Iranian Journal of Biotechnology最新文献

Background: Kremen2 is a key regulator of the Wnt/β-catenin signaling pathway, implicating in tumorigenesis. While its role in colorectal cancer (CRC) remains largely unexplored.

Objectives: This study aimed to investigate the biological function of Kremen2 in colorectal cancer and to elucidate the molecular mechanisms underlying its involvement in cancer cell proliferation and metastasis.

Materials and methods: We examined Kremen2 expression in human colon cancer cell lines (SW480 and HCT116) and compared it to normal intestinal epithelial cells (NCM460). A Kremen2-knockdown cell model was established to assess the impact on cell proliferation and migration using standard in vitro assays. The associated signaling pathways were analyzed to determine mechanistic changes following Kremen2 suppression.

Results: Kremen2 expression was significantly elevated in colon cancer cell lines compared to normal controls. Knockdown of Kremen2 in HCT116 cells led to reduced cell viability and impaired migratory ability. Mechanistically, silencing Kremen2 resulted in downregulation of the EGFR/JAK2/STAT3 signaling pathway.

Conclusions: Kremen2 promotes colorectal cancer cell proliferation and migration through activation of the EGFR/JAK2/STAT3 pathway. These findings identify Kremen2 as a potential therapeutic target in CRC.

Background: Interferon alpha-2b, a therapeutic protein, suffers from short half-life. PEGylation extends its circulation time but reduces activity. Site-specific PEGylation on cysteine residues, offers a strategy for improved conjugates.

Objectives: This study explores cysteine analogs of interferon for site-specific PEGylation and assesses their pharmacokinetic properties.

Materials and methods: Surface-exposed amino acids, identified through in silico studies, were selected for cysteine substitution based on the spatial accessibility of the introduced cysteine for site-specific PEGylation. The resulting mutant genes were synthesized, cloned into the pET26-b vector, and expressed in E. coli. The recombinant proteins were purified by immobilized metal chelate affinity chromatography (IMAC) and their biological activity was assessed via cell-based assays. PEGylation was performed using a 5 kDa Methoxy PEG Maleimide, and the resulting conjugate was purified by ion-exchange chromatography. The pharmacokinetic properties of both the PEGylated and unmodified interferon were then evaluated in rat blood circulation.

Results: The in-silico modeling of interferon α-2b identified surface-exposed residues Glu78 and Thr86 as candidates for cysteine substitution. Successful gene synthesis and cloning in E. coli was achieved, followed by protein expression in E. coli with subsequent purification exceeding 95% homogeneity. Activity analysis revealed that the IFN_E78C variant suffered a substantial loss of activity, while IFN_T86C retained 83% of the native interferon's activity. Following PEGylation, the IFN_T86C conjugate retained a high specific activity and exhibited improved pharmacokinetic properties in rats. The differential activities observed between IFN_E78C and IFN_T86C highlight the impact of amino acid positioning on protein function, as IFN_T86C demonstrated significantly better activity retention compared to IFN_E78C. Although PEGylation resulted in some reduction in biological activity, PEG5-IFN_T86C still exhibited superior pharmacokinetic stability compared to its non-PEGylated counterpart.

Conclusion: PEGylation at position 86 improves early interferon pharmacokinetic properties, but longer-term stability requires investigation of higher molecular weight or branched PEG. These findings underline the importance of strategic amino acid modifications and PEGylation in optimizing therapeutic proteins for improved efficacy and stability.

Background: Since the COVID-19 outbreak in 2019, nucleic acid testing has been central to epidemic control and clinical decision-making. However, variability in assay kits and extraction steps can affect sensitivity, specificity, and reproducibility. Systematic evaluation of detection reagents and extraction efficiency is essential to ensure reliable clinical results.

Objective: This study aimed to evaluate whether three domestic novel coronavirus nucleic acid detection kits based on fluorescence PCR meet clinical requirements for COVID-19 testing.

Materials and methods: The performance of the kits was assessed through coincidence rate, precision, detection limit, specificity, and interference resistance using standard positive, negative, and interference reference samples. Amplification efficiency of three detection reagents was compared with eight positive clinical samples. Additionally, amplification performance was examined under oscillation and non-oscillation conditions before nucleic acid extraction, and with varying input volumes.

Results: The coincidence rates for both N and ORF1ab genes in positive and negative references were 100%. Precision testing showed intra-batch coefficients of variation (CV) ≤2.26%. The lower detection limit of 5.0E+02 copies/mL was consistently detected in all repetitions. No cross-reactivity was observed with Influenza A/B, respiratory syncytial virus, parainfluenza virus, Epstein-Barr virus, or Mycobacterium tuberculosis. Interference testing with hemoglobin, bilirubin, triglyceride, oseltamivir, azithromycin, ceftriaxone, and NaCl demonstrated no impact on detection. Comparative analysis showed significant differences in amplification efficiency among the three reagents for both N and ORF1ab genes (p<0.05), with reagent 2 exhibiting faster reaction cycles and reagent 1 providing greater stability. Significant disparities were also observed between oscillated and unoscillated samples (p<0.05), and across different input volumes (notably between 100 μL vs. 200 μL and 200 μL vs. 300 μL).

Conclusions: The evaluated kits met clinical performance standards for coincidence rate, precision, detection limit, specificity, and resistance to interference. While amplification efficiencies differed among reagents and experimental conditions, all satisfied clinical diagnostic requirements. These findings underscore the importance of systematic performance verification and optimization of sample handling to ensure reliable nucleic acid detection for COVID-19.

Background: Zinc finger proteins, particularly Zinc Finger Protein 706 (ZNF706), have been implicated in various cancers, yet their functional and clinical significance in hepatocellular carcinoma (HCC) remains unclear. Identifying novel biomarkers and therapeutic targets is essential for improving HCC diagnosis and treatment.

Objectives: To investigate the functional role of ZNF706 in HCC and evaluate its potential as a diagnostic and therapeutic target with biotechnological applications.

Materials and methods: Bioinformatic analyses were performed using clinical and transcriptomic data from TCGA and GTEx databases to assess ZNF706 expression and its correlation with clinicopathological features and patient prognosis. Quantitative PCR and Western blotting validated ZNF706 expression in liver epithelial and HCC cell lines. Functional assays, including CCK-8, flow cytometry, and Transwell migration/invasion assays, were conducted following ZNF706 knockdown.

Results: ZNF706 was significantly overexpressed in HCC tissues and cell lines compared to normal controls. High ZNF706 expression correlated with advanced TNM stage, pathological stage, and poor survival outcomes. Knockdown of ZNF706 inhibited proliferation, migration, and invasion, and increased apoptosis in HCC cells. ROC analysis demonstrated high diagnostic accuracy (AUC > 0.90) for ZNF706 in distinguishing HCC and predicting prognosis.

Conclusions: ZNF706 is a promising biomarker for HCC diagnosis and prognosis and represents a potential therapeutic target for biotechnological intervention. These findings support the development of ZNF706-based diagnostic assays and targeted therapies for improved management of HCC.

Background: hepatocellular carcinoma (HCC) is a major cancer, and PTTG1 alters asparagine metabolism to promote HCC progression, but its diagnostic and prognostic significance in HCC remains unclear.

Objectives: This study aimed to evaluate the prognostic value of PTTG1-related genes in hepatocellular carcinoma by integrating Mendelian randomization, transcriptomic analysis, and single-cell sequencing approaches.

Materials and methods: This study identified PTTG1-interacting differential genes (PTTG1-IDGs) through differential analysis and protein network construction, then applied Mendelian randomization (MR) to assess their causal relationship with HCC. Univariate Cox regression and machine learning methods screened prognostic genes and constructed prognostic model.

Results: CDC45 and CENPE were prognostic genes with a causal relationship to HCC. Notably, the odds ratios (ORs) of these prognostic genes were close to 1, indicating that although the two genes had a statistically significant causal association with HCC, the independent effect of each allele on HCC risk was weak. This reflected that PTTG1-related genes played a subtle regulatory role rather than a strong direct causal role in the pathogenesis of HCC. nomogram analysis indicated that risk score and pathological T-stage were independent prognostic factors. Immune infiltration and molecular network analysis highlighted the biological value of CDC45 and CENPE. Single-cell analysis demonstrated the key role of hepatocytes in HCC, while pseudotime analysis revealed the distribution of different cell subtypes. Cell communication analysis showed enhanced interactions between PTTG1-highly expressed cells and fibroblasts, myeloid cells, and endothelial cells; experimental validation confirmed elevated expression of CDC45 and CENPE in the HCC group in addition to PTTG1.

Conclusion: Overall, CDC45 and CENPE, as prognostic genes related to PTTG1, provided new research perspectives and potential therapeutic targets for HCC treatment.

Background: Cervical cancer poses a significant threat to women's health as a leading gynecological malignancy. Among them, HeLa cells, as a classic model for cervical cancer research, have attracted much attention regarding the regulatory mechanisms of their proliferation, apoptosis, and invasion. Research indicates that microRNAs (miRNAs) are critically involved in tumorigenesis and cancer progression.

Objective: This study investigates how miR-628-5p influences the proliferation, apoptosis, and invasive potential in cervical cancer-derived HeLa cells.

Materials and methods: To start with, we quantified miR-628-5p expression in cervical carcinoma tissue using RT-qPCR, with comparisons made to adjacent normal tissues. HeLa cells derived from cervical cancer were divided into four groups: blank control, non-targeting plasmid control (miR-NC), miR-628-5p overexpression, and miR-628-5p knockdown. We evaluated miR-628-5p expression using RT-qPCR, assessed proliferation via MTT assay, analyzed invasion/migration with Transwell chambers, and quantified apoptosis by flow cytometry.

Results: Cervical cancer tissues exhibited significant miR-628-5p downregulation compared to both matched normal tissues and controls. Consistent with tumor findings, miR-628-5p levels in paracervical tissues were significantly reduced relative to normal controls. Meanwhile, Overexpression of miR-628-5p produced statistically significant reductions in cellular viability and metastatic propensity in HeLa cultures, alongside a promotion of apoptosis. miR-628-5p knockdown produced opposite effects, significantly enhancing HeLa cell viability and invasiveness while suppressing apoptosis.

Conclusion: Cervical cancer exhibits reduced miR-628-5p expression. Functional analyses revealed that miR-628-5p overexpression suppresses cervical cancer cell proliferation and invasion while promoting apoptosis. This study provides the first evidence that miR-628-5p functions as a tumor suppressor in HeLa cells.

Background: Dendritic cell (DC) maturation is traditionally triggered by pathogen-associated molecular patterns (PAMPs) through Toll-like receptors. However, emerging evidence suggests that the cytokine interleukin-1β (IL-1β) can act independently of PAMPs to drive DC activation and T cell priming.

Objectives: This study aimed to perform a comparative transcriptomic analysis of IL-1β- and R848-induced maturation in human DCs to uncover shared and distinct molecular signatures underlying these activation pathways.

Materials and methods: RNA-seq data from dataset GSE108526 were analyzed following normalization and quality control. Differential gene expression was assessed using a linear model incorporating both treatment type and timepoint as factors. Functional enrichment analyses were conducted to identify pathways and biological processes enriched under each condition.

Results: Both IL-1β and R848 activated core immune response programs, yet exhibited distinct transcriptional emphases. IL-1β preferentially upregulated genes associated with cytokine regulation, surface receptor signaling, and cell communication, whereas R848 induced stronger interferon-stimulated gene networks and antiviral defense pathways. Comparative visualization of key maturation, cytokine, and IL-2 receptor genes revealed overlapping but stimulus-specific expression profiles.

Conclusions: The findings highlight that IL-1β can substitute for PAMPs in inducing DC activation, yet orchestrates a unique immune transcriptional landscape. This suggests a specialized immunomodulatory potential for IL-1β distinct from classical TLR-mediated pathways.

Background: Coronary artery disease (CAD) is the leading cause of death in the world. Traditional imaging diagnostic techniques are difficult to be popularized due to the limitations of equipment and technical threshold. PANoptosis, as a synergistic integration form of apoptosis, necroptosis and pyroptosis, regulates multiple death pathways through PANoptosome complex, which is closely related to inflammation and organ damage.

Objective: This study aims to integrate bioinformatics methods, comprehensively analyze the association between CAD and PANoptosis, and screen the key diagnostic markers.

Materials and methods: Three gene chip datasets (GSE12288, GSE20680 and GSE20681) were obtained from the GEO database, and 7 PANoptosis related differentially expressed genes (PRDEGs) were identified by Venn diagram overlap analysis to construct a CAD diagnostic model. The functional enrichment analysis of these PRDEGs was performed, and human umbilical vein endothelial cells (HUVEC) were used for in vitro functional verification.

Results: FADD, PSTPIP2 and CASP8 were the core regulatory genes of CAD, and the logistic regression model based on three genes showed the best diagnostic efficiency (AUC=0.92). PRDEGs were mainly enriched in inflammatory factor signaling pathway, cell necrosis and pyroptosis pathway. Overexpression of FADD/CASP8 inhibited HUVEC proliferation and promoted apoptosis, while PSTPIP2 showed the opposite effect.

Conclusion: This study reveals the molecular network of PANoptosis in CAD, and the constructed FADD-PSTPIP2-CASP8 diagnostic model provides a new tool for non-invasive screening.

Background: Heavy metal (HMs) pollution is a global concern that demands sustainable remediation approaches. Urease-induced carbonate precipitation provides an eco-friendly alternative that avoids dependence on live microbial systems.

Objectives: This study evaluated urease extracted from Sporosarcina pasteurii for its ability to precipitate Pb²⁺ and Cd²⁺. A novel objective was to investigate whether pre-incubating urease with urea prior to heavy-metal exposure could enhance carbonate production and protect enzymatic activity.

Materials and methods: Urease was extracted after 20 h growth. Optimal enzymatic conditions, kinetic parameters, and precipitation efficiency were assessed under varying conditions. The effect of pre-incubation was evaluated, and the precipitates were characterized using XRD.

Results: The enzyme exhibited optimal activity at pH 8, 60 °C, and 0.25 M urea, with V_max 6.8 U mg^-¹ protein and K_m 89 mM. Pre-incubation increased carbonate production and reduced metal-induced inactivation. Maximum precipitation of Pb²⁺ and Cd²⁺ was achieved at a heavy metal concentration of 20 mM and a urea concentration of 250 mM, resulting in the formation of crystalline carbonates. The removal efficiencies were 82.2% for Cd²⁺ and 38.1% for Pb²⁺.

Conclusion: Extracted urease demonstrates selective affinity toward Cd²⁺ and Pb²⁺. Pre-incubation substantially enhances enzyme stability and precipitation efficiency, offering a cost-effective and sustainable approach for HMs remediation.