C2H2 zinc finger proteins(C2H2-ZFPs),one of the largest transcription factor families in plants,exhibit unique functional diversity in woody and horticultural species due to their modular architecture and regulatory f...C2H2 zinc finger proteins(C2H2-ZFPs),one of the largest transcription factor families in plants,exhibit unique functional diversity in woody and horticultural species due to their modular architecture and regulatory flexibility.This review synthesizes recent advances in understanding their roles in perennial growth cycles and horticultural trait regulation,with a focus on their contributions to fruit development,secondary growth,and complex environmental adaptation.Structurally,C2H2-ZFPs harbor conserved zinc-coordinating motifs(C2H2 domains)and regulatory domains,enabling precise transcriptional control of developmental processes.In woody and horticultural plants,they integrate hormonal signals(e.g.,auxin,ABA)and environmental cues(e.g.,drought,photoperiod)to regulate seasonal dormancy and flowering timing in horticultural species,cambial activity for wood formation,and fruit ripening processes.Under abiotic stress,C2H2-ZFPs act as key modulators in ABA-dependent and independent pathways,governing osmotic balance,redox homeostasis,and stress memory.Meanwhile,emerging evidence also highlights their involvement in biotic stress responses.However,dissecting their regulatory networks in woody and horticultural species remains challenging due to genomic redundancy and complex regulatory layers.Future research should leverage multi-omics approaches and CRISPR-based tools to uncover their functional redundancy and species-specific adaptations,thereby advancing sustainable forestry and precision horticulture breeding under climate change.展开更多
Jasmonic acid(JA)plays an important role in regulating leaf senescence.However,the molecular mechanisms of leaf senescence in apple(Malus domestica)remain elusive.In this study,we found that MdZAT10,a C2H2-type zinc f...Jasmonic acid(JA)plays an important role in regulating leaf senescence.However,the molecular mechanisms of leaf senescence in apple(Malus domestica)remain elusive.In this study,we found that MdZAT10,a C2H2-type zinc finger transcription factor(TF)in apple,markedly accelerates leaf senescence and increases the expression of senescence-related genes.To explore how MdZAT10 promotes leaf senescence,we carried out liquid chromatography/mass spectrometry screening.We found that MdABI5 physically interacts with MdZAT10.MdABI5,an important positive regulator of leaf senescence,significantly accelerated leaf senescence in apple.MdZAT10 was found to enhance the transcriptional activity of MdABI5 for MdNYC1 and MdNYE1,thus accelerating leaf senescence.In addition,we found that MdZAT10 expression was induced by methyl jasmonate(MeJA),which accelerated JA-induced leaf senescence.We also found that the JA-responsive protein MdBT2 directly interacts with MdZAT10 and reduces its protein stability through ubiquitination and degradation,thereby delaying MdZAT10-mediated leaf senescence.Taken together,our results provide new insight into the mechanisms by which MdZAT10 positively regulates JA-induced leaf senescence in apple.展开更多
oluble solids content(SSC) plays an important role in determining the flavor of tomato fruits. Tomato fruit SSC is transcriptionally regulated via sugar metabolism. Previous studies have predominantly focused on the r...oluble solids content(SSC) plays an important role in determining the flavor of tomato fruits. Tomato fruit SSC is transcriptionally regulated via sugar metabolism. Previous studies have predominantly focused on the role of C2H2-type zinc finger proteins in tomato growth and development. However, the specific regulatory mechanisms of C2H2 in the accumulation of soluble solids in tomato fruits are not fully understood. This study used eight tomato accessions with varying levels of SSC to study the expression of SlC2H2 family genes in red ripe fruits. The levels of SlC2H2-71 expression were found to be significantly reduced in high-SSC accessions compared to low-SSC accessions. Several Slc2h2-71 mutant lines were developed using the CRISPR-Cas9 system, which led to elevated levels of soluble solids, fructose, glucose, malic acid, and citric acid in mature red ripe fruits. However, the sucrose content in the edited Slc2h2-71 mutant lines was generally lower. RNA-seq analysis revealed that fruits from the mutant lines had altered expression of genes related to the sugar and acid metabolic pathways, which was further confirmed by quantitative real-time PCR. Specifically, the expression of SlLIN5 encoding the cell wall invertase(CWIN) was elevated. The yeast one-hybrid(Y1H) assay, 35S::UAS-GUS, dual-luciferase reporter systems and electrophoretic mobility shift assay(EMSA) demonstrated that C2H2-71 regulates tomato sugar metabolism by directly binding to the promoter region of Sl LIN5, culminating in the repression of its transcriptional activity. The activity of acid invertase exhibited a significantly higher level in the SlC2H2-71 knock-out lines compared to the control lines. In summary, the regulation of tomato fruit SSC by C2H2-71 involves the inhibition of SlLIN5 expression.展开更多
Rapeseed(Brassica napus L.)is a global oil crop.Salinity stress impedes the growth of rapeseed,especially during seed germination.The key genes mediating salinity stress response during seed germination in B.napus rem...Rapeseed(Brassica napus L.)is a global oil crop.Salinity stress impedes the growth of rapeseed,especially during seed germination.The key genes mediating salinity stress response during seed germination in B.napus remain largely unknown.Here,we found that all six paralogs of C2H2 zinc finger transcription factor WIP DOMAIN PROTEIN 2(BnaWIP2)showed increased expression during the initial 12 hours of germination,and expression was further enhanced by salinity stress.Under NaCl treatment,knocking out all six BnaWIP2 paralogs in B.napus led to significantly reduced germination,while overexpression of BnaC06.WIP2 promoted germination.Transcriptomic analysis revealed that BnaC06.WIP2 downregulated a series of genes related to abscisic acid(ABA)biosynthesis and signaling,among which BnaA05.NCED3,BnaC04.ABI5-2,BnaA03.EM6,and BnaA05.EM6 were directly repressed by BnaC06.WIP2.Further analysis showed that in germinating seeds,BnaC06.WIP2 was induced by ABA and in turn restrained ABA production,indicating that BnaC06.WIP2 forms a negative feedback loop with ABA to promote seed germination under salinity stress in B.napus.Collectively,these results enhance our understanding of the novel function of BnaWIP2 and provide valuable genetic resources for breeding salinity-tolerant rapeseed varieties.展开更多
BACKGROUND Colony-stimulating factor 3(CSF3)and its receptor(CSF3R)are known to promote gastric cancer(GC)growth and metastasis.However,their effects on the immune microenvironment remain unclear.Our analysis indicate...BACKGROUND Colony-stimulating factor 3(CSF3)and its receptor(CSF3R)are known to promote gastric cancer(GC)growth and metastasis.However,their effects on the immune microenvironment remain unclear.Our analysis indicated a potential link between CSF3R expression and the immunosuppressive receptor leukocyte immunoglobulin-like receptor B2(LILRB2)in GC.We hypothesized that CSF3/CSF3R may regulate LILRB2 and its ligands,angiopoietin-like protein 2(ANGPTL2)and human leukocyte antigen-G(HLA-G),contributing to immunosuppression.AIM To investigate the relationship between CSF3/CSF3R and LILRB2,as well as its ligands ANGPTL2 and HLA-G,in GC.METHODS Transcriptome sequencing data from The Cancer Genome Atlas were analyzed,stratifying patients by CSF3R expression.Differentially expressed genes and immune checkpoints were evaluated.Immunohistochemistry(IHC)was performed on GC tissues.Correlation analyses of CSF3R,LILRB2,ANGPTL2,and HLA-G were conducted using The Cancer Genome Atlas data and IHC results.GC cells were treated with CSF3,and expression levels of LILRB2,ANGPTL2,and HLA-G were measured by quantitative reverse transcriptase-polymerase chain reaction and western blotting.RESULTS Among 122 upregulated genes in high CSF3R expression groups,LILRB2 showed the most significant increase.IHC results indicated high expression of LILRB2(63.0%),ANGPTL2(56.5%),and HLA-G(73.9%)in GC tissues.Strong positive correlations existed between CSF3R and LILRB2,ANGPTL2,and HLA-G mRNA levels(P0.05).CSF3 increased LILRB2,ANGPTL2,and HLA-G expression in GC cells.Heterogeneous nuclear ribonucleoprotein H1 modulation significantly altered their expression,impacting CSF3’s regulatory effects.CONCLUSION The CSF3/CSF3R pathway may contribute to immunosuppression in GC by upregulating LILRB2 and its ligands,with heterogeneous nuclear ribonucleoprotein H1 playing a regulatory role.展开更多
BACKGROUND: Human gliomas are more likely to express basic fibroblast growth factor-2 (FGF-2) insulin-like growth factor-1(IGF-1), and IGF-1 receptor (IGF-1R) than normal brain tissue. These factors activate si...BACKGROUND: Human gliomas are more likely to express basic fibroblast growth factor-2 (FGF-2) insulin-like growth factor-1(IGF-1), and IGF-1 receptor (IGF-1R) than normal brain tissue. These factors activate signal transduction systems of Ras/MAPK and PI3K/Akl, which promote glioma growth. OBJECTIVE: To utilize RNA interference (RNAi) technique to down-regulate FGF-2, IGF-1, and IGF-1R gene expression, and to investigate the effects of these genes on rat C6 glioma cells, as well as the feasibility of RNAi for treating glioma. DESIGN, TIME AND SETTING: This neurooncological, randomized, controlled, in vivo and in vitro experiment, which used RNAi methodology, was performed at the Laboratory of Molecular Biology, Institute of Biochemistry, Chinese Academy of Sciences between August 2005 and February 2008. MATERIALS: Rat C6 cell lines were purchased from Shanghai Institute of Cellular Biology Affiliated to Chinese Academy of Sciences. Small interfering RNA (siRNA) was synthesized by Shanghai GenePharma. Anti-IGF-1, anti-IGF-1R, anti-FGF-2, anti-mouse and anti-rabbit IgG G1-HRP antibodies were provided by Santa Cruz Biotechnology, USA. Four to six week-old BALB/c nude mice were purchased from the Laboratory Animal Center, Chinese Academy of Sciences. METHODS: C6 glioma cells were transfected with siRNA, which was chemically synthesized in vitro to correspond to endogenous FGF-2, IGF-1, and IGF-1R genes. The inhibition ratio of targeting mRNA expression was detected by semiquantitative RT-PCR, and protein expression was determined by Western blot analysis. C6 glioma cell proliferation was observed using a growth curve C6 glioma cell apoptosis rate and cell cycle were detected by flow cytometry. C6 glioma cell growth regression was observed by transwell migration assay. In addition, nude mouse subcutaneous tumor models were used in this study. For studying the anti-tumor effects of IGF-1 and IGF-1R siRNA, two blank control groups, with six mice each, were set up: A (2.5 μg siRNA was injected one week after C6 cells were inoculated, Le., when tumor volume reached 8 mm × 8 mm) and B (siRNA was injected at the same time with C6 cells were inoculated. To study the effects of FGF-2 siRNA, the groups consisted of a blank control group, negative control group, 2.6 μg siRNA group, 4 μg siRNA group, and 5.3 μg siRNA group, with six mice each. MAIN OUTCOME MEASURES: mRNA and protein inhibition ratio of FGF-2, IGF-1, and IGF-1 R; C6 glioma cell proliferation, apoptosis, and cycle growth arrest; C6 glioma cell growth regression and subcutaneous tumorigenicity rates. RESULTS: All siRNA constructs proved to be effective. After 48 hours, transfection of 200 nmol/L siRNA resulted in a FGF-2 or IGF-1R gene inhibition ratio 〉 80% and an IGF-1 gene inhibition ratio of approximately 70%. Protein expression levels for FGF-2, IGF-1, and IGF-1R decreased in a dose-dependent manner following siRNA transfection, with an inhibition rate 〉 85%, 60%, and 50%, respectively. C6 glioma cell proliferation and apoptosis rates increased in proportion to siRNA. The apoptosis rate of C6 glioma cells induced by FGF-2, IGF-1, and IGF-1R siRNA was 39.96%, 15.07% and 22.47%, respectively (P 〈 0.01). Transfection of 200 nmol/L IGF or IGF-1R siRNA for 48 hours suppressed C6 glioma cell migration. At 30 days after intratumoral injection of 2.6, 4, and 5.3 tJg FGF-2 siRNA, tumor growth regression rate of FGF-2 siRNA was 56%, 67%, and 86%, respectively. The tumor growth regression rate was 71.88% and 45.71%, respectively, when IGF-1 or IGF-1R siRNA was intratumorally injected 1 week after C6 glioma cell transplantation. When IGF-1 or IGF-1 R siRNA was intratumorally injected during C6 glioma cell transplantation, the tumor growth regression rate was 78.13% and 74.29%, respectively. CONCLUSION: siRNA transfection downregulated gene expression of FGF-2, IGF-1, and IGF-1R In addition, siRNA treatment markedly suppressed glioma cell proliferation, growth, and migration, and concomitantly reduced subcutaneous tumorigenicity.展开更多
锌指蛋白转录因子(zinc-finger protein transcription factor, ZFP)是一种具有特定结构的蛋白,是真核生物重要转录因子之一,其通过与核内其他因子相互作用,调控各种生理反应,参与植物的生长发育和生物与非生物胁迫。目前对木薯锌指蛋...锌指蛋白转录因子(zinc-finger protein transcription factor, ZFP)是一种具有特定结构的蛋白,是真核生物重要转录因子之一,其通过与核内其他因子相互作用,调控各种生理反应,参与植物的生长发育和生物与非生物胁迫。目前对木薯锌指蛋白家族的研究依然较少。本研究利用木薯基因组数据库筛选鉴定了85个木薯C2H2型锌指蛋白转录因子家族成员,利用生物信息学手段分析该家族基因的结构特征和理化性质,结果表明同一C2H2型锌指蛋白家族的基因结构及氨基酸序列长度存在显著差异,大致可分为6个亚家族,各成员具有特征性的锌指结构域,但数量上存在差异;染色体定位显示该家族成员并非定位于木薯所有的18条染色体,在13号和16号染色体上没有C2H2型锌指蛋白家族成员定位。结合已有的转录组数据分析其在不同组织及非生物胁迫下的表达模式,发现各成员在不同组织中的表达模式不同,各组织均有高表达的基因,部分成员无组织表达差异,部分家族成员响应非生物胁迫。因此,木薯C2H2型锌指蛋白转录因子在植物生长发育和响应非生物胁迫功能方面具有重要的作用。展开更多
基金supported by the Zhejiang Science and Technology Major Program on Agricultural New Variety Breeding(Grant No.2021C02070-1)the National Natural Science Foundation of China(Grant Nos.32471890,32301615)the Open Fund of the National Key Laboratory for Development and Utilization of Forest Food Resources(SKLSS-KF2024-06)。
摘要C2H2 zinc finger proteins(C2H2-ZFPs),one of the largest transcription factor families in plants,exhibit unique functional diversity in woody and horticultural species due to their modular architecture and regulatory flexibility.This review synthesizes recent advances in understanding their roles in perennial growth cycles and horticultural trait regulation,with a focus on their contributions to fruit development,secondary growth,and complex environmental adaptation.Structurally,C2H2-ZFPs harbor conserved zinc-coordinating motifs(C2H2 domains)and regulatory domains,enabling precise transcriptional control of developmental processes.In woody and horticultural plants,they integrate hormonal signals(e.g.,auxin,ABA)and environmental cues(e.g.,drought,photoperiod)to regulate seasonal dormancy and flowering timing in horticultural species,cambial activity for wood formation,and fruit ripening processes.Under abiotic stress,C2H2-ZFPs act as key modulators in ABA-dependent and independent pathways,governing osmotic balance,redox homeostasis,and stress memory.Meanwhile,emerging evidence also highlights their involvement in biotic stress responses.However,dissecting their regulatory networks in woody and horticultural species remains challenging due to genomic redundancy and complex regulatory layers.Future research should leverage multi-omics approaches and CRISPR-based tools to uncover their functional redundancy and species-specific adaptations,thereby advancing sustainable forestry and precision horticulture breeding under climate change.
基金This research was supported by the National Natural Science Foundation of China(31772288)Shandong Natural Science Major Basic Research Foundation(ZR2020ZD43).
摘要Jasmonic acid(JA)plays an important role in regulating leaf senescence.However,the molecular mechanisms of leaf senescence in apple(Malus domestica)remain elusive.In this study,we found that MdZAT10,a C2H2-type zinc finger transcription factor(TF)in apple,markedly accelerates leaf senescence and increases the expression of senescence-related genes.To explore how MdZAT10 promotes leaf senescence,we carried out liquid chromatography/mass spectrometry screening.We found that MdABI5 physically interacts with MdZAT10.MdABI5,an important positive regulator of leaf senescence,significantly accelerated leaf senescence in apple.MdZAT10 was found to enhance the transcriptional activity of MdABI5 for MdNYC1 and MdNYE1,thus accelerating leaf senescence.In addition,we found that MdZAT10 expression was induced by methyl jasmonate(MeJA),which accelerated JA-induced leaf senescence.We also found that the JA-responsive protein MdBT2 directly interacts with MdZAT10 and reduces its protein stability through ubiquitination and degradation,thereby delaying MdZAT10-mediated leaf senescence.Taken together,our results provide new insight into the mechanisms by which MdZAT10 positively regulates JA-induced leaf senescence in apple.
基金supported by the grants from the National Key Research & Development Plan of China (2022YFF10030022022YFD1200502)+5 种基金the National Natural Science Foundation of China (32372696)the Wuhan Biological Breeding Major Project, China (2022021302024852)the HZAU–AGIS Cooperation Fund, China (SZYJY2023022)the Funds for High Quality Development of Hubei Seed Industry, China (HBZY2023B004)the Hubei Agriculture Research System, China (2024HBSTX4-06)the Funds of National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, China (Horti3Y-2024-008)。
摘要oluble solids content(SSC) plays an important role in determining the flavor of tomato fruits. Tomato fruit SSC is transcriptionally regulated via sugar metabolism. Previous studies have predominantly focused on the role of C2H2-type zinc finger proteins in tomato growth and development. However, the specific regulatory mechanisms of C2H2 in the accumulation of soluble solids in tomato fruits are not fully understood. This study used eight tomato accessions with varying levels of SSC to study the expression of SlC2H2 family genes in red ripe fruits. The levels of SlC2H2-71 expression were found to be significantly reduced in high-SSC accessions compared to low-SSC accessions. Several Slc2h2-71 mutant lines were developed using the CRISPR-Cas9 system, which led to elevated levels of soluble solids, fructose, glucose, malic acid, and citric acid in mature red ripe fruits. However, the sucrose content in the edited Slc2h2-71 mutant lines was generally lower. RNA-seq analysis revealed that fruits from the mutant lines had altered expression of genes related to the sugar and acid metabolic pathways, which was further confirmed by quantitative real-time PCR. Specifically, the expression of SlLIN5 encoding the cell wall invertase(CWIN) was elevated. The yeast one-hybrid(Y1H) assay, 35S::UAS-GUS, dual-luciferase reporter systems and electrophoretic mobility shift assay(EMSA) demonstrated that C2H2-71 regulates tomato sugar metabolism by directly binding to the promoter region of Sl LIN5, culminating in the repression of its transcriptional activity. The activity of acid invertase exhibited a significantly higher level in the SlC2H2-71 knock-out lines compared to the control lines. In summary, the regulation of tomato fruit SSC by C2H2-71 involves the inhibition of SlLIN5 expression.
基金supported by the Biological Breeding-National Science and Technology Major Project(2022ZD04010)Scientific and Technological Innovation Team of Shaanxi Province(2024RS-CXTD-69)+1 种基金Key Research and Development Program of Shaanxi Province(2021LLRH-07)a grant from the Yang Ling Seed Industry Innovation Center(K3031122024).
摘要Rapeseed(Brassica napus L.)is a global oil crop.Salinity stress impedes the growth of rapeseed,especially during seed germination.The key genes mediating salinity stress response during seed germination in B.napus remain largely unknown.Here,we found that all six paralogs of C2H2 zinc finger transcription factor WIP DOMAIN PROTEIN 2(BnaWIP2)showed increased expression during the initial 12 hours of germination,and expression was further enhanced by salinity stress.Under NaCl treatment,knocking out all six BnaWIP2 paralogs in B.napus led to significantly reduced germination,while overexpression of BnaC06.WIP2 promoted germination.Transcriptomic analysis revealed that BnaC06.WIP2 downregulated a series of genes related to abscisic acid(ABA)biosynthesis and signaling,among which BnaA05.NCED3,BnaC04.ABI5-2,BnaA03.EM6,and BnaA05.EM6 were directly repressed by BnaC06.WIP2.Further analysis showed that in germinating seeds,BnaC06.WIP2 was induced by ABA and in turn restrained ABA production,indicating that BnaC06.WIP2 forms a negative feedback loop with ABA to promote seed germination under salinity stress in B.napus.Collectively,these results enhance our understanding of the novel function of BnaWIP2 and provide valuable genetic resources for breeding salinity-tolerant rapeseed varieties.
基金Supported by Hebei Province Medical Science Research Project Plan,No.20230755.
摘要BACKGROUND Colony-stimulating factor 3(CSF3)and its receptor(CSF3R)are known to promote gastric cancer(GC)growth and metastasis.However,their effects on the immune microenvironment remain unclear.Our analysis indicated a potential link between CSF3R expression and the immunosuppressive receptor leukocyte immunoglobulin-like receptor B2(LILRB2)in GC.We hypothesized that CSF3/CSF3R may regulate LILRB2 and its ligands,angiopoietin-like protein 2(ANGPTL2)and human leukocyte antigen-G(HLA-G),contributing to immunosuppression.AIM To investigate the relationship between CSF3/CSF3R and LILRB2,as well as its ligands ANGPTL2 and HLA-G,in GC.METHODS Transcriptome sequencing data from The Cancer Genome Atlas were analyzed,stratifying patients by CSF3R expression.Differentially expressed genes and immune checkpoints were evaluated.Immunohistochemistry(IHC)was performed on GC tissues.Correlation analyses of CSF3R,LILRB2,ANGPTL2,and HLA-G were conducted using The Cancer Genome Atlas data and IHC results.GC cells were treated with CSF3,and expression levels of LILRB2,ANGPTL2,and HLA-G were measured by quantitative reverse transcriptase-polymerase chain reaction and western blotting.RESULTS Among 122 upregulated genes in high CSF3R expression groups,LILRB2 showed the most significant increase.IHC results indicated high expression of LILRB2(63.0%),ANGPTL2(56.5%),and HLA-G(73.9%)in GC tissues.Strong positive correlations existed between CSF3R and LILRB2,ANGPTL2,and HLA-G mRNA levels(P0.05).CSF3 increased LILRB2,ANGPTL2,and HLA-G expression in GC cells.Heterogeneous nuclear ribonucleoprotein H1 modulation significantly altered their expression,impacting CSF3’s regulatory effects.CONCLUSION The CSF3/CSF3R pathway may contribute to immunosuppression in GC by upregulating LILRB2 and its ligands,with heterogeneous nuclear ribonucleoprotein H1 playing a regulatory role.
基金the National Natural Science Foundation of China,No.30371459Science and Technology Development Fund of Shanghai,No.034047
摘要BACKGROUND: Human gliomas are more likely to express basic fibroblast growth factor-2 (FGF-2) insulin-like growth factor-1(IGF-1), and IGF-1 receptor (IGF-1R) than normal brain tissue. These factors activate signal transduction systems of Ras/MAPK and PI3K/Akl, which promote glioma growth. OBJECTIVE: To utilize RNA interference (RNAi) technique to down-regulate FGF-2, IGF-1, and IGF-1R gene expression, and to investigate the effects of these genes on rat C6 glioma cells, as well as the feasibility of RNAi for treating glioma. DESIGN, TIME AND SETTING: This neurooncological, randomized, controlled, in vivo and in vitro experiment, which used RNAi methodology, was performed at the Laboratory of Molecular Biology, Institute of Biochemistry, Chinese Academy of Sciences between August 2005 and February 2008. MATERIALS: Rat C6 cell lines were purchased from Shanghai Institute of Cellular Biology Affiliated to Chinese Academy of Sciences. Small interfering RNA (siRNA) was synthesized by Shanghai GenePharma. Anti-IGF-1, anti-IGF-1R, anti-FGF-2, anti-mouse and anti-rabbit IgG G1-HRP antibodies were provided by Santa Cruz Biotechnology, USA. Four to six week-old BALB/c nude mice were purchased from the Laboratory Animal Center, Chinese Academy of Sciences. METHODS: C6 glioma cells were transfected with siRNA, which was chemically synthesized in vitro to correspond to endogenous FGF-2, IGF-1, and IGF-1R genes. The inhibition ratio of targeting mRNA expression was detected by semiquantitative RT-PCR, and protein expression was determined by Western blot analysis. C6 glioma cell proliferation was observed using a growth curve C6 glioma cell apoptosis rate and cell cycle were detected by flow cytometry. C6 glioma cell growth regression was observed by transwell migration assay. In addition, nude mouse subcutaneous tumor models were used in this study. For studying the anti-tumor effects of IGF-1 and IGF-1R siRNA, two blank control groups, with six mice each, were set up: A (2.5 μg siRNA was injected one week after C6 cells were inoculated, Le., when tumor volume reached 8 mm × 8 mm) and B (siRNA was injected at the same time with C6 cells were inoculated. To study the effects of FGF-2 siRNA, the groups consisted of a blank control group, negative control group, 2.6 μg siRNA group, 4 μg siRNA group, and 5.3 μg siRNA group, with six mice each. MAIN OUTCOME MEASURES: mRNA and protein inhibition ratio of FGF-2, IGF-1, and IGF-1 R; C6 glioma cell proliferation, apoptosis, and cycle growth arrest; C6 glioma cell growth regression and subcutaneous tumorigenicity rates. RESULTS: All siRNA constructs proved to be effective. After 48 hours, transfection of 200 nmol/L siRNA resulted in a FGF-2 or IGF-1R gene inhibition ratio 〉 80% and an IGF-1 gene inhibition ratio of approximately 70%. Protein expression levels for FGF-2, IGF-1, and IGF-1R decreased in a dose-dependent manner following siRNA transfection, with an inhibition rate 〉 85%, 60%, and 50%, respectively. C6 glioma cell proliferation and apoptosis rates increased in proportion to siRNA. The apoptosis rate of C6 glioma cells induced by FGF-2, IGF-1, and IGF-1R siRNA was 39.96%, 15.07% and 22.47%, respectively (P 〈 0.01). Transfection of 200 nmol/L IGF or IGF-1R siRNA for 48 hours suppressed C6 glioma cell migration. At 30 days after intratumoral injection of 2.6, 4, and 5.3 tJg FGF-2 siRNA, tumor growth regression rate of FGF-2 siRNA was 56%, 67%, and 86%, respectively. The tumor growth regression rate was 71.88% and 45.71%, respectively, when IGF-1 or IGF-1R siRNA was intratumorally injected 1 week after C6 glioma cell transplantation. When IGF-1 or IGF-1 R siRNA was intratumorally injected during C6 glioma cell transplantation, the tumor growth regression rate was 78.13% and 74.29%, respectively. CONCLUSION: siRNA transfection downregulated gene expression of FGF-2, IGF-1, and IGF-1R In addition, siRNA treatment markedly suppressed glioma cell proliferation, growth, and migration, and concomitantly reduced subcutaneous tumorigenicity.
摘要锌指蛋白转录因子(zinc-finger protein transcription factor, ZFP)是一种具有特定结构的蛋白,是真核生物重要转录因子之一,其通过与核内其他因子相互作用,调控各种生理反应,参与植物的生长发育和生物与非生物胁迫。目前对木薯锌指蛋白家族的研究依然较少。本研究利用木薯基因组数据库筛选鉴定了85个木薯C2H2型锌指蛋白转录因子家族成员,利用生物信息学手段分析该家族基因的结构特征和理化性质,结果表明同一C2H2型锌指蛋白家族的基因结构及氨基酸序列长度存在显著差异,大致可分为6个亚家族,各成员具有特征性的锌指结构域,但数量上存在差异;染色体定位显示该家族成员并非定位于木薯所有的18条染色体,在13号和16号染色体上没有C2H2型锌指蛋白家族成员定位。结合已有的转录组数据分析其在不同组织及非生物胁迫下的表达模式,发现各成员在不同组织中的表达模式不同,各组织均有高表达的基因,部分成员无组织表达差异,部分家族成员响应非生物胁迫。因此,木薯C2H2型锌指蛋白转录因子在植物生长发育和响应非生物胁迫功能方面具有重要的作用。