1.Overexpression of microRNA-32 weakens the inhibition of polychlorinated biphenyls to the differentiation of P19 cells into cardiomyocytes
Songyu ZHANG ; Yan LI ; Jiangbo LIU ; Gang LI
Chinese Journal of Tissue Engineering Research 2016;20(23):3464-3469
BACKGROUND:Polychlorinated biphenyls inhibit the differentiation of P19 cels into cardiomyocytes. In the meanwhile, microRNAs play an important role in regulating cel differentiation.
OBJECTIVE:To explore the effect of microRNA-32(miR-32) on the polychlorinated biphenyls-inhibited differentiation of P19 cels into cardiomyocytes.
METHODS:P19 cels were co-cultured with 2.5 μmol/L polychlorinated biphenyls and 1% dimethyl sulfoxide. Afterwards, α-actinin, desmin, cTnI and GATA4 were identified by western blot assay.Wddwxrof polychlorinated biphenyls on the expression of miR-32 was evaluated by real-time PCR. Mouse eukaryotic vector expressing miR-32 was constructed by gene recombination technology,andtransfected into P19 celsfolowed byco-culturedwith2.5 μmol/Lpolychlorinated biphenylsand1% dimethyl sulfoxide. Then, expressions of differentiation-related proteins were detected by western blot assay.
RESULTS AND CONCLUSION:Polychlorinated biphenylsinhibited the differentiation of P19 cels into cardio myocytes anddecreased the miR-32 expression. Cel lines overexpressing miR-32 was successfuly established in mice.Furthermore, we found thatoverexpression of miR-32 weakens the inhibition of polychlorinated biphenyls to the differentiation of P19 cels into cardiomyocytes.
2.Functional role of protein kinase D1 in Aspergillus fumigatus-induced activation of nuclear factor-κB signal pathway and transcription.
Xiaolu NIU ; Wanfu XU ; Songyu LI ; Zhiyong KE ; Fanyyin ZENG ; Shenqiu LUO ; Fan DENG
Journal of Southern Medical University 2013;33(3):351-355
OBJECTIVETo explore the functional role of protein kinase D1 (PKD1) in the activation of nuclear factor-κB (NF-κB) signal pathway and NF-κB transcription mediated by Aspergillus fumigatus.
METHODSA549 cells and HEK293 cells were transfected with green fluorescence protein (GFP) or GFP-PKD1 followed by treatment with 1×10(5) CFU/ml Aspergillus fumigatus conidia for different time lengths. The phosphorylation levels of PKD1, IκB and p65 (pS276) in the transfected cells were measured by Western blotting. A549 cells were transfected with GFP-PKD1 or siRNA-PKD1, and the phosphorylation of IκB and p65 (pS276) was examined. Finally, NF-κB-luc and renilla luciferase reporter pRL-SV40 were cotransfected into GFP- or GFP-PKD1-transfected A549 cells before exposure of the cells to Aspergillus fumigatus conidia for 24 h, and NF-κB transcriptional activity in the cells was determined using dual-luciferase reporter assay.
RESULTSOverexpression of PKD1 significantly increased Aspergillus fumigatus conidia-stimulated phosphorylation of PKD1, IκB and p65 (pS276), whereas PKD1 knockdown by siRNA-PKD1 suppressed IκB and p65 (pS276) phosphorylation. Dual luciferase assay demonstrated that PKD1 overexpression markedly enhanced Aspergillus fumigatus-induced NF-κB transcription in A549 cells.
CONCLUSIONPKD1 may contribute to the activation of NF-κB signal pathway and NF-κB transcription induced by Aspergillus fumigatus.
Aspergillus fumigatus ; Cell Line, Tumor ; HEK293 Cells ; Humans ; I-kappa B Kinase ; metabolism ; NF-kappa B ; metabolism ; Phosphorylation ; Protein Kinase C ; metabolism ; Signal Transduction ; Transcription Factor RelA ; metabolism ; Transcription, Genetic ; Transfection
3.AXIN1-related CSRNP1 mRNA expression and its transcriptional regulation in TGF-β1-induced tumor cells.
Fan DENG ; Songyu LI ; Wanfu XU ; Zhipeng ZOU ; Zhiyong KE ; Fangyin ZENG
Journal of Southern Medical University 2013;33(8):1122-1126
OBJECTIVETo investigate AXIN1-related CSRNP1 gene expression and the mechanism of its transcriptional regulation in TGF-β1-induced tumor cells.
METHODSHuman lung carcinoma A549 cells or human prostate cancer PC3 cells were treated with TGF-β1 at different doses (0, 20, 40, and 80 ng/ml) or at 20 ng/ml for 0, 8, 12, or 24 h, and the dose and time effect of TGF-β1 on CSRNP1 mRNA expression in the tumor cells were evaluated with real-time RT-PCR. A549 cells were also treated with TGF-β1 and cycloheximide to clarify whether CSRNP1 expression induced by TGF-β1 required de novo protein synthesis. A549 cells transfected with pcDNA3.1, flag-SMAD3, or flag-SMAD3-mu, after serum starvation, were treated with or without TGF-β1 (20 ng/mL) for 24 h, and the overexpression of wild-type SMAD3 and dominant negative SMAD3-mu mutant were confirmed by Western blotting. The effect of SMAD3 or SMAD3-mu overexpression on CSRNP1 mRNA expression was also measured by real-time RT-PCR.
RESULTSIn both A549 and PC3 cells, TGF-β1 dose- and time-dependently stimulated CSRNP1 expression, which required de novo protein synthesis in A549 cells. Overexpression of wild-type SMAD3 significantly increased the expression of CSRNP1 mRNA induced by TGF-β1, while overexpression of dominant negative SMAD3 mutant remarkably reduced CSRNP1 mRNA expression in response to TGF-β1 in A549 cells.
CONCLUSIONTGF-β1 may contribute to CSRNP1 expression through SMAD3 activation and downstream signaling in tumor cells.
Apoptosis Regulatory Proteins ; genetics ; metabolism ; Axin Protein ; genetics ; metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; RNA, Messenger ; genetics ; Signal Transduction ; Smad3 Protein ; genetics ; metabolism ; Transfection ; Transforming Growth Factor beta1 ; pharmacology
4.Effect of Three Chinese Herbs Processed with Different Proportions of Glycyrrhizae Radix et Rhizoma on Pharmacokinetics of Dapsone in Rats
Zai-xing CHENG ; Zhen-zhen CAI ; Li-hong LIN ; Bao-yu ZHENG ; Hong CHEN
Chinese Journal of Experimental Traditional Medical Formulae 2020;26(8):148-155
Objective::To investigate the processing purpose of Morindae Officinalis Radix (MO), Euodiae Fructus (EF) and Polygalae Radix (PR) processed by Glycyrrhizae Radix et Rhizoma (Gly). Method::The content of dapsone in rat plasma was determined by high performance liquid chromatography (HPLC), the mobile phase was acetonitrile (A)-water (B) for gradient elution (0-5 min, 10%-25%A; 5-20 min, 25%A) and detection wavelength was set at 292 nm. PK Solution 2.0 software was used to simulate pharmacokinetic parameters. Result::Within 300 min after dapsone was administrated, compared with the control (CTL) group, the elimination of dapsone was slowed down and its plasma concentration was increased in the unprocessed product of MO (UMO) group. The elimination of dapsone was accelerated and its peak concentration (