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1.
Article in Chinese | WPRIM | ID: wpr-878916

ABSTRACT

This study cloned the transcription factor gene PnbHLH which held an open reading frame of 966 bp encoding 321 amino acids. This study constructed the overexpression vector of transcription factor PnbHLH of Panax notoginseng. The combination of PnbHLH overexpression and RNAi of the key enzyme gene PnCAS involved in the phytosterol biosynthesis was achieved in P. notoginseng cells, thus exploring the biosynthetic regulation of P. notoginseng saponins(PNS) by the synergistic effect of PnbHLH overexpression and PnCAS RNAi. The results showed that the PnbHLH transcription factor interacted with the promoters of key enzyme genes PnDS, PnSS and PnSE in the biosynthetic pathway of PNS, and then regulated the expression levels of key enzyme genes and affected the biosynthesis of saponins indirectly. Further study indicated that the synergistic effect of PnbHLH overexpression and PnCAS RNAi was a more effective approach to regulate the biosynthesis of saponins. Compared with the wild type and PnCAS RNAi cells of P. notoginseng, the contents of total saponins and monomeric saponins(Rd, Rb_1, Re, Rg_1 and R_1) were increased to some extent in the cell lines of PnbHLH overexpression and PnCAS RNAi. This indicated that the two ways of forward regulation and reverse regulation of saponin biosynthesis showed superposition effect. This study explored a more rational and efficient regulation strategy of PNS biosynthesis based on the advantages of multi-point regulation of transcription factors as well as the down-regulation of by-product synthesis of saponins.


Subject(s)
Intramolecular Transferases , Panax notoginseng , RNA Interference , Saponins , Transcription Factors/genetics
2.
Chinese Journal of Biotechnology ; (12): 1237-1248, 2021.
Article in Chinese | WPRIM | ID: wpr-878627

ABSTRACT

RNA interference (RNAi) is one of the important mechanisms to regulate gene expression in eukaryotes. One of the original functions of RNAi is to facilitate the antiviral strategy of host. Early studies reveal that invertebrates can use RNAi to resist viruses. However, if this mechanism exists in mammals is still controversial. The latest studies confirm that mammals do have the RNAi-based immunity, and researchers believe that RNAi-based antiviral immunity is a brand-new immunological mechanism that was neglected in the past. It is worthy to note that virus can also use RNAi to enhance its infectivity and immune escape in host cells. This review introduces the research history of RNAi-based antiviral immunity in animals and summarizes the main findings in this field. Last but not least, we indicate a series of unresolved questions about RNAi-based antiviral immunity, and explore the relationship between RNAi-based antiviral immunity and other innate immunological pathways. The virus-mediated RNAi pathway in animal is not only an interesting basic biology question, but also has important guiding roles in the development of antiviral drugs.


Subject(s)
Animals , Antiviral Agents , Immunity, Innate/genetics , Mammals , RNA Interference , RNA, Small Interfering/genetics , RNA, Viral
3.
Braz. oral res. (Online) ; 34: e006, 2020. tab, graf
Article in English | LILACS | ID: biblio-1055522

ABSTRACT

Abstract Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.


Subject(s)
Signal Transduction/physiology , Smad1 Protein/physiology , Induced Pluripotent Stem Cells/cytology , Ameloblasts/cytology , Phosphorylation , Time Factors , Gene Expression , Cell Differentiation/physiology , Cell Differentiation/genetics , Cells, Cultured , Blotting, Western , Fluorescent Antibody Technique , Culture Media, Serum-Free , Reverse Transcriptase Polymerase Chain Reaction , MAP Kinase Signaling System/physiology , Activin Receptors/analysis , Activin Receptors/physiology , RNA Interference , p38 Mitogen-Activated Protein Kinases/analysis , p38 Mitogen-Activated Protein Kinases/physiology , Bone Morphogenetic Protein Receptors, Type II/analysis , Bone Morphogenetic Protein Receptors, Type II/physiology , Smad1 Protein/analysis
4.
Braz. oral res. (Online) ; 34: e006, 2020. tab, graf
Article in English | LILACS | ID: biblio-1089380

ABSTRACT

Abstract Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.


Subject(s)
Signal Transduction/physiology , Smad1 Protein/physiology , Induced Pluripotent Stem Cells/cytology , Ameloblasts/cytology , Phosphorylation , Time Factors , Gene Expression , Cell Differentiation/physiology , Cell Differentiation/genetics , Cells, Cultured , Blotting, Western , Fluorescent Antibody Technique , Culture Media, Serum-Free , Reverse Transcriptase Polymerase Chain Reaction , MAP Kinase Signaling System/physiology , Activin Receptors/analysis , Activin Receptors/physiology , RNA Interference , p38 Mitogen-Activated Protein Kinases/analysis , p38 Mitogen-Activated Protein Kinases/physiology , Bone Morphogenetic Protein Receptors, Type II/analysis , Bone Morphogenetic Protein Receptors, Type II/physiology , Smad1 Protein/analysis
5.
Article in Chinese | WPRIM | ID: wpr-828429

ABSTRACT

In this study, based on the transcriptome database of suspension cells of Arnebia euchroma, we explored two candidate cytochrome P450 enzyme genes that might relate to the shikonin biosynthesis downstream pathway when CYP76B74 sequence was referenced. We constructed interference-type hairy roots of candidate genes and cultured them. We measured the fresh weight, dry weight, total naphthoquinone content, shikonin and its derivatives content and expression levels of key enzyme genes involved in shikonin biosynthesis pathway. The effects of candidate genes on the growth and shikonin production of A. euchroma hairy roots were discussed, and the possible regulatory mechanisms that candidate genes affected shikonin synthesis were discussed. Through local Blast and phylogenetic analysis, two candidate CYP450 genes(CYP76B75 and CYP76B100) with high homology to CYP76B74 in A. euchroma were screened, and corresponding interference hairy roots were constructed. Compared with the control(RNAi-control), the fresh weight of CYP76B75 interfered hairy root(RNAi-CYP76B75) and CYP76B100 interfered hairy root(RNAi-CYP76B100) were significantly reduced, while dry weight were not affected, so the dry rate increased significantly. Except for β-acetoxyisovalerylalkannin, which is high in three groups of hairy roots, the contents of shikonin, deoxyshikonin, acetylshikonin, β,β'-dimethacrylicalkannin, β-hydroxyisovalerylshikonin,β-hydroxyisovalerylshikonin, isobutyrylshikonin and total naphthoquinones showed a consistent pattern: RNAi-CYP76B75>RNAi-CYP76B100>RNAi-control. Among them, the synthesis of β-hydroxyisovalerylshikonin was most significantly promoted by interfering with the expression of CYP76B75. The content of β-hydroxyisovalerylshikonin in RNAi-CYP76B75 was 11.7 times that of RNAi-control. RESULTS:: of real-time qPCR analysis showed that compared to RNAi-control, the expression levels of AePGT gene in RNAi-CYP76B75 and RNAi-CYP76B100 were not changed significantly, and the expression levels of CYP76B74 and AeHMGR were up-regulated. In addition, the expression level of CYP76B100 in RNAi-CYP76B75 was down-regulated, whereas in RNAi-CYP76B100, the expression of CYP76B75 was significantly up-regulated. Therefore, this study confirmed that when the expression of CYP76B75 and CYP76B100 were interrupted, the growth of hairy roots were suppressed, but the synthesis of shikonin were promoted. They might increase the shikonin biosynthesis by up-regulating the expression of CYP76B74 in the hairy roots of A. euchroma.


Subject(s)
Boraginaceae , Genetics , Cytochrome P-450 Enzyme System , Naphthoquinones , Phylogeny , Plant Roots , RNA , RNA Interference
6.
Braz. j. med. biol. res ; 53(7): e9207, 2020. tab, graf
Article in English | ColecionaSUS, LILACS, ColecionaSUS | ID: biblio-1132533

ABSTRACT

The objective of this study was to investigate the relationship between PI3K/mTOR/RhoA signaling regulated cytoskeletal rearrangements and phagocytic capacity of macrophages. RAW264.7 macrophages were divided into four groups; blank control, negative control, PI3K-RNAi, and mTOR-RNAi. The cytoskeletal changes in the macrophages were observed. Furthermore, the phagocytic capacity of macrophages against Escherichia coli is reported as mean fluorescence intensity (MFI) and percent phagocytosis. Transfection yielded 82.1 and 81.5% gene-silencing efficiencies against PI3K and mTOR, respectively. The PI3K-RNAi group had lower mRNA and protein expression levels of PI3K, mTOR, and RhoA than the blank and negative control groups (Р<0.01). The mTOR-RNAi group had lower mRNA and protein levels of mTOR and RhoA than the blank and the negative control groups (Р<0.01). Macrophages in the PI3K-RNAi group exhibited stiff and inflexible morphology with short, disorganized filopodia and reduced number of stress fibers. Macrophages in the mTOR-RNAi group displayed pronounced cellular deformations with long, dense filopodia and an increased number of stress fibers. The PI3K-RNAi group exhibited lower MFI and percent phagocytosis than blank and negative control groups, whereas the mTOR-RNAi group displayed higher MFI and percent phagocytosis than the blank and negative controls (Р<0.01). Before and after transfection, the mRNA and protein levels of PI3K were both positively correlated with mTOR and RhoA (Р<0.05), but the mRNA and protein levels of mTOR were negatively correlated with those of RhoA (Р<0.05). Changes in the phagocytic capacity of macrophages were associated with cytoskeletal rearrangements and were regulated by the PI3K/mTOR/RhoA signaling pathway.


Subject(s)
Humans , Animals , Rats , Phagocytosis/physiology , Cytoskeleton/metabolism , Phosphatidylinositol 3-Kinases/metabolism , rhoA GTP-Binding Protein/metabolism , TOR Serine-Threonine Kinases/metabolism , Macrophages/metabolism , Transfection , Signal Transduction , Blotting, Western , Gene Silencing , RNA Interference , Real-Time Polymerase Chain Reaction , RAW 264.7 Cells , Genetic Vectors
7.
Journal of Experimental Hematology ; (6): 1744-1748, 2019.
Article in Chinese | WPRIM | ID: wpr-781403

ABSTRACT

OBJECTIVE@#To analyze the effect of down-regulating the CD59 gene expression by RNAi lentivirus as vector on Jurkat cell line of acute T-lineage leukemia.@*METHODS@#The expression of CD59 in Jurkat cell line of acute T-line leukemia was induced to decrease by RNAi lentivirus as vector. The transfection of RNA lentivirus and the localization of CD59 molecule were analyzed by laser confocal technique. The relative expression of CD59 gene in blank control, negative control and RNAi lentivirus transfected group was detected by real-time fluorescence quantitative PCR, and the enzyme-linked immunosorbent assay was used to detect the expression of TNF-β and IL-3 in supernatants of cultured cells in 3 groups. The expression levels of apoptosis-related molecules including Caspase-3, Survivin, BCL-2 and BCL-2-associated X protein (BAX) were measured by Western blot.@*RESULTS@#The transfection efficiency for Jurkat cells was higher than 90%. CD59 was mainly located on the cell membrane. Compared with the blank control group and the negative control group, the expression level of CD59 mRNA and protein in the RNAi lentivirus transfected group significantly decreased (P<0.05). Compared with the blank control group and the negative control group, the expression of TNF-β and IL-3 in the RNAi lentivirus transfected group were significantly higher and lower (P<0.05) respectively. The expression levels of Survivin and BCL-2 in the RNAi lentivirus transfected group were significantly lower than those in the blank control group and the negative control group, while the expression levels of Caspase-3 and BAX in the RNAi lentivirus transfected group were significantly higher than those in the blank control group and the negative control group (P< 0.05).@*CONCLUSION@#The down-regulation of CD59 gene expression induced by RNAi lenti-virus can decrease the expression of proliferation and differentiation-promoting molecule such as IL-3 and increase the expression of TNF-related factor in Jurkat cell line of acute T-lineage leukemia, which also can increase the expression of apoptosis-related proteins such as Caspase-3 and BAX, and decrease the expression of anti-apoptosis-related proteins such as Survivin and BCL-2.


Subject(s)
Apoptosis , CD59 Antigens , Cell Lineage , Cell Proliferation , Down-Regulation , Humans , Jurkat Cells , Lentivirus , Leukemia , RNA Interference , RNA, Small Interfering , Transfection
8.
Article in Chinese | WPRIM | ID: wpr-774297

ABSTRACT

OBJECTIVE@#To investigate the effect of steadily down-regulating the expression of calreticulin (CALR) on the invasion of natural killer/T-cell lymphoma SNK6 cells, and explore its possible mechanism.@*METHODS@#The sequences of specific short hairpin RNA (shRNA) targeting on human CALR were designed, and were inserted into pLKO.1-puro lentivirus vector, and the reconbinant lentivirus vector was obtained; the lentivirus particles were backed by three-plasmid system and transfected into SNK6 cells, the SNK6 cells stably down-regulating the CALR expression were sercened by puromytain, the CALR-silencing effect was verified by real-time PCR and Western blot. CCK-8 assay was used to evaluate the cell viability, The transwell invasion assays was used to analyse invasion of SNK6 cells. The mRNA expression of Calreticulin, MMP2, MMP9 and VEGF was determined by real time PCR, the protein expression of Calreticulin and GAPDH was analyzed by Western blot.@*RESULTS@#The recombinant lentiviral vector pLKO.1-puro-shCALR was successfully constructed, packed into the lentivirus, then the SNK6 cells stably down-regulating Calreticulin expression was obtained. When Calreticulin was down-rengulated in SNK6 cells, the proliferation rate was reduced and the invasion ability was decreased; the mRNA levels of VEGF and MMP-2/9 also were reduced.@*CONCLUSION@#The stable down-regnlation of CALR expression in SNK6 cells can attenuate the imvasiveness of SNK6 cells, which maybe related with transcriptional decrease of MMP2, MMP9 and VEGF.


Subject(s)
Calreticulin , Genetics , Metabolism , Cell Line, Tumor , Cell Proliferation , Down-Regulation , Genetic Vectors , Humans , Lentivirus , Matrix Metalloproteinase 2 , Matrix Metalloproteinase 9 , RNA Interference , RNA, Small Interfering , Transfection , Vascular Endothelial Growth Factor A
9.
Article in Chinese | WPRIM | ID: wpr-772684

ABSTRACT

OBJECTIVE@#This study aimed to explore the influence of Rce1 on invasion and migration of tongue squamous cell carcinoma cells by silencing the Rce1 gene with RNA interference.@*METHODS@#The tongue squamous cell carcinoma Cal-27 and SCC-4 cells were cultured in vitro. The small interfering RNA (siRNA) of the Rce1 gene was designed, and the Rcel gene expression was silenced vialiposome transfection. According to the siRNA transfected by liposome, the experimental group was divided into three groups, namely, Rce1-siRNA-1, Rce1-siRNA-2, and Rce1-siRNA-3 groups. Negative control group was transfected by siCON, and the blank control group was untransfected by siRNA. The Rce1, RhoA, and K-Ras gene expression levels in each group were analyzed by real-time quantitative polymerase chain reaction. The Rce1, RhoA, K-Ras, MMP-2, and MMP-9 protein expression levels were analyzed by Western blot. The invasiveness of tongue cancer cell Cal-27 and SCC-4 were determined by Transwell invasion assay, and cell migration assay was performed by cell scratch assay.@*RESULTS@#Real-time quantitative polymerase chain reaction and Western blot results showed that compared with the negative and blank control groups, the Rce1 gene and protein expression levels in three experimental groups decreased (P0.05). Meanwhile, the MMP-2 and MMP-9 expression levels decreased (P<0.05). Transwell invasion assay results showed that the total number of cells in the PET film of the experimental groups was significantly decreased compared with the control group (P<0.05). The cell scratch test showed that the cell closure time of the scratch in the interference group was significantly longer than those in the control and blank groups (P<0.05).@*CONCLUSIONS@#Silencing Rce1 in vitro can effectively downregulate its expression in tongue squamous cell carcinoma cells Cal-27 and SCC-4 and reduce the migration and invasion abilities of these cells.


Subject(s)
Cell Line, Tumor , Cell Movement , Cell Proliferation , Endopeptidases , Metabolism , Humans , Neoplasm Invasiveness , RNA Interference , RNA, Small Interfering , Tongue Neoplasms , Metabolism , Therapeutics , Transfection
10.
Mycobiology ; : 207-216, 2019.
Article in English | WPRIM | ID: wpr-760538

ABSTRACT

Talaromyces marneffei is the only dimorphic species in its genus and causes a fatal systemic mycosis named talaromycosis. Our previous study indicated that knockdown of AcuD gene (encodes isocitrate lyase of glyoxylate bypass) of T. marneffei by RNA interference approach attenuated the virulence of T. marneffei, while the virulence of the AcuD knockout strains was not studied. In this study, T. marneffei-zebrafish infection model was successfully established through hindbrain microinjection with different amounts of T. marneffei yeast cells. After co-incubated at 28°C, the increasing T. marneffei inoculum doses result in greater larval mortality; and hyphae generation might be one virulence factor involved in T. marneffei-zebrafish infection. Moreover, the results demonstrated that the virulence of the ΔAcuD was significantly attenuated in this Zebrafish infection model.


Subject(s)
Gene Knockout Techniques , Hyphae , Isocitrate Lyase , Microinjections , Mortality , Rhombencephalon , RNA Interference , Talaromyces , Virulence , Yeasts , Zebrafish
11.
Article in English | WPRIM | ID: wpr-739653

ABSTRACT

Suppressor of Variegation 3–9 Homolog 2 (SUV39H2) methylates the lysine 9 residue of histone H3 and induces heterochromatin formation, resulting in transcriptional repression or silencing of target genes. SUV39H1 and SUV39H2 have a role in embryonic development, and SUV39H1 was shown to suppress cell cycle progression associated with Rb. However, the function of human SUV39H2 has not been extensively studied. We observed that forced expression of SUV39H2 decreased cell proliferation by inducing G1 cell cycle arrest. In addition, SUV39H2 was degraded through the ubiquitin-proteasomal pathway. Using yeast two-hybrid screening to address the degradation mechanism and function of SUV39H2, we identified translationally controlled tumor protein (TCTP) as an SUV39H2-interacting molecule. Mapping of the interacting regions indicated that the N-terminal 60 amino acids (aa) of full-length SUV39H2 and the C-terminus of TCTP (120–172 aa) were critical for binding. The interaction of SUV39H2 and TCTP was further confirmed by co-immunoprecipitation and immunofluorescence staining for colocalization. Moreover, depletion of TCTP by RNAi led to up-regulation of SUV39H2 protein, while TCTP overexpression reduced SUV39H2 protein level. The half-life of SUV39H2 protein was significantly extended upon TCTP depletion. These results clearly indicate that TCTP negatively regulates the expression of SUV39H2 post-translationally. Furthermore, SUV39H2 induced apoptotic cell death in TCTP-knockdown cells. Taken together, we identified SUV39H2, as a novel target protein of TCTP and demonstrated that SUV39H2 regulates cell proliferation of lung cancer cells.


Subject(s)
Amino Acids , Apoptosis , Carrier Proteins , Cell Cycle , Cell Death , Cell Proliferation , Embryonic Development , Female , Fluorescent Antibody Technique , G1 Phase Cell Cycle Checkpoints , Half-Life , Heterochromatin , Histones , Humans , Immunoprecipitation , Lung Neoplasms , Lysine , Mass Screening , Pregnancy , Repression, Psychology , RNA Interference , Up-Regulation , Yeasts
12.
Article in English | WPRIM | ID: wpr-742359

ABSTRACT

PURPOSE: This study investigated the role of natriuretic peptide receptor 2 (NPR2) on cell proliferation and testosterone secretion in mouse Leydig cells. MATERIALS AND METHODS: Mouse testis of different postnatal stages was isolated to detect the expression C-type natriuretic peptide (CNP) and its receptor NPR2 by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Leydig cells isolated from mouse testis were cultured and treated with shNPR2 lentiviruses or CNP. And then the cyclic guanosine monophosphate production, testosterone secretion, cell proliferation, cell cycle and cell apoptosis in mouse Leydig cells were analyzed by ELISA, RT-qPCR, Cell Counting Kit-8, and flow cytometry. Moreover, the expression of NPR2, cell cycle, apoptosis proliferation and cell cycle related gene were detected by RT-qPCR and Western blot. RESULTS: Knockdown of NPR2 by RNAi resulted in S phase cell cycle arrest, cell apoptosis, and decreased testosterone secretion in mouse Leydig cells. CONCLUSIONS: Our study provides more evidences to better understand the function of CNP/NPR2 pathway in male reproduction, which may help us to treat male infertility.


Subject(s)
Animals , Apoptosis , Blotting, Western , Cell Count , Cell Cycle , Cell Cycle Checkpoints , Cell Proliferation , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Germ Cells , Guanosine Monophosphate , Humans , Infertility, Male , Lentivirus , Leydig Cells , Male , Mice , Natriuretic Peptide, C-Type , Polymerase Chain Reaction , Receptors, Peptide , Reproduction , Reverse Transcription , RNA Interference , S Phase , Testicular Diseases , Testis , Testosterone
13.
Electron. j. biotechnol ; 35: 25-32, sept. 2018. graf, ilus
Article in English | LILACS | ID: biblio-1047765

ABSTRACT

Background: Pollen development is an important reproductive process that directly affects pollen fertility and grain yield in rice. Argonaute (AGO) proteins, the core effectors of RNA-mediated silencing, play important roles in regulating plant growth and development. However, few AGO proteins in rice were reported to be involved in pollen development. In this study, artificial microRNA technology was used to assess the function of OsAGO17 in pollen development. Results: In this study, OsAGO17, a rice-specific gene, was specifically expressed in rice pollen grains, with the highest expression in uninucleate microspores. Downregulation of OsAGO17 by artificial microRNA technology based on the endogenous osa-miRNA319a precursor was successfully achieved. It is found that downregulation of OsAGO17 could significantly affect pollen fertility and cause pollen abortion, thus suggesting that OsAGO17 functions in rice pollen development. In addition, the downregulation of OsAGO17 mainly caused a low seed-setting rate, thereby resulting in the reduction of grain yield, whereas the downregulation of OsAGO17 did not significantly affect rice vegetative growth and other agricultural traits including number of florets per panicle, number of primary branch per panicle, and 100-grain weight. Furthermore, the result of subcellular localization analysis indicated that the OsAGO17 protein was localized to both the nucleus and the cytoplasm. Conclusion: These results represent the first report of the biological function for OsAGO17 in rice and indicate that OsAGO17 may possibly play crucial regulatory roles in rice pollen development. It helps us to better understand the mechanism of pollen development in rice.


Subject(s)
Pollen/growth & development , Oryza/growth & development , Down-Regulation , Argonaute Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , MicroRNAs , RNA Interference , Fertility , Argonaute Proteins/genetics
14.
Braz. j. med. biol. res ; 51(2): e6768, 2018. graf
Article in English | LILACS | ID: biblio-889019

ABSTRACT

This study aimed to investigate the mechanism of hypoxia-inducible factor-1 alpha (HIF-1α) mediated hypoxia-induced permeability changes in bladder endothelial cells. Models of in vitro hypoxic cell culture of bladder cancer, bladder cancer cells with low HIF-1α expression and HIF-1α RNA interference (RNAi) expression vector were established. Western blot and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of HIF-1α and vascular endothelial growth factor (VEGF) in each group. Bladder cell permeability was determined. Results showed that protein and mRNA expression of HIF-1α and VEGF at 3 and 12 h of hypoxia were significantly higher than normal control (P<0.05), and peaked at 12 h. HIF-1α and VEGF expression in the hypoxic group and hypoxic+3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1) group were significantly higher than normal control (P<0.05), while expression in the hypoxic+YC-1 group was significantly lower than the hypoxic group (P<0.05). Bladder cell permeability in the hypoxic and hypoxic+YC-1 group were significantly increased compared to normal control (P<0.05), while in the hypoxic+YC-1 group was significantly decreased compared to the hypoxic group (P<0.05). Most of the cells in the stably transfected HIF-1α RNAi expression vector pcDNA6.2-GW/EmGFP-miR-siHIF-1α expressed green fluorescence protein (GFP) under fluorescence microscope. pcDNA6.2-GW/EmGFP-miR-siHIF-1α could significantly inhibit HIF-1α gene expression (P<0.05). HIF-1α and VEGF expression in the hypoxic group and siHIF-1α hypoxic group were significantly higher than normal group (P<0.05), while expression in the siHIF-1α hypoxic group was significantly lower than the hypoxic group (P<0.05). Findings suggest that HIF-1α is an important factor in the increase of bladder cancer cell permeability.


Subject(s)
Animals , Rats , Urinary Bladder Neoplasms/metabolism , Endothelial Cells/physiology , Vascular Endothelial Growth Factor A/physiology , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , Tumor Hypoxia/physiology , Permeability , Gene Expression Regulation, Neoplastic/physiology , Blotting, Western , RNA Interference , Cell Line, Tumor , Endothelial Cells/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/analysis , Real-Time Polymerase Chain Reaction
15.
São Paulo; s.n; s.n; 2018. 174 p. graf, tab, ilus.
Thesis in Portuguese | LILACS | ID: biblio-996534

ABSTRACT

O adenocarcinoma ductal pancreático (PDAC, pancreatic ductal adenocarcinoma), o tipo mais prevalente de câncer do pâncreas, é uma neoplasia extremamente agressiva e com elevado índice de letalidade. Há uma necessidade premente de identificação de vulnerabilidades no PDAC que possam ser exploradas como alvos terapêuticos, e a utilização de modelos pré-clínicos que recapitulem a complexidade biológica e heterogeneidade clínica da doença é um aspecto central para a realização dessa tarefa. Os xenotransplantes de tecido tumoral derivado de pacientes (PDX, patient-derived tumor tissue xenografts), realizados em camundongos imunodeficientes, replicam com grande similaridade as principais características do tumor original e, assim, constituem uma ferramenta valiosa para o teste de drogas e estudos funcionais. Neste trabalho, 17 amostras cirúrgicas de PDAC humano foram implantadas subcutaneamente em camundongos nude atímicos. Sete tumores (41%) foram enxertados com sucesso e têm sido mantidos em sucessivas gerações de animais receptores. O exame histológico de seis desses xenoenxertos identificou características morfológicas compatíveis com os padrões reconhecidos no PDAC humano, assim como uma consistente similaridade de seu status de diferenciação histológica em relação aos perfis verificados nos tumoresoriginais. O cultivo in vitro de células derivadas de um dos xenotumores resultou em uma nova linhagem de câncer de pâncreas, com morfologia e cinética de crescimento comparáveis às de outras linhagens celulares de câncer pancreático. O potencial tumorigênico dessa nova linhagem foi validado in vivo, com uma consistente formação de tumores após inoculação em camundongos nude. A fim de aproveitar esse recurso para a investigação de potenciais alvos terapêuticos no PDAC, um rastreamento de vulnerabilidades moleculares foi realizado por meio de silenciamento gênico em larga-escala com RNA de interferência (RNAi). Uma biblioteca lentiviral de 4492 shRNAs (short hairpin RNAs), alvejando cerca de 350 genes envolvidos na regulação epigenética, foi empregada para a triagem de genes de suscetibilidade nas células derivadas de PDX, e em outras cinco linhagens tumorais pancreáticas (AsPC-1, BxPC-3, Capan-1, MIA PaCa-2 e PANC-1). Inicialmente, foi realizada uma série de experimentos preliminares, visando à amplificação e controle de qualidade da biblioteca de silenciamento, à produção de vetores lentivirais e à padronização das condições experimentais para a transdução e seleção das células-alvo. Apenas três das linhagens avaliadas (AsPC-1, MIA PaCa-2 e PANC-1) mostraram-se permissíveis à transdução pelos vetores lentivirais, e foram assim utilizadas no screening de alvos epigenéticos. A análise dos dados obtidos nesse ensaio está em curso e os resultados serão utilizados para a definição de potenciais alvos candidatos. Em conclusão, recursos valiosos para apoiar a pesquisa sobre o câncer de pâncreas foram desenvolvidos. A coleção de PDXs estabelecida, bem como a linhagem celular recém-derivada, constituem uma fonte permanente e estável de células de PDAC para análises moleculares e estudos funcionais que busquem elucidar aspectos da doença ainda pouco compreendidos. Adicionalmente, os reagentes gerados e a expertise adquirida com os ensaiosrealizados com a biblioteca de shRNAs contra alvos epigenéticos serão de grande utilidade em futuras investigações para identificar genes com funções importantes na manutenção do fenótipo tumoral, e consequentemente com potencial para serem explorados terapeuticamente


Pancreatic ductal adenocarcinoma (PDAC), the most prevalent type of pancreatic cancer, is a highly aggressive and lethal neoplasm. There is a pressing need to identify vulnerabilities in PDAC suited to be exploited as therapeutic targets, and the use of preclinical models recapitulating the biological complexity and clinical heterogeneity of the disease is central to this task. Patient-derived tumor tissue xenografts (PDX), established in immunodeficient mice, replicate with great similarity the main characteristics of the original tumor and thus constitute a valuable tool for drug testing and functional studies. In this work, 17 surgical samples of human PDAC were implanted subcutaneously in athymic nude mice. Seven tumors (41%) were successfully grafted and have been maintained through successive generations of recipient animals. Histological examination of six of these xenografts identified morphological characteristics compatible with the recognized patterns of human PDAC, as well as a consistent similarity of their histological differentiation status in relation to the profiles verified in the original tumors. In vitro culture of cells derived from one of these xenografts resulted in a new pancreatic cancer cell line, with morphology and growth kinetics comparable to those of other pancreatic tumor cells. The tumorigenic potential of this freshly derived cell line was validated in vivo, with a consistent tumor formation following inoculation into nude mice. To take advantage ofthis resource to investigate potential therapeutic targets in PDAC, a screening of molecular vulnerabilities was performed through large-scale gene silencing with RNA interference (RNAi). A lentiviral library containing 4492 short hairpin RNAs (shRNAs), targeting about 350 genes involved in epigenetic regulation, was employed for the search of susceptibility genes in the PDX-derived cells and in other five pancreatic tumor cell lines (AsPC-1, BxPC -3, Capan-1, MIA PaCa-2 and PANC-1). Initially, a series of preliminary experiments were carried out aiming at the amplification and quality control of the silencing library, production of lentiviral vectors and adjustment of the experimental conditions for transduction and selection of the target cells. Only three of the cell lines evaluated (AsPC-1, MIA PaCa-2 and PANC-1) were permissible for transduction by the lentiviral vectors, and were accordingly used in the screening of epigenetic targets. The analysis of data obtained in this trial is ongoing and the results will be used for definition of potential candidate targets. In conclusion, valuable resources to support research on pancreatic cancer have been developed. The established collection of PDXs as well as the newly derived cell line constitutes a permanent and stable source of PDAC cells for molecular analyzes and functional studies seeking to elucidate aspects of this disease that are still poorly understood. Additionally, both the reagents generated and the expertise gained from the RNAi assay against epigenetic targets will have inordinate usefulness in future investigations to identify genes with major functions in maintaining the malignant phenotype, and consequently with the potential to be exploited therapeutically


Subject(s)
Animals , Female , Mice , Pancreatic Neoplasms/physiopathology , Cell Line, Tumor/classification , Heterografts/metabolism , Transplantation, Heterologous/instrumentation , Gene Library , RNA, Small Interfering , RNA Interference , Epigenomics/standards
16.
Article in Chinese | WPRIM | ID: wpr-813223

ABSTRACT

To investigate the effect of manganese superoxide dismutase (MnSOD) silence on the in vitro tumorigenicity in human small cell lung cancer NCI-H446 cells and the underlying mechanisms.
 Methods: Sphere formation cells from NCI-H446 cells were obtained by suspension culture, while the expression of MnSOD and urokinase type plasminogen activator (uPAR) was analyzed by Western blot. Silence of MnSOD was performed by adenovirus infection in the second passage formation cells, and the effect of MnSOD silence on tumorigenicity in NCI-H446 cells was evaluated by sphere formation assay and soft-agar colony formation assay, while the expression of uPAR was analyzed by Western blot.
 Results: Compared with NCI-H446 cells, the sphere formation rate, colony formation rate, and the expression of MnSOD and uPAR were significantly increased in the second passage sphere formation cells in NCI-H446 cells (P<0.05). Silence of MnSOD inhibited the sphere formation rate, colony formation rate, and the expression level of uPAR in the second passage sphere formation cells in NCI-H446 cells.
 Conclusion: MnSOD may promote tumorigenicity in NCI-H446 cells by up-regulation of uPAR expression in vitro.


Subject(s)
Adenoviridae , Carcinogenesis , Cell Line, Tumor , Humans , In Vitro Techniques , Lung Neoplasms , Metabolism , RNA Interference , Receptors, Urokinase Plasminogen Activator , Genetics , Metabolism , Small Cell Lung Carcinoma , Metabolism , Spheroids, Cellular , Pathology , Superoxide Dismutase , Genetics , Metabolism , Tumor Stem Cell Assay , Up-Regulation
17.
Chinese Medical Journal ; (24): 2097-2104, 2018.
Article in English | WPRIM | ID: wpr-773921

ABSTRACT

Background@#Signal transducer and activator of transcription 3 (STAT3) was strongly expressed and activated in psoriatic keratinocytes (KCs) and correlated with the severity of psoriasis. The study aimed to investigate the effects of STAT3 small interfering RNA (siRNA) combined with ultrasonic irradiation and SonoVue microbubbles on the proliferation and apoptosis in KCs of psoriatic lesions and the relative mechanisms.@*Methods@#Psoriatic KCs were transfected under four experimental conditions: (1) STAT3 siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (LUS group); (2) STAT3 siRNA only carried by Lipofectamine 3000 (L group); (3) the negative control of siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (siRNA-NC); (4) not treated as Blank. Cell Counting Kit-8 assay was used to evaluate the cell proliferation. Cell cycle analysis was detected with cycle test Plus DNA reagent kit associated with flow cytometer. FITC Annexin V apoptosis detection kit associated with flow cytometer was applied for apoptosis analysis. Fluo calcium indicator associated with flow cytometer was used to analyze intracellular free calcium concentration ([Ca]). The expressions of cyclin D1 and Bcl-xL were detected both at the mRNA level by real-time reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by Western blotting. The obtained data were statistically evaluated by two-way analysis of variance.@*Results@#STAT3 siRNA inhibited the growth of KCs in a time-dependent manner showing the highest proliferation inhibition in LUS group with proliferation ratio of 45.38% ± 5.85% at 72h (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced an altered cell cycle distribution of KCs showing the highest increases in G2/M-phase population up to 18.06% ± 0.36% in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced late apoptosis of KCs with the highest late apoptosis percentage of 22.87% ± 1.28% in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced the elevation of [Ca]of KCs with the highest calcium fluorescence intensity mean of 1213.67 ± 60.51 in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced the downregulation of cyclin D1 and Bcl-xL expressions of KCs at mRNA and protein levels with the lowest expressions in LUS group with cyclin D1 expression of 51.81% ± 9.58% and 70.17% ± 4.22% at mRNA level and at protein level, respectively, and with Bcl-xL expression of 37.58% ± 4.92% and 64.06% ± 7.78% at mRNA level and at protein level, respectively (P < 0.05 vs. L group, siRNA-NC, or Blank).@*Conclusions@#STAT3 siRNA inhibited the growth and induced the apoptosis in psoriatic KCs likely partly through altering cell cycle distribution, elevating [Ca], and downregulating cyclin D1 and Bcl-xL expressions. Silencing the target gene STAT3 in psoriatic KCs with siRNA combined with ultrasonic irradiation and microbubbles would contribute to a significant innovation as a new clinical therapy for psoriasis.


Subject(s)
Apoptosis , Cell Line, Tumor , Cell Proliferation , Humans , Keratinocytes , Microbubbles , Phospholipids , Psoriasis , Therapeutics , RNA Interference , RNA, Small Interfering , STAT3 Transcription Factor , Metabolism , Sulfur Hexafluoride , Ultrasonics
18.
Journal of Experimental Hematology ; (6): 1637-1643, 2018.
Article in Chinese | WPRIM | ID: wpr-773043

ABSTRACT

OBJECTIVE@#To investigate the expression changes of serum transferrin receptor(sTFR) and its related mechanism in children with acute leukemia(AL).@*METHODS@#Forty-six children with acute leukemia treated in our hospital from June 2016 to June 2017 were selected and enrolled in the AL group, 40 healthy children were enrolled in the control group. The related clinical data were recorded, including age, sex and CNSL level. RNA interference technology was used to silence TFR genes of KG-1a and TCHu147 cells, MTT method and flow cytometry were used to analyze the effect of TFR gene on proliferation and cell cycle of KG-1a cells and TCHu147 cells. Western blot was used to detect the level of cyclin related to leukemic cells after siRNA interference.@*RESULTS@#The level of sTFR in AL patients was significantly higher than that of healthy people (P<0.05). The mRNA and protein expression levels of TFR in peripheral blood leukemic cells were all higher than those in healthy people (P<0.05). The level of sTFR closely related to the white blood cell(WBC) count, the proportion and absolute number of leukemic cells, hepcidin(Hepc) level, and risk grade in AL patients (P<0.05). The proliferation ability of KG-1a and TCHu147 cells after TFR siRNA interference was significantly inhibited (P<0.05). Fow cytometry showed that after the TFR siRNA interference, the ratio of KG-1a and TCHu147 cells in G/G phase was 62.51%±5.39% and 63.37%±4.27%, respectively, which increased significantly as compared with the blank and negative control group (P<0.05); the ratio of KG-1a and TCHu147 in G/M phase was 5.74%±1.34% and 7.37%±1.56%, respectively, which significantly decreased as compared with the blank control and the negative control group (P<0.05).@*CONCLUSION@#The peripheral blood leukemic cells of AL patients can synthesize more TFR protein, lead into the increase of sTFR level. It can effectively interfere the division of leukemia cells by downregulating the expression of TFR gene.


Subject(s)
Acute Disease , Cell Cycle , Cell Proliferation , Child , Humans , Leukemia , RNA Interference , RNA, Small Interfering , Receptors, Transferrin
19.
Article in English | WPRIM | ID: wpr-772994

ABSTRACT

Precision oncology aims to offer the most appropriate treatments to cancer patients mainly based on their individual genetic information. Genomics has provided numerous valuable data on driver mutations and risk loci; however, it remains a formidable challenge to transform these data into therapeutic agents. Transcriptomics describes the multifarious expression patterns of both mRNAs and non-coding RNAs (ncRNAs), which facilitates the deciphering of genomic codes. In this review, we take breast cancer as an example to demonstrate the applications of these rich RNA resources in precision medicine exploration. These include the use of mRNA profiles in triple-negative breast cancer (TNBC) subtyping to inform corresponding candidate targeted therapies; current advancements and achievements of high-throughput RNA interference (RNAi) screening technologies in breast cancer; and microRNAs as functional signatures for defining cell identities and regulating the biological activities of breast cancer cells. We summarize the benefits of transcriptomic analyses in breast cancer management and propose that unscrambling the core signaling networks of cancer may be an important task of multiple-omic data integration for precision oncology.


Subject(s)
Female , Gene Expression Profiling , Genomics , Humans , MicroRNAs , Metabolism , Precision Medicine , RNA Interference , RNA, Messenger , Metabolism , Triple Negative Breast Neoplasms , Classification , Genetics , Metabolism , Therapeutics
20.
Article in English | WPRIM | ID: wpr-772974

ABSTRACT

DNA damage response (DDR) is essential for maintaining genome stability and protecting cells from tumorigenesis. Ubiquitin and ubiquitin-like modifications play an important role in DDR, from signaling DNA damage to mediating DNA repair. In this report, we found that the E3 ligase ring finger protein 126 (RNF126) was recruited to UV laser micro-irradiation-induced stripes in a RNF8-dependent manner. RNF126 directly interacted with and ubiquitinated another E3 ligase, RNF168. Overexpression of wild type RNF126, but not catalytically-inactive mutant RNF126 (CC229/232AA), diminished ubiquitination of H2A histone family member X (H2AX), and subsequent bleomycin-induced focus formation of total ubiquitin FK2, TP53-binding protein 1 (53BP1), and receptor-associated protein 80 (RAP80). Interestingly, both RNF126 overexpression and RNF126 downregulation compromised homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSBs). Taken together, our findings demonstrate that RNF126 negatively regulates RNF168 function in DDR and its appropriate cellular expression levels are essential for HR-mediated DSB repair.


Subject(s)
Carrier Proteins , Metabolism , Cell Line, Tumor , DNA Breaks, Double-Stranded , DNA Repair , Genetics , DNA-Binding Proteins , Metabolism , Genomic Instability , HeLa Cells , Histones , Metabolism , Humans , Nuclear Proteins , Metabolism , RNA Interference , RNA, Small Interfering , Genetics , Signal Transduction , Tumor Suppressor p53-Binding Protein 1 , Metabolism , Ubiquitin , Ubiquitin-Protein Ligases , Genetics , Metabolism , Ubiquitination
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