Journal of Molecular Cell Biology

End-stage renal disease (ESRD) remains to be a major clinical challenge with persistently high morbidity and mortality, and its molecular mechanisms, particularly those shared among diverse primary kidney diseases during the progression to ESRD, have not been studied. Here we conducted a large-scale two-stage epigenome-wide association study of ESRD in two independent cohorts consisting of 704 controls and 1031 ESRD cases resulting from multiple kidney diseases. We identified 52 ESRD-associated differentially methylated CpG loci (DMLs) that showed consistent association effect between the two cohorts and across diverse kidney diseases. These 52 DMLs implicated 144 candidate genes that showed enrichment in calprotectin complex, RAGE receptor binding and herpes simplex virus 1 infection. Of the 52 DMLs, 5 DMLs were found to be associated with common complications of ESRD, and another 7 DMLs were also found to be associated with renal function decline in early-stage chronic kidney disease, demonstrating their potential as prognostic biomarkers for ESRD risk and related clinical complications. By identifying prognostic biomarkers and revealing the important roles of inflammation and immune dysregulation and renal fibrosis in renal progression to ESRD across diverse primary kidney diseases, our study has contributed greatly to improve clinical management and advance the development of novel therapies for ESRD.

Calciphylaxis, also known as calcific uremic arteriolopathy (CUA), is an orphan disease without proven therapies, we rescued it with human amnion-derived mesenchymal stem cells (hAMSCs). In a discovery cohort of 10 uremic patients and 3 CUA patients, plasma proteomic analysis showed core differentially expressed proteins (DEPs) Thrombospondin 1 (THBS1) and Latent transforming growth factor (TGF)-{beta} binding protein 1 (LTBP1) decreased significantly after 3 days of hAMSC treatment. Single-cell transcriptome sequencing of peripheral blood mononuclear cells (PBMCs) indicated megakaryocytes were the source of THBS1 in CUA patient. Same as the discovery cohort, plasma THBS1 and TGF-{beta}1 levels were increased in seven CUA patients compared to the uremic group (n=20), as measured by enzyme-linked immunosorbent assay (ELISA) in the validation cohort. They can be inhibited after hAMSC treatment and increased as the frequency of therapy decreased. THBS1 and its receptor, CD47, were increased in the CUA skin. THBS1 and TGF-{beta}1 are biomarker candidates for calciphylaxis.

After fertilization, maternally deposited mRNA is cleared, and de novo mRNA is transcribed from the zygotic genome through zygotic genome activation (ZGA), a process known as maternal-to-zygotic transition (MZT) occurring in the mouse at 2-cell (2C) stage. 2C-like cells (2CLCs) marked by MERVL expression are transcriptionally similar to 2C embryos spontaneously emerge from mouse embryonic stem cells (mESCs). Although the emergence of 2CLCs completely depends on DUX function, a recent knockout study clearly showed that DUX is dispensable for mouse embryos, suggesting that DUX-independent molecular pathways are not recapitulated in 2CLCs. We present here that the disruption of C-terminal binding protein 1/2 (Ctbp1/2) activates DUX-dependent and -independent molecular pathway associated with the development of early mouse embryos mediated by the upregulation of Preferentially expressed antigen of melanoma family-like 7 (PRAMEL7). Furthermore, the abnormality of the gene expression profile caused by Dux KO is partially rescued by the overexpression of PRAMEL7 in mESCs. Our study provides new insights into the DUX-independent molecular pathway for the activation of early embryonic genes in mESCs.

The nuclear mitochondrial DNA (NUMT) is found in cancer cells, but the mitochondrial DNAs entering the nuclei in normal cells have not been captured. Here, we utilized super-resolution optical imaging to capture the phenomenon by the probe PicoGreen and found mitochondrial DNAs and mitochondria accumulated in the nucleoli by four probes and overexpressing the MRPL58-DsRed. Our results provide an new explanation for mtDNA carryover and lay the foundation for the involvement of nuclear export of nucleoli in de novo mitochondrial biogenesis in another of our unpublished articles.

Triple-nucleotide chain of RNA:DNA hybrid structure R-loop is formed during transcription process which is closely associated with transcriptional regulation, transcriptional termination, epigenetic modifications, and structure of chromatin. Dysregulation of R-loop formation or resolution may disturb normal DNA replication or RNA transcription, leading to associated progress of disease. Herein, we found A-kinase-anchoring protein 8 (AKAP8) bind with R-loop structure in cell. Knock down of AKAP8 showed perturbation of balance of genomic R-loop formation and gene transcription. Evidences was shown that AKAP8 interacted with R-loop resolution protein ATP-dependent DEAD box RNA helicase DDX5 and increased chromatin associated DDX5 level. Finally, AKAP8 promoted UCP2 transcription and resolved R-loop level of its promoter region, and contributed to cell growth of adenocarcinomic human alveolar basal epithelial cell line A549, which may provide clues for poor prognosis of lung adenocarcinoma in high AKAP8 expression patients.

For insights into the fact that liver-specific knockout of Nrf1 leads to development of non-alcoholic steatohepatitis and spontaneous hepatoma, we previously found that loss of Nrf1 (i.e., a full-length isoform encoded by Nfe2l1) promotes HepG2-derived tumor growth in xenograft mice, but malgrowth of the xenograft tumor is significantly suppressed by knockout of Nrf2 (encoded by Nfe2l2). The mechanism underlying such marked distinctions in their pathologic phenotypes remains elusive, however, to date. Herein, we mined the transcriptome data of liver cancer from the TCGA database to establish a prognostic model of liver cancer and then calculated the predicted risk score of each cell line. The results indicated that knockout of Nrf1 significantly increased the risk score in HepG2 cells, whereas the risk score was reduced by knockout of Nrf2. Of note, stanniocalcin 2 (STC2, a biomarker of liver cancer, that is up-expressed in hepatocellular carcinoma (HCC) tissues with a reduction in the overall survival ratio of those patients) was augmented in Nrf1Nrf2-/- cells, but diminished in Nrf2-/- cells. Thereby, it is inferable that STC2 is likely involved in mediating the distinction between Nrf1Nrf2-/- and Nrf2-/-. Further investigation revealed that HIF1A is an upstream regulator of STC2 in caNrf2{Delta}N, rather than Nrf1Nrf2-/-, cells, and regulation of STC2 and HIF1A in Nrf1Nrf2-/- is determined by Nrf2, but the regulation of STC2 by Nrf2 may be independent on HIF1A. In turn, STC2 can regulate Nrf2 via the putative calcium-mediated Keap1-p62 signaling so to form a feedback regulatory loop. Such potential function of STC2 was further corroborated by a series of experiments combined with transcriptomic sequencing. The results unraveled that STC2 manifests as a dominant tumor-promoter, because the STC2-leading increases in clonogenicity of hepatoma cells and malgrowth of relevant xenograft tumor were almost completely abolished in STC2-/-cells. Together, these demonstrate that STC2 could be paved as a novel potent therapeutic target, albeit as a diagnostic marker, for hepatocellular carcinoma.

Palatine tonsil (hereinafter referred to as "tonsil") plays role in the immune systems first line of defense against foreign pathogens. Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a worldwide pandemic since the infection was first reported in China in December 2019. The aim of this study was to establish tonsil epithelial cell-derived organoids and to examine their feasibility as an ex vivo model for SARS-CoV-2 infection. Using an optimized protocol, we achieved 3D tonsil organoid culture from human tonsil tissue that reflects the distinctive characteristics of the tonsil epithelium, such as its cellular composition, histologic properties, and molecular biological features. Notably, we verified that SARS-CoV-2 can infect tonsil organoids with a robust replication efficiency. Furthermore, treatment with remdesivir, an antiviral agent, effectively protected them from viral infection. Therefore, tonsil organoids could be available for investigation of SARS-CoV-2 infection-mediated pathology and for preclinical screening of novel antiviral drug candidates. One-sentence SummaryThis study established tonsil epithelial cell-derived organoids and demonstrated their feasibility as an ex vivo model for SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is spread primary via respiratory droplets and infects the lungs. Currently widely used cell lines and animals are unable to accurately mimic human physiological conditions because of the abnormal status of cell lines (transformed or cancer cells) and species differences between animals and humans. Organoids are stem cell-derived self-organized three-dimensional culture in vitro and model the physiological conditions of natural organs. Here we demonstrated that SARS-CoV-2 infected and extensively replicated in human embryonic stem cells (hESCs)-derived lung organoids, including airway and alveolar organoids. Ciliated cells, alveolar type 2 (AT2) cells and rare club cells were virus target cells. Electron microscopy captured typical replication, assembly and release ultrastructures and revealed the presence of viruses within lamellar bodies in AT2 cells. Virus infection induced more severe cell death in alveolar organoids than in airway organoids. Additionally, RNA-seq revealed early cell response to SARS-CoV-2 infection and an unexpected downregulation of ACE2 mRNA. Further, compared to the transmembrane protease, serine 2 (TMPRSS2) inhibitor camostat, the nucleotide analog prodrug Remdesivir potently inhibited SARS-CoV-2 replication in lung organoids. Therefore, human lung organoids can serve as a pathophysiological model for SARS-CoV-2 infection and drug discovery.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes coronavirus disease 2019 (COVID-19), the respiratory illness responsible for the COVID-19 pandemic. SARS-CoV-2 is a positive-stranded RNA virus belongs to Coronaviridae family. The viral genome of SARS-CoV-2 contains around 29.8 kilobase with a 5'-cap structure and 3'-poly-A tail, and shows 79.2% nucleotide identity with human SARS-CoV-1, which caused the 2002-2004 SARS outbreak. As the successor to SARS-CoV-1, SARS-CoV-2 now has circulated across the globe. There is a growing understanding of SARS-CoV-2 in virology, epidemiology, and clinical management strategies. In this study, we verified the existence of two 18-22 nt small viral RNAs (svRNAs) derived from the same precursor in human specimens infected with SARS-CoV-2, including nasopharyngeal swabs and formalin-fixed paraffin-embedded (FFPE) explanted lungs from lung transplantation of COVID-19 patients. We then simulated and confirmed the formation of these two SARS-CoV-2-Encoded small RNAs in human lung epithelial cells. And the potential pro-inflammatory effects of the splicing and maturation process of these two svRNAs in human lung epithelial cells were also explored. By screening cytokine storm genes and the characteristic expression profiling of COVID-19 in the explanted lung tissues and the svRNAs precursor transfected human lung epithelial cells, we found that the maturation of these two small viral RNAs contributed significantly to the infection associated lung inflammation, mainly via the activation of the CXCL8, CXCL11 and type I interferon signaling pathway. Taken together, we discovered two SARS-CoV-2-Encoded small RNAs and investigated the pro-inflammatory effects during their maturation in human lung epithelial cells, which might provide new insight into the pathogenesis and possible treatment options for COVID-19.

To investigate the role of nuclear receptor interaction protein (NRIP) in myoblast fusion, both the primary myoblasts from muscle-specific NRIP-knockout mice and NRIP-null C2C12 cells (KO19 cells) exhibited a significant deficit in the fusion index during myogenesis; on the other hand, overexpressed NRIP in KO19 cells could rescue myotube formation. Furthermore, NRIP was found to interact with actin directly and reciprocally that is an invadosome component for myoblast fusion. Endogenous NRIP colocalized with components of invadosome such as F-actin, Tks5, and cortactin at the tips of cells during C2C12 differentiation, and exogenous NRIP was enriched with actin at the tip of attacking cells during myogenic fusion, implying that NRIP is a novel invadosome component. Using time-lapse microscopy and cell-cell fusion assays further confirmed NRIP directly participates in cell fusion through actin. Moreover, to map the domain of NRIP-actin binding, NRIP interacted with actin either through WD40 domains directly for binding or indirectly through the IQ domain for -actinin 2 binding with actin. NRIP with actin binding was strongly correlated with invadosome formation and myotube fusion. Collectively, NRIP acts as a novel actin-binding protein through its WD40 or the IQ to form invadosomes that trigger myoblast fusion.

Although increasing studies has demonstrated that cell competition widely involved in the growth and homeostasis of multicellular organisms is closely linked to tumorigenesis and development, the mechanistic contributions to the association between tumor cell competition-driven heterogeneity and drug resistance remains ill-defined. In our study, lenvitinib-resistant hepatocellular carcinoma (HCC) cells display obviously competitive growth dominance against sensitive cells through reprogramming energy metabolism. Mechanistically, when BCL2 interacting protein3 (BNIP3) overexpression activates mitophagy activity in lenvatinib-resistant HCC cells, energy imbalance signal caused by reduced mitochondrial oxidative phosphorylation levels provokes the phosphorylation of AMP-activated protein kinase (AMPK) sensor; subsequently, enabled AMPK specifically targets enolase 2 (ENO2) to enhance glycolysis and eventually promots the competitive capacity and dominant growth. Of note, BNIP3 deficiency shows certain inhibition of cell competition outcome. Our findings emphasize a vital role for BNIP3-AMPK-ENO2 signaling in maintaining the competitive outcome of lenvitinib-resistant HCC cells via regulating energy metabolism; meanwhile this work recognaizes BNIP3 as a promising target to overcome HCC drug resistance.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is raging across the world, leading to a global mortality rate of 3.4% (estimated by World Health Organization in March 2020). As a potential vaccine and therapeutic target, the nucleocapsid protein of SARS-CoV-2 (nCoVN) functions in packaging the viral genome and viral self-assembly. To investigate the biological effects of nCoVN to human stem cells, genetically engineered human induced pluripotent stem cells (iPSC) expressing nCoVN (iPSC-nCoVN) were generated by lentiviral expression systems, in which the expression of nCoVN could be induced by the doxycycline. The proliferation rate of iPSC-nCoVN was decreased. Unexpectedly, the morphology of iPSC started to change after nCoVN expression for 7 days. The pluripotency marker TRA-1-81 were not detectable in iPSC-nCoVN after a four-day induction. Meanwhile, iPSC-nCoVN lost the ability for differentiation into cardiomyocytes with a routine differentiation protocol. The RNA-seq data of iPSC-nCoVN (induction for 30 days) and immunofluorescence assays illustrated that iPSC-nCoVN were turning to fibroblast-like cells. Our data suggested that nCoVN disrupted the pluripotent properties of iPSC and turned them into other types of cells, which provided a new insight to the pathogenic mechanism of SARS-CoV-2.

Regeneration after tissues injury is often associated with cell fate plasticity, which restores damaged or lost cells. The de-differentiation of corneal epithelial cells (CECs) into functional stem cells after the ablation of innate stem cells, known as limbal epithelial stem cells (LESCs), remains controversial. In this study, we showed the functional maintenance of corneal epithelium after the ablation of innate stem cells, and the regeneration of functional LESCs, which maintained corneal transparency, prevented corneal conjunctivalization and participated in the wound healing. Subsequent intravital lineage tracing revealed that CECs could de-differentiate into active or quiescent LESCs, which functioned as well as their innate counterparts. Furthermore, the de-differentiation of CECs required an intact limbal niche, and the outcome of the competition between conjunctival and corneal epithelium for the limbal niche determined whether the de-differentiation would occur or not. Mechanically, the suppression of YAP signal promoted the de-differentiation of CECs after the ablation of innate stem cells, while the persistent activation of YAP prevented the de-differentiation of CECs after an additional alkali burn to the limbal stroma. These results will pave the way for an alternative approach to treat limbal stem cell deficiency (LSCD) by modulating the de-differentiation of CECs in vivo.

Human embryonic stem cells (hESCs) derived lung organoids (HLOs) provide a promising model to study human lung development and disease. However, whether HLO cells could reconstitute airway epithelial structure in vivo remains unclear. Here we established an orthotopic xenograft system for hESCs-derived HLOs, enabling stable reconstruction of human airway epithelium in vivo. Removal of the mouse airway epithelium by naphthalene (NA) treatment enabled xenografted organoid cells survival, differentiation, and reconstruction of airway pseudostratified epithelium in immune-compromised NSG mice. Compared to unsorted pool cells, CD47high cells generated more ciliated cells and possessed thicker pseudostratified epithelium. RNA-seq data revealed that CD47high cells highly expressed epithelial cell, lung progenitor, lung proximal cell and embryonic lung development associated genes. These data reveal that HLOs hold cell therapy potential in regenerative medicine by long-term reconstituting airway epithelium.

MicroRNAs (miRNAs) control organogenesis in mammals but their role in specific cell types is not fully explored. miRNAs exert their function by binding mRNAs and inhibiting translation. Skin is an excellent model to study the role of miRNAs in primary cells of epidermal (keratinocytes) and mesodermal (fibroblasts) origin, because the growth of these cells is tightly controlled at translation. Previous research demonstrated that miRNA-29 family functions during skin repair, however, the exact mRNA targets and the downstream mechanisms of miRNA-29-mediated regulation of cell growth is missing. Here, we use miRNA crosslinking and immunoprecipitation (miRNA-CLIP) method to find the direct targets of miRNA-29 in keratinocytes and fibroblasts from human skin. We uncover previously unrecognized roles of miRNA-29 in protein folding and RNA processing, common to all cell types tested, and determine the cell-specific role of miRNA-29. Using modified anti-sense oligonucleotides (ASO) in 2D and 3D cultures of keratinocytes and fibroblasts, we enhanced cell-to-matrix adhesion and found an autocrine and paracrine mechanism of miRNA-29-dependent cell growth. Our results include a comprehensive list and functional analyses of mRNAs directly bound by miRNA-29 keratinocytes and fibroblasts, determined by miRNA-CLIP and ASO-mediated inhibition of miRNA-29 followed by RNA-seq. We reveal a full transcriptome of human keratinocytes with enhanced adhesion to the wound matrix, which supports regeneration of the epidermis and is regulated by miRNA-29. The functions of miRNA-29 identified in this study can provide a new approach to improve cutaneous repair by restoring and enhancing the endogenous mechanisms through the stage-specific delivery of miRNA-29 ASO.

The outbreak of COVID-19 has caused serious epidemic events in China and other countries. With the rapid spread of COVID-19, it is urgent to explore the pathogenesis of this novel coronavirus. However, the foundational research of COVID-19 is very weak. Although angiotensin converting enzyme 2 (ACE2) is the reported receptor of SARS-CoV-2, information about SARS-CoV-2 invading airway epithelial cells is very limited. Based on the analysis of the Human Protein Atlas database, we compared the virus-related receptors of epithelial-derived cells from different organs and found potential key molecules in the local microenvironment for SARS-CoV-2 entering airway epithelial cells. In addition, we found that these proteins were associated with virus reactive proteins in host airway epithelial cells, which may promote the activation of the immune system and the release of inflammatory factors. Our findings provide a new research direction for understanding the potential microenvironment required by SARS-CoV-2 infection in airway epithelial, which may assist in the discovery of potential drug targets against SARS-CoV-2 infection.

Spatial cellular organization patterns (COPs) in tumor microenvironment influence the tumor progression and therapeutic response, however, little is known about the cellular composition and functional potential of these multicellular structures during lung adenocarcinoma progression. Here, we integrate spatial transcriptomics with single cell RNA sequencing to characterize the local tumor and immunological landscape of samples from 8 patients with early-stage lung adenocarcinoma at different pathological stages. We identified ten COPs that show distinct associations with local immune states and clinical outcomes, including survival and therapy response. The local infiltration levels of regulatory and dysfunctional immune cells are increased with pathological progression. Cell-to-cell interactions between malignant cells and tumor microenvironment (TME) cells were involved in protumor immune state remodeling. Finally, we detected a group of malignant cells that were specifically located at the tumor boundary, representing a more aggressive state, were involved in the invasion of invasive adenocarcinoma (IAC). Altogether, these results can improve our understanding of the local microenvironment characteristics that underlie LUAD progression and may facilitate the identification of drug targets to prevent invasive progression and biomarkers for diagnosis.

Membrane-less organelles (MLOs) perform diverse and important functions inside cells. However, how they interact with each other to carry out these functions collectively is unknown. Here we devised a multi-spectral imaging technique called "Rainbow Nucleus" to simultaneously visualize five nuclear MLOs using live-cell imaging. We find that while some interactions are stable, such as those between the histone locus bodies and Cajal bodies, others are transient, such as those between PML bodies and Cajal bodies. Furthermore, interactions among MLOs are not random: functionally related MLOs interact more frequently than unrelated MLOs, and these interactions completely rewire when we inhibit transcription. Our study provides first glimpses into how different MLOs interact with each other under different conditions, and lays the foundation for future cellular engineering efforts that modulate MLOs interactome to treat diseases.

Antibody-dependent enhancement (ADE) has been reported in several virus infections including dengue fever virus, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronavirus infection. To study whether ADE is involved in COVID-19 infections, in vitro pseudotyped SARS-CoV-2 entry into Raji cells, K562 cells, and primary B cells mediated by plasma from recovered COVID-19 patients were employed as models. The enhancement of SARS-CoV-2 entry into cells was more commonly detected in plasma from severely-affected elderly patients with high titers of SARS-CoV-2 spike protein-specific antibodies. Cellular entry was mediated via the engagement of Fc{gamma}RII receptor through virus-cell membrane fusion, but not by endocytosis. Peptide array scanning analyses showed that antibodies which promote SARS-CoV-2 infection targeted the variable regions of the RBD domain. To further characterize the association between the spike-specific antibody and ADE, an RBD-specific monoclonal antibody (7F3) was isolated from a recovered patient, which potently inhibited SARS-Cov-2 infection of ACE-2 expressing cells and also mediated ADE in Raji cells. Site-directed mutagenesis the spike RBD domain reduced the neutralization activity of 7F3, but did not abolish its binding to the RBD domain. Structural analysis using cryo-electron microscopy (Cryo-EM) revealed that 7F3 binds to spike proteins at a shift-angled pattern with one "up" and two "down" RBDs, resulting in partial overlapping with the receptor binding motif (RBM), while a neutralizing monoclonal antibody that lacked ADE activity binds to spike proteins with three "up" RBDs, resulting in complete overlapping with RBM. Our results revealed that ADE mediated by SARS-CoV-2 spike-specific antibodies could result from binding to the receptor in slightly different pattern from antibodies mediating neutralizations. Studies on ADE using antibodies from recovered patients via cell biology and structural biology technology could be of use for developing novel therapeutic and preventive measures for control of COVID-19 infection.

At the end of 2019, the SARS-CoV-2 induces an ongoing outbreak of pneumonia in China1, even more spread than SARS-CoV infection2. The entry of SARS-CoV into host cells mainly depends on the cell receptor (ACE2) recognition and spike protein cleavage-induced cell membrane fusion3,4. The spike protein of SARS-CoV-2 also binds to ACE2 with a similar affinity, whereas its spike protein cleavage remains unclear5,6. Here we show that an insertion sequence in the spike protein of SARS-CoV-2 enhances the cleavage efficiency, and besides pulmonary alveoli, intestinal and esophagus epithelium were also the target tissues of SARS-CoV-2. Compared with SARS-CoV, we found a SPRR insertion in the S1/S2 protease cleavage sites of SARS-CoV-2 spike protein increasing the cleavage efficiency by the protein sequence aligment and furin score calculation. Additionally, the insertion sequence facilitates the formation of an extended loop which was more suitable for protease recognition by the homology modeling and molicular docking. Furthermore, the single-cell transcriptomes identified that ACE2 and TMPRSSs are highly coexpressed in AT2 cells of lung, along with esophageal upper epithelial cells and absorptive enterocytes. Our results provide the bioinformatics evidence for the increased spike protein cleavage of SARS-CoV-2 and indicate its potential target cells.