OBJECTIVETo investigate the effect of ANGPTL4 gene silencing on the migration of human colon cancer cells in vitro.

METHODSThe expression of ANGPTL4 in human colorectal cancer cell lines was detected by semi-quantitative RT-PCR. Following stable transfection with a short-hairpin RNA (shRNA) targeting ANGPTL4 gene in HT29 cells, ANGPTL4 mRNA and protein expressions were detected by semi-quantitative RT-PCR and direct ELISA, respectively, and the changes in cell migration ability and cell morphology were observed with transwell and immunofluorescence assays.

RESULTSANGPTL4 was expressed in most of the colorectal cancer cell lines. Compared with the control groups, HT29 cells with shRNA-mediated ANGPTL4 gene silencing showed significantly decreased expression of ANGPTL4 mRNA and protein (P<0.05) and lowered cell migration ability possibly due to decreased pseudopodia formation.

CONCLUSIONANGPTL4 was expressed in most colorectal cancer cell lines. Decreased ANGPTL4 gene expression can inhibit the cell migration and pseudopodia formation in HT29 cells.

Angiopoietin-like 4 Protein ; Angiopoietins ; genetics ; Cell Movement ; Colorectal Neoplasms ; genetics ; pathology ; Gene Silencing ; HT29 Cells ; Humans ; RNA, Small Interfering ; genetics

OBJECTIVE: To investigate the association between angiopoietin-like 4 (ANGPTL4) and aldolase A (ALDOA) in human melanoma cell. 
 METHODS: Overexpression or knockdown of ANGPTL4 was performed in WM-115 or WM-266-4 cells, respectively. The expression of ANGPTL4 and ALDOA was measured by RT-PCR and Western blot, respectively. The promoter activity of ALDOA gene was determined by luciferase assay.
 RESULTS: The promoter activity of ALDOA gene and the expression of ALDOA (mRNA and protein) were increased or decreased in the melanoma cells with overexpression or knockdown of ANGPTL4, which was blocked by selective protein kinase C (PKC) inhibitor or restored by PKC agonist, respectively.
 CONCLUSION ANGPTL4 promotes ALDOA expression in human melanoma cell in a PKC dependent manner.
Angiopoietin-like 4 Protein ; Angiopoietins ; genetics ; metabolism ; Blotting, Western ; Cell Line, Tumor ; Fructose-Bisphosphate Aldolase ; metabolism ; Gene Knockdown Techniques ; Humans ; Melanoma ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism

OBJECTIVE: To detect whether Danlou Tablet (DLT) regulates the hypoxia-induced factor (HIF)-1α-angiopoietin-like 4 (Angptl4) mRNA signaling pathway and explore the role of DLT in treating chronic intermittent hypoxia (CIH)-induced dyslipidemia and arteriosclerosis. METHODS: The mature adipocytes were obtained from 3T3-L1 cell culturation and allocated into 8 groups including control groups (Groups 1 and 5, 0.1 mL of cell culture grade water); DLT groups (Groups 2 and 6, 0.1 mL of 1,000 µg/mL DLT submicron powder solution); dimethyloxalylglycine (DMOG) groups (Groups 3 and 7, DMOG and 0.1 mL of cell culture grade water); DMOG plus DLT groups (Groups 4 and 8, DMOG and 0.1 mL of 1,000 µg/mL DLT submicron powder solution). Groups 1-4 used mature adipocytes and groups 5-8 used HIF-1 α-siRNA lentivirus-transfected mature adipocytes. After 24-h treatment, real-time polymerase chain reaction and Western blot were employed to determine the mRNA and protein expression levels of HIF-1 α and Angptl4. In animal experiments, the CIH model in ApoE^-/- mice was established. Sixteen mice were complete randomly divided into 4 groups including sham group, CIH model group [intermittent hypoxia and normal saline (2 mL/time) gavage once a day]; Angptl4 Ab group [intermittent hypoxia and Angptl4 antibody (30 mg/kg) intraperitoneally injected every week]; DLT group [intermittent hypoxia and DLT (250 mg/kg) once a day], 4 mice in each group. After 4-week treatment, enzyme linked immunosorbent assay was used to detect the expression levels of serum total cholesterol (TC) and triglyceride (TG). Hematoxylin-eosin and CD68 staining were used to observe the morphological properties of arterial plaques. RESULTS: Angptl4 expression was dependent on HIF-1 α, with a reduction in mRNA expression and no response in protein level to DMOG or DLT treatment in relation to siHIF-1 α -transfected cells. DLT inhibited HIF-1 α and Angptl4 mRNA expression (P<0.05 or P<0.01) and reduced HIF-1 α and Angptl4 protein expressions with DMOG in mature adipocytes (all P<0.01), as the effect on HIF-1 α protein also existed in the presence of siHIF-1 α (P<0.01). ApoE^-/- mice treated with CIH had increased TG and TC levels (all P<0.01) and atherosclerotic plaque. Angptl4 antibody and DLT both reduce TG and TC levels (all P<0.01), as well as reducing atherosclerotic plaque areas, narrowing arterial wall thickness and alleviating atherosclerotic lesion symptoms to some extent. CONCLUSION DLT had positive effects in improving dyslipidemia and arteriosclerosis by inhibiting Angptl4 protein level through HIF-1 α-Angptl4 mRNA signaling pathway.
Angiopoietin-Like Protein 4/genetics* ; Animals ; Apolipoproteins E ; Atherosclerosis/metabolism* ; Drugs, Chinese Herbal ; Dyslipidemias/genetics* ; Hypoxia/metabolism* ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Mice ; Plaque, Atherosclerotic ; Powders ; RNA, Messenger/genetics* ; Signal Transduction ; Triglycerides ; Water

Recent advances in hematopoietic stem cells (HSCs) expansion by growth factors including angiopoietin-like proteins (Angptls) have opened up the possibility to use HSCs in regenerative medicine. However, the unavailability of true in vitro HSCs expansion by these growth factors has limited the understanding of the cellular and molecular mechanism of HSCs expansion. Here, we report the functional role of mouse Angptls 1, 2, 3, 4, 6 and 7 and growth factors SCF, TPO, IGF-2 and FGF-1 on purified mouse bone-marrow (BM) Lineage(-)Sca-1(+)(Lin-Sca-1(+)) HSCs. The recombinant retroviral transduced-CHO-S cells that secrete Angptls in serum-free medium were used alone or in combination with growth factors (SCF, TPO, IGF-2 and FGF-1). None of the Angptls stimulated HSC proliferation, enhanced or inhibited HSCs colony formation, but they did support the survival of HSCs. By contrast, any of the six Angptls together with saturating levels of growth factors dramatically stimulated a 3- to 4.5-fold net expansion of HSCs compared to stimulation with a combination of those growth factors alone. These findings lead to an understanding of the basic function of Angptls on signaling pathways for the survival as well as expansion of HSCs in the bone marrow niche.
Angiopoietin-like 4 Protein ; Angiopoietin-like Proteins ; Angiopoietins ; genetics ; metabolism ; Animals ; Antigens, Ly ; metabolism ; Bone Marrow Cells ; cytology ; CHO Cells ; Cell Differentiation ; drug effects ; Cell Lineage ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Cricetinae ; Cricetulus ; Culture Media, Conditioned ; pharmacology ; Hematopoietic Stem Cells ; cytology ; metabolism ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Membrane Proteins ; metabolism ; Mice ; Transfection

OBJECTIVETo study in vitro and in vivo protein expression and biological function of gene pp1158, a hepatocellular carcinoma (HCC)-related gene.

METHODSpp1158 was expressed with fusion expression vector pET-32a in E. Coli-BL21 (DE3), and rabbit anti-pp1158 fusion protein polyclonal antibody was prepared. The biological function and differential expressions of pp1158 were studied by in vitro colony formation assay nude mouse in vivo tumor formation assay of transfected BEL7402 cell line and immunohistochemistry and Western blot. Differential expression of pp1158 in human fetal tissues were examined by Northern blot.

RESULTSIn vitro experiment showed that pp1158 inhibited colony formation rate of transfected BEL 7402 cells, with an inhibition rate of 58.3% (P < 0.01). Tumor formation assay indicated that tumor formation of pCMV-Script-1158 transfected BEL 7402 cell line was significantly inhibited (P < 0.05) as compared with that of the control group. Immunohistochemical assay showed that pp1158 was expressed in human tissue in the following sequence: normal liver >/= noncancerous liver tissue > HCC. Western blot indicated that a 60kD protein (pp1158 protein) was expressed in BEL 7402 cells transfected with pCMV-Script-pp1158 DNA, while it was detected in BEL 7402 cells transfected with pCMV-Script vector DNA. Northern blot showed pp1158 was expressed in the placenta at a very high level, heart, liver, muscle, pancreas and lung but expressed poorly in the brain and kidney.

CONCLUSIONpp1158 is a new candidate tumor suppressor gene of HCC.

Angiopoietin-like 4 Protein ; Angiopoietins ; Animals ; Blotting, Northern ; Blotting, Western ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Female ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; Humans ; Intercellular Signaling Peptides and Proteins ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Neoplasm Proteins ; genetics ; metabolism ; Neoplasm Transplantation ; Neoplasms, Experimental ; genetics ; metabolism ; pathology ; Proteins ; genetics ; RNA, Messenger ; genetics ; metabolism ; Transplantation, Heterologous ; Tumor Cells, Cultured

BACKGROUNDThis study was designed to obtain a recombinant retroviral vector containing the human hepatocellular carcinoma-related gene ANGPTL4 (angiopoietin-like 4) cDNA and to evaluate the anti-tumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene transfection.

METHODSANGPTL4 cDNA was cloned in vitro from normal human liver cells HL-7702 by using RT-PCR, and then subcloned into the plasmid vector pMSCV and sequenced. The retroviral plasmid vectors pMSCV-ANGPTL4, pVSV, and pGAG-POL were co-transfected into the packaging cell line 293 EBNA under mediation of lipofectamine. A high-titer retrovirus was obtained as a result, and HepG2 cells were infected with this retrovirus in vitro. Flow cytometry and fluorescence microscopy were used to detect expression of green fluorescence protein (GFP). The expression of ANGPTL4 mRNA in HepG2-ANGPTL4 cells was investigated using RT-PCR. The formation of tumors in nude mice and MTT assays were used to detect the growth of HepG2-ANGPTL4 cells in vivo and in vitro, respectively.

RESULTSThe cDNA sequence of the cloned ANGPTL4 gene was consistent with the recently reported sequence. Thus, the recombinant retroviral vector pMSCV-ANGPTL4 was constructed successfully. The titer of the packaged recombinant retrovirus was 1.4 x 10(6) infective viral grains/ml, and the rate of HepG2-ANGPTL4 cells expressing GFP was 68.45%, with an average intensity of fluorescence 31.67 times greater in HepG2-ANGPTL4 cells than in HepG2 cells. The expression of ANGPTL4 mRNA in HepG2-ANGPTL4 cells was higher than in HepG2-pMSCV cells (154% higher) or HepG2 cells (161% higher). The proliferation rate of HepG2-ANGPTL4 cells in vitro was obviously lower than those of HepG2-pMSCV cells and HepG2 cells (P <0.01). The mean volume and weight of tumors seeded from HepG2-ANGPTL4 cells were obviously lower than the mean volume or weight of tumors seeded from HepG2 cells and HepG2-pMSCV cells (P <0.01).

CONCLUSIONA stable ANGPTL4-transfected human liver cancer cell line HepG2-ANGPTL4 has been created. The transfer of the human ANGPTL4 gene mediated by a retroviral vector is a possibly effective approach for liver cancer therapy.

Angiopoietin-like 4 Protein ; Angiopoietins ; Animals ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; Liver Neoplasms ; therapy ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; NIH 3T3 Cells ; RNA, Messenger ; analysis ; Recombination, Genetic ; Retroviridae ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo study the effect of Astragali Radix on the denervated tibial muscle atrophy in rats, and discuss its mechanism.

METHODTotally 60 SPF-grade Sprague-Dawley rats were selected in the common peroneal nerve crush model, and then randomly divided into 6 groups: Astragali Radix high-dose, medium-dose, low-dose groups, the Mecobalamin group, the model group, and the sham operation group. They were administered with drugs after the operation. At 18 d, the pathological section staining and morphological analysis were performed. The wet-weight ratio and section area of tibial muscles were also measured. The real-time fluorescence quantification was adopted to detect the differential expression between Angptl4 and PI3K genes.

RESULT(1) Wet-weight ratio: The wet-weight ratio in Astragali Radix high-dose, medium-dose groups was much higher than that in the model group (P < 0.05 or P < 0.01). (2) Section area: The sham operation group was higher, with regular morphology; Whereas the model group showed significant decrease, with chaotic structure and obvious connective tissue proliferation; Astragali Radix groups and the mecobalamin group showed relatively small section areas, with chaotic structure and unobvious connective tissue proliferation. Compared with the model group, Astragali Radix groups showed significant increase (P < 0.01). (3) Motor end plate: The sham operation group was in uniform brownish black color and oval or round shape; Astragali Radix medium-dose and high-dose group and the mecobalamin group showed rough line edges; Astragali Radix medium-dose and low-dose groups and the model group showed decline in the number, with irregular morphology, rough line edges and a light color. (4) Angptl4 and PI3K: Compared with the model group, the Astragali Radix high-dose group showed significant increase (P < 0.05).

CONCLUSIONAstragali Radix has a significant effect in preventing and treating denervated tibial muscle atrophy. It may delay the muscle atrophy by increasing Angptl4 and PI3K gene expressions.

Angiopoietin-like 4 Protein ; Angiopoietins ; genetics ; Animals ; Astragalus Plant ; Astragalus membranaceus ; chemistry ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Gene Expression Regulation ; drug effects ; Male ; Muscular Atrophy ; drug therapy ; genetics ; pathology ; prevention & control ; Phosphatidylinositol 3-Kinases ; genetics ; Rats ; Rats, Sprague-Dawley ; Tibia