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1.
Int. j. morphol ; 40(3): 735-741, jun. 2022. ilus, tab
Article in English | LILACS-Express | LILACS | ID: biblio-1385656

ABSTRACT

SUMMARY: This study is to investigate the regulation of Notch1 and Foxp1 by miR-34a in the development of psoriasis vulgaris. RT-PCR was used to compare the levels of miR-34a in the skin lesions of 20 patients with psoriasis vulgaris and 20 normal skin tissues. Immunohistochemistry was used to detect the expression of Notch1 and Foxp1 in 51 patients with psoriasis vulgaris, which were further compared with that in 29 normal control tissues. In addition, in HaCaT cells, we used miR-34a mimics and inhibitors to overexpress and inhibit miR-34a, respectively, and detected the mRNA and protein levels of miR-34a, Notch1, and Foxp1. The level of miR-34a in the skin lesions of patients with psoriasis vulgaris was significantly higher than that in normal skin tissues (t=2.192, P<0.05). The positive rate of Notch1 in the skin lesions of patients with psoriasis vulgaris was 76.47 %, which was significantly higher than that in normal skin tissues (13.79 %) (t=29.215, P<0.01). The positive rate of FOXP1 in the psoriasis vulgaris group was 92.16 %, which was also significantly higher than that in the normal skin group (65.52 %) (t=9.087, P<0.01). In addition, overexpression of miR-34a significantly promoted the expression of Notch1 and Foxp1. However, inhibition of miR-34a significantly reduced Notch1 and Foxp1 levels. miR- 34a is highly expressed in the skin tissues of patients with psoriasis vulgaris, and may participate in the development of psoriasis vulgaris by regulating Notch1 and Foxp1.


RESUMEN: El objetivo de este estudio fue investigar la regulación de Notch1 y Foxp1 por miR-34a en el desarrollo de la psoriasis vulgar. Se utilizó RT-PCR con el fin de comparar los niveles de miR-34a en las lesiones cutáneas de 20 pacientes con psoriasis vulgar y 20 tejidos de piel normales. Se utilizó inmunohistoquímica para detectar la expresión de Notch1 y Foxp1 en 51 pacientes con psoriasis vulgar, que se compararon además con la de 29 tejidos normales control. Además, en las células HaCaT, usamos miméticos e inhibidores de miR-34a para sobreexpresar e inhibir miR-34a, respectivamente, y detectamos los niveles de ARNm y proteína de miR-34a, Notch1 y Foxp1. El nivel de miR- 34a en las lesiones cutáneas de pacientes con psoriasis vulgar fue significativamente mayor que en los tejidos normales de la piel (t=2,192, P<0,05). La tasa de positividad de Notch1 en las lesiones cutáneas de pacientes con psoriasis vulgar fue del 76,47 %, que fue significativamente mayor que la de los tejidos normales de la piel (13,79 %) (t=29,215, P<0,01). La tasa positiva de FOXP1 en el grupo de psoriasis vulgar fue del 92,16 %, que también fue significativamente mayor que la del grupo de piel normal (65,52 %) (t=9,087, P<0,01). Además, la sobreexpresión de miR-34a promovió significativamente la expresión de Notch1 y Foxp1. Sin embargo, la inhibición de miR-34a redujo de manera importante los niveles de Notch1 y Foxp1. miR-34a se expresa en gran medida en los tejidos de la piel en pacientes con psoriasis vulgar y puede participar en el desarrollo de la psoriasis vulgar mediante la regulación de Notch1 y Foxp1.


Subject(s)
Humans , Psoriasis/genetics , MicroRNAs/genetics , Forkhead Transcription Factors/genetics , Receptor, Notch1/genetics , Psoriasis/metabolism , Immunohistochemistry , Transfection , Blotting, Western , Reverse Transcriptase Polymerase Chain Reaction , MicroRNAs/metabolism , Forkhead Transcription Factors/metabolism , Receptor, Notch1/metabolism
2.
Article in Chinese | WPRIM | ID: wpr-941034

ABSTRACT

OBJECTIVE@#To construct an adenovirus vector expressing artificial splicing factor capable of regulating alternative splicing of Yap1 in cardiomyocytes.@*METHODS@#The splicing factors with different sequences were constructed against Exon6 of YAP1 based on the sequence specificity of Pumilio1. The PCR fragment of the artificially synthesized PUF-SR or wild-type PUFSR was cloned into pAd-Track plasmid, and the recombinant plasmids were transformed into E. coli DH5α for plasmid amplification. The amplified plasmids were digested with Pac I and transfected into 293A cells for packaging to obtain the adenovirus vectors. Cultured neonatal rat cardiomyocytes were transfected with the adenoviral vectors, and alternative splicing of YAP1 was detected using quantitative and semi-quantitative PCR; Western blotting was performed to detect the signal of the fusion protein Flag.@*RESULTS@#The transfection efficiency of the adenovirus vectors was close to 100% in rat cardiomyocytes, and no fluorescent protein was detected in the cells with plasmid transfection. The results of Western blotting showed that both the negative control and Flag-SR-NLS-PUF targeting the YAPExon6XULIE sequence were capable of detecting the expression of the protein fused to Flag. The results of reverse transcription-PCR and PCR demonstrated that the artificial splicing factor constructed based on the 4th target sequence of YAP1 effectively regulated the splicing of YAP1 Exon6 in the cardiomyocytes (P < 0.05).@*CONCLUSION@#We successfully constructed adenovirus vectors capable of regulating YAP1 alternative splicing rat cardiomyocytes.


Subject(s)
Adenoviridae/metabolism , Alternative Splicing , Animals , Animals, Newborn , Escherichia coli/metabolism , Genetic Vectors , Myocytes, Cardiac/metabolism , Plasmids , RNA Splicing Factors/metabolism , Rats , Transfection
3.
Chinese Journal of Burns ; (6): 119-129, 2022.
Article in Chinese | WPRIM | ID: wpr-935986

ABSTRACT

Objective: To explore the effects of P311 on the angiogenesis ability of human microvascular endothelial cell 1 (HMEC-1) in vitro and the potential molecular mechanism. Methods: The experimental research method was used. HMEC-1 was collected and divided into P311 adenovirus group and empty adenovirus group according to the random number table (the same grouping method below), which were transfected correspondingly for 48 h. The cell proliferation activity was detected using the cell counting kit 8 on 1, 3, and 5 days of culture. The residual scratch area of cells at post scratch hour 6 and 11 was detected by scratch test, and the percentage of the residual scratch area was calculated. The blood vessel formation of cells at 8 h of culture was observed by angiogenesis experiment in vitro, and the number of nodes and total length of the tubular structure were measured. The protein expressions of vascular endothelial growth factor receptor 2 (VEGFR2), phosphorylated VEGFR2 (p-VEGFR2), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated ERK1/2 (p-ERK1/2) in cells were detected by Western blotting. HMEC-1 was collected and divided into P311 adenovirus+small interfering RNA (siRNA) negative control group, empty adenovirus+siRNA negative control group, P311 adenovirus+siRNA-VEGFR2 group, and empty adenovirus+siRNA-VEGFG2 group, which were treated correspondingly. The protein expressions of VEGFR2, p-VEGFR2, ERK1/2, and p-ERK1/2 in cells were detected by Western blotting at 24 h of transfection. The blood vessel formation of cells at 24 h of transfection was observed by angiogenesis experiment in vitro, and the number of nodes and total length of the tubular structure were measured. HMEC-1 was collected and divided into P311 adenovirus+dimethylsulfoxide (DMSO) group, empty adenovirus+DMSO group, P311 adenovirus+ERK1/2 inhibitor group, and empty adenovirus+ERK1/2 inhibitor group, which were treated correspondingly. The protein expressions of ERK1/2 and p-ERK1/2 in cells were detected by Western blotting at 2 h of treatment. The blood vessel formation of cells at 2 h of treatment was observed by angiogenesis experiment in vitro, and the number of nodes and total length of the tubular structure were measured. The sample number at each time point in each group was 6. Data were statistically analyzed with independent sample t test, analysis of variance for repeated measurement, one-way analysis of variance, and least significant difference test. Results: Compared with that of empty adenovirus group, the proliferation activity of cells in P311 adenovirus group did not show significant difference on 1, 3, and 5 days of culture (with t values of -0.23, -1.30, and -1.52, respectively, P>0.05). The residual scratch area percentages of cells in P311 adenovirus group were significantly reduced at post scratch hour 6 and 11 compared with those of empty adenovirus group (with t values of -2.47 and -2.62, respectively, P<0.05). At 8 h of culture, compared with those of empty adenovirus group, the number of nodes and total length of the tubular structure of cells in P311 adenovirus group were significantly increased (with t values of 4.49 and 4.78, respectively, P<0.01). At 48 h of transfection, compared with those of empty adenovirus group, the protein expressions of VEGFR2 and ERK1/2 of cells in P311 adenovirus group showed no obvious changes (P>0.05), and the protein expressions of p-VEGFR2 and p-ERK1/2 of cells in P311 adenovirus group were significantly increased (with t values of 17.27 and 16.08, P<0.01). At 24 h of transfection, the protein expressions of p-VEGFR2 and p-ERK1/2 of cells in P311 adenovirus+siRNA negative control group were significantly higher than those in empty adenovirus+siRNA negative control group (P<0.01). The protein expressions of VEGFR2, p-VEGFR2, and p-ERK1/2 of cells in P311 adenovirus+siRNA negative control group were significantly higher than those in P311 adenovirus+siRNA-VEGFR2 group (P<0.01). The protein expressions of VEGFR2 and p-ERK1/2 of cells in empty adenovirus+siRNA negative control group were significantly higher than those in empty adenovirus+siRNA-VEGFR2 group (P<0.05 or P<0.01). At 24 h of transfection, the number of nodes of the tubular structure in cells of P311 adenovirus+siRNA negative control group was 720±62, which was significantly more than 428±38 in empty adenovirus+siRNA negative control group and 364±57 in P311 adenovirus+siRNA-VEGFR2 group (with P values both <0.01). The total length of the tubular structure of cells in P311 adenovirus+siRNA negative control group was (21 241±1 139) μm, which was significantly longer than (17 005±1 156) μm in empty adenovirus+siRNA negative control group and (13 494±2 465) μm in P311 adenovirus+siRNA-VEGFR2 group (with P values both <0.01). The number of nodes of the tubular structure in cells of empty adenovirus+siRNA negative control group was significantly more than 310±75 in empty adenovirus+siRNA-VEGFR2 group (P<0.01), and the total length of the tubular structure of cells in empty adenovirus+siRNA negative control group was significantly longer than (11 600±2 776) μm in empty adenovirus+siRNA-VEGFR2 group (P<0.01). At 2 h of treatment, the protein expression of p-ERK1/2 of cells in P311 adenovirus+DMSO group was significantly higher than that in empty adenovirus+DMSO group and P311 adenovirus+ERK1/2 inhibitor group (with P values both <0.01), and the protein expression of p-ERK1/2 of cells in empty adenovirus+DMSO group was significantly higher than that in empty adenovirus+ERK1/2 inhibitor group (P<0.05). At 2 h of treatment, the number of nodes of the tubular structure in cells of P311 adenovirus+DMSO group was 726±72, which was significantly more than 421±39 in empty adenovirus+DMSO group and 365±41 in P311 adenovirus+ERK1/2 inhibitor group (with P values both <0.01). The total length of the tubular structure of cells in P311 adenovirus+DMSO group was (20 318±1 433) μm, which was significantly longer than (16 846±1 464) μm in empty adenovirus+DMSO group and (15 114±1 950) μm in P311 adenovirus+ERK1/2 inhibitor group (with P values both <0.01). The number of nodes of the tubular structure in cells of empty adenovirus+DMSO group was significantly more than 317±67 in empty adenovirus+ERK1/2 inhibitor group (P<0.01), and the total length of the tubular structure of cells in empty adenovirus+DMSO group was significantly longer than (13 188±2 306) μm in empty adenovirus+ERK1/2 inhibitor group (P<0.01). Conclusions: P311 can enhance the angiogenesis ability of HMEC-1 by activating the VEGFR2/ERK1/2 signaling pathway.


Subject(s)
Adenoviridae/genetics , Cell Line , Endothelial Cells , Endothelium, Vascular , Humans , Neovascularization, Physiologic , Nerve Tissue Proteins , Oncogene Proteins , Signal Transduction , Transfection , Vascular Endothelial Growth Factor A
4.
Article in Chinese | WPRIM | ID: wpr-935804

ABSTRACT

Objective: To construct a recombinant lentiviral vector for mouse miR-204 overexpression, and to verify the targeted regulation of miR-204 and DVL3 in silica (SiO(2)) -induced mouse lung epithelial cells (MLE-12 cells) . Methods: In October 2019, the pre-miR-204 gene was amplified from the mouse genome by the polymerase chain reaction (PCR) method. After sequencing, the amplified product was cloned into the pLenti-CMV-EGFP lentiviral vector. The positive clones were identified by PCR screening and sequencing. The miR-204 overexpressed lentiviral vector was transfected into 293T cells, and lentiviral packaging and titer determination were performed. The experiment was divided into SiO(2) control group, virus control group, and miR-204 virus group, and the expressions of miR-204 and DVL3 gene were detected by real-time PCR. Results: The miR-204 lentiviral expression vector Lv-miR-204-5p was constructed and identified correctly by PCR and sequencing, and a virus dilution with a titer of 9.57×10(8) IU/ml was obtained. The results of real-time PCR showed that the expression of miR-204 in MLE-12 cells of the miR-204 virus group was higher than that of SiO(2) control group and virus control group, and the expression of DVL3 gene was lower than that of SiO(2) control group and virus control group, the differences were statistically significant (P<0.05) . Conclusion: Overexpression of miR-204 by lentiviral vector may inhibit the expression of DVL3 gene in silica-induced mouse lung epithelial cells.


Subject(s)
Animals , Epithelial Cells , Genetic Vectors , Lentivirus/metabolism , Lung , Mice , MicroRNAs/metabolism , Silicon Dioxide/toxicity , Transfection
5.
Chinese Journal of Biotechnology ; (12): 1086-1095, 2022.
Article in Chinese | WPRIM | ID: wpr-927765

ABSTRACT

ERα-36 is a novel subtype of estrogen receptor α which promotes tumor cell proliferation, invasion and drug resistance, and it serves as a therapeutic target. However, only small-molecule compounds targeting ERα-36 are under development as anticancer drugs at present. Gene therapy approach targeting ERα-36 can be explored using recombinant adenovirus armed with decoy receptor. The recombinant shuttle plasmid pDC316-Ig κ-ERα-36-Fc-GFP was constructed via genetic engineering to express an Ig κ-signaling peptide-leading secretory recombinant fusion protein ERα-36-Fc. The recombinant adenovirus Ad-ERα-36-Fc-GFP was subsequently packaged, characterized and amplified using AdMaxTM adenovirus packaging system. The expression of fusion protein and functional outcome of Ad-ERα-36-Fc-GFP transduction were further analyzed with triple-negative breast cancer MDA-MB-231 cells. Results showed that the recombinant adenovirus Ad-ERα-36-Fc-GFP was successfully generated. The virus effectively infected MDA-MB-231 cells which resulted in expression and secretion of the recombinant fusion protein ERα-36-Fc, leading to significant inhibition of EGFR/ERK signaling pathway. Preparation of the recombinant adenovirus Ad-ERα-36-Fc-GFP provides a basis for further investigation on cancer gene therapy targeting ERα-36.


Subject(s)
Adenoviridae/genetics , Cell Proliferation , Estrogen Receptor alpha/metabolism , Recombinant Proteins , Transfection
6.
Article in Chinese | WPRIM | ID: wpr-936347

ABSTRACT

OBJECTIVE@#To construct a HEK293 cell line stably overexpressing TrxR1 as a cell model for functional study of TrxR1 and screening of TrxR1-targeting drugs.@*METHODS@#TrxR1 gene was amplified by PCR and ligated with the lentivirus expression vector pLVX-Puro, which was transformed into Escherichia coli and identified by Sanger dideoxy sequencing. HEK293 cells were infected with the recombinant lentivirus vector (pLVX-Puro-TXNRD1) and screened with Puromycin for cell clones with stable TrxR1 overexpression (HEK293-TrxR1-OE cells). HEK293-TrxR1-OE cells, along with HEK293 cells infected with pLVX-Puro vector (HEK293-NC) and normal HEK293 cells, were tested for mRNA and protein expression levels of TrxR1 using RT-qPCR and Western blotting. TrxR1 enzyme activity in the cells was evaluated with insulin endpoint assay and TRFS-green probe imaging. The sensitivity of the cells to auranofin, a specific TrxR1 inhibitor, was determined with CCK8 assay.@*RESULTS@#TrxR1 gene was successfully inserted into the lentiviral vector pLVX-Puro as confirmed by DNA sequencing. The enzyme activity and mRNA and protein expression levels of TrxR1 were significantly higher in HEK293-TrxR1-OE cells than in HEK293 and HEK293-NC cells (P < 0.005). The inhibitory effects of auranofin on proliferation and cellular TrxR1 enzyme activity were significantly attenuated in HEK293-TrxR1-OE cells as compared with HEK293 and HEK293-NC cells (P < 0.005).@*CONCLUSION@#We successfully obtained a HEK293 cell line with stable TrxR1 overexpression, which shows resistance to auranofin and can be used for screening TrxR1 targeting drugs.


Subject(s)
Auranofin , Cell Line, Tumor , Genetic Vectors , HEK293 Cells , Humans , Lentivirus/genetics , RNA, Messenger , Transfection
7.
Article in Chinese | WPRIM | ID: wpr-936333

ABSTRACT

OBJECTIVE@#To construct a luciferase reporter gene vector carrying human nuclear factor of activated T cells 2 (NFATc2) gene promoter and examine the effects of metformin and lipopolysaccharide (LPS) on the transcriptional activity of NFATc2 gene.@*METHODS@#The promoter sequence of human NFATc2 gene was acquired from UCSC website for PCR amplification. NFATc2 promoter fragment was inserted into pGL3-basic plasmid double cleaved with Kpn Ⅰ and Hind Ⅲ. The resultant recombinant plasmid pGL3-NFATC2-promoter was co-transfected with the internal reference plasmid pRL-TK in 293F cells, and luciferase activity in the cells was detected. Reporter gene vectors of human NFATc2 gene promoter with different fragment lengths were also constructed and assayed for luciferase activity. The changes in transcription activity of NFATc2 gene were assessed after treatment with different concentrations of metformin and LPS for 24 h. We also examined the effect of mutation in RUNX2-binding site in NFATC2 gene promoter on the regulatory effects of metformin and LPS on NFATc2 transcription.@*RESULTS@#We successfully constructed pGL3-NFATc2-promoter plasmids carrying different lengths (2170 bp, 2077 bp, 1802 bp, 1651 bp, 1083 bp, 323 bp) of NFATc2 promoter sequences as verified by enzymatic digestion and sequencing. Transfection of 293F cells with the plasmid carrying a 1651 bp NFATc2 promoter (pGL3-1651 bp) resulted in the highest transcriptional activity of NFATc2 gene, and the luciferase activity was approximately 3.3 times that of pGL3-2170 bp (1.843 ± 0.146 vs 0.547 ± 0.085). Moderate (5 mmol/L) and high (10 mmol/L) concentrations of metformin significantly upregulated the transcriptional activity of pGL3-1651 bp by up to 2.5 and 3 folds, respectively. LPS at different doses also upregulated the transcriptional activity of pGL3-1651 bp by at least 1.6 folds. The mutation in the RUNX2 binding site on pGL3-1651 bp obviously reduced metformin- and LPS-induced enhancement of pGL3-1651bp transcription by 1.7 and 2 folds, respectively.@*CONCLUSION@#pGL3-NFATc2-promoter can be transcribed and activated in 293F cells, and LPS and metformin can activate the transcription of pGL3- NFATc2-promoter in a RUNX2-dependent manner.


Subject(s)
Core Binding Factor Alpha 1 Subunit/genetics , Humans , Lipopolysaccharides/pharmacology , Luciferases/genetics , Metformin/pharmacology , NFATC Transcription Factors/genetics , Promoter Regions, Genetic , T-Lymphocytes , Transcription, Genetic/drug effects , Transfection
8.
Article in Chinese | WPRIM | ID: wpr-921928

ABSTRACT

OBJECTIVE@#To explore the effects of siRNA hsa-circ-0000885 modified bone marrow mesenchymal stem cells (BMSCs) on osteogenic differentiation, cell proliferation and apoptosis in order to provide new ideas and methods for the clinical treatment of osteoporosis (OP).@*METHODS@#From September 2018 to February 2020, 13 patients with osteoporosis admitted to our hospital were selected as the research objects, including 11 females and 2 males, with an age of (65.45±10.77) years old. After obtaining the informed consent of patients, peripheral blood tissues were extracted. Then the expression level of cir-cRNA in peripheral blood mononuclear cells(PBMC) was detected by circ RNA chip. The expression of circ RNA was silenced by siRNA technology. The BMSCs were transfected with lentivirus. According to the siRNA interference plasmid hsa-circ-0000885, the cells were divided into the blank group, the empty vector group and the siRNA interference group. After 72 hours of treatment, the cell cycle was detected by flow cytometry, the apoptosis level was detected by AV-PI kit, and the osteogenic differentiation ability of BMSCs was detected by ALP staining.@*RESULTS@#The expression of hsa-circ-0000885 in PBMC of patients with osteoporosis was significantly higher than that of healthy controls (@*CONCLUSION@#The lentivirus mediated siRNA hsa-circ-0000885 plasmid transfected into BMSCs and osteoclast co culture system can promote cell proliferation, inhibit apoptosis and promote osteogenic differentiation of BMSCs, which can be used as a potential therapeutic target for OP patients.


Subject(s)
Aged , Apoptosis/genetics , Cell Differentiation , Cell Proliferation/genetics , Cells, Cultured , Coculture Techniques , Female , Humans , Lentivirus , Leukocytes, Mononuclear , Mesenchymal Stem Cells , Middle Aged , Osteoclasts , Osteogenesis/genetics , RNA, Small Interfering/genetics , Transfection
9.
Article in Chinese | WPRIM | ID: wpr-888337

ABSTRACT

OBJECTIVE@#To construct and identify adenovirus vector co-expressing hBMP2 and hVEGF165 fusion protein which labeled with green fluorescence protein, and laying the foundtion of the effect of hBMP2 and hVEGF165 gene inducing BMMSCs differentiation to osteoblast and bone defect repaired in the body.@*METHODS@#BMP2 and VEGF165 gene was amplified from cDNA library by PCR and inserted to the polyclonal site of adenovirus shuttle plasmid pAd-MCMV-GFP. Ad-BMP2- VEGF165 was recombinated and propagated in HEK293 cells by co-transfecting with the constructed recombinant shuttle plasmid pAd-MCMV-BMP2-VEGF165 and adenovirus helper plasmid pBHGloxΔ E1, 3Cre. The recombinant adenovirus was purified and virustiter was determined, and then to research GFP expression and to calculate the adenovirus transfection rate in rabbit BMMSCs.@*RESULTS@#The recombinant adenovirus vector Ad-BMP2-VEGF165 was successfully constructed by the methods of gene analyzing, colony PCR, Western blotting and observing GFP expression, and the titer of the adenovirus was 1×10@*CONCLUSION@#Recombinant adenovirus vector containing hBMP2 and hVEGF165 gene was successfully constructed and its high titer was obtained.


Subject(s)
Adenoviridae/genetics , Animals , Bone Marrow Cells , Genetic Vectors/genetics , HEK293 Cells , Humans , Mesenchymal Stem Cells , Rabbits , Transfection
10.
Article in Chinese | WPRIM | ID: wpr-880076

ABSTRACT

OBJECTIVE@#To construct an acute myeloid leukemia cell line stably expressing CD123-CLL1 so as to provide an "in vitro" model for studying the role of CD123 and CLL-1 in leukemia and the treatment targeting CD123 and CLL-1.@*METHODS@#The recombinant plasmid of lentivirus was constructed by synthesizing CD123 and CLL-1 sequences and PCR homologous recombination. The lentivirus vector was packaged by three-plasmid packaging system. After collecting the supernatant of lentivirus, the virus titer was determined by quantitative PCR. K562 leukemia cells were collected and transtected with virus supernatant. Leukemia cell line stably expressing the target gene were screened by purinomycin. The expression levels of CD123 and CLL-1 were detected by RT-PCR and flow cytometry.@*RESULTS@#The lentiviral vector was successfully constructed, and identified by agarose gel electrophoresis and gene sequencing, then the virus titer of the supernatant was up to 5.81×10@*CONCLUSION@#Lentiviral vector expressing CD123-CLL1 has been successfully constructed, and K562 leukemia cell line stably expressing CD123 and CLL-1 has been successfully obtained.


Subject(s)
Cell Line, Tumor , Genetic Vectors , Humans , Interleukin-3 Receptor alpha Subunit , K562 Cells , Lentivirus/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Plasmids , Transfection
11.
Article in English | WPRIM | ID: wpr-878451

ABSTRACT

OBJECTIVES@#The effect of isoprenylcysteine carboxymethyltransferase (ICMT) silencing on the migration and invasion of tongue squamous cell carcinoma was investigated by constructing the small interfering RNA (siRNA) of ICMT.@*METHODS@#Through liposomal transfection, siRNA was transfected into human tongue squamous cell carcinoma CAL-27 and SCC-4 cells (ICMT-siRNA group) with a negative control group (transfected with NC-siRNA) and a blank control group (transfected with a transfection reagent but not with siRNA). Quantitative real-time polymerase chain reaction was performed to analyze the mRNA expression of ICMT and RhoA in each group of cells after transfection and to measure the silencing efficiency. Western blot was applied to examine the expression levels of ICMT, total RhoA, membrane RhoA, ROCK1, matrix metalloproteinase (MMP)-2, and MMP-9 proteins in each group. The migration and invasion abilities were evaluated via wound healing and Transwell motility assays.@*RESULTS@#After CAL-27 and SCC-4 cells were transfected with ICMT-siRNA, the expression levels of ICMT genes and proteins decreased significantly in the experimental group compared with those in the negative and blank control groups (@*CONCLUSIONS@#The migration and invasion abilities of CAL-27 and SCC-4 cells were reduced significantly after the transfection of ICMT-siRNA, and the involved mechanism might be related to the RhoA-ROCK signaling pathway.


Subject(s)
Carcinoma, Squamous Cell , Cell Line, Tumor , Cell Movement , Cell Proliferation , Humans , Neoplasm Invasiveness , Protein Methyltransferases , RNA, Small Interfering , Tongue , Tongue Neoplasms , Transfection , rho-Associated Kinases
12.
Braz. j. med. biol. res ; 54(2): e9944, 2021. tab, graf
Article in English | LILACS, ColecionaSUS | ID: biblio-1142581

ABSTRACT

The aim of this study was to inhibit adipogenic differentiation by transfecting two growth factors, platelet-derived growth factor (PDGF-BB) and bone morphogenic protein 2 (BMP-2), into modified rat bone marrow mesenchymal stem cells (rBMSCs) and then compounded with platelet-rich plasma (PRP). To achieve rBMSCs, the osteoporosis model of rats was established, and then the rBMSCs from the rats were isolated and identified. Co-transfection of rBMSCs with PDGF-BB-GFP and BMP-2 and detection of PDGF-BB/BMP-2 expression in transfected BMSCs was assessed by qRT-PCR and western blot, respectively. Moreover, the effect of the two growth factors transfection of rBMSCs on adipogenic differentiation was evaluated by oil red O staining and western blot, respectively. Finally, construction of the two growth factors transfection of rBMSCs compounded with PRP and detection of adipogenic differentiation were assessed by oil red O staining, CCK-8, and western blot, respectively. In vitro studies revealed that the two growth factors transfection of rBMSCs compounded with PRP promoted cell viability and inhibited adipogenic differentiation and could be promising for inhibiting adipogenic differentiation.


Subject(s)
Animals , Rats , Cell Differentiation , Adipose Tissue/cytology , Platelet-Rich Plasma , Bone Morphogenetic Protein 2/genetics , Mesenchymal Stem Cells/cytology , Becaplermin/genetics , Transfection , Cells, Cultured
13.
Braz. j. med. biol. res ; 54(5): e10743, 2021. tab, graf
Article in English | LILACS | ID: biblio-1180738

ABSTRACT

Amphiphilic copolymers have a wide variety of medical and biotechnological applications, including DNA transfection in eukaryotic cells. Still, no polymer-primed transfection of prokaryotic cells has been described. The reversible addition-fragmentation chain transfer (RAFT) polymer synthesis technique and the reversible deactivation radical polymerization variants allow the design of polymers with well-controlled molar mass, morphology, and hydrophilicity/hydrophobicity ratios. RAFT was used to synthesize two amphiphilic copolymers containing different ratios of the amphiphilic poly[2-(dimethyl-amino) ethyl methacrylate] and the hydrophobic poly [methyl methacrylate]. These copolymers bound to pUC-19 DNA and successfully transfected non-competent Escherichia coli DH5α, with transformation efficiency in the range of 103 colony-forming units per µg of plasmid DNA. These results demonstrate prokaryote transformation using polymers with controlled amphiphilic/hydrophobic ratios.


Subject(s)
Polymers , DNA/genetics , Bacteria , Transfection , Cations
14.
Braz. j. med. biol. res ; 53(1): e9144, Jan. 2020. graf
Article in English | LILACS | ID: biblio-1055480

ABSTRACT

Wound scarring remains a major challenge for plastic surgeons. Transforming growth factor (TGF)-β plays a key role in the process of scar formation. Previous studies have demonstrated that truncated TGF-β type II receptor (t-TGF-βRII) is unable to continue signal transduction but is still capable of binding to TGF-β, thereby blocking the TGF-β signaling pathway. Hepatocyte growth factor (HGF) is a multifunctional growth factor that promotes tissue regeneration and wound healing. Theoretically, the combination of HGF and t-TGF-βRII would be expected to exert a synergistic effect on promoting wound healing and reducing collagen formation. In the present study, lentivirus-mediated transfection of the two genes (t-TGF-βRII/HGF) into fibroblasts in vitro and in a rat model in vivo was used. The results demonstrated that the expression of t-TGF-βRII and HGF in NIH-3T3 cells was successfully induced. The expression of both molecules significantly reduced collagen I and III expression, and also inhibited fibroblast proliferation. Furthermore, histological examination and scar quantification revealed less scarring in the experimental wound in a rat model. Moreover, on macroscopic inspection, the experimental wound exhibited less visible scarring compared with the control. Therefore, the present study demonstrated that the combination gene therapy of t-TGF-βRII and HGF promoted wound healing, with less scarring and more epithelial tissue formation, not only by suppressing the overgrowth of collagen due to its antifibrotic effect, but also by promoting tissue regeneration.


Subject(s)
Animals , Rabbits , Rats , Transfection , Collagen/metabolism , Cicatrix/metabolism , Hepatocyte Growth Factor/metabolism , Transforming Growth Factor beta2/metabolism , Cicatrix/pathology , Rats, Sprague-Dawley , Models, Animal , Cell Proliferation
15.
Article in Chinese | WPRIM | ID: wpr-827180

ABSTRACT

OBJECTIVE@#To transinfect SD adipose tissue-derived stem cell (ADSC) in vitro with a recombinant adenoviral vector containing human B-domain-deleted FVIII (BDDhFⅧ), so as to lay the foundation for the treatment of hemophilia A by using ADSC combined with BDDhFⅧ gene.@*METHODS@#ADSCs were isolated from the inguinal adipose tissue of SD rats and passed to third passage for identification. Third passage ADSCs were transfected in vitro with recombinant adenovirus vector Ad-BDDhFⅧ-GFP. The experiments were divided into Ad-BDDhFⅧ-GFP-transfected ADSCs group (A), Ad-GFP-transfected ADSC group (B), and untransfected ADSC group (C). CCK-8 method was used to detect the proliferation of transfected cells in three groups, and the expression level of hFⅧ antigen in cell supernatant was detected by ELISA. RT-PCR and Western blot respectively were used to detect the mRNA and protein expression of BDDhFⅧ in the three groups after transfection.@*RESULTS@#The growth curve of third passage cells isolated and cultured showed an inverted "S" shap; the flow cytometry detection showed the positive expression of CD29, CD90, CD44, and the negative expression of CD45 in third passage cells. After the adipogenic and osteogenic induction, the cells could transformed to adipogenic and osteogenic directions. CCK-8 detection showed that the proliferation of cells in 3 groups not was influenced. ELISA showed that the expression of hFⅧAg in group A was significantly higher than that in group B and C (P<0.05). RT-PCR showed that compared with group A, there was no target band in B and C groups, and BDDhFⅧ gene was not expressed. The results in group A were consistent with the length of amplified fragments, and BDDhFⅧ target gene was expressed. Western blot analysis showed that the expression of hFⅧ protein in group A was significantly higher than that in group B and C. (P<0.05).@*CONCLUSION@#Recombinant adenovirus Ad-BDDhFⅧ-GFP can effectively transfect rat ADSC in vitro, which lays an experimental foundation for gene therapy of hemophilia A.


Subject(s)
Adenoviridae , Adipose Tissue , Animals , Cell Differentiation , Cells, Cultured , Factor VIII , Humans , Rats , Rats, Sprague-Dawley , Stem Cells , Transfection
16.
Journal of Experimental Hematology ; (6): 1144-1151, 2020.
Article in Chinese | WPRIM | ID: wpr-827149

ABSTRACT

OBJECTIVE@#To explore the effect of regulating A20 expression on NF-κB and biological characteristics of Jurkat cells with glucocorticoid (GC) resistance.@*METHODS@#CCRF CEM and Jurkat cells were treated with dexamethasone (DEX) at concentrations of 100、10、1、0.1、0.01 and 0.001 μmol/L, and cultured for 24、48 and 72 h. The proliferation inhibition rate of Jurkat cell was detected by CCK-8. A20 plasmid was constructed, A20-siRNA was designed and synthesized, and transfected into Jurkat cells by liposome. CCK-8 was used to detect the proliferation rates of Jurkat cells in different concentrations of DEX group, DEX combined with A20 plasmid group and A20-siRNA group. The mRNA expression level of NF-κB was detected by RT-qPCR, the protein expression level of NF-κB was detected by Western blot, and the apoptosis of Jurkat cells was examined by flow cytometry.@*RESULTS@#The inhibitory effects of DEX at different concentrations on the growth of CCRF CEM cells were time-dependent (r=0.984, P<0.05) and concentration-dependent (r=0.966, P<0.05). At the point of 24 hour, the IC approached 1 μmol/L in CCRF CEM cells. Great large differences began to appear between 1 and 10 μmol/L, the proliferation rate of Jurkat cells treated with 1 μmol/L DEX did not show a significant change. Therefore, 1 μmol/L was selected as control group. The cell proliferation rate of A20 plasmid transfection combined with different concentrations of DEX group was lower than that of DEX group and A20-siRNA combined with DEX group. After transfection of A20 plasmid, the expression level of NF-κB was significantly lower than that of control group (P<0.05), and the apoptotic rate was significantly higher than that of control group (P<0.05). After transfection of Jurkat cells with A20-siRNA, the expression level of NF-κB was significantly higher than that of control group (P<0.05). The apoptotic rate of cells in A20-siRNA group was not significantly changed (P>0.05).@*CONCLUSION@#Jurkat cells are resistant to DEX. A20 overexpression combined with DEX can increase sensitivity of Jurkat cells with GC resistance and decrease the proliferation rate of Jurkat cells, down-regulate the expression level of NF-κB and promote the apoptosis of Jurkat cells.


Subject(s)
Apoptosis , Cell Proliferation , Humans , Jurkat Cells , NF-kappa B , RNA, Small Interfering , Transfection , Tumor Necrosis Factor alpha-Induced Protein 3
17.
Article in English | WPRIM | ID: wpr-811138

ABSTRACT

BACKGROUND: Recent studies have shown that microRNAs (miRNAs) are involved in the process of cardiomyocyte apoptosis. We have previously reported that granulocyte-colony stimulating factor (G-CSF) ameliorated diastolic dysfunction and attenuated cardiomyocyte apoptosis in a rat model of diabetic cardiomyopathy. In this study, we hypothesized a regulatory role of cardiac miRNAs in the mechanism of the anti-apoptotic effect of G-CSF in a diabetic cardiomyopathy rat model.METHODS: Rats were given a high-fat diet and low-dose streptozotocin injection and then randomly allocated to receive treatment with either G-CSF or saline. H9c2 rat cardiomyocytes were cultured under a high glucose (HG) condition to induce diabetic cardiomyopathy in vitro. We examined the extent of apoptosis, miRNA expression, and miRNA target genes in the myocardium and H9c2 cells.RESULTS: G-CSF treatment significantly decreased apoptosis and reduced miR-34a expression in diabetic myocardium and H9c2 cells under the HG condition. G-CSF treatment also significantly increased B-cell lymphoma 2 (Bcl-2) protein expression as a target for miR-34a. In addition, transfection with an miR-34a mimic significantly increased apoptosis and decreased Bcl-2 luciferase activity in H9c2 cells.CONCLUSION: Our results indicate that G-CSF might have an anti-apoptotic effect through down-regulation of miR-34a in a diabetic cardiomyopathy rat model.


Subject(s)
Animals , Apoptosis , Diabetic Cardiomyopathies , Diet, High-Fat , Down-Regulation , Glucose , Granulocyte Colony-Stimulating Factor , In Vitro Techniques , Luciferases , Lymphoma, B-Cell , MicroRNAs , Models, Animal , Myocardium , Myocytes, Cardiac , Rats , Streptozocin , Transfection
18.
Article in Chinese | WPRIM | ID: wpr-828638

ABSTRACT

OBJECTIVE@#To study the role of microRNA-17-5p (miR-17-5p) in the pathogenesis of pediatric nephrotic syndrome (NS) and its effect on renal podocyte apoptosis via the activin A (ActA)/Smads pathway.@*METHODS@#An analysis was performed on 55 children with NS (NS group) who were admitted from March 2018 to March 2019. Fifty healthy children who underwent physical examination during the same period of time were enrolled as the control group. The mRNA expression of miR-17-5p in peripheral blood was measured and compared between the two groups. Human renal podocytes were transfected with antisense oligonucleotide recombinant plasmid containing miR-17-5p (inhibition group) or control vector containing nonsense random sequence (negative control group), and untreated human renal podocytes were used as the blank group. These groups were compared in terms of cell apoptosis and the mRNA and protein expression of miR-17-5p, ActA, and Smads after transfection.@*RESULTS@#The NS group had a significantly higher level of miR-17-5p in peripheral blood than the control group (P<0.001). Compared with the blank and negative control groups, the inhibition group had significantly lower apoptosis rate and relative mRNA expression of miR-17-5p and significantly higher relative mRNA and protein expression of ActA, Smad2, and Smad3 (P<0.001).@*CONCLUSIONS@#There is an increase in the content of miR-17-5p in peripheral blood in children with NS. Low expression of miR-17-5p can inhibit the apoptosis of human renal podocytes, which may be associated with the upregulation of the mRNA and protein expression of ActA, Smad2 and Smad3.


Subject(s)
Apoptosis , Child , Humans , MicroRNAs , Genetics , Nephrotic Syndrome , Genetics , Podocytes , Transfection
19.
Article in Chinese | WPRIM | ID: wpr-941991

ABSTRACT

OBJECTIVE@#To analyze the subcellular localization of GTPase of immunity-associated protein 2 (GIMAP2) for the further functional study.@*METHODS@#In the study, we first obtained the protein sequences of GTPase of immunity-associated protein 2 (GIMAP2) from National Center for Biotechnology Information (NCBI) database, and then performed a prediction analysis of its transmembrane structure, nuclear localization signal (NLS), nuclear export signal (NES) and subcellular localization through bioinformatics online tools. GIMAP2 gene amplified by PCR was inserted into the expression vector pQCXIP-mCherry-N1 and positive clones were selected by ampicillin resistance. After using methods to extract and purify, the sequenced recombinant plasmid pQCXIP-GIMAP2-mCherry, together with the retroviral packaging plasmids VSVG and Gag/pol, was transferred into HEK293FT cells by liposomes for virus packaging. The virus supernatant was collected 48 h after transfection and directly infected the human breast cancer cell line MDA-MB-436. Immunofluorescence staining was constructed to detect the localization of endogenous and exogenous GIMAP2 in MDA-MB-436 cells. Meanwhile, green fluorescent chemical dyes were used to label mitochondria, endoplasmic reticulum, and lipid droplets in living MDA-MB-436 cells stably expressing the GIMAP2-mCherry fusion protein. Images for the three dye-labeled organelles and GIMAP2-mCherry fusion protein were captured by super-resolution microscope N-SIM.@*RESULTS@#Bioinformatics analysis data showed that GIMAP2 protein composed of 337 amino acids might contain two transmembrane helix (TM) structures at the carboxyl terminus, of which TMs were estimated to contain 40-41 expected amino acids, followed by the residual protein structures toward the cytoplasmic side. NES was located at the 279-281 amino acids of the carboxyl terminus whereas NLS was not found. GIMAP2 might locate in the lumen of the endoplasmic reticulum. Sequencing results indicated that the expression vector pQCXIP-GIMAP2-mCherry was successfully constructed. Fluorescent staining confirmed that GIMAP2-mCherry fusion protein, co-localized well with endogenous GIMAP2, expressed successfully in the endoplasmic reticulum and on the surface of lipid droplets in MDA-MB-436 cells.@*CONCLUSION@#GIMAP2 localizes in the endoplasmic reticulum and on the surface of LDs, suggesting potential involvement of GIMAP2 in lipid metabolism.


Subject(s)
Amino Acid Sequence , Cytoplasm , GTP Phosphohydrolases , Humans , Membrane Proteins , Nuclear Export Signals , Nuclear Localization Signals , Recombinant Fusion Proteins , Transfection
20.
Chinese Journal of Biotechnology ; (12): 763-771, 2020.
Article in Chinese | WPRIM | ID: wpr-826900

ABSTRACT

The recombinant adenoviruses expressing miR-22 (Ad-miR-22) was constructed and the effect of Ad-miR-22 on insulin signal pathway and glucose uptake in HepG2 cells was analyzed. MiR-22 gene was amplified by PCR from human hepatocytes and cloned into the pAdTrack-CMV vector to generate the shuttle plasmid pAdT-22. The positive colonies were confirmed by PCR and sequencing. The resultant shuttle plasmid was linearized with Pme I, followed by co-transformation into competent BJ5183 cells containing an adenoviral backbone plasmid (pAdEasy-1) to create the recombinant plasmid pAd-miR-22. After digested with Pac I, the linearized pAd-miR-22 was transfected into 293A packaging cell line to generate recombinant adenoviruses Ad-miR-22. HepG2 cells were infected with Ad-miR-22 or control Ad-GFP (adenoviruses expressing green fluorescent protein), and then the miR-22 expression levels were analyzed by qPCR. The result shows that adenovirus-mediated overexpression of miR-22 significantly decreased insulin-induced glucose uptake in HepG2 cells. Moreover, overexpression of miR-22 markedly decreased insulin-induced phosphorylation of GSK-3β. miR-22 also increased the mRNA levels of gluconeogenic genes in HepG2 cells. Furthermore, Western blotting results indicate that the protein expression of SIRT1 decreased in Ad-miR-22 infected HepG2 cells as compared with Ad-GFP infected HepG2 cells. In summary, overexpressing of miR-22 significantly increased gluconeogenesis while decreased glucose uptake in HepG2 cells. The effect of miR-22 on glucose metabolism may be mediated by SIRT1.


Subject(s)
Adenoviridae , Genetics , Glucose , Metabolism , Glycogen Synthase Kinase 3 beta , Metabolism , Hep G2 Cells , Humans , MicroRNAs , Genetics , Metabolism , Signal Transduction , Genetics , Transfection
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