Objective:To investigate the effects of silencing BAK1 and BCL2 gene expression on proliferation,invasion and metastasis of hepatocellular carcinoma(HCC)HepG2 cells.Methods:30 HCC HepG2 cells were randomly divided int...Objective:To investigate the effects of silencing BAK1 and BCL2 gene expression on proliferation,invasion and metastasis of hepatocellular carcinoma(HCC)HepG2 cells.Methods:30 HCC HepG2 cells were randomly divided into groups and received the corresponding treatments,namely,control group,NC-siRNA group,BAK1-siRNA group,BCL2-siRNA group and BAK1+BCL2 group,with 6 strains in each group.ThenqRT-PCR,CCK8,Transwell chamber invasion and scratch assay were used to detect the expression,proliferation,invasion and metastasis of BAK1 and BCL2 genes in HepG2 cells.Results:The mRNA expression,cell proliferation rate,cell migration rate and cell invasion ability of BAK1 and BCL2 in HepG2 cells were lowest in the BAK1+BCL2 siRNA group,followed by BCL2-siRNA group,BAK1-siRNA group,NC-shRNA group and control group(P<0.05).The proliferation rate of HepG2 cells in the BAK1+BCL2 siRNA group decreased significantly with time(P<0.05).Conclusion:Silencing the expression of BAK1 and BCL2 genes can inhibit the proliferation and invasion of HCC HepG2 cells and promote their apoptosis.展开更多
Cell-surface-localized leucine-rich-repeat receptorlike kinases(LRR-RLKs)are crucial for plant immunity.Most LRR-RLKs that act as receptors directly recognize ligands via a large extracellular domain(ECD),whereas LRR-...Cell-surface-localized leucine-rich-repeat receptorlike kinases(LRR-RLKs)are crucial for plant immunity.Most LRR-RLKs that act as receptors directly recognize ligands via a large extracellular domain(ECD),whereas LRR-RLK that serve as regulators are relatively small and contain fewer LRRs.Here,we identified LRR-RLK regulators using high-throughput tobacco rattle virus(TRV)-based gene silencing in the model plant Nicotiana benthamiana.We used the cell-death phenotype caused by INF1,an oomycete elicitin that induces pattern-triggered immunity,as an indicator.By screening 33 small LRR-RLKs(≤6 LRRs)of unknown function,we identified ELICITIN INSENSITIVE RLK 1(NbEIR1)as a positive regulator of INF1-induced immunity and oomycete resistance.Nicotiana benthamiana mutants of eir1 generated by CRISPR/Cas9-editing showed significantly compromised immune responses to INF1 and were more vulnerable to the oomycete pathogen Phytophthora capsici.NbEIR1 associates with BRI1-ASSOCIATED RECEPTOR KINASE 1(NbBAK1)and a downstream component,BRASSINOSTEROIDSIGNALING KINASE 1(NbBSK1).NbBSK1 also contributes to INF1-induced defense and P.capsici resistance.Upon INF1 treatment,NbEIR1 was released from NbBAK1 and NbBSK1 in vivo.Moreover,the silencing of NbBSK1 compromised the association of NbEIR1 with NbBAK1.We also showed that NbEIR1 regulates flg22-induced immunity and associates with its receptor,FLAGELLIN SENSING 2(NbFLS2).Collectively,our results suggest that NbEIR1 is a novel regulatory element for BAK1-dependent immunity.NbBSK1-NbEIR1 association is required for maintaining the NbEIR1/NbBAK1 complex in the resting state.展开更多
The mechanisms that balance plant growth and stress responses are poorly understood, but they appear to involve abscisic acid(ABA) signaling mediated by protein kinases. Here, to explore these mechanisms, we examined ...The mechanisms that balance plant growth and stress responses are poorly understood, but they appear to involve abscisic acid(ABA) signaling mediated by protein kinases. Here, to explore these mechanisms, we examined the responses of Arabidopsis thaliana protein kinase mutants to ABA treatment. We found that mutants of BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1(BAK1) were hypersensitive to the effects of ABA on both seed germination and primary root growth. The kinase OPEN STOMATA 1(OST1) was more highly activated by ABA in bak1 mutant than the wild type. BAK1 was not activated by ABA treatment in the dominant negative mutant abi1-1 or the pyr1 pyl4 pyl5 pyl8 quadruple mutant, but it was more highly activated by this treatment in the abi1-2 abi2-2 hab1-1 loss-of-function triple mutant than the wild type. BAK1 phosphorylates OST1 T146 and inhibits its activity. Genetic analyses suggested that BAK1 acts at or upstream of core components in the ABA signaling pathway, including PYLs, PP2 Cs,and Sn RK2 s, during seed germination and primary root growth. Although the upstream brassinosteroid(BR) signaling components BAK1 and BR INSENSITIVE 1(BRI1) positively regulate ABAinduced stomatal closure, mutations affecting downstream components of BR signaling, including BRASSINOSTEROID-SIGNALING KINASEs(BSKs)and BRASSINOSTEROID-INSENSITIVE 2(BIN2), did not affect ABA-mediated stomatal movement. Thus,our study uncovered an important role of BAK1 in negatively regulating ABA signaling during seed germination and primary root growth, but positively modulating ABA-induced stomatal closure, thus optimizing the plant growth under drought stress.展开更多
Arabidopsis thallana CERK1 is an essential receptor-like kinase in the chitin signal transduction pathway. The juxtamembrane (JM) domain of CERK1 regulates the kinase activity of this receptor. Here we demonstrate tha...Arabidopsis thallana CERK1 is an essential receptor-like kinase in the chitin signal transduction pathway. The juxtamembrane (JM) domain of CERK1 regulates the kinase activity of this receptor. Here we demonstrate that the JM domains of LysM RLKs, CERK1,and OsCERK1 play a functionally conserved role in the activation of chitin signaling in Arabidopsis. The Ctrmini of the JM domains of both CERK1 and OsCERK1 are indispensable for their function. Moreover, after replacing the JM domain of CERK1 with that of the non-homologous RLK, BAK1 (CJBa) or FL52 (CJFI), the chimeric CERK1 receptors maintained their ability to ac.tivate chitin signaling in Arabidopsis. Interestingly, theheterologous expression of CJBa and CJFI did not induce cell death in Nicotiand benthamiand leaves. These results suggest that the JM domains of CERK1, BAK1, and FLS2 play a conserved role in chitin signaling via a mechanism not related to sequence homology.展开更多
文摘目的 探讨miR-125b通过靶向抑制Bak1表达影响人髓系白血病(AML)细胞增殖的机制及意义。方法选择miR-125b mimics、miR-125b inhibitor、miR-125b NC分别转染人AML细胞株THP-1(miR-125b mimics组、miR-125b inhibitor组、NC组)。采用荧光定量PCR检测miR-125b mRNA相对表达量, MTT法检测细胞增殖,流式细胞仪检测细胞凋亡,Western-blot法检测蛋白表达水平,利用生物信息学分析和荧光素酶双报告系统明确miR-125b的下游靶基因。结果 miR-125b mimics转染后THP-1细胞中miR-125b表达水平显著上调( P <0.05),miR-125b inhibitor转染后细胞中miR-125b水平则显著降低( P <0.05)。转染24 h、48 h后,MTT法检测显示miR-125b mimics组的细胞增殖活性显著低于miR-125b inhibitor组与NC组( P <0.05),细胞凋亡率显著高于miR-125b inhibitor组与NC组( P <0.05),后两组对比差异无统计学意义( P >0.05)。转染48 h后,Western-blot法检测显示miR-125b mimics组的 Bak1蛋白表达水平显著高于miR-125b inhibitor组与NC组( P <0.05),三组PI3K、AKT蛋白表达水平对比差异无统计学意义( P >0.05)。在HEK293细胞株中,miR-125b mimic转染48 h后可以显著降低Bak1萤火虫荧光素酶活性( P <0.05),而对于含有突变型载体的Bak1萤火虫荧光素酶活性无显著影响( P >0.05)。结论 过表达miR-125b可通过靶向抑制Bak1的活性,抑制人AML细胞株的增殖活性,促进细胞凋亡。
基金Science and technology introduction and innovation project of Xinjiang uygur autonomous region people's hospital(No.20170305).
文摘Objective:To investigate the effects of silencing BAK1 and BCL2 gene expression on proliferation,invasion and metastasis of hepatocellular carcinoma(HCC)HepG2 cells.Methods:30 HCC HepG2 cells were randomly divided into groups and received the corresponding treatments,namely,control group,NC-siRNA group,BAK1-siRNA group,BCL2-siRNA group and BAK1+BCL2 group,with 6 strains in each group.ThenqRT-PCR,CCK8,Transwell chamber invasion and scratch assay were used to detect the expression,proliferation,invasion and metastasis of BAK1 and BCL2 genes in HepG2 cells.Results:The mRNA expression,cell proliferation rate,cell migration rate and cell invasion ability of BAK1 and BCL2 in HepG2 cells were lowest in the BAK1+BCL2 siRNA group,followed by BCL2-siRNA group,BAK1-siRNA group,NC-shRNA group and control group(P<0.05).The proliferation rate of HepG2 cells in the BAK1+BCL2 siRNA group decreased significantly with time(P<0.05).Conclusion:Silencing the expression of BAK1 and BCL2 genes can inhibit the proliferation and invasion of HCC HepG2 cells and promote their apoptosis.
基金Acknowledgments This study was supported by the Chinese Academy of Sciences and National Natural Science Foundation of China （Grants 30425029, 30421001, 90717001）. We greatly thank Prof Hong Ma （Penn. State University, USA） for critical reading and writing improvement and Prof Nam-Hai Chua （The Rockefeller University, USA） for helpful comments. We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants, and Prof Sheng Luan （University of California, Berkeley, USA） for providing the construct pATC940. We thank Prof Hong-Quan Yang （SIPPE, CAS） for providing LexA yeast two-hybrid system and Prof Zhi-Yong Wang （The Stanford University, USA） for providing the BRI1 antibody. We thank Mr Xiao-Shu Gao for the help on Confocal Laser Scanning Microscopy.
基金supported by the National Natural Science Foundation of China(32100155 and 32072507)the Natural Science Foundation of Jiangsu Province(BK20221000)+1 种基金the fellowship of China Postdoctoral Science Foundation(2021M701740)the Jiangsu Funding Program for Excellent Postdoctoral Talent(2022ZB343 and 2022ZB768)。
文摘Cell-surface-localized leucine-rich-repeat receptorlike kinases(LRR-RLKs)are crucial for plant immunity.Most LRR-RLKs that act as receptors directly recognize ligands via a large extracellular domain(ECD),whereas LRR-RLK that serve as regulators are relatively small and contain fewer LRRs.Here,we identified LRR-RLK regulators using high-throughput tobacco rattle virus(TRV)-based gene silencing in the model plant Nicotiana benthamiana.We used the cell-death phenotype caused by INF1,an oomycete elicitin that induces pattern-triggered immunity,as an indicator.By screening 33 small LRR-RLKs(≤6 LRRs)of unknown function,we identified ELICITIN INSENSITIVE RLK 1(NbEIR1)as a positive regulator of INF1-induced immunity and oomycete resistance.Nicotiana benthamiana mutants of eir1 generated by CRISPR/Cas9-editing showed significantly compromised immune responses to INF1 and were more vulnerable to the oomycete pathogen Phytophthora capsici.NbEIR1 associates with BRI1-ASSOCIATED RECEPTOR KINASE 1(NbBAK1)and a downstream component,BRASSINOSTEROIDSIGNALING KINASE 1(NbBSK1).NbBSK1 also contributes to INF1-induced defense and P.capsici resistance.Upon INF1 treatment,NbEIR1 was released from NbBAK1 and NbBSK1 in vivo.Moreover,the silencing of NbBSK1 compromised the association of NbEIR1 with NbBAK1.We also showed that NbEIR1 regulates flg22-induced immunity and associates with its receptor,FLAGELLIN SENSING 2(NbFLS2).Collectively,our results suggest that NbEIR1 is a novel regulatory element for BAK1-dependent immunity.NbBSK1-NbEIR1 association is required for maintaining the NbEIR1/NbBAK1 complex in the resting state.
基金supported by grants from the National Science Foundation of China (31730007, 31921001)the Beijing Outstanding University Discipline Program
文摘The mechanisms that balance plant growth and stress responses are poorly understood, but they appear to involve abscisic acid(ABA) signaling mediated by protein kinases. Here, to explore these mechanisms, we examined the responses of Arabidopsis thaliana protein kinase mutants to ABA treatment. We found that mutants of BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1(BAK1) were hypersensitive to the effects of ABA on both seed germination and primary root growth. The kinase OPEN STOMATA 1(OST1) was more highly activated by ABA in bak1 mutant than the wild type. BAK1 was not activated by ABA treatment in the dominant negative mutant abi1-1 or the pyr1 pyl4 pyl5 pyl8 quadruple mutant, but it was more highly activated by this treatment in the abi1-2 abi2-2 hab1-1 loss-of-function triple mutant than the wild type. BAK1 phosphorylates OST1 T146 and inhibits its activity. Genetic analyses suggested that BAK1 acts at or upstream of core components in the ABA signaling pathway, including PYLs, PP2 Cs,and Sn RK2 s, during seed germination and primary root growth. Although the upstream brassinosteroid(BR) signaling components BAK1 and BR INSENSITIVE 1(BRI1) positively regulate ABAinduced stomatal closure, mutations affecting downstream components of BR signaling, including BRASSINOSTEROID-SIGNALING KINASEs(BSKs)and BRASSINOSTEROID-INSENSITIVE 2(BIN2), did not affect ABA-mediated stomatal movement. Thus,our study uncovered an important role of BAK1 in negatively regulating ABA signaling during seed germination and primary root growth, but positively modulating ABA-induced stomatal closure, thus optimizing the plant growth under drought stress.
基金supported by the National Natural Science Foundation of China(31571249 and 31425003)。
文摘Arabidopsis thallana CERK1 is an essential receptor-like kinase in the chitin signal transduction pathway. The juxtamembrane (JM) domain of CERK1 regulates the kinase activity of this receptor. Here we demonstrate that the JM domains of LysM RLKs, CERK1,and OsCERK1 play a functionally conserved role in the activation of chitin signaling in Arabidopsis. The Ctrmini of the JM domains of both CERK1 and OsCERK1 are indispensable for their function. Moreover, after replacing the JM domain of CERK1 with that of the non-homologous RLK, BAK1 (CJBa) or FL52 (CJFI), the chimeric CERK1 receptors maintained their ability to ac.tivate chitin signaling in Arabidopsis. Interestingly, theheterologous expression of CJBa and CJFI did not induce cell death in Nicotiand benthamiand leaves. These results suggest that the JM domains of CERK1, BAK1, and FLS2 play a conserved role in chitin signaling via a mechanism not related to sequence homology.
