Bile acids regulate anorexigenic neuropeptide through p-STAT3-SOCS3 signaling in mouse hypothalamic cells / 南方医科大学学报

OBJECTIVE@#To explore the effects of taurolithocholic acid (tLCA) and chenodeoxycholic acid (CDCA) on the expression of aorexigenic neuropeptide in mouse hypothalamus GT1-7 cells.@*METHODS@#Mouse hypothalamic GT1-7 cells were treated with culture medium containing 10% FBS (control group, =3) or with 10 nmol/L, 100 nmol/L, 1 μmol/L and 10 μmol/L tLCA (tLCA group, =3) or CDCA (CDCA group, =3) for 12, 24 or 48 h. Real-time PCR was performed to determine the expression levels of proopiomelanocortin (POMC) mRNA in the cells, and the production levels of α-melanocyte-stimulating hormone (α-MSH) were assessed using an ELISA kit. Signal transduction and activator of transcription 3 phosphorylation (p-STAT3), threonine kinase phosphorylation (p-AKT), suppressor of cytokine signaling 3 (SOCS3), G protein-coupled bile acid receptor-1 (TGR5) and farnesoid X receptor (FXR) protein were detected by Western blotting.@*RESULTS@#Western blotting results showed that mouse hypothalamic GT1-7 cells expressed two bile acid receptors, TGR5 and FXR, whose expressions were regulated by bile acids. Real-time PCR showed that the expression of POMC mRNA was significantly increased in the cells after treatment with 10 μmol/L tLCA or CDCA for 24 h. POMC-derived anorexigenic peptide α-MSH increased significantly in GT1-7 cells after treatment with 10 μmol/L tLCA or CDCA for 24 h. Treatment of the cells with tLCA or CDCA significantly increased the expressions of intracellular signaling proteins including p-STAT3, p-AKT and SOCS3.@*CONCLUSIONS@#Mouse hypothalamic GT1-7 cells express bile acid receptors TGR5 and FXR. Bile acids tLCA or CDCA can promote the expression of POMC mRNA and increase the production of the anorexigenic peptide α-MSH. The intracellular signaling proteins p-AKT, p-STAT3 and SOCS3 are likely involved in bile acid-induced anorexigenic peptide production.

Construction of MicroRNA-29b Overexpression Lentiviral Vector and Study on Its Biological Characteristics / 现代生物医学进展

Objective:To construct a lentiviral vector overexpression of micrRNA-29b and investigate the biological characteristics in mouse neuronal cell lines GT1-7.Methods:We chemically synthesized two oligonucleotide single-stranded,complete the comple-mentary by bridging extension into DNA double-stranded to form miR-29b precursor structure.The restriction enzyme digested vector plasmid FUGW was ligated to the precursor structure ofmiR-29b by homologous recombination to construct the corresponding lentiviral vector of microRNA-29b overexpression,and the stable cells were obtained in the mouse neuronal cell line GT1-7 by bleomycin drag screening.RT-PCR was used to detect the expression level of related genes at mRNA transcription level,Results:The recombinant lentiviral expression plasmid f-F-miR-29b was successfully constructed,and the expression level was about 30 times higher than that of the control group.The expressions of DCX,Vdac1 and pten were inhibited,have no changes in sex developmental related genes LH-β,kiss-l,Inshulin,IGF-I,GPR54,GnRH and leptin-R.Conclusion:Using the method of lentivirus screening,the microRNA-29b overexpressing stably transformed cells was successfully obtained in mouse neuron GT1-7 cells,which laid a foundation for the study of biological characteristics ofmicroRNA-29b.