Objective: To construct the genetically engineered macrophages expressing Smad6 and Smad7 genes with adeno-associated virus (AAV). Methods: The plasmids containing pcDNA3-Smad6/Flag and pcDNA3-Smad7/Flag were digested...Objective: To construct the genetically engineered macrophages expressing Smad6 and Smad7 genes with adeno-associated virus (AAV). Methods: The plasmids containing pcDNA3-Smad6/Flag and pcDNA3-Smad7/Flag were digested with BamHⅠ and XhoⅠ, respectively. Then the Smad6/Flag and Smad7/Flag gene segments obtained were cloned into plasmid pAAV-MCS respectively to construct the recombinant pAAV-Smad6/Flag and pAAV-Smad7/Flag plasmids. The resulting recombinant plasmids (pAAV-Smad6/Flag or pAAV-Smad7/Flag) or pAAV-LacZ plasmid were co-transfected into the HEK 293cells with pHelper and pAAV-RC by calcium-phosphate precipitation method. Recombinant AAV-2 viral particles were prepared from infected HEK293 cells and then were used to infect mouse macrophages. The expressions of Smad6 and Smad7 in macrophages were detected by immunocytochemical staining and expression of b-galactosidase was evaluated by X-gal staining. Results: The recombinant AAV vector containing Smad6 or Smad7 genes was successfully constructed. More than 95% macrophage cells expressed X-gal and Smad6 and Smad7 genes at 72 h after infection. Conclusion: These results indicate that the genetically engineered macrophages can express Smad6 and Smad7 proteins effectively, laying the foundation for the studies of TGF-β-induced diseases in vivo and highlighting the feasibility of macrophage-based gene therapy.展开更多
Inhibitory Smads(I-Smads),which belong to the Smad family and inhibit bone morphogenic protein 2(BMP2)signaling by a variety of mechanisms,can suppress innate immunity responses in vertebrates.However,there are no rep...Inhibitory Smads(I-Smads),which belong to the Smad family and inhibit bone morphogenic protein 2(BMP2)signaling by a variety of mechanisms,can suppress innate immunity responses in vertebrates.However,there are no reports for the role of Smad6 in immunity in mollusks.In this study,we showed that Smad6 of the pearl oyster Pinctada fucata martensii was located in the Smad6 cluster of the phylogenetic tree;mRNA expression of Smad6 and Smad3 was up-regulated after lipopolysaccharide and polyinosinic:polycytidylic challenge;and transcript levels of Smad6 and Smad3 showed opposite patterns during wound healing.Under salinity stress,water inflow and outflow in the gills appear to be regulated by BMP2-Smads signals,and BMP2-Smads signaling may be closely related to the immune response.Our results indicate that Smad6 is involved in immunity,that it plays a positive role in the response to immune challenge and an inhibitory role during wound healing,and that Smad6 and Smad3 may work against each other.展开更多
BACKGROUND Pancreatic adenocarcinoma(PAAD)is a cancerous tumor with an extremely poor 5-year survival rate.The exploration of biomarkers for the diagnosis and treatment of PAAD is crucial in clinical practice.Krü...BACKGROUND Pancreatic adenocarcinoma(PAAD)is a cancerous tumor with an extremely poor 5-year survival rate.The exploration of biomarkers for the diagnosis and treatment of PAAD is crucial in clinical practice.Krüppel-like factors(KLFs)are involved in a variety of biological functions in cells.According to multiple studies,KLF16 behave as an oncogene in prostate,breast and gastric cancers.However,no research has been done on the significance of KLF16 in PAAD.AIM To explore the molecular mechanisms of KLF16 in PAAD.METHODS KLF16 was identified in the tumor specimens and normal tissues by GEPIA database and verified by quantitative real-time PCR(qRT-PCR).Knockdown or exogenous expression of KLF16,combined with in vitro and in vivo assays,was performed to show the functional significance of KLF16.The molecular mechanism of KLF16 was demonstrated by qRT-PCR,western blotting,immunoprecipitation assay and flow cytometry.RESULTS We showed that KLF16 was highly expressed in PAAD patients based on the GEPIA database.KLF16 silencing suppressed while KLF16 overexpression promoted the malignant function of PAAD cells.Based on RNA sequencing,we discovered that KLF16 potentiated the expression of SMAD6 in PAAD cells.SMAD6 transcript abundance was increased and positively correlated with KLF16 expression in PAAD samples.In addition,inhibiting SMAD6 was able to mitigate the effects of KLF16 overexpression on PAAD cell processes,suggesting the importance of SMAD6 in the development of KLF16-triggered PAAD.CONCLUSION KLF16/SMAD6 axis might be explored as a therapeutic target for PAAD therapy.展开更多
The BMP signaling pathway plays a crucial role in regulating early embryonic development and tissue homeostasis.SMAD6 encodes a negative regulator of BMP,and rare variants of SMAD6 are recurrently found in individuals...The BMP signaling pathway plays a crucial role in regulating early embryonic development and tissue homeostasis.SMAD6 encodes a negative regulator of BMP,and rare variants of SMAD6 are recurrently found in individuals with birth defects.However,we observed that a subset of rare pathogenic variants of SMAD6 consistently exhibited positive regulatory effects instead of the initial negative effects on the BMP signaling pathway.We sought to determine whether these SMAD6 variants have common pathogenic mechanisms.Here,we showed that pathogenic SMAD6 variants accompanying this functional reversal exhibit similar increases in deamidation.Mechanistically,increased deamidation of SMAD6 variants promotes the accumulation of the BMP receptor BMPR1A and the formation of new complexes,both of which lead to BMP signaling pathway activation.Specifically,two residues,N262 and N404,in SMAD6 were identified as the crucial sites of deamidation,which was catalyzed primarily by glutamine-fructose-6-phosphate transaminase 2(GFPT2).Additionally,treatment of cells harboring SMAD6 variants with a deamidase inhibitor restored the inhibitory effect of SMAD6 on the BMP signaling pathway.Conversely,when wild-type SMAD6 was manually simulated to mimic the deamidated state,the reversed function of activating BMP signaling was reproduced.Taken together,these findings show that deamidation of SMAD6 plays a crucial role in the functional reversal of BMP signaling activity,which can be induced by a subset of various SMAD6 variants.Our study reveals a common pathogenic mechanism shared by these variants and provides a potential strategy for preventing birth defects through deamidation regulation,which might prevent the off-target effects of gene editing.展开更多
文摘Objective: To construct the genetically engineered macrophages expressing Smad6 and Smad7 genes with adeno-associated virus (AAV). Methods: The plasmids containing pcDNA3-Smad6/Flag and pcDNA3-Smad7/Flag were digested with BamHⅠ and XhoⅠ, respectively. Then the Smad6/Flag and Smad7/Flag gene segments obtained were cloned into plasmid pAAV-MCS respectively to construct the recombinant pAAV-Smad6/Flag and pAAV-Smad7/Flag plasmids. The resulting recombinant plasmids (pAAV-Smad6/Flag or pAAV-Smad7/Flag) or pAAV-LacZ plasmid were co-transfected into the HEK 293cells with pHelper and pAAV-RC by calcium-phosphate precipitation method. Recombinant AAV-2 viral particles were prepared from infected HEK293 cells and then were used to infect mouse macrophages. The expressions of Smad6 and Smad7 in macrophages were detected by immunocytochemical staining and expression of b-galactosidase was evaluated by X-gal staining. Results: The recombinant AAV vector containing Smad6 or Smad7 genes was successfully constructed. More than 95% macrophage cells expressed X-gal and Smad6 and Smad7 genes at 72 h after infection. Conclusion: These results indicate that the genetically engineered macrophages can express Smad6 and Smad7 proteins effectively, laying the foundation for the studies of TGF-β-induced diseases in vivo and highlighting the feasibility of macrophage-based gene therapy.
基金Supported by the Natural Science Foundation of Guangdong Province,China(No.2019A1515011968)the Key Special Project for Introduced Talents Team of the Southern Marine Science and Engineering Guangdong Laboratory(Guangzhou)(No.GML2019ZD0401)+1 种基金the Earmarked Fund for the Modern Agro-industry Technology Research System(No.CARS-49)the Science and Technology Planning Project of Guangdong Province,China(No.2020B1212060058)。
文摘Inhibitory Smads(I-Smads),which belong to the Smad family and inhibit bone morphogenic protein 2(BMP2)signaling by a variety of mechanisms,can suppress innate immunity responses in vertebrates.However,there are no reports for the role of Smad6 in immunity in mollusks.In this study,we showed that Smad6 of the pearl oyster Pinctada fucata martensii was located in the Smad6 cluster of the phylogenetic tree;mRNA expression of Smad6 and Smad3 was up-regulated after lipopolysaccharide and polyinosinic:polycytidylic challenge;and transcript levels of Smad6 and Smad3 showed opposite patterns during wound healing.Under salinity stress,water inflow and outflow in the gills appear to be regulated by BMP2-Smads signals,and BMP2-Smads signaling may be closely related to the immune response.Our results indicate that Smad6 is involved in immunity,that it plays a positive role in the response to immune challenge and an inhibitory role during wound healing,and that Smad6 and Smad3 may work against each other.
基金approved by the Ethics Committee of Beijing Friendship Hospital,Capital Medical University(No.2018-P2-015-02).
文摘BACKGROUND Pancreatic adenocarcinoma(PAAD)is a cancerous tumor with an extremely poor 5-year survival rate.The exploration of biomarkers for the diagnosis and treatment of PAAD is crucial in clinical practice.Krüppel-like factors(KLFs)are involved in a variety of biological functions in cells.According to multiple studies,KLF16 behave as an oncogene in prostate,breast and gastric cancers.However,no research has been done on the significance of KLF16 in PAAD.AIM To explore the molecular mechanisms of KLF16 in PAAD.METHODS KLF16 was identified in the tumor specimens and normal tissues by GEPIA database and verified by quantitative real-time PCR(qRT-PCR).Knockdown or exogenous expression of KLF16,combined with in vitro and in vivo assays,was performed to show the functional significance of KLF16.The molecular mechanism of KLF16 was demonstrated by qRT-PCR,western blotting,immunoprecipitation assay and flow cytometry.RESULTS We showed that KLF16 was highly expressed in PAAD patients based on the GEPIA database.KLF16 silencing suppressed while KLF16 overexpression promoted the malignant function of PAAD cells.Based on RNA sequencing,we discovered that KLF16 potentiated the expression of SMAD6 in PAAD cells.SMAD6 transcript abundance was increased and positively correlated with KLF16 expression in PAAD samples.In addition,inhibiting SMAD6 was able to mitigate the effects of KLF16 overexpression on PAAD cell processes,suggesting the importance of SMAD6 in the development of KLF16-triggered PAAD.CONCLUSION KLF16/SMAD6 axis might be explored as a therapeutic target for PAAD therapy.
基金supported by the National Key Research and Development Program of China(2021YFC2701101)the National Natural Science Foundation of China(82150008,81930036)+1 种基金Commission for Science and Technology of Shanghai Municipality(20JC1418500)Open Fund Project of Guangdong Academy of Medical Sciences(YKY-KF202202)。
文摘The BMP signaling pathway plays a crucial role in regulating early embryonic development and tissue homeostasis.SMAD6 encodes a negative regulator of BMP,and rare variants of SMAD6 are recurrently found in individuals with birth defects.However,we observed that a subset of rare pathogenic variants of SMAD6 consistently exhibited positive regulatory effects instead of the initial negative effects on the BMP signaling pathway.We sought to determine whether these SMAD6 variants have common pathogenic mechanisms.Here,we showed that pathogenic SMAD6 variants accompanying this functional reversal exhibit similar increases in deamidation.Mechanistically,increased deamidation of SMAD6 variants promotes the accumulation of the BMP receptor BMPR1A and the formation of new complexes,both of which lead to BMP signaling pathway activation.Specifically,two residues,N262 and N404,in SMAD6 were identified as the crucial sites of deamidation,which was catalyzed primarily by glutamine-fructose-6-phosphate transaminase 2(GFPT2).Additionally,treatment of cells harboring SMAD6 variants with a deamidase inhibitor restored the inhibitory effect of SMAD6 on the BMP signaling pathway.Conversely,when wild-type SMAD6 was manually simulated to mimic the deamidated state,the reversed function of activating BMP signaling was reproduced.Taken together,these findings show that deamidation of SMAD6 plays a crucial role in the functional reversal of BMP signaling activity,which can be induced by a subset of various SMAD6 variants.Our study reveals a common pathogenic mechanism shared by these variants and provides a potential strategy for preventing birth defects through deamidation regulation,which might prevent the off-target effects of gene editing.
