BACKGROUND A series of long non-coding RNAs(lncRNAs)have been reported to play a crucial role in cancer biology.Some previous studies report that lncRNA CDKN2B-AS1 is involved in some human malignancies.However,its ro...BACKGROUND A series of long non-coding RNAs(lncRNAs)have been reported to play a crucial role in cancer biology.Some previous studies report that lncRNA CDKN2B-AS1 is involved in some human malignancies.However,its role in hepatocellular carcinoma(HCC)has not been fully deciphered.AIM To decipher the role of CDKN2B-AS1 in the progression of HCC.METHODS CDKN2B-AS1 expression in HCC was detected by quantitative real-time polymerase chain reaction.The malignant phenotypes of Li-7 and SNU-182 cells were detected by the CCK-8 method,EdU method,and flow cytometry,respectively.RNA immunoprecipitation was executed to confirm the interaction between CDKN2B-AS1 and E2F transcription factor 1(E2F1).Luciferase reporter assay and chromatin immunoprecipitation were performed to verify the binding of E2F1 to the promoter of G protein subunit alpha Z(GNAZ).E2F1 and GNAZ were detected by western blot in HCC cells.RESULTS In HCC tissues,CDKN2B-AS1 was upregulated.Depletion of CDKN2B-AS1 inhibited the proliferation of HCC cells,and the depletion of CDKN2B-AS1 also induced cell cycle arrest and apoptosis.CDKN2B-AS1 could interact with E2F1.Depletion of CDKN2B-AS1 inhibited the binding of E2F1 to the GNAZ promoter region.Overexpression of E2F1 reversed the biological effects of depletion of CDKN2B-AS1 on the malignant behaviors of HCC cells.CONCLUSION CDKN2B-AS1 recruits E2F1 to facilitate GNAZ transcription to promote HCC progression.展开更多
POU transcription factor OCT4 not only plays an essential role in maintaining the pluripotent and self-renewing state of embryonic stem （ES） cells but also acts as a cell fate determinant through a gene dosage effec...POU transcription factor OCT4 not only plays an essential role in maintaining the pluripotent and self-renewing state of embryonic stem （ES） cells but also acts as a cell fate determinant through a gene dosage effect. However, the molecular mechanisms that control the intracellular OCT4 protein level remain elusive. Here, we report that human WWP2, an E3 ubiquitin （Ub）-protein ligase, interacts with OCT4 specifically through its WW domain and enhances Ub modification of OCT4 both in vitro and in vivo. We first demonstrated that endogenous OCT4 in hu- man ES cells can be post-translationally modified by Ub. Furthermore, we found that WWP2 promoted degradation of OCT4 through the 26S proteasome in a dosage-dependent manner, and the active site cysteine residue of WWP2 was required for both its enzymatic activity and proteolytic effect on OCT4. Remarkably, our data show that the en- dogenous OCT4 protein level was significantly elevated when WWP2 expression was downregulated by specific RNA interference （RNAi）, suggesting that WWP2 is an important regulator for maintaining a proper OCT4 protein level in human ES cells. Moreover, northern blot analysis showed that the WWP2 transcript was widely present in diverse human tissues/organs and highly expressed in undifferentiated human ES cells. However, its expression level was quickly decreased after human ES cells differentiated, indicating that WWP2 expression might be developmentally regulated. Our findings demonstrate that WWP2 is an important regulator of the OCT4 protein level in human ES cells.展开更多
BACKGROUND Heart diseases are the primary cause of death all over the world.Following myocardial infarction,billions of cells die,resulting in a huge loss of cardiac function.Stem cell-based therapies have appeared as...BACKGROUND Heart diseases are the primary cause of death all over the world.Following myocardial infarction,billions of cells die,resulting in a huge loss of cardiac function.Stem cell-based therapies have appeared as a new area to support heart regeneration.The transcription factors GATA binding protein 4(GATA-4)and myocyte enhancer factor 2C(MEF2C)are considered prominent factors in the development of the cardiovascular system.AIM To explore the potential of GATA-4 and MEF2C for the cardiac differentiation of human umbilical cord mesenchymal stem cells(hUC-MSCs).METHODS hUC-MSCs were characterized morphologically and immunologically by the presence of specific markers of MSCs via immunocytochemistry and flow cytometry,and by their potential to differentiate into osteocytes and adipocytes.hUC-MSCs were transfected with GATA-4,MEF2C,and their combination to direct the differentiation.Cardiac differentiation was confirmed by semiquant itative real-time polymerase chain reaction and immunocytochemistry.RESULTS hUC-MSCs expressed specific cell surface markers CD105,CD90,CD44,and vimentin but lack the expression of CD45.The transcription factors GATA-4 and MEF2C,and their combination induced differentiation in hUC-MSCs with significant expression of cardiac genes i.e.,GATA-4,MEF2C,NK2 homeobox 5(NKX2.5),MHC,and connexin-43,and cardiac proteins GATA-4,NKX2.5,cardiac troponin T,and connexin-43.CONCLUSION Transfection with GATA-4,MEF2C,and their combination effectively induces cardiac differentiation in hUC-MSCs.These genetically modified MSCs could be a promising treatment option for heart diseases in the future.展开更多
5-Aminolevulinic acid(ALA)can inhibit abscisic acid(ABA)-induced stomatal closure.However,the molecular mechanism is unclear.In this study,we found that ALA upregulated the MdPP2AC expression and PP2A activity in the ...5-Aminolevulinic acid(ALA)can inhibit abscisic acid(ABA)-induced stomatal closure.However,the molecular mechanism is unclear.In this study,we found that ALA upregulated the MdPP2AC expression and PP2A activity in the apple(Malus domestica Borkh.cv.‘Fuji’)leaves.With the promoter of MdPP2AC as bait,a diacylglycerol kinase MdDGK3-like was selected by the Yeast One Hybrid(Y1H)from the cDNA library of the epidermis of apple leaves treated by exogenous ALA.Additional to binding the promoter of MdPP2AC,MdDGK3-like was found to inhibit the transcription activity of MdPP2AC promoter,while ALA significantly eliminated the role of MdDGK3-like.In tobacco leaves,MdDGK3-like was localized in the nucleus of stomatal guard cells.Therefore,MdDGK3-like might act as a transcription factor negatively regulating MdPP2AC expression and causing stomatal closure.To further identify MdDGK3-like functions,several transiently transgenic apple leaves(including overexpression and interference)were established.The results revealed that overexpression of MdDGK3-like promoted stomatal closure by increasing Ca^(2+)and H_(2)O_(2)and decreasing flavonol levels in the guard cells.Conversely,MdDGK3-like(i)led the stomatal opening with lower levels of Ca^(2+)and H_(2)O_(2)but higher flavonols.Based on these,we proposed a new hypothesis that ALA up-regulated MdPP2AC expression via negatively regulating the expression of MdDGK3-like to up-regulate PP2A expression and the enzyme activity,which improved the stomatal aperture.Since it was the first time that MdDGK3-like was showed to act as a transcription factor,the proposed model provided a new insight onto the mechanisms of ALA-induced stomatal opening.展开更多
文摘BACKGROUND A series of long non-coding RNAs(lncRNAs)have been reported to play a crucial role in cancer biology.Some previous studies report that lncRNA CDKN2B-AS1 is involved in some human malignancies.However,its role in hepatocellular carcinoma(HCC)has not been fully deciphered.AIM To decipher the role of CDKN2B-AS1 in the progression of HCC.METHODS CDKN2B-AS1 expression in HCC was detected by quantitative real-time polymerase chain reaction.The malignant phenotypes of Li-7 and SNU-182 cells were detected by the CCK-8 method,EdU method,and flow cytometry,respectively.RNA immunoprecipitation was executed to confirm the interaction between CDKN2B-AS1 and E2F transcription factor 1(E2F1).Luciferase reporter assay and chromatin immunoprecipitation were performed to verify the binding of E2F1 to the promoter of G protein subunit alpha Z(GNAZ).E2F1 and GNAZ were detected by western blot in HCC cells.RESULTS In HCC tissues,CDKN2B-AS1 was upregulated.Depletion of CDKN2B-AS1 inhibited the proliferation of HCC cells,and the depletion of CDKN2B-AS1 also induced cell cycle arrest and apoptosis.CDKN2B-AS1 could interact with E2F1.Depletion of CDKN2B-AS1 inhibited the binding of E2F1 to the GNAZ promoter region.Overexpression of E2F1 reversed the biological effects of depletion of CDKN2B-AS1 on the malignant behaviors of HCC cells.CONCLUSION CDKN2B-AS1 recruits E2F1 to facilitate GNAZ transcription to promote HCC progression.
基金Acknowledgments We are grateful to Dr DA Melton （Harvard University） for shar- ing his human ES cells with us. The study was supported by grants from the National High Technology Research and Development Program of China （2006CB943900）, the National Natural Science Foundation of China （General Program, 30500088）, the Shang- hai Jiao Tong University School of Medicine, and the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The study was also supported by the Shanghai Leading Academic Deciline Project （S30201）.
文摘POU transcription factor OCT4 not only plays an essential role in maintaining the pluripotent and self-renewing state of embryonic stem （ES） cells but also acts as a cell fate determinant through a gene dosage effect. However, the molecular mechanisms that control the intracellular OCT4 protein level remain elusive. Here, we report that human WWP2, an E3 ubiquitin （Ub）-protein ligase, interacts with OCT4 specifically through its WW domain and enhances Ub modification of OCT4 both in vitro and in vivo. We first demonstrated that endogenous OCT4 in hu- man ES cells can be post-translationally modified by Ub. Furthermore, we found that WWP2 promoted degradation of OCT4 through the 26S proteasome in a dosage-dependent manner, and the active site cysteine residue of WWP2 was required for both its enzymatic activity and proteolytic effect on OCT4. Remarkably, our data show that the en- dogenous OCT4 protein level was significantly elevated when WWP2 expression was downregulated by specific RNA interference （RNAi）, suggesting that WWP2 is an important regulator for maintaining a proper OCT4 protein level in human ES cells. Moreover, northern blot analysis showed that the WWP2 transcript was widely present in diverse human tissues/organs and highly expressed in undifferentiated human ES cells. However, its expression level was quickly decreased after human ES cells differentiated, indicating that WWP2 expression might be developmentally regulated. Our findings demonstrate that WWP2 is an important regulator of the OCT4 protein level in human ES cells.
基金Supported by the Higher Education Commission(HEC),Pakistan Scholarship for Ph.D.Studies to Razzaq SS,No.520-148390-2BS6-011.
文摘BACKGROUND Heart diseases are the primary cause of death all over the world.Following myocardial infarction,billions of cells die,resulting in a huge loss of cardiac function.Stem cell-based therapies have appeared as a new area to support heart regeneration.The transcription factors GATA binding protein 4(GATA-4)and myocyte enhancer factor 2C(MEF2C)are considered prominent factors in the development of the cardiovascular system.AIM To explore the potential of GATA-4 and MEF2C for the cardiac differentiation of human umbilical cord mesenchymal stem cells(hUC-MSCs).METHODS hUC-MSCs were characterized morphologically and immunologically by the presence of specific markers of MSCs via immunocytochemistry and flow cytometry,and by their potential to differentiate into osteocytes and adipocytes.hUC-MSCs were transfected with GATA-4,MEF2C,and their combination to direct the differentiation.Cardiac differentiation was confirmed by semiquant itative real-time polymerase chain reaction and immunocytochemistry.RESULTS hUC-MSCs expressed specific cell surface markers CD105,CD90,CD44,and vimentin but lack the expression of CD45.The transcription factors GATA-4 and MEF2C,and their combination induced differentiation in hUC-MSCs with significant expression of cardiac genes i.e.,GATA-4,MEF2C,NK2 homeobox 5(NKX2.5),MHC,and connexin-43,and cardiac proteins GATA-4,NKX2.5,cardiac troponin T,and connexin-43.CONCLUSION Transfection with GATA-4,MEF2C,and their combination effectively induces cardiac differentiation in hUC-MSCs.These genetically modified MSCs could be a promising treatment option for heart diseases in the future.
基金supported by the National Natural Science Foundation of China(Grant No.32172512)the Jiangsu Special Fund for Frontier Foundation Research of Carbon Peaking and Carbon Neutralization(Grant No.BK20220005)+1 种基金Jiangsu Agricultural Science and Technology Innovation Fund[Grant No.CX(20)2023]a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions。
文摘5-Aminolevulinic acid(ALA)can inhibit abscisic acid(ABA)-induced stomatal closure.However,the molecular mechanism is unclear.In this study,we found that ALA upregulated the MdPP2AC expression and PP2A activity in the apple(Malus domestica Borkh.cv.‘Fuji’)leaves.With the promoter of MdPP2AC as bait,a diacylglycerol kinase MdDGK3-like was selected by the Yeast One Hybrid(Y1H)from the cDNA library of the epidermis of apple leaves treated by exogenous ALA.Additional to binding the promoter of MdPP2AC,MdDGK3-like was found to inhibit the transcription activity of MdPP2AC promoter,while ALA significantly eliminated the role of MdDGK3-like.In tobacco leaves,MdDGK3-like was localized in the nucleus of stomatal guard cells.Therefore,MdDGK3-like might act as a transcription factor negatively regulating MdPP2AC expression and causing stomatal closure.To further identify MdDGK3-like functions,several transiently transgenic apple leaves(including overexpression and interference)were established.The results revealed that overexpression of MdDGK3-like promoted stomatal closure by increasing Ca^(2+)and H_(2)O_(2)and decreasing flavonol levels in the guard cells.Conversely,MdDGK3-like(i)led the stomatal opening with lower levels of Ca^(2+)and H_(2)O_(2)but higher flavonols.Based on these,we proposed a new hypothesis that ALA up-regulated MdPP2AC expression via negatively regulating the expression of MdDGK3-like to up-regulate PP2A expression and the enzyme activity,which improved the stomatal aperture.Since it was the first time that MdDGK3-like was showed to act as a transcription factor,the proposed model provided a new insight onto the mechanisms of ALA-induced stomatal opening.
