BMC Molecular and Cell Biology最新文献

Background: The differentiation of mesodermal cells (MCs) in the early stage of embryonic development contributes to the organogenesis of several core organs. However, the single-cell molecular architecture of MCs and the key molecular events during the differentiation remain unclear.

Methods: We performed single-cell RNA sequencing (RNA sequencing) and single-cell assay for transposase-accessible chromatin (ATAC-Seq) to analyze the developmental features of MCs to heart, kidney, spleen, liver, and brain in human embryos at gestational ages 7-17 weeks.

Results: We found that EGR1 might be relevant to the differentiation of heterogeneous MC sub-clusters. Meanwhile, RPL10P9⁺PTMAP5⁺ MCs had the closest expression profiling with endocardial cells. NDUFA4L2⁺A2M⁺ MCs presented the potentials to form endothelial cells (ECs) and hematopoietic stem cells, and MEF2C might be involved in this process.

Conclusions: These findings provide insights into the molecular architecture and lineage progression of MCs during early human embryonic organogenesis, offering a valuable reference for regenerative medicine and organ bioengineering.

Objectives: We fabricated a pre-vascularized bioprinted hydrogel construct via coculture of human umbilical vein endothelial cells (HUVECs) and dental pulp stem cells (DPSCs) to investigate the role of ephrinB2 in 3D capillary-like cord self-assemble.

Materials and methods: After bioprinting gelatin methacrylate (GelMA) hydrogels, we performed a morphology observation of the inside capillary-like cords, quantitative analysis of branch points and cord length, and examination of pericyte markers in DPSCs. Next, we assessed the influence of DPSCs: HUVECs ratio and degree of functionalization (DoF) of GelMA on the cord formation. To investigate the role of ephrinB2, we used either EphB4/ephrinB2 inhibitor peptide, or knocked down or upregulated ephrinB2 in DPSCs, and evaluated their effects on these cords' generation. Finally, bioprinted hydrogels were subcutaneously transplanted into mice to assess in vitro vascularization.

Results: Abundant capillary-like cords containing lumen were found in bioprinted hydrogels. DPSCs wrapped outside the cords and expressed pericyte markers. A 1:3 DPSCs: HUVECs ratio and a 30% DoF of GelMA led to significant increase in branch points and cord length by over 2-fold and 3-fold, respectively. Adding EphB4/ephrinB2 inhibitor peptide or knockdown of ephrinB2 in DPSCs impaired capillary-like cord generation, whereas overexpression of ephrinB2 in DPSCs promoted cord formation. In in vivo experiments, a significant over 3-fold increase in vessel number was observed in hydrogels when ephrinB2 was upregulated in DPSCs compared with wild-type DPSCs.

Conclusions: Overexpression of ephrinB2 in DPSCs promoted in vitro capillary-like cord formation and in vivo vascularization in a 3D bioprinted hydrogel.

Background: Methyltransferase-like 16 (METTL16) is involved in regulating kidney disease progression. This study aimed to investigate the effect of METTL16 on the progression of diabetic kidney disease (DKD) and its potential mechanism.

Methods: SV40-MES13 cells were transfected with METTL16 siRNA (siMETTL16), scramble control (siNC), or RAP1B siRNA (siRAP1B) under low glucose (LG) or high glucose (HG) conditions. Subsequently, RNA immunoprecipitation (RIP) assays, cell viability assays, EdU staining, TUNEL staining, ROS staining, and MDA detection were carried out.

Results: In SV40-MES13 cells, cell viability, the number of EdU-positive cells, the MDA level, METTL16 mRNA, and protein levels were greater in the HG groups than in the control group. Moreover, cell viability, the number of EdU-positive cells, and VIMENTIN protein levels were lower, whereas the apoptosis rate and ROS and MDA levels were greater in the HG + siMETTL16 group than in the HG + siNC group. RAP1B mRNA and protein levels and N⁶-methyladenosine modification levels were greater in the HG group than in the control group but lower in the HG + siMETTL16 group than in the HG + siNC group. Furthermore, the METTL16 protein bound to the RAP1B mRNA. Compared with those in the HG + siNC group, cell viability, the number of EdU-positive cells, and VIMENTIN protein levels were decreased, whereas the ROS and MDA levels were increased in the HG + siRAP1B group.

Conclusion: METTL16 knockdown attenuates cell viability and fibrosis by reducing m6A modification and the expression of RAP1B in high glucose-treated mesangial cells, suggesting the potential of METTL16 as a treatment target for DKD.

Background: CAPN-1 and CAPN-2, two ubiquitously expressed calpains, have been implicated in cancer progression, but their distinct roles in breast cancer remain poorly defined. This study aims to define the opposing roles of CAPN-1 and CAPN-2 in breast cancer progression, with a focus on their regulatory impact on cell proliferation. Since these calpains may have different functions in the mammary gland, we aimed to investigate the possible antagonistic roles of CAPN-1 and CAPN-2 in breast cancer progression, focusing on their expression patterns and functional impact on cell proliferation.

Methods and results: We analyzed breast cancer cell lines using immunoblotting and real-time cellular assays, showing that HCC1937 cells exhibit an opposite expression pattern of CAPN-1 and CAPN-2 compared to non-cancerous breast cells. CAPN-1 promoted cancer cell survival and negatively regulated CAPN-2 at both the protein and mRNA levels, whereas CAPN-2 suppressed proliferation. Additionally, the calpain activator AG-08 triggered cell death through CAPN-2 but not CAPN-1. In silico analysis confirmed higher CAPN-1 and lower CAPN-2 expression levels in breast cancer samples compared to normal tissue.

Conclusions: These findings indicate that CAPN-1 and CAPN-2 may exert antagonistic roles in breast cancer, but importantly, this effect was restricted to HCC1937 cells, representing a basal A TNBC subtype. Validation in additional basal A models and patient-derived samples will be essential to confirm these results. Our study, therefore, provides preliminary, model-specific insights into calpain regulation in TNBC and suggests that future therapeutic strategies should carefully account for subtype heterogeneity.

Background: Cilia are small, hair-like structures on the surface of most eukaryotic cells. They are composed of distinct substructures: the basal body, the transition zone, and the axoneme. Proper ciliary function is crucial for human health, and defects can result in a group of disorders known as ciliopathies. Many ciliopathy-associated mutations affect genes encoding proteins of the ciliary transition zone, a key structural and regulatory region at the base of the cilium. Understanding the molecular composition and interactions within subciliary compartments, such as the transition zone, is essential to elucidate their role in ciliary function and disease.

Results: Protein interaction studies have played a central role in uncovering the functional landscape of subciliary compartments. In this context, the in situ proximity ligation assay (in situ PLA) has emerged as a valuable tool to investigate whether two proteins are located in close proximity (less than 40 nm) within the cellular environment, implying potential interaction. In situ PLA uses primary antibodies to recognise target proteins, followed by secondary antibodies conjugated with oligonucleotides (PLA probes). When two probes are sufficiently close, added circle-forming oligonucleotides can hybridise and ligate to form a circular DNA strand. This DNA circle serves as a template for rolling circle amplification, which is then detected through hybridisation with fluorescently labelled oligonucleotides. The resulting signals can be visualised using fluorescence microscopy, enabling precise spatial mapping of protein proximities in cells.

Conclusions: The in situ PLA technique offers a powerful means of detecting protein proximities in subciliary compartments with high spatial resolution. This method supports the identification of novel protein interactions and contributes to a deeper understanding of subciliary architecture and its disruption in ciliopathies.