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OBJECTIVE@#To construct a PA28γ overexpression cell line and determine its effects after infecting an oral squa-mous cell carcinoma (OSCC) cell line.@*METHODS@#The PA28γ gene was cloned into the pLOV.CMV.cherry.2A.EF1a.PuroR lentiviral vector by polymerase chain reaction (PCR), and PCR and DNA sequencing alignment analysis were used for identification. Then, 293T cells were used to package viral diseases. Infected OSCC cells were used to construct a cell line with stable PA28γ overexpression. Finally, the level of PA28γ expression in the OSCC cell line was detected through Western blot.@*RESULTS@#The successful construction of PA28γ recombinant lentiviral vectors was confirmed by DNA sequencing. The results of immunofluorescence showed that the PA28γ overexpression lentivirus successfully infected the OSCC cells and showed cherry red fluorescence. The results of Western blot demonstrated that the constructed cells with stable PA28γ overexpression significantly increased the expression of PA28γ.@*CONCLUSIONS@#The PA28γ overexpression lentiviral vector can significantly increase its protein expression in OSCC cells. We provide a stable OSCC cell line for further study on the effect of PA28γ in OSCC.
Subject(s)
Humans , Autoantigens , Carcinoma, Squamous Cell , Cell Line, Tumor , Genetic Vectors , Lentivirus , Mouth Neoplasms , Proteasome Endopeptidase Complex , TransfectionABSTRACT
To investigate the effect of miR-331-3p on the proliferation of porcine renal epithelial cells (PK15) and its mechanism, the pcDNA 3.1(+) overexpression vector of miRNA-331-3p (pcDNA 3.1(+)-miR-331-3p) was constructed. PK15 cells were divided into four groups, including experimental group, experimental control group, inhibitor group and inhibitor control group. Experimental group and experimental control group were transfected with pcDNA 3.1(+)-miR-331-3p and pcDNA 3.1(+), respectively. Inhibitor group and inhibitor control group were transfected with miR-331-3p inhibitor and miR-331-3p negative control (miR-331-3p NC), respectively. Above all, CCK-8 reagent was used to plot the cell proliferation curve and Propidium (PI) staining was used to detect the proportion of cell stages. Secondly, its expression change were detected by quantitative real-time PCR that included the growth inhibitory protein family member 5 (ING5), cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 3 (CDK3), cyclin-dependent kinase 4 (CDK4), Cyclin B and cyclin-dependent kinase inhibitor 1A (CDKN1A). The results showed that the expression of miRNA-331-3p was significantly increased in the experimental group. The cell proliferation curve showed that the number of cells in experimental group was significantly higher than that in experimental control group or inhibitor control group at 48 h and 72 h (P<0.05). Simultaneously, Inhibitor group was significantly lower than experimental control group or inhibitor control group in the number of cells at 48 h and 72 h (P<0.05), but there was no significant difference between the experimental group and the control group. Compared with the experimental control group, the proportion of cells of experimental group in G0/G1 phase decreased, the proportion of S phase and G2/M phase increased, and the inhibitor control group showed the opposite trend. Simultaneously, the expression levels of CDK2, CDK3, CDK4 and Cyclin B genes in the experimental group were significantly increased, while ING5 and CDKN1A genes inhibiting proliferation showed a significant downward trend. These results demonstrate that the miR-331-3p overexpression vector was successfully constructed, and miR-331-3p has the ability to promote the proliferation of PK15 cells. The study lays a solid foundation for further research for its role in pig growth and development.
Subject(s)
Animals , Cell Line , Cell Proliferation , Genetics , Epithelial Cells , Cell Biology , Genetic Vectors , MicroRNAs , Genetics , SwineABSTRACT
Objective Circular RNA is a research hotspot of non-coding RNAs. The purpose of this study was to investigate the basic characteristics and expression effect of the overexpression vectors of circular RNA hsa_circ_0082626 transcribed from antiviral gene ZC3HAV1. Methods The basic characteristics of hsa_circ_0082626 were studied by the verification of reverse cleavage site, RNase digestion assay and intracellular distribution location with extraction of cytoplasmic and nuclear RNA. In the RNase digestion assay, samples were divided into RNase R treatment group and control group. RNase. 20 U (2 U/μg) of RNase R was added to the R treatment group, and the control group was replaced with an equivalent amount of ddH2O to detect changes in expression levels after RNase treatment. The cells were divided into 2 groups: overexpression group and negative control group. At 24, 48 and 72 h after transfection, cells were collected to detect the expression of circular RNA by RT-qPCR. Results Compared with the control group, the expression of ZC3HAV1, CDR1as and GAPDH in the RNase R treatment group was increased (0.144±0.002 vs 1.000±0.016, 0.772±0.058 vs 1.000±0.122, 0.077±0.009 vs 1.000±0.164, P<0.05). Hsa_circ_0082626 could resist the treatment of RNase R and was mainly distributed in cytoplasm. The expression level of hsa_circ_0082626 in the overexpression group was significantly higher than that in the negative control group, and the expression level was the highest at 48 h after transfection. Conclusion The characteristics of hsa_circ_0082626 reverse cleavage site, RNase resistance and expression in cells were successfully analyzed, which proved that hsa_circ_0082626 does have a circular structure. The overexpression vector of hsa_circ_0082626 was successfully constructed to provide an experimental basis for the biological function and mechanism of RNA hsa_circ_0082626.
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Objective To investigate the effect of Smad3 on cell migration of A549 and HeLa cells.Methods Primers for pCMV-Myc-Smad3 plasmid construction and siRNA targeting Smad 3 were designed and synthesized .pCMV-Myc-Smad3 plasmid was constructed with molecular cloning techniques .Overexpression of Smad 3 with Myc-tag or silencing of endogenous Smad3, and then scratch assay was used to detect the migration ability of A 549 and HeLa cells in vitro. Results pCMV-Myc-Smad3 plasmid was successfully constructed .Overexpression of Smad 3 significantly up-regulated the migration rate of A549 and HeLa cells.Conversely, in the same cells, silencing of endogenous Smad3 or treatment with Smad3 inhibitor, SIS3, down-regulated the migration rate .Conclusions Smad3 promotes cell migration of A549 and HeLa cells.
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Objective: To separate the full length cDNA of gene encoding the key enzyme AK in aspartate metabolic pathways of Carthamus tinctorius and to construct plant overexpression vector. Methods: According to annotation on transcriptome library of C. tinctorius and core fragments and expression analysis data of CtAK identified by qRT-PCR, we separated the full length cDNA of AK gene of C. tinctorius (CtAK) using RACE technology and constructed plant expression vector using recombinant DNA technology. Results: Bioinformatics analysis showed the full length CtAK was 1 703 bp and ORF area was 1 626 bp, encoding 541 amino acid residues. The function structure domain analysis showed the gene might be a monofunction feedback inhibition sensitive AK1 from plant. We successfully constructed plant expression vector pCAMBIA3301-CTP-AK1 which contained 35 S promoter and Bar resistance genes and chloroplast transit peptide by recombinant DNA technology. Conclusion: The gene encoding CtAK1 is obtained and the plant overexpression vector is constructed, which lays the foundation for researching on biological function and mechanism of action in amino acid metabolism regulation of C. tinctorius.