The Role of Bone Morphogenetic Protein 9 in Nonalcoholic Fatty Liver Disease in Mice.

Background and Aims: It's reported that bone morphogenetic protein 9 (BMP9) played an important role in lipid and glucose metabolism, but the role of BMP9 in nonalcoholic fatty liver disease (NAFLD) is unclear. Here, we evaluated the therapeutic efficacy of recombined BMP9 in NAFLD mice and investigated the potential mechanism. Methods: The effects of recombinant BMP9 on NAFLD were assessed in HFD-induced NAFLD mice. C57BL/6 mice were administrated with high-fat diet (HFD) for 12 weeks. In the last 4 weeks, mice were treated with PBS or recombined BMP9 once daily. Insulin sensitivity was evaluated by glucose tolerance test (GTT) and insulin tolerance test (ITT) at the end of the 12th week. Then NAFLD related indicators were assessed by a variety of biological methods, including histology, western blotting, real-time PCR, RNA-seq and assay for transposase-accessible chromatin using sequencing (ATAC-seq) analyses. Results: BMP9 reduced obesity, improved glucose metabolism, alleviated hepatic steatosis and decreased liver macrophages infiltration in HFD mice. RNA-seq showed that Cers6, Cidea, Fabp4 involved in lipid and glucose metabolism and Fos, Ccl2, Tlr1 involved in inflammatory response downregulated significantly after BMP9 treatment in HFD mouse liver. ATAC-seq showed that chromatin accessibility on promoters of Cers6, Fabp4, Ccl2 and Fos decreased after BMP9 treatment in HFD mouse liver. KEGG pathway analysis of dysregulated genes in RNA-seq and integration of RNA-seq and ATAC-seq showed that TNF signaling pathway and Toll-like receptor signaling pathway decreased in BMP9 treated HFD mouse liver. Conclusion: Our data revealed that BMP9 might alleviate NAFLD via improving glucose and lipid metabolism, decreasing inflammatory response and reshaping chromatin accessibility in HFD mouse liver. BMP9 downregulate genes related to lipid metabolism, glucose metabolism and inflammation expression, at least partially via decreasing promoter chromatin accessibility of Cers6, Fabp4, Fos and Tlr1. BMP9 may also reduce the expression of liver Ccl2, thereby changing the number or composition of liver macrophages, and ultimately reducing liver inflammation. The effect of BMP9 on NAFLD might be all-round, and not limit to lipid and glucose metabolism. Therefore, the underlying mechanism needs to be studied in detail further.

Long non-coding RNA LINC01503 promotes colorectal cancer cell proliferation and invasion by regulating miR-4492/FOXK1 signaling.

Increasing evidence indicates that long non-coding RNAs (lncRNAs) are closely associated with the progression of human cancer, including colorectal cancer (CRC). A previous study suggested that lncRNA LINC01503 promotes squamous cell carcinoma progression. However, the function of LINC01503 in CRC has remained elusive. The present study indicated that LINC01503 was significantly upregulated in CRC tissues compared with that in adjacent normal tissues as detected by reverse transcription-quantitative polymerase chain reaction. It was demonstrated that knockdown of long intergenic non-protein coding RNA (LINC)01503 markedly inhibited the proliferation and invasion of CRC cells, whereas overexpression of LINC01503 had the opposite effects, as indicated by Cell Counting kit-8 and Transwell assays. Mechanistically, it was revealed that LINC01503 serves as a sponge for microRNA (miR)-4492, which targets forkhead box K1 (FOXK1) in CRC cells. In addition, luciferase reporter assays demonstrated the direct binding of miR-4492 mimics to LINC01503 and to a sequence in the 3'-untranslated region of FOXK1. Furthermore, it was demonstrated that overexpression of LINC01503 reduced the availability of miR-4492 in CRC cells. Furthermore, miR-4492 mimics inhibited FOXK1 expression, while simultaneous overexpression of LINC01503 abolished this effect. Finally, it was demonstrated that restoration of FOXK1 abolished the inhibitory effect of LINC01503 knockdown on CRC cell proliferation and invasion. Taken together, the present results suggested that LINC01503 promotes CRC progression via acting as a competing endogenous RNA for miR-4492/FOXK1.

[Inhibition of bFGF gene expression and tumor angiogenesis of orthotopic implantation of human gastric carcinoma by N-desulfated heparin].

OBJECTIVE: To investigate the effect of N-desulfated heparin on tumor metastasis, tumor angiogenesis and basic fibroblast growth factor(bFGF) gene expression of orthotopically implanted human gastric carcinoma in NOD-SCID mice. METHODS: Human gastric cancer SGC-7901 tissues were orthotopically implanted into the stomach of the NOD-SCID mice. Twenty mice were randomly divided into two groups which received either intravenous injection of 0.9% NaCl solution(0.9%NaCl solution group) or 10 mg/kg N-desulfated heparin (N-desulfated heparin group) twice a week for three weeks. Mice were sacrificed six weeks after tumor implantation. Tissues from stomach and other organs were obtained for histopathological evaluation. The intratumoral microvessel density (MVD) in tumor was evaluated immunohistochemically. Real time PCR was used to detect bFGF mRNA expression. RESULTS: The tumor metastasis rates were 9/10 in 0.9% NaCl solution group and 2/10 in N-desulfated heparin group(P<0.05).MVD was 9.1+/-3.4 in 0.9% NaCl solution group and 4.7+/-1.8 in N-desulfated heparin group (t=3.617,P<0.05). bFGF mRNA expression was lower in N-desulfated heparin group(2.60+/-0.56%)than that in 0.9% NaCl solution group(30.65+/-6.84%). CONCLUSION: N-desulfated heparin can inhibit the metastasis of gastric cancer through inhibiting tumor bFGF gene expression and tumor angiogenesis with no obvious anticoagulant activity.

Effect of 2-(8-hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl) propionic acid in combination with carboplatin on gastric carcinoma growth in vivo.

AIM: To investigate the effects of 2-(8-hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl) propionic acid (NM-3) alone and in combination with carboplatin on tumor growth and apoptosis in mouse models of human gastric cancer constructed by subcutaneous implantation of histologically intact tumor tissue. METHODS: Human gastric cancer SGC-7901 tissues were implanted into the dorsal subcutis of nude mice. One week after tumors reached to a volume of 50-100 mm(3) for around 1 wk, these mice were randomly divided into 8 groups (n = 10). NM-3 was injected peritoneally at the dose of 10 mg/kg, 20 mg/kg or 40 mg/kg every other day for 5 wk, combined with carboplatin (5 mg/kg) every third day for 4 wk. As controls of combined treatment, another 4 groups of mice were injected with either NM-3 at 10 mg/kg, 20 mg/kg or 40 mg/kg, or with carboplatin alone (5 mg/kg). The control mice received normal saline. Tumor weight, tumor growth inhibition (TGI), and intratumoral microvessel density (MVD) were evaluated. Apoptosis of human gastric cancer was detected by TUNEL method and flow cytometry analysis, respectively. RESULTS: The mean tumor volume (692.40 +/- 58.43 mm(3), 548.30 +/- 66.02 mm(3), 382.13 +/- 43.52 mm(3)) after treatment with carboplatin combined NM-3 at the dose of 10 mg/kg, 20 mg/kg or 40 mg/kg was lower than that after treatment with either NM-3 at the dose of 10 mg/kg, 20 mg/kg or 40 mg/kg or with carboplatin alone. Compared with the normal saline group, NM-3 administered at 10 mg/kg, 20 mg/kg or 40 mg/kg significantly reduced the tumor weight in these groups (P < 0.05). Carboplatin used alone at 5 mg/kg showed minimal effects. But NM-3 in combination with carboplatin had greater effects of tumor weight than either NM-3 or carboplatin alone. NM-3 alone at the dose 10 mg/kg or in combination with carboplatin had no obvious effects on body changes. Two mice died of diarrhea in each of the two groups treated with 40 mg/kg NM-3 or with 40 mg/kg NM-3 in combination with carboplatin. A significant increase in apoptosis was observed in the NM-3 treated groups, and the effect was more significant in the groups treated with carboplatin in combination with NM-3 at 10 mg/kg, 20 mg/kg and 40 mg/kg, than in the control group. The induction of apoptosis was positively associated with the dose of NM-3. NM-3 significantly reduced the neo-microvascular formation of gastric cancer. The MVD was lower in the groups treated with NM-3 or with NM-3 in combination with carboplatin than in the group treated with carboplatin or in the normal saline group (P < 0.05). CONCLUSION: The results suggest that the inhibitory effect of NM-3 on gastric cancer growth is mediated through decreased angiogenesis and the increased induction of apoptosis. Furthermore, NM-3 alone at the dose of 10 mg/kg or in combination with carboplatin has no obvious effects on body changes, indicating that NM-3 in combination with carboplatin may be effective in the treatment of gastric cancer. The toxicity of NM-3 needs further studies.

Effect of non-anticoagulant N-desulfated heparin on expression of vascular endothelial growth factor, angiogenesis and metastasis of orthotopic implantation of human gastric carcinoma.

AIM: To investigate the effect of N-desulfated heparin on tumor metastasis and angiogenesis, and expression of vascular endothelial growth factor (VEGF) of orthotopic implantation of human gastric carcinoma in male severe combined immune deficiency (SCID) mice. METHODS: Human gastric cancer SGC-7901 cells were orthotopically implanted into the stomach of SCID mice. The mice were randomly divided into normal saline group and N-desulfated heparin group. One week after operation, the mice in N-desulfated heparin group received i.v. injections of N-desulfated heparin (Shanghai Institute of Cell Biology, Chinese Academy of Sciences, 10 mg/kg.d) twice weekly for 3 wk. The mice in normal saline group received i.v. injections of normal saline (100 microL) twice weekly for 3 wk. The mice were sacrificed six weeks after implantation. Tumor metastasis was evaluated histologically for metastasis under microscope. Intratumoral microvessel density (MVD) and VEGF expression were evaluated immuohistochemically. VEGF mRNA expression in gastric tissue of SCID mice was detected by real time PCR. RESULTS: The tumor metastasis rate was 80% in normal saline group and 20% in N-desulfated heparin group (P < 0.05). MVD was 8.0 +/- 3.1 in normal saline group and 4.3 +/- 1.8 in N-desulfated heparin group (P < 0.05). VEGF positive immunostaining was found in cytoplasm of cancer cells. The rate of VEGF positive expression was higher in normal saline group than in N-desulfated heparin treated group (90% vs 20%, P < 0.05). VEGF mRNA expression was significantly inhibited by N-desulfated heparin and was higher in normal saline group than in N-desulfated heparin group (Ct value 19.51 +/- 1.01 vs 22.55 +/- 1.36, P < 0.05). N-desulfated heparin significantly inhibited the expression of VEGF mRNA in cancer cells. No bleeding occurred in N-desulfated heparin group. CONCLUSION: N-desulfated heparin can inhibit metastasis of gastric cancer by suppressing tumor VEGF expression and tumor angiogenesis, but has no obvious anticoagulant activity.