Current Biotechnology

Recent Advances in Biosynthesis and Application of Chlorogenic Acid from Tobacco
Qiang JI, Na WU, Chao ZHENG, Zhikang SUN, Han WU, Xuanwen LI, Jie HAO
2025, 15(6): 933-943. DOI: 10.19586/j.2095-2341.2025.0094
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Chlorogenic acid （CGA） is the most abundant polyphenolic compound in tobacco leaves， playing a crucial role in tobacco growth and development as well as cigarette smoke quality. It can serve as a key indicator to evaluate tobacco leaf quality. The biosynthesis and regulation mechanisms of CGA in tobacco have long been the focus of attention among tobacco researchers， while studies on the biological activities of CGA have also extended to fields including medicine， healthcare， and food science. This paper comprehensively reviewed the synthesis pathway， biological activities， and applications of CGA in tobacco， along with the factors influencing CGA biosynthesis in tobacco and the key enzyme genes involved in its synthesis pathway. The aim of this review was to provide a theoretical foundation for the in-depth research， development， and application of CGA metabolic regulation and biological activities in tobacco and other plants.

Research Advances on the Functional Activities of Naematelia aurantialba
Zimo ZHU, Linran LIU, Yuhang HU, Jie LI, Weijie ZHU, Xu CHEN, Longjiao ZHU, Wentao XU
2025, 15(6): 944-951. DOI: 10.19586/j.2095-2341.2025.0133
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Naematelia aurantialba， a valuable edible and medicinal fungus known for its pleasant taste and texture， is rich in various bioactive compounds such as polysaccharides， proteins， amino acids， vitamins， and essential minerals. Research has demonstrated that Naematelia aurantialba possesses multiple biological activities， including hypoglycemic， hypolipidemic， anti-inflammatory， antioxidant， and immunomodulatory effects， indicating its significant potential for applications in the food industry and biomedical fields. This review systematically compiled these functional properties and elaborates on the structure-activity relationships of its active constituents. Furthermore， it addressed current research challenges and offered perspectives on the industrial development of Naematelia aurantialba， aiming to provide a reference for further studies and comprehensive utilization.

The Ecological Niche Protection Function Mediated by Microbial Secondary Metabolites
Yujing WANG, Jia WU, Honge LI, Shanshan LI, Xiaoni YANG, Qingqing GUO, Wenran HU
2025, 15(6): 952-959. DOI: 10.19586/j.2095-2341.2025.0146
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Microbes， as indispensable components of ecosystems， such as bacteria and fungi existed in nearly all natural environments can produce secondary metabolites with varying chemical structures and ecological functions，which endowed the producing strains with ecological niche protection functions. The article reviewed the research progress on the adaptability of secondary metabolites to microorganisms in ecological environments， with a focus on their functions in microbial attack and defense， quorum sensing， interspecies cooperative pathogenicity， virulence， regulation of morphological differentiation， and resistance to ultraviolet radiation. The ecological functions of secondary metabolites in microbial interactions and nutrient acquisition processes， as well as their potential application value in agriculture and medicine， were summarized. It also proposes the use of specific ecological environment microbial resources to mine active secondary metabolites， which would help meet the demand for new compounds with high activity and low toxicity in agriculture and medical fields.

The Research Progress of mRNA Drugs
Wen XUE, Yifan JIANG, Yu JIA, Liping YANG
2025, 15(6): 960-968. DOI: 10.19586/j.2095-2341.2025.0088
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In recent years， mRNA drugs have accelerated their expansion into the pharmaceutical field as a disruptive biomedical technology. Owing to advantages such as short development cycles， rapid response capabilities， and significant therapeutic potential for previously “undruggable” targets， mRNA drugs offer new solutions to major medical challenges including viral epidemics， cancer， and rare diseases. With continuous technological advances， researchers have made breakthroughs in key areas such as molecular design and delivery systems. To date， more than 85 mRNA drug candidates have entered Phase Ⅱ/Ⅲ clinical trials globally， greatly boosting confidence in their therapeutic prospects. This article detailed structural modifications of in vitro transcribed mRNA， delivery systems for mRNA drugs， and their research progress and development trends across various disease areas， aiming to serve as a reference for research engaged in mRNA drug development.

The Appliation Advance of Fab Synthetic Libraries with Phage Display in Antibody Discovery
Xue MIN, Weihong TAO
2025, 15(6): 969-976. DOI: 10.19586/j.2095-2341.2025.0063
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Antibodies have attracted significant attention in disease treatment， especially in cancer therapy， owing to their high target selectivity. However， traditional antibody discovery methods， such as hybridoma technology， are complex to operate and dependent on animal models， rendering them increasingly inadequate to meet the needs of modern large-scale antibody screening. Consequently， there is an urgent need to develop novel high-throughput antibody screening approaches. Fab synthetic phage libraries offer advantages including lower cost， higher efficiency， and the capability to generate custom-specific antibodies， which is conducive to the identification of new disease targets and large-scale antibody screening. This review systematically summarized the research progress and clinical applications of Fab synthetic phage display libraries in terms of library design， display systems， and screening methods， with the aim of providing references for optimizing antibody screening and engineering.

Research Advances in Targeting Histone Acetylation for the Prevention and Treatment of Ischemic Stroke
Shengli WANG, Yiru CHEN, Yunxiang GUAN
2025, 15(6): 977-984. DOI: 10.19586/j.2095-2341.2025.0082
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Ischemic stroke （IS） is a neurological disorder caused by cerebrovascular occlusion or stenosis leading to inadequate cerebral blood supply， resulting in brain tissue damage that severely affects patient survival and prognosis. Histone acetylation， a post-translational modification dynamically regulated by histone acetyltransferase （HAT） and histone deacetylase （HDAC）， influences IS pathogenesis by modulating gene expression related to microglial polarization， apoptosis， and ferroptosis. Studies demonstrate that interventions targeting histone acetylation through natural compounds， small-molecule drugs， and biopharmaceuticals can effectively mitigate neuronal damage and exert neuroprotective effects. This review focused on histone acetylation to elucidate its regulatory mechanisms in IS progression and its potential as a therapeutic target， aiming to provide theoretical foundations for the subsequent clinical IS treatment.

Research Progress on the Regulation of Osteoporosis by Oxidative Stress Based on the Nrf2 Signaling Pathway
Qingfeng YANG, Siyu ZHAO, Xiaxuan CHEN, Xiang ZHANG, Yaozhong LIU, Yiwei JIANG, Jiarui LIU
2025, 15(6): 985-991. DOI: 10.19586/j.2095-2341.2025.0093
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Osteoporosis （OP） is a metabolic bone disease characterized by reduced bone mass and deterioration of bone microarchitecture. An imbalance in bone remodeling represents the core pathological basis of OP， with oxidative stress playing a critical role in disrupting bone homeostasis and contributing to the development and progression of the disease. This article provided a systematic review of the structure and function of Nrf2， the biological effects of oxidative stress， and their interplay in the pathological mechanisms of OP. It is expected to promote future research combining single-cell omics， gene editing， and clinical translation， deepen the understanding of the regulatory mechanism of Nrf2， and promote the development of OP diagnosis and treatment technologies targeting Nrf2.

An AHP-based Comprehensive Assessment of Full-chain Risks in the Commercialization of Biotechnological Breeding Corn in China
Bingxin HUANGFU, Ruiqi ZHANG, Teng WANG, Guangfeng WU, Lingxian ZHANG, Jingang LIANG, Xiaoyun HE
2025, 15(6): 992-1002. DOI: 10.19586/j.2095-2341.2025.0096
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Corn is a major food crop worldwide. The development of biobreeding technologies， particularly those based on transgenics and gene editing， holds promise for improving yield and stress resistance. In China， the industrialization of genetically modified corn has not yet been achieved and remains at the pilot stage. To assess the risks in its industrialization process， the analytic hierarchy process （AHP）， combined with expert opinions， was used to quantitatively analyze the key risk factors in the full industrial chain of bio-bred maize in China. The results indicated that the failure of transformant development was the most critical risk， while seed production management， inbred line selection， and trait introgression also require focused prevention and control. Risks during planting and processing were highly interconnected， with issues such as admixture， seed sourcing， and qualification requirements being particularly prominent. Although the regulatory process was considered relatively controllable， improvements were still needed in labeling and testing accuracy. This study provides a quantitative foundation for risk management and policy development in the industrialization of biobreeding technologies.

Study on Screening of Odorant Degradation Enzyme Genes in Dioryctria sylvestrella and its Expression Level in Different Tissues
Kaipeng ZHANG, Li WANG, Li TANG, Jing LI, Qi CHEN
2025, 15(6): 1003-1019. DOI: 10.19586/j.2095-2341.2025.0145
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This study aimed to identify， classify， and analyze the tissue expression patterns of odorant-degrading enzyme （ODE）-related genes in Dioryctria sylvestrella （Lepidoptera： Pyralidae）， thereby providing a theoretical basis for developing green and efficient control technologies against this pest. Four families of ODE-related genes were screened from the transcriptome data of female and male antennae of D. sylvestrella， including carboxylesterases （CXEs）， aldehyde oxidases （AOXs）， glutathione S-transferases （GSTs）， and cytochrome P450 monooxygenases （CYPs）. These genes were subjected to bioinformatics analysis， homologous sequence alignment， phylogenetic tree construction， and standardized naming. Candidate genes with relatively high FPKM values in antennae were selected for subsequent validation， and reverse transcription-quantitative polymerase chain reaction （RT-qPCR） was employed to analyze the tissue-specific expression profiles of the selected genes in different adult tissues. A total of 137 candidate ODE-related genes were identified， including 39 DsylCXEs， 6 DsylAOXs， 32 DsylGSTs， and 60 DsylCYPs. RT-qPCR results showed that 18 DsylODEs exhibited significantly higher expression levels in antennae than in other tissues. Among these antennae-enriched ODE genes， glutathione S-transferases （GSTs） and cytochrome P450 monooxygenases （CYPs） were the dominant families. This study is the first to screen and identify potential D. sylvestrella ODE genes with strong odor-degrading capabilities， which established a foundational framework for further investigating the odor degradation mechanisms involved in the olfactory recognition process of D. sylvestrella through sequence characterization and gene expression profiling.

Functional Analysis of the Wheat TaWRKY13 Transcription Factor in ABA-mediated Leaf Senescence
Hualiang QIAO, Jiao WANG, Bo JIAO, Hong LIU, Junming LI, Geng WANG, Chunjiang ZHOU, Shuo ZHOU
2025, 15(6): 1020-1030. DOI: 10.19586/j.2095-2341.2025.0085
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Transcription factor TaWRKY13 of wheat is a key gene regulating leaf senescence， and analyzing its mechanism of action can affect crop yield and quality. Using agrobacterium-mediated transformation， we obtained wheat overexpressing TaWRKY13 and observed that TaWRKY13 promotes leaf senescence. Exogenous abscisic acid （ABA） treatment revealed that TaWRKY13 responds to ABA and accelerates the senescence process in TaWRKY13-overexpressing plants. Real-time quantitative PCR， ChIP-PCR， and electrophoretic mobility shift assay （EMSA） results demonstrated that TaWRKY13 influences ABA dynamics by directly binding to TaNCED5， thereby altering the leaf senescence process. In summary， TaWRKY13 directly binds to TaNCED5 to regulate ABA synthesis， affecting ABA content and thus influencing the process of leaf senescence. The results can provide a theoretical basis for elucidating the aging mechanism of leaves in wheat.

Establishment of Specific Qualitative PCR Detection Method of Transgenic Maize Bt11
Jin TIAN, Fangrui GAO, Ling LI, Ziyan CHEN, Yifan CHEN, Hua ZHANG, Hong CHEN, Haoqian WANG, Jingang LIANG
2025, 15(6): 1031-1039. DOI: 10.19586/j.2095-2341.2025.0058
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This study aims to establish a specific qualitative PCR detection method for the precise identification of the Bt11 transgenic insect-resistant maize， in order to meet the requirements of biosafety management of genetically modified （GM） organisms and ensure the safety and authenticity of GM maize products. The genomic DNA of Bt11 was extracted using the QIAGEN DNeasy^? Plant Maxi kit， and specific primers were designed and screened based on the flanking sequences of the inserted foreign gene segment in Bt11. The detection conditions were optimized by adjusting the annealing temperature and primer concentration， specificity， detection limit， and reproducibility of the method were tested. The primer pair Bt11-RB-F3/R3 was ultimately selected， with an annealing temperature of 60 ℃ and a primer concentration of 0.4 μmol·L^-1. The selected primer pair Bt11-RB-F3/R3 specifically amplified a 260 bp target fragment without any non-specific amplification. The method was able to stably detect the Bt11 transgenic event in samples with a copy number ratio of 0.1%， establishing a detection limit of 0.1%. The reproducibility test demonstrated that the detection results across different operators， time periods， and PCR instruments are consistent and meet the expected outcomes. This study successfully established a scientific and reliable detection method for Bt11 transgenic maize， which is capable of precisely and efficiently identifying the Bt11 transgenic event. This method provides strong technical support for biosafety management institutions and holds significant importance for strengthening the regulatory system of GM biosafety.

Solid-state Fermentation of Marine Fungus Meira nashicola ZJ353 and the Isolation and Identification of its Metabolites
Jingwen CAO, Yanchun BAO, Yuanchao MAO, Xinyuan YANG, Haidong CUI, Xiyan HOU, Liming JIN
2025, 15(6): 1040-1049. DOI: 10.19586/j.2095-2341.2025.0072
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In recent years， the development of new antibiotics from the ocean has become a research hotspot. Using 21 fungi strains isolated from arctic sea mud as the research objects， three strains of fungi with inhibitory effects on Staphylococcus aureus were screened out by the agar diffusion method， among which the strain numbered ZJ353 exhibited the best antibacterial activity. Through morphological identification and 26S rDNA species identification， it was named Meira nashicola ZJ353. The solid-state fermentation conditions were optimized， and the metabolites were isolated and identified to determine the optimal fermentation conditions： rice medium， initial water addition of the medium at 90%， fermentation time of 11 days， and fermentation temperature of 30 ℃. Then， the M. nashicola ZJ353 was expanded for cultivation， and its fermentation products were extracted by ultrasonic treatment with ethyl acetate， a compound monomer was isolated through column chromatography on silica gel and HPLC. Using infrared spectroscopy， precise mass time-of-flight liquid chromatography-mass spectrometry， ¹H NMR and ¹³C NMR spectra， the structure of the compound was analyzed， and the molecular formula of the compound was determined to be C₁₆H₂₂O₄. The results provides a certain theoretical basis for the development of antibacterial substances from marine microorganisms.

Screening, Identification of a Novel Lignocellulolytic Enzyme-degrading Strain and its Enzyme Characteristics
Nan DING, Yang DING, Haotian YANG, Chang LIU, Shichen HUANG, Minjie FU
2025, 15(6): 1050-1056. DOI: 10.19586/j.2095-2341.2025.0069
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Lignocellulose， as an abundant and renewable bioresource， holds significant societal and ecological value for achieving carbon neutrality goals. Harnessing highly efficient lignocellulose-degrading microorganisms represents a promising solution to enhance conversion efficiency. Lignocellulases are a large class of enzymes that can decompose cellulose. Studying the composition and characteristics of this enzyme system is the theoretical basis for promoting the use of microbial decomposition of lignocellulose technology. In this study， we isolated a fungal strain （designated Aspergillus ochraceus ZJ-20） from soil using sodium carboxymethyl cellulose as the sole carbon source. Preliminary characterization revealed optimal activity at 28 ℃ and pH7.0， with glucose demonstrating superior performance as carbon source. Nitrogen source specificity was observed： ammonium sulfate maximized filter paper activity （FPA）， peptone enhanced endoglucanase production， and potassium nitrate optimized β-glucosidase activity. Under optimal conditions， the activities of filter paper enzyme， cellulase， and β-glucosidase of this strain increased by 3.00， 2.66， and 2.82 times， respectively， compared to the initial enzyme activity. The successful screening of A. ochraceus ZJ-20 in this study provides high-quality bacterial resources for the resource utilization of lignocellulose， offers important technical support for reducing the cost of biorefinery and promote the development of green energy under the dual carbon goal.

Molecular Mechanisms Underlying Membrane Composition-dependent Regulation of Electroporation
Yuxuan PENG, Mingyun SHI, Zhongying JIANG, Jing MA
2025, 15(6): 1057-1063. DOI: 10.19586/j.2095-2341.2025.0081
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Transmembrane potential-mediated electroporation is a critical physical process in biomedical applications， where membrane components serve as key regulatory factors. This study employed all-atom molecular dynamics （MD） simulations to explore the microscopic mechanisms of potential imbalance-driven electroporation， focusing on the effects of membrane thickness， transmembrane ion-pair number difference， and cholesterol content. The results demonstrated a linear positive correlation between the hydrophobic core thickness of the membrane and the critical transmembrane potential difference for electroporation： compared with DLPC bilayers （2.91 nm）， the critical potential difference of DPPC bilayers （3.30 nm） increased by 25%. An increase in the transmembrane ion-pair number difference promoted the expansion of aqueous pore size and accelerated ion transport. Cholesterol enhanced lipid ordering and acyl chain order parameters， thereby increasing membrane rigidity and elevating the energy barrier for pore formation. Under the condition of high ion-pair difference （8 pairs）， cholesterol could enhance the membrane compressibility modulus to accelerate aqueous pore formation， while significantly shortening the pore lifetime. These findings establish a quantitative model for the regulation of electroporation by three factors （membrane thickness， cholesterol content， and transmembrane potential）， providing a theoretical basis for the design of targeted electroporation therapeutic technologies.

Establishment and Verification of a Capillary Electrophoresis-laser Induced Fluorescence Method for Assessing Linear mRNA Integrity in Gene Therapy Vaccines
Wenyan CUI, Yue YANG, Wenzhong CHEN, Yan ZUO, Liangliang CHEN, Xiaodong HU
2025, 15(6): 1064-1070. DOI: 10.19586/j.2095-2341.2025.0083
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A linear mRNA integrity capillary electrophoresis-laser induced fluorescence （CE-LIF） method for gene therapy vaccines has been established and validated， in order to provide a reliable detection method for characterization research， quality control and process stability monitoring of gene therapy vaccine products. Linear mRNA integrity was analyzed using CE-LIF， with an injection time of 3 s， a voltage of -2 kV， a separation voltage of 9 kV and an analysis time of 35 min. Using sample GL2327-4-120A， the method was validated for specificity， precision， accuracy， linearity， limit of quantitation （LOQ） and limit of detection （LOD）. The results showed that the resolution between peaks in a mixture of GL2327-4-120A and GL2327-4-0A was 2.86. For six replicate analyses of sample， the relative standard deviation （RSD） of the main peak migration time was 0.05%， and the RSD of the corrected peak area was 0.32%. The recovery rate of the peak area of the GL2327-4-120A samples spiked with the test sample was within the range of 90% to 115%. The mRNA concentration showed a good linear relationship within the range of 0.30~60.0 μg·mL^-1， and the linear regression equation was y = 217 601x - 216 294， R2 = 0.994 9. The LOQ was determined to be 0.30 μg·mL^-1 and the limit of detection （LOD） was 0.06 μg·mL^-1. The established CE-LIF method has good specificity， precision， and accuracy， and can be used to analyze the linear mRNA integrity in gene therapy vaccines.

Establishment and Verification of an Ion-pair Reversed-phase Chromatography Method for mRNA Purity Testing in HPV Vaccines
Wenyan CUI, Yue YANG, Wenzhong CHEN, Yan ZUO, Xiaodong HU, Yongjian CHENG
2025, 15(6): 1071-1076. DOI: 10.19586/j.2095-2341.2025.0092
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The rapid development of multivalent mRNA therapeutic vaccines against human papillomavirus （HPV） has raised higher requirements for the quality control of long-chain nucleic acids molecules. Currently， Pharmacopoeiaof the People’s Republic of China and the United States Phanmacopoeia lack specific analytical conditions for separation of relevant substances in biological produds， with limited industry reports available. To establish a purity method for long-chain mRNA， the chromatographic conditions were systematically optimized using a poly-A-deficient impurity reference standard to determine optimal separation. The method was validated for linearity， limit of quantitation （LOQ）， limit of detection （LOD）， specificity， and precision. Optimal separation was achieved using a Thermo DNA Pac RP column （100 mm×2.1 mm， 4 μm） with a mobile phase of 400 mmol·L^-1 triethylamine acetic acid（TEAA）-methanol under gradient elution， at a flow rate of 0.4 mL·min^-1， column temperature of 80 ℃， and detection wavelength of 260 nm. Under these conditions， resolution between the main mRNA peak and the known impurity peak exceeded 1.5， and the LOQ and LOD were 4.0 μg·mL^-1 and 0.8 μg·mL^-1， respectively. Excellent linearity （R2=0.999 6） was demonstrated over the range of 4.0 μg·mL^-1 to 2 000.0 μg·mL^-1. Repeatability and intermediate precision both showed relative standard deviation （RSD）1.0%. This optimized and validated ion-pairing reversed-phase ultra performance liquid chromatography （IP-RP-HPLC） method significantly improves the resolution for separating long-chain nucleic acid related substances， providing reliable data for mRNA vaccine process development， quality control， and batch consistency studies.

Construction of the Bone Metastasis Model in B6-hRANKL Mice and Exploration of the Efficacy of hRANKL Monoclonal Antibody
Lili BI, Yunxia ZHAO, Kui JIANG, Yuanyuan WANG, Ruiping LI
2025, 15(6): 1077-1085. DOI: 10.19586/j.2095-2341.2025.0098
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To evaluate the pharmacological efficacy of hRANKL monoclonal antibody and explore the underlying pharmacodynamic mechanisms of tumor bone metastasis， the B6-hRANKL mouse model of osteolytic bone metastasis was established by injecting LLC1-luc into the tibial bone marrow cavity. LLC1-luc cells were injected into the tibial bone marrow cavity of B6-hRANKL mice. The model efficacy was evaluated through mouse live imaging， bone mineral density X-ray imaging， tartrate-resistant acid phosphatase 5b （TRACP 5b） detection， and histological analysis. After model establishment， eligible mice were randomly divided into 0.9% sodium chloride control group and the drug administration group. On the basis of the model evaluation indicators， immunohistochemistry was used to verify the distribution of the drug in bone tissue. The results showed that the osteolytic bone metastasis model（B6-hRANKL-LLC1-luc model）of tumor was successfully established， exhibiting significant bone density reduction （BMD decrease in LLC1-luc cell group vs. physiological saline group， P0.05）， elevated TRACP5b activity （P0.05）， and tumor cell bone infiltration. Although the hRANKL monoclonal antibody treatment group demonstrated drug distribution in femur and tibia bones， it failed to improve bone destruction markers. The above results suggestted that the B6-hRANKL-LLC1-luc model established in this study can be utilized for investigating osteolytic bone metastasis mechanisms. However， hRANKL monoclonal antibody did not demonstrate the expected efficacy in this model， which may be attributed to the biological characteristics of the tumor， mechanism of action compatibility issues， and inherent limitations of the model itself.

Effect of miR-5589-5p on Apoptosis and Autophagy of Hepatocellular Carcinoma Cells by Targeting FOXK1
Ce JIANG, Yi LIU, Yalei ZHAO, Xiangrong CHANG
2025, 15(6): 1086-1093. DOI: 10.19586/j.2095-2341.2025.0056
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This study aimed to investigate the role and mechanism of miR-5589-5p on apoptosis and autophagy in hepatocellular carcinoma （HCC） cells. Bioinformatics analysis was employed to explore the differential expression of miR-5589-5p and FOXK1 in human HCC and their association with the prognosis of patients with HCC. miR-5589-5p mimics were used to upregulate miR-5589-5p levels. The levels of miR-5589-5p， protein expression levels of LC3， p62， Beclin-1， FOXK1， Bax， and Bcl-2 were detected. Cell apoptosis and autophagy were detected. Dual-luciferase reporter gene assay and RNA immunoprecipitation （RIP） were utilized to verify the binding of miR-5589-5p to the 3'-UTR of FOXK1. The results showed that miR-5589-5p was lowly expressed in human HCC （P0.05）， and a lower expression level of miR-5589-5p correlated with a poorer prognosis in HCC （P0.05）. In contrast， FOXK1 was highly expressed in HCC （P0.05）， and a higher expression level of FOXK1 was associated with a worse prognosis （P0.05）. Overexpression of miR-5589-5p significantly promoted cell apoptosis and autophagy. miR-5589-5p was confirmed to target the 3'-UTR of FOXK1. Overexpression of FOXK1 significantly inhibited the aforementioned effects of miR-5589-5p mimics （P0.05）. In conclusion， miR-5589-5p is lowly expressed in HCC and promotes apoptosis and autophagy in HCC cells by regulating FOXK1. This study lays an experimental foundation for further exploring the feasibility of miR-5589-5p as a predictive marker or therapeutic target for HCC.

Risk Modeling of Cancer-associated Fibroblasts and Associated Prognostic Features in Breast Cancer Patients Based on Single-cell RNA-seq and Bulk RNA-seq Data
Xiaoyi ZHAI, Zhongqi DIAO, Yi CHEN, Wenjia GUO
2025, 15(6): 1094-1107. DOI: 10.19586/j.2095-2341.2025.0079
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Breast cancer （BRCA） is one of the most common cancers in women worldwide， and its development is closely related to the interaction of stromal cells in the tumor microenvironment （TME）. As a key component of the TME， cancer associated fibroblasts （CAF） play an important role in tumor growth， metastasis and immunity. In order to investigate the value of CAF functional subpopulations in the treatment and prognosis of breast cancer patients， single-cell RNA sequencing （scRNA-seq） data from the Gene Expression Omnibus （GEO） and The Cancer Genome Atlas （TCGA） databases were integrated with the clinical information of breast cancer， and differentially expressed genes （DEGs） were analyzed in the TCGA database to identify the DEGs in the normal and tumor samples. CAF-related prognostic genes were selected by Pearson correlation coefficients and a one-way Cox regression analysis. Based on this foundation， a CAF-related risk profile was constructed by the least absolute shrinkage and selection operator （Lasso） regression model， and a column chart was created by combining the clinicopathological variables to establish a prognostic model based on CAF subgroup profiles， which provides a new theoretical support for the accurate assessment of clinical prognosis， optimization of immunotherapy strategies， and guidance of individualized drug use.

Exploratory Multi-cohort Integrative Analysis of B-cell Activation States in Triple-negative Breast Cancer
Zhongqi DIAO, Xiaoyi ZHAI, Yi CHEN, Wenjia GUO
2025, 15(6): 1108-1119. DOI: 10.19586/j.2095-2341.2025.0111
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Triple-negative breast cancer （TNBC） lacks ER/PR/HER2 targets， exhibits high aggressiveness， and is associated with poor prognosis. Tumor?infiltrating B-cells （TIL-B） have emerged as key modulators of both antitumor immunity and immune escape， yet their specific roles in TNBC and impact on immune checkpoint inhibitor （ICI） efficacy remain incompletely understood. We analyzed two publicly available single-cell RNA-seq datasets of TNBC from GEO to identify and annotate TIL-B subpopulations. Samples were stratified into “activated” and “suppressed” B-cell groups via unsupervised clustering. Immune infiltration， cell-cell communication networks， and survival differences between these groups were assessed in the TCGA-TNBC cohort and validated across independent GEO datasets. LASSO and Cox regression were employed to screen key prognostic genes and to build a four-gene risk model. The activated B-cell group exhibited significantly enhanced intercellular immune signaling， higher infiltration of CD8⁺ T cells and NK cells， and improved overall survival （P=0.016）. The prognostic model comprising JCHAIN， F11R， IGHG3， and CD24 achieved AUCs of 0.82， 0.72， and 0.81 for 1-， 3-， and 5-year survival predictions in the TCGA cohort， respectively， and was robustly validated in independent datasets. By prospectively profiling B-cell activation states in TNBC， we reveal their potential roles in antitumor immunity and ICI response. The four-gene prognostic risk model offers a robust tool for individualized immunotherapeutic decision?making and B-cell-targeted interventions.

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