Objective To clone and express human laminin alpha4 chain LG1 module(hLN?4LG1)protein holding biological activity.Methods The cDNA encoding hLN?4LG1 was amplified by RT-PCR,then inserted into pMD-18T vector and sequenced.The hLN?4LG1 cDNA fragment was subcloned into pET-28a vector and expressed in BL21(DE3) strain.The hLN?4LG1 protein was detected by Western blotting and purified by Ni-NTA column.The biological activity of target protein was detected through cell adhesion and expansion test.Results The cDNA fragment of hLN?4LG1 was cloned successfully.While BL21(DE3)/pET-28a-LG1 bacterium was induced with IPTG,a new protein band with a relative molecular weight of 26 000 was shown.Purified hLN?4LG1 protein could promote the expansion and adhesion of A549 cells obviously,as compared with the control group.Conclusion The hLN?4LG1 was cloned and expressed successfully,on which the cells can adhere and expand.

Objective To express and detect the antigenicity of human laminin alpha4 LG3-4 module (hLN?4LG3-4) protein by gene engineering techniques. Methods The cDNA encoding hLN?4LG3-4 was amplified by RT-PCR from human placenta, then inserted into pMD-18T vector by T/A cloning and sequenced. Prokaryotic expression vector pET-28a-LG3-4 was constructed by recombinant DNA technique. The hLN?4LG3-4 fusion protein expressed in BL21(DE3)/pET system was identified by SDS-PAGE, purified by Ni-NTA resin, and assayed by Western blotting. Results The cDNA fragment of hLN?4LG3-4 was cloned successfully. While BL21 (DE3)/pET28a-LG3-4 bacterium was induced with IPTG, a new protein band with a relative molecular weight of 44 000 was shown on SDS-PAGE profile. hLN?4LG3-4 fusion protein of high purity (95%) was obtained and specific protein band was detected by Western blotting. Conclusion The hLN?4LG3-4 fusion protein was successfully expressed.

OBJECTIVE: To investigate the effect of sodium valproate (VPA) on activation of miR-34c-5p/ATG4B signaling pathway and autophagy in SH-SY5Y cells. METHODS: Routinely cultured SH-SY5Y cells were treated with VPA at different doses for 24 h, and the changes in the mRNA levels of ATG4B and miR-34c-5p and the protein expression of ATG4B were assessed using qRTPCR and immunoblotting, respectively. The effect of transfection with a plasmid containing ATG4B promoter on the promoter activity of ATG4B in VPA-treated SH-SY5Y cells was assessed using the reporter gene assay. The stability of ATG4B mRNA was analyzed with qPCR in SH-SY5Y cells treated with VPA alone or with VPA combined with the transcription inhibitor actinomycin D. The expression level of miR-34c-5p was detected using qPCR in SH-SY5Y cells treated with VPA alone or with VPA combined with miR-34c-5p mimics or antagonist, and the role of miR-34c-5p in VPA-induced ATG4B down-regulation was evaluated. The changes in the level of autophagy were evaluated by detecting LC3-Ⅱ expression in the cells after treatment with VPA or VPA combined with miR-34c-5p antagonist. RESULTS: VPA dose-dependently down-regulated the expression of ATG4B at both the mRNA and protein levels in SH-SY5Y cells. VPA treatment did not significantly affect the promoter activity of ATG4B, but obviously lowered the mRNA stability of ATG4B in SH-SY5Y cells. VPA treatment up-regulated the expression of miR-34c-5p, and the miR-34c-5p antagonist reversed VPA-induced down-regulation of ATG4B in SH-SY5Y cells. VPA also down-regulated the expression level of LC3-Ⅱ in SH-SY5Y cells. CONCLUSIONS VPA suppresses autophagy in SH-SY5Y cells possibly via activating miR-34c-5p/ATG4B signaling pathway.
Autophagy ; drug effects ; Autophagy-Related Proteins ; genetics ; metabolism ; Cell Line ; Cysteine Endopeptidases ; genetics ; metabolism ; Dactinomycin ; pharmacology ; Down-Regulation ; Genes, Reporter ; Humans ; MicroRNAs ; antagonists & inhibitors ; metabolism ; Microtubule-Associated Proteins ; metabolism ; RNA, Messenger ; metabolism ; Signal Transduction ; drug effects ; Transfection ; Valproic Acid ; administration & dosage ; antagonists & inhibitors ; pharmacology