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Objective To investigate the impact of miR-200c overexpression on colon cancer cell proliferation ability and the related mechanism.Methods MicroRNAs which may combined with the transcription factor AP-2α were screened and forecasted by the bioinformatics database,while its eukaryotic expression plasmids and specific inhibitor were synthesized.Plasmids PEZX-miR-200c,PEZX-NC,pmirGLO-AP-2α3'UTR,pmir-GLO and the specific inhibitors miR-67-inhibtor,miR-200c-inhibitor were transfected in vitro into colon cancer HCT-116 and SW480 cells and the HEK293T cell by Lipofectamine2000.The expression of AP-2α mRNA and protein in colon cancer cells was analyzed by qRT-PCR,Westem blot and immunocytochemical staining.CCK-8 assay and flow cytometry were adopted to observe the effect of miR-200c on colon cancer cells proliferation and apoptosis.Dual-Luciferase assay experiments were performed to observe the relative luciferase activity induced by miR-200c.Results The proliferation activity was significantly decreased in anti-miR-200c/SW480 group,while in PEZX-miR-200c/HCT-116 group,it was higher than that in PEZX-NC/HCT-116 group.The apoptosis ability was significantly increased in anti-miR-200c/SW480 group [(78±0.7) ％ vs (66±1.1) ％,P ＜ 0.05].The expression of AP-2o both in mRNA and protein levels was decreased in PEZX-miR-200c/HCT-116 group,while the protein level was increased in Anti-miR-200c/SW480 group.The relative luciferase activity inhibited by miR-200c was decreased in HEK-293T cells transfected with PEZX-miR-200c and pmir-GLO-AP-2α3' UTR (0.51±0.09 vs 0.98±0.04,P ＜ 0.01).Conclusion MicroRNA-200c could promote cell proliferation ability by targeting transcriptional factor AP-2α in human colorectal cancer cells.
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BACKGROUND:A group of nuclease-like proteins were previously purified from Eisenia foetida tissues, exploring primary structures of these proteins wil help to uncover basic structure characteristics of them and provide foundations for the study addressing the relationship of their structures and functions. OBJECTIVE:To explore primary structures of nuclease-like proteins EWD1 and EWD2. METHODS:Edman degradation method was used to sequence the N-terminal amino acids of EWD1 and EWD2, acid hydrolisis method was used to analyze amino acid compositions of EWD1 and EWD2, LC-MS/MS was used to analyze some peptide sequences within the proteins, and MALDI-TOF-MS was used to calculate the number of the disulfide bonds and the contents of polysaccharides. RESULTS AND CONCLUSION:Among the amino acid compositions in EWD1 and EWD2, the sum contents of aspartate and asparagines were the highest (al nearly 10%), the contents of hydrophobic amino acids were also high, and the contents of cysteine was low. The EWD1 and EWD2 had similar amino acid compositions with other nucleases. Edman degradation results showed that, the N-terminal sequences of the large subunit of EWD1 were in turn as fol ows:D, E, W, V, Y, P;the N-terminal sequences of EWD2 were as fol ows:L, L, G, P, Y, K, P, K, C. The results of LC-MS/MS indicated the two proteins were novel proteins;MALDI-TOF-MS results showed that 8 cysteine residues formed 4 disulfide bonds in EWD1, 6 cysteine residues formed 3 disulfide bonds in EWD2. EWD1 and EWD2 were al glycoproteins, the content of polysaccharides was 17.3%in EWD1 and 15.6%in EWD2.
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Objective To investigate the effect of AP-2α on the chemoresistance to oxaliplatin of colorectal carcinoma cell and its related mechanism.Methods Plasmid of GV102-AP-2α-RNAi (experimental group) and control plasmid GV102-NC (negative control group) were transfected into HCT-116 using Lipofectamine 2000 respectively.The AP-2α expression levels of mRNA and protein of experimental group,control group and HCT-116 blank group were detected by qRT-PCR and Western blot.Cell proliferation assay was performed using the CCK-8 and the apoptosis assays were preformed with Annexin V-PE Apoptosis Kit.Results The AP-2α expression levels of mRNA and protein both decreased after transfection of AP-2α-RNAi plasmid,moreover,the effect produced by subsequence 1 was the most significant.After treatment by oxaliplatin,AP-2α protein levels increased with time while mRNA did not change significantly.Western blot results suggested that the level of AP-2α protein in experimental group which was maintained in oxaliplatin was lower than the negative control group.CCK-8 results suggested that cell proliferation ability was significantly higher for the experimental group maintained in oxaliplatin [(88±3) ％] than the negative control group maintained in oxaliplatin [(57±3) ％] and the blank group maintained in oxaliplatin [(73t4) ％].Flow cytometry showed that the apoptosis rate of the experimental group maintained in oxaliplatin [(15.07±1.20) ％] was lower than the control group maintained in oxaliplatin [(24.93±0.90) ％] and the blank group maintained in oxaliplatin [(23.71±1.32) ％].Conclusion AP-2α may be related to the sensitivity of colon cancer cells to oxaliplatin.
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According to the problem existing in PBL teaching of biochemistry, we reformed teaching approach, adjusted curriculum content, reassembled teaching material system, emphasized clinical practice, strengthened the experiment technique, expanded open class, created essential condition, built independent learning atmosphere actively, which promoted teaching quality enormously and demonstrated great advantage of PBL teaching method. Meanwhile, it made the curriculum reform of biochemistry carried out deeply.
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Objective To study inhibitory effects of transcription factor activator protein-2α(AP-2α)on proliferation of colon cancer cells in vitro and its mechanism. Methods The peDNA3.1 (+)-AP-2α recombinant plasmid was constructed. Plasmid pcDNA3.1(+)- AP-2α and pcDNA3.1(+)was transfected into SW620 cell by liposome mediation for transient expression, and proliferative activities of SW620 cell were evaluated by MTT assay. The change in the mRNA and protein expression level of ER-β before and after transfection was detected using the methods of Real-Time PCR and Western blotting respectively. Results The mRNA and protein expressions of AP-2α could be enhanced by transfecting of AP-2α gene in SW620 cell. MTT assay indicated: the proliferation velocity of SW620 cell for transfection of the pcDNA3.1(+)-AP-2α plasmid was apparently inhibited. The expression of ER-β in SW620 cell increased significantly after AP-2α gene transfection. Compared with control group, the difference was significant (P＜0.05). Conclusion Overexpression of AP-2α inhibits the proliferation of SW620 cell in vitro, which is probably related with activation of ER-β.
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Objective To inhibit the expression of transcription factor special protein 1(Sp1) through RNA interference (RNAi) technique and to investigate its impact on the proliferation ability of colorectal cancer cell line SW620. Methods The recombinant plasmid of Sp1 RNAi (pGenesil-1-Sp1) was constructed and transfected into SW620 cells by Lipofectamine. The transfcction efficiency was observed under fluorescence confocal microscopy. Expression levels of Sp1 mRNA and protein from SW620 after transfection were examined by real time PCR and Western blot respectively, after transduction of the recombinant plasmid into the SW620. The proliferation ability of SW620 cell line was evaluated by MTT assay. Results The expression plasmid (pGenesil-1-Sp1) against Sp1 was successfully constructed, recombinant vectors could reduce the expressions of Sp1 mRNA and protein in SW620, the ratio of inhibition of the expression of Sp1 mRNA and protein was 68.47 % and 73.82 % in 48th hour respectively. Compared with the control group, the difference was significant (P <0.05). MTT showed that the proliferation ability of SW620 cell was degraded. Conclusion Silencing Sp1 gene by the RNAi technology can actively inhibit the proliferation of SW620 cell. The successful application of Spl SiRNA extends the list of available therapeutic modalitics in the treatment of human colon cancer.
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Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous group of tumors composed of large B cells. It is the most common type of non-Hodgkin lymphoma. As its variety of clinical, morphological and genetic characters, the classification and prognosis are still debated. This article aims to elucidate t(14;18) translocation and c-myc gene rearrangement is of great significance to the clinical diagnosis, classification, treatment and prognosis of DLBCL.
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Objective To evaluate the proliferation and invasion potential of human colon cancer SW480 cells transfected by AP-2α gene in vitro. Methods pcDNA3.1 (+)-AP-2α was created by cloning AP-2α eDNA into the EcoRI site of poDNA3.1 (+). LipofectamineTM 2000 Reagent was used to mediate the transfection of pcDNA3.1(+)-AP-2α and pcDNA3.1 (+), and normal SW480 celia were cultured as a negative control. The mRNA and protein level of AP-2α in the cells of each group were detected 48 h after being transfected with plasmids above by RT-PCR and Western blotting analysis. The cell proliferation and invasion potential were examined by colony formation assay and Transwell invasion assay respectively. Results Lack of AP-2α protein expression in SW480 cells was verified by western blot analysis. After being transfected with AP-2α gene, the mRNA amount and protein expression increased dramatically, while the colony formation efficiency decreased(P <0.05), the cell proliferation in soft agar was inhibited, and the ability of its invasion dropped off(P <0.05) in vitro. Conclusion AP-2α gene suppresses the proliferation and invasion potential of human colon cancer SW480 cells in vitro.
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Objective To investigate whether prostaglandin E2(PGE2) can promote the ability of adhesion.migration and invasion of colorectal cancer cells SW480 and its mechanism.Methods Extrinsic source PGE2 and the antagonist of EP1 SC19220 were added to the culture media. MTr assay was used to identify the adhesion ability of SW480 cells. Migration ability was tested by transwell plate.The invasion ability was tested bv ECM gel coated transweU plate. RT-PCR and western blotting were used to detect the mRNA and protein level of vascular endothelial growth factor (VEGF). Results The adhesion.invasion and migration ratio of SW480 ceils were all increased significantly after treated with PGEh the A values of adhesion ceils increased from 0.207±0.009 to 0.417±0.088. Migration cells increased from 6.33±0.33 to 43.33±0.88.invasion cells increased from 3.67±0.34 to 26.33±0.89(P＜0.05).The adhesion.migration and invasion cells of PGE2+SC 19220 group decreased significantly compared to PGE2 group.The A values of adhesion cells decreased from 0.417±0.088 to 0.140±0.006. Migration cells decreased from 43.33±0.88 to 28.00±0.58.invasion cells decreased from 26.33±0.89 to 5.67±0.33 (P＜0.05).The results of RT-PCR and western blotting showed that the expression of VEGF mRNA and protein increased in a dose dependent manner after PGE2 treatment. Conclusion The ability of adhesion. Migration and invasion of SW480increased after PGE2 was added to the culture media.It may be related to the upregulation of VEGF.
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Humanin (HN, its analogue [Gly14]-Humanin, HNG) was originally identified as an endogenous peptide that protects neuronal cells from apoptosis induced by various types of Alzheimer's disease-related insults. But the relative low content of this peptide in its natural sources limits its further characterization. An expression vector pET32a/HNG was corstructed and transformed it into E. coli BL21 trxB (DE3). HNG was expressed as a fusion protein in the soluble fraction and was purified by nickel affinity chromatography. Subsequently, the purified fusion protein was cleaved by enterokinase and was further purified by reverse-phase HPLC. A 23 mg recombinant HNG (rHNG) from 1 L bacterial culture was purified. The molecular weight of rHNG determined by ESI-MS was 2876.5 Da which was the expected size for correctly processed peptide. The N-terminal amino acid sequence of rHNG determined by Edman degradation method is identical to the theoretical sequence. Neuroprotective bioassay studies of rHNG exhibited its potential neuroprotective effect comparable to that of the natural HNG peptide.
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Objective To investigate and analyse the effect of PBL teaching method on the clincal medical students of different level,furthermore,to provide an effective guide for biochemistry teaching method reform.Methods Comparing the results of traditional teaching method and PBL teaching method in biochemistry on the clinical medical students of different level,and analyzing the applicable of PBL to various students.Results The seven-year students had a significant higher score in sum record by using PBL teaching method(P
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To adapt the requirements of cultivating high-quality medical talents,we implemented autonomous management model on selecting courses,research subjects,classic discussion,experiments and practice. This reformation is effective. One step further,we illustrated its significance from medical view: doctors,patients and diseases. Meanwhile,it was pointed out that the autonomous management is an inevitable trend for management development in the future.
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AIM: To study the effects of [Gly~(14)]-humanin,one of the strongest derivate of humanin,on proliferation and differentiation of neural stem cells(NSCs) and the protective action of cell death or apoptosis induced by ?-amyloid protein 1-42.METHODS: NSCs were treated with different concentrations of [Gly~(14)]-humanin and ?-amyloid protein 1-42.RESULTS: 10 nmol/L [Gly~(14)]-humanin made NSCs resistant to the apoptotic action induced by A?P1-42 and prevented NSCs from death induced by 25 ?mol/L ?-amyloid protein 1-42.The differentiated neural stem cells yield more neuronal cells than that in control groups when 10 nmol/L [Gly~(14)]-humanin was added to the culture media.The number of cells increased and the cultures grown with a manner of floating cell clones likes that cultured in the presence of mitogen when 100 nmol/L [Gly~(14)]-humanin was added to the differentiation culture media.CONCLUSION: The [Gly~(14)]-humanin significantly promoted the proliferation and neuronal differentiation of neural stem/progenitor cells and also inhibited the toxic action of ?-amyloid protein 1-42 on cultured neural stem/progenitor cells.