WGCNA reveals a biomarker for cancer-associated fibroblasts to predict prognosis in cervical cancer.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are crucial components of the cervical cancer tumor microenvironment, playing a significant role in cervical cancer progression, treatment resistance, and immune evasion, but whether the expression of CAF-related genes can predict clinical outcomes in cervical cancer is still unknown. In this study, we sought to analyze genes associated with CAFs through weighted gene co-expression network analysis (WGCNA) and to create a predictive model for CAFs in cervical cancer. METHODS: We acquired transcriptome sequencing data and clinical information on cervical cancer patients from the TCGA and GEO databases. Weighted gene co-expression network analysis was conducted to identify genes related to CAFs. We developed a prognostic model based on CAF genes in cervical cancer using LASSO Cox regression analysis. Single-cell sequencing data analysis and in vivo experiments for validation of hub genes in CAFs. RESULTS: A prognostic model for cervical cancer was developed based on CAF genes including COL4A1, LAMC1, RAMP3, POSTN, and SERPINF1. Cervical cancer patients were divided into low and high risk groups based on the optimal cutoff value. Patients in the high risk group had significantly worse prognosis. Single-cell RNA sequencing data revealed that hub genes in the CAFs risk model were expressed mainly in fibroblasts. The real-time fluorescence quantitative PCR results revealed a significant difference in the expression levels of COL4A1, LAMC1, POSTN, and SERPINF1 between the cancer group and the normal group (p < 0.05). Consistently, the results of the immunohistochemical tests exhibited notable variations in COL4A1, LAMC1, RAMP3, POSTN, and SERPINF1 expression between the cancer and normal groups (p < 0.001). CONCLUSION: The CAF risk model for cervical cancer constructed in this study can be used to predict prognosis, while the CAF hub genes can be utilized as crucial markers for cervical cancer prognosis.

Designing Microneedle Patch for Prophylaxis of Postoperative Atrial Fibrillation.

Postoperative atrial fibrillation (POAF) is a common complication following cardiac surgery, which often occurs within 30 postoperative days, especially peaking at 2-3 days. Antiarrhythmic medications such as amiodarone are recommended in clinical practice for the prophylaxis and treatment of POAF. However, conventional oral administration is hindered due to delayed drug action and high risks of systemic toxicity, and emerging localized delivery strategies suffer from a limited release duration (less than 30 days). Herein, we develop a microneedle (MN) patch for localized delivery of amiodarone to the atria in a "First Rapid and Then Sustained" dual-release mode. Specifically, this patch is composed of a needle array integrated with an amiodarone-loaded reservoir for a sustained and steady release for over 30 days; and an amiodarone-containing coating film deposited on the needle surface via the Langmuir-Blodgett technique for a rapid release at the first day. Upon this design, only one MN patch enables a higher drug accumulation in the atrial tissue at the first day than oral administration and simultaneously remains therapeutical levels for over 30 days, despite at a significantly reduced drug dosage (5.08 mg in total versus ∼10 mg per day), thereby achieving ideal preventive effects and safety in a rat model. Our findings indicate that this MN device provides a robust and efficient delivery platform for long-term prophylaxis of POAF.

Core-shell structured alginate-based hydrogel beads modified by starch and protocatechuic acid: Preparation, characterization, phenolic slow release and stable antioxidant potential.

A novel core-shell structured alginate-based hydrogel bead modified by co-gelatinizing with starch and protocatechuic acid (PA), was designed to modulate physical properties of beads, release behavior and antioxidant stability of encapsulated bioactives. Core was fabricated by ionotropic gelation, and its formulation (ratio of sodium alginate/starch) was determined by particle size/starch distribution, texture and bioactive encapsulation capacity of core. Then, coating core with shell-forming solution co-gelatinized with different doses of PA, and subsequently cross-linked with Ca2+ to obtain core-shell structured beads. Surface microstructure, mechanical characteristics, and swelling ratio of beads were affected by concentrations of PA. Besides, core-shell structure containing PA could enhance delivery and sustained release of encapsulated phenolic bioactives during in vitro digestion, and improve their antioxidant potential stability. Furthermore, interaction between PA and polysaccharide components was elucidated by FTIR and TGA. The present information was beneficial for the advancement of functional food materials and bioactive delivery systems.

Gene expression profiling and the isocitrate dehydrogenase mutational landscape of temozolomide­resistant glioblastoma.

Glioblastoma multiforme (GBM) is an aggressive brain cancer that occurs more frequently than other brain tumors. The present study aimed to reveal a novel mechanism of temozolomide resistance in GBM using bioinformatics and wet lab analyses, including meta-Z analysis, Kaplan-Meier survival analysis, protein-protein interaction (PPI) network establishment, cluster analysis of co-expressed gene networks, and hierarchical clustering of upregulated and downregulated genes. Next-generation sequencing and quantitative PCR analyses revealed downregulated [tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 1 (TIE1), calcium voltage-gated channel auxiliary subunit α2Δ1 (CACNA2D1), calpain 6 (CAPN6) and a disintegrin and metalloproteinase with thrombospondin motifs 6 (ADAMTS6)] and upregulated [serum amyloid (SA)A1, SAA2, growth differentiation factor 15 (GDF15) and ubiquitin specific peptidase 26 (USP26)] genes. Different statistical models were developed for these genes using the Z-score for P-value conversion, and Kaplan-Meier plots were constructed using several patient cohorts with brain tumors. The highest number of nodes was observed in the PPI network was for ADAMTS6 and TIE1. The PPI network model for all genes contained 35 nodes and 241 edges. Immunohistochemical staining was performed using isocitrate dehydrogenase (IDH)-wild-type or IDH-mutant GBM samples from patients and a significant upregulation of TIE1 (P<0.001) and CAPN6 (P<0.05) protein expression was demonstrated in IDH-mutant GBM in comparison with IDH-wild-type GBM. Structural analysis revealed an IDH-mutant model demonstrating the mutant residues (R132, R140 and R172). The findings of the present study will help the future development of novel biomarkers and therapeutics for brain tumors.

An updated patent review of stimulator of interferon genes agonists (2021 - present).

INTRODUCTION: Stimulator of Interferon Genes (STING) is an innate immune sensor. Activation of STING triggers a downstream response that results in the expression of proinflammatory cytokines (TNF-α, IL-1ß) via nuclear factor kappa-B (NF-κB) or the expression of type I interferons (IFNs) via an interferon regulatory factor 3 (IRF3). IFNs can eventually result in promotion of the adaptive immune response including activation of tumor-specific CD8+ T cells to abolish the tumor. Consequently, activation of STING has been considered as a potential strategy for cancer treatment. AREAS COVERED: This article provides an overview on structures and pharmacological data of CDN-like and non-nucleotide STING agonists acting as anticancer agents (January 2021 to October 2023) from a medicinal chemistry perspective. The data in this review come from EPO, WIPO, RCSB PDB, CDDI. EXPERT OPINION: In recent years, several structurally diverse STING agonists have been identified. As an immune enhancer, they are used in the treatment of tumors, which has received extensive attention from scientific community and pharmaceutical companies. Despite the multiple challenges that have appeared, STING agonists may offer opportunities for immunotherapy.

Co-occurrence of ST412 Klebsiella pneumoniae isolates with hypermucoviscous and non-mucoviscous phenotypes in a short-term hospitalized patient.

Hypermucoviscosity (HMV) is a phenotype that is commonly associated with hypervirulence in Klebsiella pneumoniae. The factors that contribute to the emergence of HMV subpopulations remain unclear. In this study, eight K. pneumoniae strains were recovered from an inpatient who had been hospitalized for 20 days. Three of the isolates exhibited a non-HMV phenotype, which was concomitant with higher biofilm formation than the other five HMV isolates. All eight isolates were highly susceptible to serum killing, albeit HMV strains were remarkably more infective than non-HMV counterparts in a mouse model of infection. Whole genome sequencing (WGS) showed that the eight isolates belonged to the K57-ST412 lineage. Average nucleotide identity (FastANIb) analysis indicated that eight isolates share 99.96% to 99.99% similarity and were confirmed to be the same clone. Through comparative genomics analysis, 12 non-synonymous mutations were found among these isolates, eight of which in the non-HMV variants, including rmpA (c.285delG) and wbaP (c.1305T > A), which are assumed to be associated with the non-HMV phenotype. Mutations in manB (c.1318G > A), dmsB (c.577C > T) and tkt (c.1928C > A) occurred in HMV isolates only. RNA-Seq revealed transcripts of genes involved in energy metabolism, carbohydrate metabolism and membrane transport, including cysP, cydA, narK, tktA, pduQ, aceB, metN, and lsrA, to be significantly dysregulated in the non-HMV strains, suggesting a contribution to HMV phenotype development. This study suggests that co-occurrence of HMV and non-HMV phenotypes in the same clonal population may be mediated by mutational mechanisms as well as by certain genes involved in membrane transport and central metabolism. IMPORTANCE: K. pneumoniae with a hypermucoviscosity (HMV) phenotype is a community-acquired pathogen that is associated with increased invasiveness and pathogenicity, and underlying diseases are the most common comorbid risk factors inducing metastatic complications. HMV was earlier attributed to the overproduction of capsular polysaccharide, and more data point to the possibility of several causes contributing to this bacterial phenotype. Here, we describe a unique event in which the same clonal population showed both HMV and non-HMV characteristics. Studies have demonstrated that this process is influenced by mutational processes and genes related to transport and central metabolism. These findings provide fresh insight into the mechanisms behind co-occurrence of HMV and non-HMV phenotypes in monoclonal populations as well as potentially being critical in developing strategies to control the further spread of HMV K. pneumoniae.

Dihydroartemisinin-driven selective anti-lung cancer proliferation by binding to EGFR and inhibition of NRAS signaling pathway-induced DNA damage.

Chemotherapeutic agents can inhibit the proliferation of malignant cells due to their cytotoxicity, which is limited by collateral damage. Dihydroartemisinin (DHA), has a selective anti-cancer effect, whose target and mechanism remain uncovered. The present work aims to examine the selective inhibitory effect of DHA as well as the mechanisms involved. The findings revealed that the Lewis cell line (LLC) and A549 cell line (A549) had an extremely rapid proliferation rate compared with the 16HBE cell line (16HBE). LLC and A549 showed an increased expression of NRAS compared with 16HBE. Interestingly, DHA was found to inhibit the proliferation and facilitate the apoptosis of LLC and A549 with significant anti-cancer efficacy and down-regulation of NRAS. Results from molecular docking and cellular thermal shift assay revealed that DHA could bind to epidermal growth factor receptor (EGFR) molecules, attenuating the EGF binding and thus driving the suppressive effect. LLC and A549 also exhibited obvious DNA damage in response to DHA. Further results demonstrated that over-expression of NRAS abated DHA-induced blockage of NRAS. Moreover, not only the DNA damage was impaired, but the proliferation of lung cancer cells was also revitalized while NRAS was over-expression. Taken together, DHA could induce selective anti-lung cancer efficacy through binding to EGFR and thereby abolishing the NRAS signaling pathway, thus leading to DNA damage, which provides a novel theoretical basis for phytomedicine molecular therapy of malignant tumors.

Dihydroartemisinin-driven TOM70 inhibition leads to mitochondrial destabilization to induce pyroptosis against lung cancer.

Enhancement of malignant cell immunogenicity to relieve immunosuppression of lung cancer microenvironment is essential in lung cancer treatment. In previous study, we have demonstrated that dihydroartemisinin (DHA), a kind of phytopharmaceutical, is effective in inhibiting lung cancer cells and boosting their immunogenicity, while the initial target of DHA's intracellular action is poorly understood. The present in-depth analysis aims to reveal the influence of DHA on the highly expressed TOM70 in the mitochondrial membrane of lung cancer. The affinity of DHA and TOM70 was analyzed by microscale thermophoresis (MST), pronase stability, and thermal stability. The functions and underlying mechanism were investigated using western blots, qRT-PCR, flow cytometry, and rescue experiments. TOM70 inhibition resulted in mtDNA damage and translocation to the cytoplasm from mitochondria due to the disruption of mitochondrial homeostasis. Further ex and in vivo findings also showed that the cGAS/STING/NLRP3 signaling pathway was activated by mtDNA and thereby malignant cells underwent pyroptosis, leading to enhanced immunogenicity of lung cancer cells in the presence of DHA. Nevertheless, DHA-induced mtDNA translocation and cGAS/STING/NLRP3 mobilization were synchronously attenuated when TOM70 was replenished. Finally, DHA was demonstrated to possess potent anti-lung cancer efficacy in vitro and in vivo. Taken together, these data confirm that TOM70 is an important target for DHA to disturb mitochondria homeostasis, which further activates STING and arouses pyroptosis to strengthen immunogenicity against lung cancer thereupon. The present study provides vital clues for phytomedicine-mediated anti-tumor therapy.

Exosomes regulate doxorubicin resistance in breast cancer via miR-34a-5p/NOTCH1.

Breast cancer (BRCA) is the most common cancer among women. Adriamycin (ADR), also known as doxorubicin (Dox), is a commonly used chemotherapeutic agent for BRCA patients, however, the susceptibility of tumor cells to develop resistance to Dox has severely limited its clinical use. One new promising therapeutic target for breast cancer patients is exosomes. The objective of this study was to investigate the role of exosomes in regulating Dox resistance in BRCA. In this study, the exosomes from both types of cells were extracted by differential centrifugation. The effect of exosomes on drug resistance was assessed by laser confocal microscopy, MTT assay, and qRT-PCR. The miRNA was transfected into cells using Lipofectamine 2000, which was then evaluated for downstream genes and changes in drug resistance. Exosomes from MCF-7 cells (MCF-7/exo) and MCF-7/ADR cells (ADR/exo) were effectively extracted in this study. The ADR/exo was able to endocytose MCF-7 cells and make them considerably more resistant to Dox. Moreover, we observed a significant difference in miR-34a-5p expression in MCF-7/ADR and ADR/exo compared to MCF-7 and MCF-7/exo. Among the miR-34a-5p target genes, NOTCH1 displayed a clear change with a negative correlation. In addition, when miR-34a-5p expression was elevated in MCF-7/ADR cells, the expression of miR-34a-5p in ADR/exo was also enhanced alongside NOTCH1, implying that exosomes may carry miRNA into and out of cells and perform their function. In conclusion, exosomes can influence Dox resistance in breast cancer cells by regulating miR-34a-5p/NOTCH1. These findings provide novel insights for research into the causes of tumor resistance and the enhancement of chemotherapy efficacy in breast cancer.

Unzippable Siamese Nanoparticles for Programmed Two-Stage Cancer Immunotherapy.

Epigenetic drugs (epi-drugs) can destruct cancer cells and initiate both innate and adaptive immunity, yet they have achieved very limited success in solid tumors so far, partly attributing to their concurrent induction of the myeloid-derived suppressor cell (MDSC) population. Here, dissociable Siamese nanoparticles (SIANPs) are developed for tumor cell-targeted delivery of epi-drug CM-272 and MDSC-targeted delivery of small molecule inhibitor Ibrutinib. The SIANPs are assembled via interparticle DNA annealing and detached via tumor microenvironment-triggered strand separation. Such binary regulation induces endogenous retrovirus expression and immunogenic cell death in tumor cells while restraining the immunosuppressive effects of MDSCs, and synergistically promotes dendritic cell maturation and CD8+ T cell activation for tumor inhibition. Significantly, immune microenvironment remodeling via SIANPs further overcomes tumor resistance to immune checkpoint blockade therapy. This study represents a two-pronged approach for orchestrating immune responses, and paves a new way for employing epi-drugs in cancer immunotherapy.

Potential of polyphenols from Ligustrum robustum (Rxob.) Blume on enhancing the quality of starchy food during frying.

The increasing concerns about health have led to a growing demand for high-quality fried foods. The potential uses of Ligustrum robustum (Rxob.) Blume, a traditional tea in China, as natural additives to enhance the quality of starchy food during frying was studied. Results indicated that L. robustum polyphenols extract (LREs) could improve the quality of fried starchy food, according to the tests of color, moisture content, oil content, texture property, and volatile flavor. The in vitro digestion results demonstrated that LRE reduced the final glucose content from 11.35 ± 0.17 to 10.80 ± 0.70 mmol/L and increased the phenolic content of fried starch foods from 1.23 ± 0.04 to 3.76 ± 0.14 mg/g. The appearance and polarizing microscopy results showed that LRE promoted large starch bulges on the surface of fried starchy foods. Meanwhile, X-ray diffraction results showed that LRE increased the intensity of characteristic diffraction peak of fried starch with a range of 21.8%-28%, and Fourier transform infrared results showed that LRE reduced the damage to short-range order structure of starch caused by the frying process. In addition, LRE increased the aggregation of starch granules according to the SEM observation and decreased the enthalpy of starch gelatinization based on the differential scanning calorimetry results. The present results suggest that LREs have the potential to be utilized as a natural additive for regulating the quality of fried starchy food in food industries. PRACTICAL APPLICATION: The enhancement of L. robustum polyphenols on the quality of starchy food during frying was found, and its mechanisms were also explored. This work indicated that L. robustum might be used as a novel economic natural additive for producing high-quality fried foods.

Bilateral keratitis associated with afatinib therapy.

This case discussed a significant ocular side effect, bilateral keratitis, which could be induced by afatinib, an irreversible epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI). We explored the disease progression of a 52-year-old, stage IV nasopharyngeal carcinoma male patient, who was under afatinib treatment and had experienced progressive bilateral eye dryness and tenderness on increasing afatinib from 40 mg every other day to 40 mg daily. Clinical examination noted bilateral visual acuity reduction, diffuse superficial punctate keratopathy in the right eye, and a central epithelial defect in the left eye. Seidel test results were negative for both eyes, with no corneal infiltration, lagophthalmos, anterior chamber cell precipitation, or retinal lesion. Symptoms subsequently resolved after reducing the frequency of afatinib used, along with intensive ocular hydration. In summary, this case highlighted afatinib's potential link to bilateral keratitis, and early afatinib dose adjustment with supportive medication could significantly reverse the condition.

H2-Promoted Benign Coke Strategy for Dimethyl Ether Carbonylation with Long-Term Stability and High Activity.

Zeolite-catalyzed dimethyl ether (DME) carbonylation provides a novel route to producing methyl acetate (MeOAc). Mordenite (MOR) has drawn significant interest because of its remarkable MeOAc selectivity in DME carbonylation, albeit with limited catalytic stability. Herein, novel MOR-based DME carbonylation catalysts, distinguished by long-term stability and high activity were successfully developed, based on an H2-promoted benign coke strategy. Both the H2 cofeeds and the presence of metal species with hydrogenation capability are demonstrated to be crucial for the regulation of coke depositions. The coke deposits can potentially cover the acid sites in the 12-MR main channels, thereby mitigating the occurrence of undesirable methanol-to-hydrocarbon side reactions. Meanwhile, the elimination of ultralarge coke species under the assistance of H2 and Cu species could ensure smooth mass transfer within the catalyst, contributing to its remarkable catalytic performance. The most highlighted DME carbonylation performance was achieved on coke-mediated CuZn-HMOR with a high MeOAc yield of 0.4-0.5 g·gcat-1·h-1 for over 520 h (over 50× enhancement versus HMOR), exhibiting promising industrial application potential. The current strategy is expected to inspire further research into zeolite-catalyzed reactions, which could be potentially improved by the presence of benign coke.

Current and future advances in practice: aromatase inhibitor-induced arthralgia.

Aromatase inhibitors (AIs) have shown great success as adjuvant therapy for post-menopausal women with hormone receptor-positive breast cancers. AI-induced arthralgia (AIA) is a frequent AI toxicity contributing to non-adherence and discontinuation. This review aims to understand current knowledge of AIA. The mean incidence of AIA was 39.1% and the mean discontinuation of AI therapy due to AIA was 9.3%. Most of the AIAs were non-inflammatory. A shorter time since the last menstrual period and pre-existing joint pain were risk factors. Vitamin D3 supplementation may be a preventative measure and treatment with duloxetine, acupuncture and/or exercise is supported by large randomized controlled trials. There was consistent improvement in AIAs with switching to an alternate AI, and this could additionally allow continuation of cancer treatment with AI. Further research is needed to identify predictive biomarkers, better characterize AIA subcategories and study more reliable therapeutic options.

A novel tumor mutation-related long non-coding RNA signature for predicting overall survival and immunotherapy response in lung adenocarcinoma.

Background: Immunotherapy has changed the treatment landscape for lung cancer. This study aims to construct a tumor mutation-related model that combines long non-coding RNA (lncRNA) expression levels and tumor mutation levels in tumor genomes to detect the possibilities of the lncRNA signature as an indicator for predicting the prognosis and response to immunotherapy in lung adenocarcinoma (LUAD). Methods: We downloaded the tumor mutation profiles and RNA-seq expression database of LUAD from The Cancer Genome Atlas (TCGA). Differentially expressed lncRNAs were extracted based on the cumulative number of mutations. Cox regression analyses were used to identify the prognostic lncRNA signature, and the prognostic value of the five selected lncRNAs was validated by using survival analysis and the receiver operating characteristic (ROC) curve. We used qPCR to validate the expression of five selected lncRNAs between human lung epithelial and human lung adenocarcinoma cell lines. The ImmuCellAI, immunophenoscore (IPS) scores and Tumor Immune Dysfunction and Exclusion (TIDE) analyses were used to predict the response to immunotherapy for this mutation related lncRNA signature. Results: A total of 162 lncRNAs were detected among the differentially expressed lncRNAs between the Tumor mutational burden (TMB)-high group and the TMB-low group. Then, five lncRNAs (PLAC4, LINC01116, LINC02163, MIR223HG, FAM83A-AS1) were identified as tumor mutation-related candidates for constructing the prognostic prediction model. Kaplan‒Meier curves showed that the overall survival of the low-risk group was significantly better than that of the high-risk group, and the results of the GSE50081 set were consistent. The expression levels of PD1, PD-L1 and CTLA4 in the low-risk group were higher than those in the high-risk group. The IPS scores and TIDE scores of patients in the low-risk group were significantly higher than those in the high-risk group. Conclusion: Our findings demonstrated that the five lncRNAs (PLAC4, LINC01116, LINC02163, MIR223HG, FAM83A-AS1) were identified as candidates for constructing the tumor mutation-related model which may serve as an indicator of tumor mutation levels and have important implications for predicting the response to immunotherapy in LUAD.

The Role of Immunotherapy in Triple-Negative Breast Cancer (TNBC).

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype, generally associated with a high risk of recurrence and poor prognosis. Our understanding of the heterogeneity of TNBC has increased over the past decade, and with it a recognition that some TNBCs are immunogenically active. This finding has led to the investigation of immunotherapy-based approaches for treatment of both early and advanced-stage TNBC. In this review, we provide an overview of the biologic rationale for immunotherapy use in TNBC, and review data from seminal trials which have culminated in the approval of immunotherapy for both early and advanced TNBC. Identification of predictive biomarkers to aid in treatment selection, development of novel treatment combinations to combat resistance, and refinement of therapeutic targets enables continued improvement in outcomes with immunotherapy for TNBC.

Overexpression of SQUAMOSA promoter binding protein-like 4a (NtSPL4a) alleviates Cd toxicity in Nicotiana tabacum.

Squamosa Promoter Binding Protein-Like (SPL) plays a crucial role in regulating plant development and combating stress, yet its mechanism in regulating resistance to Cd toxicity remains unclear. In this study, we cloned a nuclear-localized transcription factor, NtSPL4a, from the tobacco cultivar TN90. Transient co-expression results showed that miR156 significantly reduced the expression of NtSPL4a by binding to the 3'-UTR of its transcript. We obtained transgenic tobacco overexpressing NtSPL4a (including the 3'-UTR) and NtSPL4aΔ (lacking the 3'-UTR) through Agrobacterium-mediated genetic transformation. Compared to the wild type (WT), overexpression of NtSPL4a/NtSPL4aΔ shortened the flowering time and exhibited a more developed root system. The transgenic tobacco showed significantly reduced Cd content, being 85.1% (OE-NtSPL4a) and 46.7% (OE-NtSPL4aΔ) of WT, respectively. Moreover, the upregulation of NtSPL4a affected the mineral nutrient homeostasis in transgenic tobacco. Additionally, overexpression of NtSPL4a/NtSPL4aΔ effectively alleviated leaf chlorosis and oxidative stress induced by Cd toxicity. One possible reason is that the overexpression of NtSPL4a/NtSPL4aΔ can effectively promote the accumulation of non-enzymatic antioxidants. A comparative transcriptomic analysis was performed between transgenic tobacco and WT to further unravel the global impacts brought by NtSPL4a. The tobacco overexpressing NtSPL4a had 183 differentially expressed genes (77 upregulated, 106 downregulated), while the tobacco overexpressing NtSPL4aΔ had 594 differentially expressed genes (244 upregulated, 350 downregulated) compared to WT. These differentially expressed genes mainly included transcription factors, metal transport proteins, flavonoid biosynthesis pathway genes, and plant stress-related genes. Our study provides new insights into the role of the transcript factor SPL in regulating Cd tolerance.