entrapment efficiency was determined with the high-performance liquid chromatography. The stability of SLN was also studied. RESULTS: TET-SLNs prepared by these two methods were sheet-shaped and irregular, but the SLNs prepared by high pressure homogenization were smaller. The mean diameter of SLN prepared by ultrasonication was (92 +/- 6) nm with Zeta potential of (-21.11 +/- 2.12) mV in distilled water, and the mean entrapment efficiency was 95.27%. The mean diameter of TET-SLN prepared by high pressure homogenization was (47 +/- 3) nm with Zeta potential of (-32.99 +/- 2.54) mV, and up to 97.82% of TET was incorporated. The diameter of SLN prepared by high pressure homogenization and ultrasonication were (52 +/- 5) nm and (168 +/- 12) nm after 90 days of storage at room temperature. CONCLUSION: Compared with ultrasonication, high pressure homogenization is a better method to prepare TET-SLN, which is smaller, steadier and highly incorporated.

Preparation of solid lipid nanoparticles loaded with traditional Chinese medicine by high-pressure homogenization.

OBJECTIVE: To investigate the preparation of solid lipid nanoparticles (SLN) loaded with traditional Chinese medicines by high-pressure homogenization, and study the physicochemical characteristics of the particles produced by this method. METHODS: The model traditional Chinese medicines, silibinin (SIL) and tetrandrine (TET), were incorporated into SLN separately by high-pressure homogenization. Transmission electron microscope was employed to study the shape of the particles. Particle characterization system and zeta potential analyzer were used to study the diameter and zeta potential of SLN in the suspension. The entrapment efficiency and drug loading were determined with the sephadex gel chromatography and high-performance liquid chromatography. The stability of SLN was also studied. RESULTS: The SIL-SLNs prepared by high-pressure homogenization were spherical and regular. The mean diameter and zeta potential of SIL-SLN in distilled water were 157+/-8 nm and -35.36+/-2.68 mV, respectively. The entrapment efficiency was 95.64%, and the drug loading was 4.63%. The TET-SLN was platelet-shaped, irregular and smaller. The mean diameter and zeta potential of TET-SLN were 47+/-3 nm and -32.99+/-2.54 mV, respectively, with drug loading of 4.76%, and up to 97.82% of TET was incorporated. SIL-SLN and TET-SLN had good stability. CONCLUSION: High-pressure homogenization is feasible for preparing SLN loaded with traditional Chinese medicines.

Preparation and characterization of solid lipid nanoparticles loaded traditional Chinese medicine.

Ultrasonication was employed to prepare solid lipid nanoparticles (SLN). The model traditional Chinese medicine, tetrandrine (TET), was incorporated into SLN. The TET-loaded SLN (TET-SLN) were spherical in the photograph of transmission electron microscope (TEM). The particle size measured by laser diffraction (LD) was found to be 157.3+/-8.2nm. Zeta potential analyzer suggested the zeta potential of TET-SLN was -29.36+/-3.68mV in distilled water. The entrapment efficiency (EE%) was determined with the sephadex gel chromatogram and high-performance liquid chromatogram (HPLC), and up to 90.59% of TET was incorporated. Stability evaluation showed relatively long-term stability with only slight particle growth (P>0.05) after storage at room temperature for 4 weeks. Therefore, ultrasonication is demonstrated to be a simple, available and effective method to prepare high quality SLN loaded traditional Chinese medicine.

Effects of interferon-alpha on expression of hepatic stellate cell and transforming growth factor-beta1 and alpha-smooth muscle actin in rats with hepatic fibrosis.

AIM: To investigate the effect of interferon-alpha (IFN-alpha) on preventing or reversing hepatic fibrosis in rat experimental model induced by CCl(4). METHODS: One hundred and ten Sprague-Dawley rats were divided into five groups: group A (normal controls, n=18), group B (fibrotic model controls, n = 22), group C (IFN-alpha prevention, n=22) initially treated with intra-muscular injection of IFN-alpha in saline daily at the doses of 1X105 U for 6 wk, group D (IFN-alpha treatment, n = 24) treated with intra-muscular injection of IFN-alpha in saline daily at the doses of 1X105 U for 6 wk after the first 6 wk, group E (0.9% sodium chloride treatment control, n = 24) treated with intra-muscular injection of 0.01 mL/kg daily for 6 wk after the first 6 wk. At the end of the experiment, all rats of each group were killed. Samples of the liver obtained by biopsy were subjected to histological, immunohistochemical and electron microscopic studies for the expressions of transforming growth factor-beta1 (TGF-beta1) and alpha-smooth muscle actin (alpha-SMA). RESULTS: The expressions of TGF-beta1, the number of activated hepatic stellate cells and alpha-SMA in hepatic tissue of group C were significantly less than those of group B (P<0.01). The degree of fibrosis score in group B was also significantly less than that of group C under light microscope (P<0.01). CONCLUSION: IFN-alpha can inhibit the production of TGF-beta1, decrease HSC activation and stimulate its apoptosis.

Effects of selenium on peripheral blood mononuclear cell membrane fluidity, interleukin-2 production and interleukin-2 receptor expression in patients with chronic hepatitis.

AIM: To study the effect of selenium on peripheral blood mononuclear cell (PBMC) membrane fluidity and immune function in patients with chronic hepatitis. METHODS: PBMCs were pretreated with selenium (1.156x10(-7) mol/L) for 6 h in vitro or extracted directly from patients after administration of selenium-yeast continuously for 8-12 wk (200 microg/d), and then exposed to Con-A for 48 h. The membrane fluidity, interleukin-2 (IL-2) production and interleukin-2 receptor (IL-2R) expression in PBMCs and malondialdehyde (MDA) concentration in medium and lipid peroxide (LPO) in plasma were determined. RESULTS: The PBMC membrane fluidity, IL-2 production and IL-2R expression in patients with chronic hepatitis were significantly lower than those in healthy blood donators (particle adhesive degree R, 0.17+/-0.01 vs 0.14+/-0.01, P<0.01; IL-2, 40.26+/-9.55 vs 72.96+/-11.36, P<0.01; IL-2R, 31.05+/-5.09 vs 60.58+/-10.56, P<0.01), and the MDA concentration in medium in patients with chronic hepatitis was significantly higher than that in healthy blood donators (1.44+/-0.08 vs 0.93+/-0.08, P<0.01). Both in vitro and in vivo administration of selenium could reverse the above parameters. CONCLUSION: Supplement of selenium can suppress lipid peroxidation, and improve PBMC membrane fluidity and immune function in patients with chronic hepatitis.

Effects of estradiol on liver estrogen receptor-alpha and its mRNA expression in hepatic fibrosis in rats.

AIM: Estradiol treatment regulates estrogen receptor (ER) level in normal rat liver. However, little information is available concerning the role of estrogen in regulating liver ER in hepatic fibrosis in rats. The present study was conducted to determine whether estradiol treatment in CCl4-induced liver fibrosis of female and ovariectomized rats altered liver ERalpha and its mRNA expression, and to investigate the possible mechanisms. METHODS: Seventy female rats were divided into seven groups with ten rats in each. The ovariectomy groups were initiated with ovariectomies and the sham operation groups were initiated with just sham operations. The CCl4 toxic fibrosis groups received 400 mL/L CCl4 subcutaneously at a dose of 2 mL/kg twice weekly. Estrogen groups were treated subcutaneously with estradiol 1 mg/kg, the normal control group and an ovariectomy group received injection of peanut oil vehicle twice weekly. At the end of 8 weeks, all the rats were killed to detect their serum and hepatic indicators, their hepatic collagen content, and liver ER and ER mRNA expression. RESULTS: Estradiol treatment in both ovariectomy and sham ovariectomy groups reduced liver levels of ALT (from 658 +/- 220 nkat/L to 311 +/- 146 nkat/L and 540 +/- 252 nkat/L to 314 +/- 163 nkat/L, P<0.05) and AST (from 697 +/- 240 nkat/L to 321 +/- 121 nkat/L and 631 +/- 268 nkat/L to 302 +/- 153 nkat/L, P<0.05), increased serum nitric oxide (NO) level (from 53.7 +/- 17.1 micromol/L to 93.3 +/- 24.2 micromol/L and 55.3 +/- 23.1 micromol/L to 87.5 +/- 23.6 micromol/L, P<0.05) and hepatic nitric oxide synthase (NOS) activity (from 1.73 +/- 0.71 KU/g to 2.49 +/- 1.20 KU/g and 1.65 +/- 0.46 KU/g to 2.68 +/- 1.17 KU/g, P<0.05), diminished the accumulation of hepatic collagen, decreased centrolobular necrotic areas as well as the inflammatory reaction in rats subjected to CCl4. The positive signal of ER and ER mRNA distributed in parenchymal and non-parenchymal hepatic cells, especially near the hepatic centrolobular and periportal areas. Ovariectomy decreased ER level (from 10.2 +/- 3.2 to 4.3 +/- 1.3) and ER mRNA expression (from 12.8 +/- 2.1 to 10.9 +/- 1.3) significantly (P<0.05). Hepatic ER and ER mRNA concentrations were elevated after treatment with estradiol in both ovariectomy (15.8 +/- 2.4, 20.8 +/- 3.1) and sham ovariectomy (18.7 +/- 3.8, 23.1 +/- 3.7) fibrotic groups (P<0.05). CONCLUSION: The increase in hepatic ER and mRNA expression may be part of the molecular mechanisms underlying the suppressive effect of estradiol on liver fibrosis induced by CCl4 administration.

Correlation between expression of cyclooxygenase-2 and angiogenesis in human gastric adenocarcinoma.

AIM: To evaluate the expression of cyclooxygenase (COX-2) and the relationship with tumor angiogenesis and advancement in gastric adenocarcinoma. METHODS: Immunohistochemical stain was used for detecting the expression of COX-2 in 45 resected specimens of gastric adenocarcinoma; the monoclonal antibody against CD34 was used for displaying vascular endothelial cells, and microvascular density (MVD) was detected by counting of CD34-positive vascular endothelial cells. Paracancerous tissues were examined as control. RESULTS: Immunohistological staining with COX-2-specific polyclonal antibody showed cytoplasmic staining in the cancer cells, some atypical hyperplasia and intestinal metaplasia,as well as angiogenic vasculature present within the tumors and prexisting vasculature adjacent to cancer lesions. The rate of expression of COX-2 and MVD index in gastric cancers were significantly increased, compared with those in the paracancerous tissues (77.78 vs 33.33 %, 58.13+/-19.99 vs 24.02+/-10.28, P<0.01, P<0.05, respectively). In 36 gastric carcinoma specimens with lymph node metastasis, the rate of COX-2 expression and MVD were higher than those in the specimens without metostasis (86.11 vs 44.44 %, 58.60+/-18.24 vs 43.54+/-15.05, P<0.05, P<0.05, respectively). The rate of COX-2 expression and MVD in the specimens with invasive serosa were significantly higher than those in the specimens without invasion to serosa (87.88 vs 50.0 %, 57.01+/-18.79 vs 42.35+/-14.65, P<0.05, P<0.05). Moreover, MVD in COX-2-positive specimens was higher than that in COX-2-negative specimens (61.29+/-14.31 vs 45.38+/-12.42,P<0.05). COX-2 expression was positively correlated with MVD (r=0.63, P<0.05). CONCLUSION: COX-2 expression might correlate with the occurance and advancement of gastric carcinoma and is involved in tumor angiogenesis in gastric carcinoma. It is likely that COX-2 by inducing angiogenesis can be one of mechanisms which promotes invasion and metastasis of gastric carcinoma. It may become a new therapeutic target for anti-angiogenesis.

Estrogen reduces CCL4- induced liver fibrosis in rats.

AIM: Chronic liver diseases, such as fibrosis or cirrhosis, are more common in men than in women. This gender difference may be related to the effects of sex hormones on the liver. The aim of the present work was to investigate the effects of estrogen on CCL(4)-induced fibrosis of the liver in rats. METHODS: Liver fibrosis was induced in male, female and ovariectomized rats by CCL(4) administration. All the groups were treated with estradiol(1 mg/kg) twice weekly. And tamoxifen was given to male fibrosis model. At the end of 8 weeks, all the rats were killed to study serum indicators and the livers. RESULTS: Estradiol treatment reduced aspartate aminotransferase(AST), alanine aminotransferase (ALT), hyaluronic acid(HA) and type IV collagen(CIV) in sera, suppressed hepatic collagen content, decreased the areas of hepatic stellate cells (HSC) positive for alpha-smooth muscle actin (alpha-SMA), and lowered the synthesis of hepatic type I collagen significantly in both sexes and ovariectomy fibrotic rats induced by CCL(4) administration. Whereas, tamoxifen had the opposite effect. The fibrotic response of the female liver to CCL(4) treatment was significantly weaker than that of male liver. CONCLUSION: Estradiol reduces CCL(4)-induced hepatic fibrosis in rats. The antifibrogenic role of estrogen in the liver may be one reason for the sex associated differences in the progression from hepatic fibrosis to cirrhosis.