BACKGROUND Gastric cancer(GC)is a common gastrointestinal malignancy worldwide.Based on cancer-related mortality,the current prevention and treatment strategies for GC still show poor clinical results.Therefore,it is ...BACKGROUND Gastric cancer(GC)is a common gastrointestinal malignancy worldwide.Based on cancer-related mortality,the current prevention and treatment strategies for GC still show poor clinical results.Therefore,it is important to find effective drug treatment targets.AIM To explore the molecular mechanism of 18β-glycyrrhetinic acid(18β-GRA)regulating the miR-345-5p/TGM2 signaling pathway to inhibit the proliferation of GC cells.METHODS CCK-8 assay was used to determine the effect of 18β-GRA on the survival rate of GES-1 cells and AGS and HGC-27 cells.Cell cycle and apoptosis were detected by flow cytometry,cell migration was detected by a wound healing assay,the effect of 18β-GRA on subcutaneous tumor growth in BALB/c nude mice was investigated,and the cell autophagy level was determined by MDC staining.TMT proteomic analysis was used to detect the differentially expressed autophagy-related proteins in GC cells after 18β-GRA intervention,and then the protein-protein interaction was predicted using STRING(https://string-db.org/).MicroRNAs(miRNAs)transcriptome analysis was used to detect the miRNA differential expression profile,and use miRBase(https://www.mirbase/)and TargetScan(https://www.targetscan.org/)to predict the miRNA and complementary binding sites.Quantitative real-time polymerase chain reaction was used to detect the expression level of miRNA in 18β-GRA treated cells,and western blot was used to detect the expression of autophagy related proteins.Finally,the effect of miR-345-5p on GC cells was verified by mir-345-5p overexpression.RESULTS 18β-GRA could inhibit GC cells viability,promote cell apoptosis,block cell cycle,reduce cell wound healing ability,and inhibit the GC cells growth in vivo.MDC staining results showed that 18β-GRA could promote autophagy in GC cells.By TMT proteomic analysis and miRNAs transcriptome analysis,it was concluded that 18β-GRA could down-regulate TGM2 expression and up-regulate miR-345-5p expression in GC cells.Subsequently,we verified that TGM2 is the target of miR-345-5p,and that overexpression of miR-345-5p significantly inhibited the protein expression level of TGM2.Western blot showed that the expression of autophagy-related proteins of TGM2 and p62 was significantly reduced,and LC3II,ULK1 and AMPK expression was significantly increased in GC cells treated with 18β-GRA.Overexpression of miR-345-5p not only inhibited the expression of TGM2,but also inhibited the proliferation of GC cells by promoting cell apoptosis and arresting cell cycle.CONCLUSION 18β-GRA inhibits the proliferation of GC cells and promotes autophagy by regulating the miR-345-5p/TGM2 signaling pathway.展开更多
Adenine phosphoribosyltransferase (APRT) is the major enzyme that converts adenine into adenosine-3'-phosphate (AMP). APRT-deficient mutant caused by APRT gene mutation results in the male sterility in Arabidopsis...Adenine phosphoribosyltransferase (APRT) is the major enzyme that converts adenine into adenosine-3'-phosphate (AMP). APRT-deficient mutant caused by APRT gene mutation results in the male sterility in Arabidopsis thaliana L. In order to confirm the existence of rice APRT gene and to investigate its association with thermo-sensitive genic male sterile (TGMS) phenotype of rice, a APRT gene was identified from BLAST search of the rice genome database using APRT gene sequences from other plant species as probes. Further, the gene was cloned from rice and named APRT(GenBank accession number AY238894) using the combination of bioinformatic and experimental approaches. The rice APRT was located in the 56 000 bp to 63 000 bp region of a rice bacterial artificial chromosome (BAC) clone (AL606604) on chromosome 4 and was deduced by software from the positive DNA clone. Its cDNA was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using primers designed according to the sequence of the putative gene. The full-length cDNA was obtained by rapid amplification of cDNA ends (RACE) procedure and was sequenced. Open reading frame (ORF) analysis indicated that the rice APRT gene encodes a peptide of 212 amino acid residues, including seven exons and six introns. Using reverse position specific BLAST (RPS-BLAST), the APRT domain was identified in the polypeptide. The homology comparison demonstrated that the polypeptide exhibits 54.9%, 54.9%, 49.6% and 59.5% identity with that from Hordeum vulgare, Ttriticum aestivum, and A. thaliana (APRT types 1 and 2), respectively. Comparing the sequence of APRT gene from TGMS mutant lines 'Annong S-1' (Oryza sativa subsp. indica) with that from its corresponding wild type 'Annong F' (Oryza sativa. subsp. indica), we found that there are five single nucleotid polymorphism (SNP) sites in the gene of 'Annong S-1', which locate mainly in the second intron. However, the result of cDNA sequencing showed that these SNP sites do not damage the successful splicing of intron 2. Qualitative RT-PCR and Northern blot indicated that the gene tran-scription in the 'Annong S-1' young panicles that were verified to be the thermo-sensitive organ at the early stage of pollen fertility alternation is down-regulated by high temperature stress (28 V), which is the critical temperature causing 'Annong S-1' fertility conversion. These results revealed that the change of expression pattern of APRT in young particles of 'Annong S-1' in high temperature conditions is perhaps related to the TGMS of 'Annong S-1'.展开更多
[Objective] Changxuan 3S was thermo-sensitive genicmale sterile（TGMS）rice selected from irradiated seeds of Peiai 64S by 350 Gy^（60）Coγ-ray.The aim of the study was to confirm GA3 spraying dosage of Changxuan 3S ...[Objective] Changxuan 3S was thermo-sensitive genicmale sterile（TGMS）rice selected from irradiated seeds of Peiai 64S by 350 Gy^（60）Coγ-ray.The aim of the study was to confirm GA3 spraying dosage of Changxuan 3S with eui gene in its hybrid seed production.[Method] Changxuan 3S possessing eui gene and its parent Peiai 64S were chosen as materials.Comparison studies on sensitivity to GA3 in their hybrid seed production were carried out.[Result] The suitable stage for spraying GA3 in the hybrid seed production of Changxuan 3S was at 10% of panicles headed;The optimal dosage was 90 g/hm2 with 2 split sprayings,the first spraying of 45 g/hm2 at heading of 10% panicles and the second one of 45 g/hm2 on the following day.Under the condition of spraying GA3 at the rate of 90 g/hm2,the panicle neck exsertions of Changxuan 3S was ＋1.78 cm,and exserted stigma rate and seed setting rate of Changxuan 3S were 96.87% and 36.44%,being 21.46% and 16.33% more than those of Peiai 64S,respectively.The theoretical yield of ＂Changxuan 3S/9311＂ reached 2 931.90 kg/hm2,which was increased by 1 259.40 kg/hm2 comparing with ＂Peiai 64S/9311＂.[Conclusion] Compared with Peiai 64S,Changxuan 3S is more sensitive to GA3,which results in no or little using GA3 in seed production of Changxuan 3S.Moreover,Changxuan 3S showed higher yield potential than Peiai 64S.展开更多
Nb-RE微合金化TGM2高速钢(%:0.88～0.89C、4.14～4.16Cr、4.73～4.76Mo、6.09～6.12W、1.85～1.86V、0.05～0.10Nb、0.05～0.06RE)由25 t EAF-30 t LF(VD,加Nb-RE)-1 t ESR工艺冶炼。试验结果表明,经Nb-RE微合金化后,TGM2高速钢Φ96 mm...Nb-RE微合金化TGM2高速钢(%:0.88～0.89C、4.14～4.16Cr、4.73～4.76Mo、6.09～6.12W、1.85～1.86V、0.05～0.10Nb、0.05～0.06RE)由25 t EAF-30 t LF(VD,加Nb-RE)-1 t ESR工艺冶炼。试验结果表明,经Nb-RE微合金化后,TGM2高速钢Φ96 mm材淬火晶粒尺寸明显细化,晶粒度由原来未微合金化钢的9.5级提高至10～10.5级;淬、回火后硬度HRC为65.2～65.8,600℃4 h红硬性HRC为62.1～62.3,Nb-RE TGM2钢制成刀具的切削寿命较原TGM2钢提高20%。展开更多
With the AMMI (additive main effects and multiplicative interaction) analysis model, thedetermination of the sensitivity to temperature among different TGMS (thermo-sensitivegenic male sterile) lines was performed. To...With the AMMI (additive main effects and multiplicative interaction) analysis model, thedetermination of the sensitivity to temperature among different TGMS (thermo-sensitivegenic male sterile) lines was performed. To assess the genetic differences due to hightemperature stress at the fertility-sensitive stage (10-20d before heading), sevengenotypes (six TGMS lines and the control Pei-Ai64S) were grown from May 4 at sevendifferent stages with 10d intervals. The temperatures at the fertility-sensitive stagesinvolved twelve levels from<20 to>℃ under the regime natural conditions in Hangzhou,China. There was considerable variation in pollen fertility among genotypes in responseto high temperature. Five genotypes identified as TGMS lines as their percentages offertile pollens were lower than or close to that of the control except for the unstableline RTS19 (V6). When the temperatures at the fertility-sensitive stage were at Ⅰ-Ⅳ,Ⅴ-Ⅵ and Ⅶ-Ⅻ, the percentages of fertile pollens varied in the ranges of 46.46-48.49%,19.62-22.79% and 3.49-5.87%, respectively. The critical temperatures of sterility andfertility in the five TGMS lines were 25.1 and 23.0℃, respectively. Considering theamounts and directions of main effect and their IPCA (interaction principal componentsanalysis), we can classify the lines and temperature levels into different groups, anddescribe the characteristics of genotypetemperature interaction, offering the informationand tools for the development and utility of thermo-sensitive male sterile lines.Several TGMS rice lines with their reproductive sensitivity to high temperature that canbe screened using the AMMI model may add valuable germplasm to the breeding program ofhybrid rice.展开更多
基金Supported by the Ningxia Natural Science Foundation,No.2022AAC03144.
文摘BACKGROUND Gastric cancer(GC)is a common gastrointestinal malignancy worldwide.Based on cancer-related mortality,the current prevention and treatment strategies for GC still show poor clinical results.Therefore,it is important to find effective drug treatment targets.AIM To explore the molecular mechanism of 18β-glycyrrhetinic acid(18β-GRA)regulating the miR-345-5p/TGM2 signaling pathway to inhibit the proliferation of GC cells.METHODS CCK-8 assay was used to determine the effect of 18β-GRA on the survival rate of GES-1 cells and AGS and HGC-27 cells.Cell cycle and apoptosis were detected by flow cytometry,cell migration was detected by a wound healing assay,the effect of 18β-GRA on subcutaneous tumor growth in BALB/c nude mice was investigated,and the cell autophagy level was determined by MDC staining.TMT proteomic analysis was used to detect the differentially expressed autophagy-related proteins in GC cells after 18β-GRA intervention,and then the protein-protein interaction was predicted using STRING(https://string-db.org/).MicroRNAs(miRNAs)transcriptome analysis was used to detect the miRNA differential expression profile,and use miRBase(https://www.mirbase/)and TargetScan(https://www.targetscan.org/)to predict the miRNA and complementary binding sites.Quantitative real-time polymerase chain reaction was used to detect the expression level of miRNA in 18β-GRA treated cells,and western blot was used to detect the expression of autophagy related proteins.Finally,the effect of miR-345-5p on GC cells was verified by mir-345-5p overexpression.RESULTS 18β-GRA could inhibit GC cells viability,promote cell apoptosis,block cell cycle,reduce cell wound healing ability,and inhibit the GC cells growth in vivo.MDC staining results showed that 18β-GRA could promote autophagy in GC cells.By TMT proteomic analysis and miRNAs transcriptome analysis,it was concluded that 18β-GRA could down-regulate TGM2 expression and up-regulate miR-345-5p expression in GC cells.Subsequently,we verified that TGM2 is the target of miR-345-5p,and that overexpression of miR-345-5p significantly inhibited the protein expression level of TGM2.Western blot showed that the expression of autophagy-related proteins of TGM2 and p62 was significantly reduced,and LC3II,ULK1 and AMPK expression was significantly increased in GC cells treated with 18β-GRA.Overexpression of miR-345-5p not only inhibited the expression of TGM2,but also inhibited the proliferation of GC cells by promoting cell apoptosis and arresting cell cycle.CONCLUSION 18β-GRA inhibits the proliferation of GC cells and promotes autophagy by regulating the miR-345-5p/TGM2 signaling pathway.
文摘Adenine phosphoribosyltransferase (APRT) is the major enzyme that converts adenine into adenosine-3'-phosphate (AMP). APRT-deficient mutant caused by APRT gene mutation results in the male sterility in Arabidopsis thaliana L. In order to confirm the existence of rice APRT gene and to investigate its association with thermo-sensitive genic male sterile (TGMS) phenotype of rice, a APRT gene was identified from BLAST search of the rice genome database using APRT gene sequences from other plant species as probes. Further, the gene was cloned from rice and named APRT(GenBank accession number AY238894) using the combination of bioinformatic and experimental approaches. The rice APRT was located in the 56 000 bp to 63 000 bp region of a rice bacterial artificial chromosome (BAC) clone (AL606604) on chromosome 4 and was deduced by software from the positive DNA clone. Its cDNA was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using primers designed according to the sequence of the putative gene. The full-length cDNA was obtained by rapid amplification of cDNA ends (RACE) procedure and was sequenced. Open reading frame (ORF) analysis indicated that the rice APRT gene encodes a peptide of 212 amino acid residues, including seven exons and six introns. Using reverse position specific BLAST (RPS-BLAST), the APRT domain was identified in the polypeptide. The homology comparison demonstrated that the polypeptide exhibits 54.9%, 54.9%, 49.6% and 59.5% identity with that from Hordeum vulgare, Ttriticum aestivum, and A. thaliana (APRT types 1 and 2), respectively. Comparing the sequence of APRT gene from TGMS mutant lines 'Annong S-1' (Oryza sativa subsp. indica) with that from its corresponding wild type 'Annong F' (Oryza sativa. subsp. indica), we found that there are five single nucleotid polymorphism (SNP) sites in the gene of 'Annong S-1', which locate mainly in the second intron. However, the result of cDNA sequencing showed that these SNP sites do not damage the successful splicing of intron 2. Qualitative RT-PCR and Northern blot indicated that the gene tran-scription in the 'Annong S-1' young panicles that were verified to be the thermo-sensitive organ at the early stage of pollen fertility alternation is down-regulated by high temperature stress (28 V), which is the critical temperature causing 'Annong S-1' fertility conversion. These results revealed that the change of expression pattern of APRT in young particles of 'Annong S-1' in high temperature conditions is perhaps related to the TGMS of 'Annong S-1'.
基金Supported by Technology Research and Development Program of Hunan Provincial Science&Technology Department(2008FJ3066)Study on Hormones Regulation of Uppermost Internode Elongation of Eui Tgms Rice Changxuan 3S and GA3 Spraying Dosoge in ItsHybrid Seed Production,Key Program of Hunan Provincial Department of Education(09A066)+1 种基金Natural Science Foundation of Hunan Province(07JJ6032)Study on Expansion Inuppermost Internode of Rice Male Sterile Line with Eui Geng~~
文摘[Objective] Changxuan 3S was thermo-sensitive genicmale sterile（TGMS）rice selected from irradiated seeds of Peiai 64S by 350 Gy^（60）Coγ-ray.The aim of the study was to confirm GA3 spraying dosage of Changxuan 3S with eui gene in its hybrid seed production.[Method] Changxuan 3S possessing eui gene and its parent Peiai 64S were chosen as materials.Comparison studies on sensitivity to GA3 in their hybrid seed production were carried out.[Result] The suitable stage for spraying GA3 in the hybrid seed production of Changxuan 3S was at 10% of panicles headed;The optimal dosage was 90 g/hm2 with 2 split sprayings,the first spraying of 45 g/hm2 at heading of 10% panicles and the second one of 45 g/hm2 on the following day.Under the condition of spraying GA3 at the rate of 90 g/hm2,the panicle neck exsertions of Changxuan 3S was ＋1.78 cm,and exserted stigma rate and seed setting rate of Changxuan 3S were 96.87% and 36.44%,being 21.46% and 16.33% more than those of Peiai 64S,respectively.The theoretical yield of ＂Changxuan 3S/9311＂ reached 2 931.90 kg/hm2,which was increased by 1 259.40 kg/hm2 comparing with ＂Peiai 64S/9311＂.[Conclusion] Compared with Peiai 64S,Changxuan 3S is more sensitive to GA3,which results in no or little using GA3 in seed production of Changxuan 3S.Moreover,Changxuan 3S showed higher yield potential than Peiai 64S.
文摘Nb-RE微合金化TGM2高速钢(%:0.88～0.89C、4.14～4.16Cr、4.73～4.76Mo、6.09～6.12W、1.85～1.86V、0.05～0.10Nb、0.05～0.06RE)由25 t EAF-30 t LF(VD,加Nb-RE)-1 t ESR工艺冶炼。试验结果表明,经Nb-RE微合金化后,TGM2高速钢Φ96 mm材淬火晶粒尺寸明显细化,晶粒度由原来未微合金化钢的9.5级提高至10～10.5级;淬、回火后硬度HRC为65.2～65.8,600℃4 h红硬性HRC为62.1～62.3,Nb-RE TGM2钢制成刀具的切削寿命较原TGM2钢提高20%。
基金supported by the National Natural Science Foundation of China(39870421)the Key Research Project of Zhejiang Province,China(2003C22007 and 8812).
文摘With the AMMI (additive main effects and multiplicative interaction) analysis model, thedetermination of the sensitivity to temperature among different TGMS (thermo-sensitivegenic male sterile) lines was performed. To assess the genetic differences due to hightemperature stress at the fertility-sensitive stage (10-20d before heading), sevengenotypes (six TGMS lines and the control Pei-Ai64S) were grown from May 4 at sevendifferent stages with 10d intervals. The temperatures at the fertility-sensitive stagesinvolved twelve levels from<20 to>℃ under the regime natural conditions in Hangzhou,China. There was considerable variation in pollen fertility among genotypes in responseto high temperature. Five genotypes identified as TGMS lines as their percentages offertile pollens were lower than or close to that of the control except for the unstableline RTS19 (V6). When the temperatures at the fertility-sensitive stage were at Ⅰ-Ⅳ,Ⅴ-Ⅵ and Ⅶ-Ⅻ, the percentages of fertile pollens varied in the ranges of 46.46-48.49%,19.62-22.79% and 3.49-5.87%, respectively. The critical temperatures of sterility andfertility in the five TGMS lines were 25.1 and 23.0℃, respectively. Considering theamounts and directions of main effect and their IPCA (interaction principal componentsanalysis), we can classify the lines and temperature levels into different groups, anddescribe the characteristics of genotypetemperature interaction, offering the informationand tools for the development and utility of thermo-sensitive male sterile lines.Several TGMS rice lines with their reproductive sensitivity to high temperature that canbe screened using the AMMI model may add valuable germplasm to the breeding program ofhybrid rice.
