The p53/ZEB1-PLD3 feedback loop regulates cell proliferation in breast cancer.

Breast cancer is the most prevalent cancer globally, endangering women's physical and mental health. Phospholipase D3 (PLD3) belongs to the phosphodiesterase family (PLD). PLD3 is related to insulin-mediated phosphorylation of the AKT pathway, suggesting that it may play a role in the occurrence and development of malignant tumors. This study may further explore the molecular mechanism of PLD3 inhibiting breast cancer cell proliferation. In this study, we demonstrated that PLD3 and miR-6796 are co-expressed in breast cancer. PLD3 can bind with CDK1 and inhibit its expression, leading to mitotic arrest and inhibiting breast cancer proliferation. Wild-type p53 regulates PLD3 and miR-6796 expression by competitively binding to the PLD3 promoter with ZEB1. DNMT3B, as the target gene of miR-6796, is recruited into the PLD3 promoter by combining with ZEB1 to regulate the DNA methylation of the PLD3 promoter and ultimately affect PLD3 and miR-6796 expression. In conclusion, we revealed the role and molecular mechanism of PLD3 and its embedded miR-6796 in breast cancer proliferation, providing clues and a theoretical foundation for future research and development of therapeutic targets and prognostic markers for breast cancer.

PAX5-miR-142 feedback loop promotes breast cancer proliferation by regulating DNMT1 and ZEB1.

BACKGROUND: Breast cancer is one of the most common malignancies occurred in female around the globe. Recent studies have revealed the crucial characters of miRNA and genes, as well as the essential roles of epigenetic regulation in breast cancer initiation and progression. In our previous study, miR-142-3p was identified as a tumor suppressor and led to G2/M arrest through targeting CDC25C. However, the specific mechanism is still uncertain. METHODS: We identified PAX5 as the upstream regulator of miR-142-5p/3p through ALGGEN website and verified by series of assays in vitro and in vivo. The expression of PAX5 in breast cancer was detected by qRT-PCR and western blot. Besides, bioinformatics analysis and BSP sequencing were performed to analyze the methylation of PAX5 promoter region. Finally, the binding sites of miR-142 on DNMT1 and ZEB1 were predicted by JASPAR, and proved by luciferase reporter assay, ChIP analysis and co-IP. RESULTS: PAX5 functioned as a tumor suppressor by positive regulation of miR-142-5p/3p both in vitro and in vivo. The expression of PAX5 was regulated by the methylation of its promoter region induced by DNMT1 and ZEB1. In addition, miR-142-5p/3p could regulate the expression of DNMT1 and ZEB1 through binding with their 3'UTR region, respectively. CONCLUSION: In summary, PAX5-miR-142-DNMT1/ZEB1 constructed a negative feedback loop to regulate the progression of breast cancer, which provided emerging strategies for breast cancer therapy.

CMTM7 inhibits breast cancer progression by regulating Wnt/ß-catenin signaling.

BACKGROUND: Breast cancer is the major cause of death in females globally. Chemokine-like factor like MARVEL transmembrane domain containing 7 (CMTM7) is reported as a tumor suppressor and is involved in epidermal growth factor receptor degradation and PI3K/AKT signaling in previous studies. However, other molecular mechanisms of CMTM7 remain unclear. METHODS: The expression level of CMTM7 in breast cancer cells and tissues was detected by qRT-PCR and western blot, and the methylation of CMTM7 promoter was detected by BSP sequencing. The effect of CMTM7 was verified both in vitro and in vivo, including MTT, colony formation, EdU assay, transwell assay and wound healing assay. The interaction between CMTM7 and CTNNA1 was investigated by co-IP assay. The regulation of miR-182-5p on CMTM7 and TCF3 on miR-182-5p was detected by luciferase reporter assay and ChIP analysis. RESULTS: This study detected the hypermethylation levels of the CMTM7 promoter region in breast cancer tissues and cell lines. CMTM7 was performed as a tumor suppressor both in vitro and in vivo. Furthermore, CMTM7 was a direct miR-182-5p target. Besides, we found that CMTM7 could interact with Catenin Alpha 1 (CTNNA1) and regulate Wnt/ß-catenin signaling. Finally, transcription factor 3 (TCF3) can regulate miR-182-5p. We identified a feedback loop with the composition of miR-182-5p, CMTM7, CTNNA1, CTNNB1 (ß-catenin), and TCF3, which play essential roles in breast cancer progression. CONCLUSION: These findings reveal the emerging character of CMTM7 in Wnt/ß-catenin signaling and bring new sights of gene interaction. CMTM7 and other elements in the feedback loop may serve as emerging targets for breast cancer therapy.

ZNF384-ZEB1 feedback loop regulates breast cancer metastasis.

BACKGROUND: Breast cancer has become the most frequently diagnosed cancer worldwide. Increasing evidence indicated that zinc finger proteins (ZNFs), the largest family of transcription factors, contribute to cancer development and progression. Although ZNF384 is overexpressed in several types of human cancer, the role of ZNF384 in breast cancer remains unknown. Therefore, our research focused on ZNF384 regulation of the malignant phenotype of breast cancer and the underlying molecular mechanisms. METHODS: CCK-8 and colony formation assays were used to evaluate cell proliferation. Transwell and scratch assays were used to evaluate the cell migration and invasion. Chromatin immunoprecipitation (ChIP)-qPCR and luciferase reporter assays were used to confirm the target relationship between ZNF384 and zinc finger E-box binding homeobox 1 (ZEB1). Xenografts were used to monitor the targets in vivo effects. RESULTS: We noted that ZNF384 was significantly overexpressed in breast cancer and highlighted the oncogenic mechanism of ZNF384. ZNF384 transactivated ZEB1 expression and induced an epithelial and mesenchymal-like phenotype, resulting in breast cancer metastasis. Furthermore, ZNF384 may be a target of miR-485-5p, and ZEB1 can up-regulate ZNF384 expression by repressing miR-485-5p expression. Together, we unveiled a feedback loop of ZNF384-ZEB1 in breast cancer metastasis. CONCLUSIONS: The findings suggest that ZNF384 can serve as a prognostic factor and a therapeutic target for breast cancer patients.

miR-92a-3p promotes breast cancer proliferation by regulating the KLF2/BIRC5 axis.

BACKGROUND: Breast cancer remains the most common malignancy in females around the world. Recently, a growing number of studies have focused on gene dysregulation. In our previous study, Krüppel-like factors (KLFs) were found to play essential roles in breast cancer development, among which KLF2 could function as a tumor suppressor. Nevertheless, the underlying molecular mechanism remains unclear. METHODS: miR-92a-3p was identified as the upstream regulator of KLF2 by starBase v.3.0. The regulation of KLF2 by miR-92a-3p was verified by a series of in vitro and in vivo assays. Further exploration revealed that Baculoviral IAP Repeat Containing 5 (BIRC5) was the target of KLF2. ChIP assay, dual-luciferase reporter analysis, quantitative real-time PCR, and western blot were performed for verification. RESULTS: miR-92a-3p functioned as a tumor promoter by inhibiting KLF2 by binding to its 3'-untranslated region (3'-UTR). In addition, KLF2 could transcriptionally suppress the expression of BIRC5. CONCLUSION: Collectively, our results uncovered the miR-92a-3p/KLF2/BIRC5 axis in breast cancer and provided a potential mechanism for breast cancer development, which may serve as promising strategies for breast cancer therapy.

Global Fold Switching of the RafH Protein: Diverse Structures with a Conserved Pathway.

It is generally believed that a protein's sequence uniquely determines its structure, the basis for a protein to perform biological functions. However, as a representative metamorphic protein, RfaH can be encoded by a single amino acid sequence into two distinct native state structures. Its C-terminal domain (CTD) either takes an all-α-helical configuration to pack tightly with its N-terminal domain (NTD), or the CTD disassociates from the NTD, transforms into an all-ß-barrel fold, and further attaches to the ribosome, leaving the NTD exposed to bind RNA polymerases. Therefore, the RfaH protein couples transcription and translation processes. Although previous studies have provided a preliminary understanding of its function, the full course of the conformational change of RfaH-CTD at the atomic level is elusive. We used teDA2, a feature space-based enhanced sampling protocol, to explore the transformation of RfaH-CTD. We found that it undergoes a large-scale structural rearrangement, with characteristic spectra as the fingerprint, and a global unfolding transition with a tighter and energetically moderate molten globule-like nucleus formed in between. The formation of this nucleus limits the possible intermediate conformations, facilitates the formation of secondary and tertiary structures, and thus ensures the efficiency of transformation. The key features along the transition path disclosed from this work are likely associated with the evolution of RfaH, such that encoding a single sequence into multiple folds with distinct biological functions is energetically unhindered.

Comparative transcriptomic analysis-based identification of the regulation of foreign proteins with different stabilities expressed in Pichia pastoris.

Introduction: The industrial yeast Pichia pastoris is widely used as a cell factory to produce proteins, chemicals and advanced biofuels. We have previously constructed P. pastoris strains that overexpress protein disulfide isomerase (PDI), which is a kind of molecular chaperone that can improve the expression of an exogenous protein when they are co-expressed. Chicken cystatin (cC) is a highly thermostable cysteine protease inhibitor and a homologous protein of human cystatin C (HCC). Wild-type cC and the two mutants, I66Q and ΔW (a truncated cC lacking the á-helix 2) represent proteins with different degrees of stability. Methods: Wild-type cC, I66Q and ΔW were each overexpressed in P. pastoris without and with the coexpression of PDI and their extracellular levels were determined and compared. Transcriptomic profiling was performed to compare the changes in the main signaling pathways and cell components (other than endoplasmic reticulum quality control system represented by molecular chaperones) in P. pastoris in response to intracellular folding stress caused by the expression of exogenous proteins with different stabilities. Finally, hub genes hunting was also performed. Results and discussion: The coexpression of PDI was able to increase the extracellular levels of both wild-type cC and the two mutants, indicating that overexpression of PDI could prevent the misfolding of unstable proteins or promote the degradation of the misfolded proteins to some extent. For P. pastoris cells that expressed the I66Q or ΔW mutant, GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses of the common DEGs in these cells revealed a significant upregulation of the genes involved in protein processing, but a significant downregulation of the genes enriched in the Ribosome, TCA and Glycolysis/Gluconeogenesis pathways. Hub genes hunting indicated that the most downregulated ribosome protein, C4QXU7 in this case, might be an important target protein that could be manipulated to increase the expression of foreign proteins, especially proteins with a certain degree of instability. Conclusion: These findings should shed new light on our understanding of the regulatory mechanism in yeast cells that responds to intracellular folding stress, providing valuable information for the development of a convenient platform that could improve the efficiency of heterologous protein expression in P. pastoris.

HS3ST3A1 and CAPN8 Serve as Immune-Related Biomarkers for Predicting the Prognosis in Thyroid Cancer.

Background: Thyroid cancer (TC) tends to be a common malignancy worldwide and results in various outcomes due to its different subtypes. The tumor microenvironment (TME) was demonstrated to play crucial roles in various malignancies, including thyroid cancer. This study combined the ESTIMATE and CIBERSORT algorithms, identified four TME-related genes, and evaluated their correlation with clinical characteristics. These findings revealed the malignant performance of TME in TC, and the TME-related DEGs might serve as prognostic biomarkers, which can be utilized for the prediction of immunotherapy effects in patients with TC. Methods: The clinical and gene expression profiles of TC patients were collected from the TCGA dataset. The ESTIMATE algorithm was utilized to estimate stromal and immune scores and predict the level of stromal and immune cell infiltration. The differential expressed genes related to TME were filtered by the "limma" package in R, and the PPI network was constructed by a string website. KEGG pathway and GO analyses were performed to investigate the biological progression and molecular functions of TME-related DEGs. Then, univariate Cox regression analysis was employed to screen four genes correlated with clinical characteristics. GSEA was conducted to assess their roles in the TME of TC. To further investigate the association between TME-related genes and tumor-infiltrating immune cells (TIICs), the CIBERSORT algorithm was performed. Finally, the malignancy behaviors of the two genes were verified by RT-qPCR, IHC, MTT, colony formation, and transwell assays. Results: Four TME-related DEGs, LRRN4CL, HS3ST3A1, PCOLCE2, and CAPN8, were identified and were significantly predictive of poor overall survival. KEGG and GO pathway analysis established that the TME-related DEGs were involved in immune responses and pathways in cancer. Furthermore, the malignancy behaviors of HS3ST3A1 and CAPN8 were verified by cellular functional experiments. These results revealed that the TME-related genes HS3ST3A1 and CAPN8 were able to serve as predictors of prognosis in patients with TC. Conclusion: HS3ST3A1 and CAPN8 may serve as valuable prognostic biomarkers and TME indicators, which can be utilized for the prediction of immunotherapy effects and provide novel treatment strategies for patients with TC.

The Morphology of Hydroxyapatite Nanoparticles Regulates Cargo Recognition in Clathrin-Mediated Endocytosis.

The clathrin-associated protein adaptin-2 (AP2) is a distinctive member of the hetero-tetrameric clathrin adaptor complex family. It plays a crucial role in many intracellular vesicle transport pathways. The hydroxyapatite (HAp) nanoparticles can enter cells through clathrin-dependent endocytosis, induce apoptosis, and ultimately inhibit tumor metastasis. Exploring the micro process of the binding of AP2 and HAp is of great significance for understanding the molecular mechanism of HAp's anti-cancer ability. In this work, we used molecular modeling to study the binding of spherical, rod-shaped, and needle-shaped HAps toward AP2 protein at the atomic level and found that different nanoparticles' morphology can determine their binding specificity through electrostatic interactions. Our results show that globular HAp significantly changes AP2 protein conformation, while needle-shaped HAP has more substantial binding energy with AP2. Therefore, this work offers a microscopic picture for cargo recognition in clathrin-mediated endocytosis, clarifies the design principles and possible mechanisms of high-efficiency nano-biomaterials, and provides a basis for their potential anti-tumor therapeutic effects.

Hydrogen peroxide modification affects the structure and physicochemical properties of dietary fibers from white turnip (Brassica Rapa L.).

Turnip (Brassica rapa L.) is widely consumed as a vegetable and traditional Chinese medicine with high dietary fiber content. Soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) were obtained from white turnips, and the IDF was modified with alkaline hydrogen peroxide to obtain modified IDF (MIDF) and modified SDF (MSDF). The compositional, structural, and functional properties of the four samples were investigated. After modification, the modified dietary fibers (MDFs) showed smaller particle sizes and lower contents of pectin and polyphenol than those of unmodified dietary fibers (DFs) The results of scanning electron microscopy (SEM), Fourier transformed infrared (FT-IR) spectroscopy, X-ray diffraction (XRD) and differential scanning calorimetry (DSC) showed that compared to the DFs, the MDFs were smaller and had more exposed hydroxyl groups. Analysis of the microrheological behaviors showed that the MDFs had higher viscosity than that of the DFs, with a looser structure for the MSDF and a stable structure for the MIDF. Therefore, due to structural changes, the physical and functional properties of the MDFs were improved compared to those of the unmodified DFs. Pearson correlation analysis showed that the particle size was positively correlated with the pectin content. The water holding capacity (WHC), oil adsorption capacity (OAC) and water swelling capacity (WSC) showed positive correlations with each other. This work indicated that white turnip could be a potential new source of DFs, which presented desirable functional properties after modification.

Defining the mechanism of PDI interaction with disulfide-free amyloidogenic proteins: Implications for exogenous protein expression and neurodegenerative disease.

Protein disulfide isomerase (PDI) is an important molecular chaperone capable of facilitating protein folding in addition to catalyzing the formation of a disulfide bond. To better understand the distinct substrate-screening principles of Pichia pastoris PDI (Protein disulfide isomerase) and the protective role of PDI in amyloidogenic diseases, we investigated the expression abundance and intracellular retention levels of three archetypal amyloidogenic disulfide bond-free proteins (Aß42, α-synuclein (α-Syn) and SAA1) in P. pastoris GS115 strain without and with the overexpression of PpPDI (P. pastoris PDI). Intriguingly, amyloidogenic Aß42 and α-Syn were detected only as intracellular proteins whereas amyloidogenic SAA1 was detected both as intracellular and extracellular proteins when these proteins were expressed in the PpPDI-overexpressing GS115 strain. The binding between PpPDI and each of the three amyloidogenic proteins was investigated by molecular docking and simulations. Three different patterns of PpPDI-substrate complexes were observed, suggesting that multiple modes of binding might exist for the binding between PpPDI and its amyloidogenic protein substrates, and this could represent different specificities and affinities of PpPDI toward its substrates. Further analysis of the proteomics data and functional annotations indicated that PpPDI could eliminate the need for misfolded proteins to be partitioned in ER-associated compartments.

Insights into the mechanism of how Morin suppresses amyloid fibrillation of hen egg white lysozyme.

This communication describes the inhibitory effect of Morin on the fibrillation of Hen Egg White Lysozyme (HEWL), a generic amyloid-forming model protein. This effect was dose-dependent and stronger than other small molecules we have tested previously. Spectrofluorometric and computational studies support a model suggesting that Morin inhibits amyloid fibril formation of HEWL by binding to the aggregation prone cleft region of the ß-domain of HEWL, thereby stabilizing the molecule in its native-like state. Interestingly, transmission electron microscopy observations suggest that, along with increases in Morin concentration, the observed amorphous aggregates became larger and morphologically different. We propose that following occupation of the binding cleft, excess Morin adheres and coats the HEWL protein surface, thereby minimizing the interaction between the protein surface and water molecules.