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Objective: To explore the effect of C7orf42 on cell proliferation of rat hepatocyte line BRL-3A in vitro. Methods: The expression of C7orf42 was knocked down by siRNA, and MTT and EdU assay were used to discover the effect of C7orf42 on cell proliferation at 48 hours after transfection. Flow cytometry was used to observe the effect of cell cycle progression. Real-tme PCR and Western blotting were used to detect the changes in the expression of cell proliferation-associated gene. Results: MTT results showed that the cell viability of the interference group (C7BRL-siR3) was significantly lower than that of the negative control group (NC) at 48 hours after transfection (P < 0. 05). Meanwhile, the percentage of EdU-labeling cells was also significantly decreased (P < 0. 05). At the same time, the flow cytometry results showed that the number of cells in division phase (S + G2/M) of the interference group was significantly reduced in parallel (P < 0. 05). Further, the interference group down-regulated the expression levels of cell proliferation-related genes and proteins of JUN, MYC, CCND1 and CCNA2. Conclusion: C7orf42 may promote cell proliferation via regulating the expression of JUN, MYC, CCND1 and CCNA2 in rat hepatocyte line BRL-3A.
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Objective To investigate the expression and possible mechanism of miR-21 and Ski-related novel protein N( SnoN) in the renal fibrosis diabetic process.Methods The animal model was established by tail-vein injection of Streptozotocin,and the other group were normal control ( NC) group.After 10 weeks, the rats were sacrificed to measure biochemical parameters and renal index , and to observe the changes of pathomorphology by HE staining as well.Meanwhile, immunohistochemistry and Western blot were employed to examine protein ex-pression of E-cadherin,α-smooth muscle actin(α-SMA), fibronectin(FN), collagen-Ⅰ(Col-Ⅰ), collagen-Ⅲ(Col-Ⅲ), transforming growth factor-β1(TGF-β1), Smad3, p-Smad3(Ser423/425) and SnoN in the renal tissue. In addition, the expression of SonN mRNA and miR-21 were detected by qPCR.Results In DM group,the ex-pressions of Col-Ⅰ, Col-Ⅲ and FN in renal interstitium were increased ( P <0.05 ) , TGF-β1 increased (P<0.05),while E-cadherin decreased(P<0.05).Compared with NC group, the expression of α-SMA,p-Smad3 (Ser423/425) protein increased in DM group(P<0.05),while the protein level of SnoN decreased but the level of SnoN mRNA increased ( P <0.05 ) .Moreover, the level of miR-21 markedly increased in DM group ( P <0.05 ) .Conclusions TGF-β1 may up-regulate the expression of miR-21 but restrain the translational expression of SnoN, aggravating fibrosis.
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Objective Identify novel protein kinase Cε(nPKCε)-interacted proteins in the cortex of hypoxic preconditioned mice.Methods Immunoprecipitation (IP) and two-dimensional electrophoresis (2-DE) combining with ImageMaster 2D Platinum software were applied to analyze the differential expressions of nPKCe-interacted proteins;the target protein spots were identified by matrix-associated laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and Western blot.Results Compared with control group,there were 34 upregulated protein spots and 20 downregulated protein spots in cytosolic fraction,while 27 upregulated prtein spots and 28 downregulated protein spots were determined in particulate fraction of cerebral cortex of HPC mice.The levels of nPKCε-interacted HSP 70 and 14-3-3γ/protein expressions increased significantly in both cytosolic and particulate fractions;but the protein level of nPKCε-interacted HSP60 increased only in particulate fraction of cerebral cortex of HPC mice.Conclusion nPKCε might be involved in the development of cerebral HPC via the regulation of its interacted proteins such as HSP60,HSP70 and 14-3-3γ.
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The combination of tandem spectrometry and database searching is one of the most popular technologies for protein identification.However,only those proteins in the searching database could be identified,and current database is far from completeness.So it is necessary to mining the MS/MS data comprehensively,in which novel protein identification is the most important one.The definition of novel protein could be divided into three levels according to their annotations of sequences and functions.As a part of protein identification,the main approaches used to identify novel protein are basing on the following two different ways:de novo sequencing combined with similarity search and searching against nucleotide acid databases such as EST or genome databases.Several mature or newly developed methods and techniques were summarized,and the problems and strategies discussed here would be helpful for the related researches.