Biology subjects

Shi, L.

Publications and source records attributed to Shi, L..

At least 217 records · Page 12Linked to original sources

bioRxiv2021

Computational modeling of C-terminal tails to predict the calcium-dependent secretion of ER resident proteins

The lumen of the endoplasmic reticulum (ER) has resident proteins that are critical to perform the various tasks of the ER such as protein maturation and lipid metabolism. These ER resident proteins typically have a carboxy-terminal ER retention sequence (ERS). The canonical ERS is Lys-Asp-Glu-Leu (KDEL) and when an ER resident protein moves from the ER to the Golgi, KDEL receptors (KDELRs) in the Golgi recognize the ERS and return the protein to the ER lumen. Depletion of ER calcium leads to the mass departure of ER resident proteins in a process termed exodosis, which is also regulated by KDELRs. Here, by combining computational prediction with machine learning-based models and experimental validation, we identify carboxy tail sequences of ER resident proteins divergent from the canonical "KDEL" ERS. Using molecular modeling and simulations, we demonstrated that two representative non-canonical ERS can stably bind to the KDELR. Collectively, we developed a method to predict whether a carboxy-terminal sequence acts as a putative ERS that would undergo secretion in response to ER calcium depletion and interact with the KDELRs. Identification of proteins that undergo exodosis will further our understanding of changes in ER proteostasis under physiological and pathological conditions where ER calcium is depleted.

cell biology↗

bioRxiv2021

Identification of regulatory networks associated with anti-HIV/AIDS genes via integrated transcriptome, epigenome and proteome analyses

There are individuals naturally resistant to HIV. To identify anti-HIV genes and regulatory networks that enable the native ability to resist HIV, we reanalyzed the transcriptome of HIV resistant and susceptible individuals based on previous efforts, and performed regulatory network prediction using HIV-infection related DNA methylation, miRNA and Chip-SEQ data. We totally found 25 potential anti-HIV genes and 23 of them are newly identified. They are enriched in pathways of immunity, neurological system and cell signaling. 4 anti-HIV genes show DNA hypermethylation signatures and 4 are possibly bounded by the HIV-1 Trans-Activator of Transcription protein (Tat). We found a potential HIV-resistance correlated miRNA hsa-miR-3074-5p possibly regulating an anti-HIV hub gene JUN. Our findings provide novel insights for AIDS treatments and approaches to HIV vaccine design.

bioinformatics↗

bioRxiv2021

Structure of SOQ1 lumenal domains identifies potential disulfide exchange for negative regulation of photoprotection, qH

Non-photochemical quenching (NPQ) plays an important role for phototrophs in decreasing photo-oxidative damage. qH is a sustained component of NPQ and depends on the plastid lipocalin (LCNP). A thylakoid membrane-anchored protein SUPPRESSOR OF QUENCHING1 (SOQ1) prevents qH formation by inhibiting LCNP. SOQ1 suppresses qH with its lumen-located C-terminal Trx-like and NHL domains. Here we report crystal structures and biochemical characterization of SOQ1 lumenal domains. Our results show that the Trx-like and NHL domains are stably associated, with the potential redox-active motif located at their interface. Residue E859 essential for SOQ1 function is pivotal for mediating the inter-domain interaction. Moreover, the C-terminal region of SOQ1 forms an independent {beta}-stranded domain, which possibly interacts with the Trx-like domain through disulfide exchange. Furthermore, SOQ1 is susceptible to cleavage at the loops connecting the neighboring domains both in vitro and in vivo, which could be a regulatory process for its suppression function of qH.

plant biology↗

bioRxiv2021

BEdeepoff: an in silico tool for off-target prediction of ABE and CBE base editors

Base editors, including adenine base editors (ABEs) and cytosine base editors (CBEs), are valuable tools for introducing point mutations, but they frequently induce unwanted off-target mutations. Here, we performed a high-throughput gRNA-target library screening to measure editing efficiencies at integrated genomic off-targets and obtained datasets of 48,632 and 52,429 off-targets for ABE and CBE, respectively. We used the datasets to train deep learning models, resulting in ABEdeepoff and CBEdeepoff which can predict editing efficiencies at off-targets. These tools are freely accessible via online web server http://www.deephf.com/#/bedeep.

bioinformatics↗

bioRxiv2021

BEdeepon: an in silico tool for prediction of base editor efficiencies and outcomes

Base editors enable direct conversion of one target base into another in a programmable manner, but conversion efficiencies vary dramatically among different targets. Here, we performed a high-throughput gRNA-target library screening to measure conversion efficiencies and outcome product frequencies at integrated genomic targets and obtained datasets of 60,615 and 73,303 targets for ABE and CBE, respectively. We used the datasets to train deep learning models, resulting in ABEdeepon and CBEdeepon which can predict on-target efficiencies and outcome sequence frequencies. The software is freely accessible via online web server http://www.deephf.com/#/bedeep/bedeepon.

bioinformatics↗

bioRxiv2021

Psychomotor impairments and therapeutic implications revealed by a mutation linked with Infantile Parkinsonism-Dystonia

Parkinson disease (PD) is a progressive, neurodegenerative disorder affecting over 6.1 million people worldwide. Although the cause of PD remains unclear, studies of highly-penetrant mutations identified in early-onset familial parkinsonism have contributed to our understanding of the molecular mechanisms underlying disease pathology. Dopamine (DA) transporter (DAT) deficiency syndrome (DTDS) is a distinct type of infantile parkinsonism-dystonia that shares key clinical features with PD, including motor deficits (progressive bradykinesia, tremor, hypomimia) and altered DA neurotransmission. Here, we define structural, functional, and behavioral consequences of a Cys substitution at R445 in human DAT (hDAT R445C), identified in a patient with DTDS. We found that this R445 substitution disrupts a phylogenetically conserved intracellular (IC) network of interactions that compromise the hDAT IC gate. This is demonstrated by both Rosetta molecular modeling and fine-grained simulations using hDAT R445C, as well as EPR analysis and X-ray crystallography of the bacterial homolog leucine transporter. Notably, the disruption of this IC network of interactions supported a channel-like intermediate of hDAT and compromised hDAT function. We demonstrate that Drosophila melanogaster expressing hDAT R445C show impaired hDAT activity, which is associated with DA dysfunction in isolated brains and with abnormal behaviors monitored at high-speed time resolution. We show that hDAT R445C Drosophila exhibit motor deficits, lack of motor coordination (i.e. flight coordination) and phenotypic heterogeneity in these behaviors that is typically associated with DTDS and PD. These behaviors are linked with altered dopaminergic signaling stemming from loss of DA neurons and decreased DA availability. We rescued flight coordination through enhanced DAT surface expression via the lysosomal inhibitor chloroquine. Together, these studies shed light on how a DTDS-linked DAT mutation underlies DA dysfunction and, more broadly, the clinical phenotypes shared by DTDS and PD.

neuroscience↗

bioRxiv2021

Whole Genome and Exome Sequencing Reference Datasets from A Multi-center and Cross-platform Benchmark Study

With the rapid advancement of sequencing technologies in the past decade, next generation sequencing (NGS) analysis has been widely applied in cancer genomics research. More recently, NGS has been adopted in clinical oncology to advance personalized medicine. Clinical applications of precision oncology require accurate tests that can distinguish tumor-specific mutations from errors or artifacts introduced during NGS processes or data analysis. Therefore, there is an urgent need to develop best practices in cancer mutation detection using NGS and the need for standard reference data sets for systematically benchmarking sequencing platforms, library protocols, bioinformatics pipelines and for measuring accuracy and reproducibility across platforms and methods. Within the SEQC2 consortium context, we established paired tumor-normal reference samples, a human triple-negative breast cancer cell line and a matched normal cell line derived from B lymphocytes. We generated whole-genome (WGS) and whole-exome sequencing (WES) data using 16 NGS library preparation protocols, seven sequencing platforms at six different centers. We systematically interrogated somatic mutations in the paired reference samples to identify factors affecting detection reproducibility and accuracy in cancer genomes. These large cross-platform/site WGS and WES datasets using well-characterized reference samples will represent a powerful resource for benchmarking NGS technologies, bioinformatics pipelines, and for the cancer genomics studies.

bioinformatics↗

bioRxiv2021

Single-cell sequencing characterizes intratumoral profile of immune cells in old mice

It has long been thought that aging is a major risk factor for cancer incidence. However, accumulating evidence indicates increased resistance of old animals to tumor growth. A systematic understanding of how old individuals defend against tumor invasion is currently lacking. Here we investigated the differences of age-associated alterations in tumor-infiltrating immune cells between young and old mice using single-cell RNA analysis. Our results showed that a higher proportion of cytotoxic CD8+ T cells, nTregs, cDC, and M1-type macrophages, while a higher percentage of exhausted CD8+ T cells, iTregs, pDC, and M2-type macrophages were found in young mice. Importantly, TCR diversity analysis showed top 10 TCR clones consisted primarily of exhausted CD8+ T cells in young mice whereas tip clones were predominantly cytotoxic CD8+ T cells in old mice. Consistently, trajectory inference demonstrated that CD8+ T cells preferentially differentiated into cytotoxic cells in old mice in contrast to exhausted cells in young mice. Meanwhile, we confirmed the main distinctions between young and old mice by flow cytometry. Collectively, our data revealed that a significantly higher proportion of effector immune cells in old mice defend against tumor progression, providing a framework for the immunotherapy of elderly patients with tumors.

immunology↗

bioRxiv2021

Prediction of occult tumor progression via platelet RNAs in a mouse melanoma model: a potential new platform of cancer screening for early detection of cancer

Cancer screening provides the opportunity to detect cancer early, ideally before symptom onset and metastasis, and offers an increased opportunity for a better prognosis. The ideal biomarkers for cancer screening should discriminate individuals who have not developed invasive cancer yet but are destined to do so from healthy subjects1,2. However, most cancers lack effective screening recommendations. Therefore, further studies on novel screening strategies are urgently required. Here, our proof-of-concept study shows blood platelets could be a platform for liquid biopsy-based early cancer detection. By using a simple suboptimal inoculation melanoma mouse model, we identified differentially expressed RNAs in platelet signatures of mice injected with a suboptimal number of cancer cells (eDEGs) compared with mice with macroscopic melanomas and negative controls. These RNAs were strongly enriched in pathways related to immune response and regulation. Moreover, 36 genes selected from the eDEGs via bioinformatics analyses were verified in a mouse validation cohort via quantitative real-time PCR. LASSO regression was employed to generate the prediction models with gene expression signatures as the best predictors for occult tumor progression in mice. The prediction models showed great diagnostic efficacy and predictive value in our murine validation cohort, and could discriminate mice with occult tumors from control group (area under curve (AUC) of 0.935 (training data) and 0.912 (testing data)) (gene signature including Cd19, Cdkn1a, S100a9, Tap1, and Tnfrsf1b) and also from macroscopic tumor group (AUC of 0.920 (training data) and 0.936 (testing data)) (gene signature including Ccr7, Cd4, Kmt2d, and Ly6e). Our study provides evidence for potential clinical relevance of blood platelets as a platform for liquid biopsy-based early detection of cancer. Furthermore, the eDEGs are mostly immune-related, not tumor-specific. Hence it is possible platelets-based liquid biopsy could enable simultaneous early detection of cancers from multiple organs of origin3. It is also feasible to determine the origin of cancer since platelet profiles are influenced by tumor type3.

cancer biology↗

bioRxiv2021

Hybrid Clustering of Long and Short-read for Improved Metagenome Assembly

Next-generation sequencing has enabled metagenomics, the study of the genomes of microorganisms sampled directly from the environment without cultivation. We previously developed a proof-of-concept, scalable metagenome clustering algorithm based on Apache Spark to cluster sequence reads according to their species of origin. To overcome its under-clustering problem on short-read sequences, in this study we developed a new, two-step Label Propagation Algorithm (LPA) that first forms clusters of long reads and then recruits short reads to these clusters. Compared to alternative label propagation strategies, this hybrid clustering algorithm (hybrid-LPA) yields significantly larger read clusters without compromising cluster purity. We show that adding an extra clustering step before assembly leads to improved metagenome assemblies, predicting more complete genomes or gene clusters from a synthetic metagenome dataset and a real-world metagenome dataset, respectively. These results suggest that hybrid-LPA is a good alternative to current metagenome assembly practice by providing benefits in both scalability and accuracy on large metagenome datasets. Availability and implementationhttps://bitbucket.org/zhong_wang/hybridlpa/src/master/. Contactzhongwang@lbl.gov

bioinformatics↗

bioRxiv2021

Identifying long non-coding RNAs and characterizing their functional roles in swine mammary gland from colostrogenesis to lactogenesis

Long non-coding RNAs (lncRNAs) play important regulatory roles in milk biological processes. While, little is known about the identification and function of swine lncRNAs in response to mammary gland development. Here, we identified 286 differentially expressed (DE) lncRNAs in mammary gland at different stages from 14 days prior to (-) parturition to day 1 after (+) parturition using the published RNA-seq data. The expression pattern of these DE lncRNAs was examined, and most of lncRNAs expressions were strongly changed from Day -2 to Day +1. Functional annotation revealed that the DE lncRNAs were mainly involved in the mammary gland developing, lactation developing, milk composition metabolism and colostrum function. By performing the weighted gene co-expression network analysis (WGCNA), we identified 7 out of 12 lncRNA-mRNA modules, including 35 lncRNAs and 319 mRNAs, which were highly associated with the mammary gland at Day -14, Day -2 and Day +1. Integrated analysis of the DE lncRNAs expression pattern examination, targets prediction, function annotation and WGCNA suggested that 18 lncRNAs (such as, XLOC_020627, ENSSSCG00000051193, XLOC_025150, ENSSSCG00000042618, XLOC_963181, ENSSSCG00000051701, XLOC_018030, and XLOC_025146) and their 20 target genes (such as, ACTN4, ADCY1, CSN3, SMO, CSN2, PRKAG2, FIBCD1, and GALNT7), were considered as the promising candidates for swine parturition and colostrum occurrence processes. Our research provided the insights into lncRNA profiles and their regulating mechanisms from colostrogenesis to lactogenesis in swine.

genetics↗

bioRxiv2021

CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is highly contagious presenting a significant public health issue. Current therapies used to treat coronavirus disease 2019 (COVID-19) include monoclonal antibody cocktail, convalescent plasma, antivirals, immunomodulators, and anticoagulants, though the current therapeutic options remain limited and expensive. The vaccines from Pfizer and Moderna have recently been authorized for emergency use, which are invaluable for the prevention of SARS-CoV-2 infection. However, their long-term side effects are not yet to be documented, and populations with immunocompromised conditions (e.g., organ-transplantation and immunodeficient patients) may not be able to mount an effective immune response. In addition, there are concerns that wide-scale immunity to SARS-CoV-2 may introduce immune pressure that could select for escape mutants to the existing vaccines and monoclonal antibody therapies. Emerging evidence has shown that chimeric antigen receptor (CAR)- natural killer (NK) immunotherapy has potent antitumor response in hematologic cancers with minimal adverse effects in recent studies, however, the potentials of CAR-NK cells in preventing and treating severe cases of COVID-19 has not yet been fully exploited. Here, we improve upon a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2 and its D614G mutant. CAR-NK cells were generated using the scFv domain of S309 (henceforward, S309-CAR-NK), a SARS-CoV and SARS-CoV-2 neutralizing antibody that targets the highly conserved region of SARS-CoV-2 spike (S) glycoprotein, therefore would be more likely to recognize different variants of SARS-CoV-2 isolates. S309-CAR-NK cells can specifically bind to pseudotyped SARS-CoV-2 virus and its D614G mutant. Furthermore, S309-CAR-NK cells can specifically kill target cells expressing SARS-CoV-2 S protein in vitro and show superior killing activity and cytokine production, compared to that of the recently published CR3022-CAR-NK cells. Thus, these results pave the way for generating off-the-shelf S309-CAR-NK cells for treatment in high-risk individuals as well as provide an alternative strategy for patients unresponsive to current vaccines.

immunology↗

bioRxiv2020

Coordinated regulation of ribosomes and proteasomes by PRMT1 in the maintenance of neural stemness of cancer cells and neural stem cells

Our studies suggest that neural stemness contributes to cell tumorigenicity. The basic cell physiological machineries and developmental programs, such as cell cycle, ribosomes, proteasomes, epigenetic factors, etc., which are upregulated in and promote cancers, are enriched in embryonic neural cells. How these machineries are coordinated is unknown. Here, we show that loss of neural stemness in cancer cells or neural stem cells leads to simultaneous downregulation of components of ribosomes and proteasomes, which are responsible for protein synthesis and degradation, respectively, and downregulation of major epigenetic factors. Inhibition of PRMT1 causes neuron-like differentiation, downregulation of a similar set of proteins, and alteration of subcellular localization of ribosome and proteasome components. PRMT1 interacts with these components, catalyzes arginine methylation of them and protects them from degradation, thereby maintaining a high level of expression of epigenetic factors that maintain neural stemness. PRMT1 inhibition results in repression of cell tumorigenicity. Therefore, PRMT1 coordinates ribosomes and proteasomes to match the needs for high protein production and protein homeostasis in cells with fast cell cycle and proliferation.

cell biology↗

bioRxiv2020

DNA methylation proxies for 16 plasma proteins predict the incidence of 7 leading causes of morbidity

Protein biomarkers have been identified across many age-related morbidities. However, characterising epigenetic influences could further inform disease predictions. Here, we leverage epigenome-wide data to study links between the DNAm signatures of the circulating proteome and incident diseases. Using data from four cohorts, we trained and tested epigenetic scores (EpiScores) for 953 plasma proteins, identifying 109 scores that explained between 1% and 58% of the variance in protein levels after adjusting for known protein quantitative trait loci (pQTL) genetic effects. By projecting these EpiScores into an independent sample, (Generation Scotland; n=9,537) and relating them to incident morbidities over a follow-up of 14 years, we uncovered 137 EpiScore - disease associations. These associations were largely independent of immune cell proportions, common lifestyle and health factors and biological aging. Notably, we found that our diabetes-associated EpiScores highlighted previous top biomarker associations from proteome-wide assessments of diabetes. These EpiScores for protein levels can therefore be a valuable resource for disease prediction and risk stratification.

genomics↗

bioRxiv2020

Upregulated functionality of mitochondria-associated ER membranes in a mouse model of traumatic brain injury

Traumatic brain injury (TBI) can lead to neurodegenerative diseases such as Alzheimers disease (AD) through mechanisms that remain incompletely characterized. Similar to AD, TBI models present with cellular metabolic alterations and modulated cleavage of amyloid precursor protein (APP). Specifically, AD and TBI tissues display increases in amyloid-{beta} as well as its precursor, APP C-terminal fragment of 99 a.a. (C99). Our recent data in cell models of AD indicate that C99, due to its affinity for cholesterol, induces the formation of transient lipid raft domains in the ER known as mitochondria-associated ER membranes ("MAM" domains). The formation of these domains recruits and activates specific lipid metabolic enzymes that regulate cellular cholesterol trafficking and sphingolipid turnover. Increased C99 levels in AD cell models promote MAM formation and significantly modulate cellular lipid homeostasis. Here, these phenotypes were recapitulated in the controlled cortical impact (CCI) model of TBI in adult mice. Specifically, the injured cortex and hippocampus displayed significant increases in C99 and MAM activity, as measured by phospholipid synthesis, sphingomyelinase activity and cholesterol turnover. In addition, our cell type-specific lipidomics analyses revealed significant changes in microglial lipid composition that are consistent with the observed alterations in MAM-resident enzymes. Altogether, we propose that alterations in the regulation of MAM and relevant lipid metabolic pathways could contribute to the epidemiological connection between TBI and AD. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/381756v2_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@12d0989org.highwire.dtl.DTLVardef@1cd1eb2org.highwire.dtl.DTLVardef@19a56caorg.highwire.dtl.DTLVardef@1b7f3e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2020

Integrative Transcriptomic Analysis Reveals the Immune Mechanism for A CyHV-3-Resistant Common Carp Strain

Anti-disease breeding is becoming the most promising solution to cyprinid herpesvirus-3 (CyHV-3) infection, the major threat to common carp aquaculture. Virus challenging studies suggested that a breeding strain of common carp is resistant to this disease. This study illustrates the immune mechanisms involved in anti-CyHV-3 ability. An integrative analysis of the protein-coding genes and long non-coding RNAs (lncRNAs) using transcriptomic data was performed. Tissues from the head kidney of common carp were extracted at day 0 (the healthy control) and day 7 after CyHV-3 infection (the survivors), and used to analyze the transcriptome through both Illumina and PacBio sequencing. Following analysis of the GO terms and KEGG pathways involved, the immune-related terms and pathways were merged. In order to dig out details in the immune aspect, The DEGs were filtered using the current common carp immune gene library. Immune gene categories and their corresponding genes in different comparison groups were revealed. Also, the immunological Gene Ontology terms for lncRNA modulation were retained. The weighted gene co-XSexpression network analysis was used to reveal the regulation of immune genes by lncRNA. The results demonstrated that the breeding carp strain develops marked resistance to CyHV-3 through a specific innate immune mechanism. The featured biological processes were autophagy, phagocytosis, cytotoxicity, and virus blockage by lectins and MUC3. Moreover, the immune suppressive signals, such as suppression of IL21R on STAT3, PI3K mediated inhibition on inflammation by dopamine upon infection, as well as the inhibition of NLRC3 on STING during a steady state. Possible susceptible factors for CyHV-3, such as ITGB1, TLR18, and CCL4, were also revealed from the non-breeding strain. The results of this study also suggested that Nramp and PAI regulated by LncRNA could facilitate virus infection and proliferation for infected cells respectively, while T cell leukemia homeobox 3 (TLX3) as well as galectin 3 function by lncRNA may play a role in the resistance mechanism. Therefore, immune factors that are immunogenetically insensitive or susceptible to CyHV-3 infection have been revealed.

immunology↗

bioRxiv2020

All-trans Retinoic Acid Regulates miR-106a-5p Inhibition of Autophagic in Developing Cleft Palates

BackgroundAll-trans retinoic acid (atRA) results in cleft palate, but the cellular and molecular mechanisms underlying the teratogenic effects on palatal development have not been fully elucidated. Autophagy interruption has been reported to seriously affect embryonic-cell differentiation and development. This study aimed to verify whether atRA-induced cleft palate occurs because atRA blocks autophagy and stemness of embryonic palatal mesenchyme (MEPM) cells, which are maintained via the phosphatase and tensin homolog (PTEN)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) autophagic signaling pathway, and inhibits osteogenic-differentiation potential of MEPM cells, which could lead to the development of cleft palate. MethodsTo assess the stemness and pluripotency of MEPM cells, we analyzed their surfacemarkers using immunofluorescence (IF) and flow cytometry (FCM). Differentiation potentials, such as osteogenic, adipogenic, and chondrogenic differentiation, were induced. We also investigated the role of the PTEN/Akt/mTOR autophagic signaling pathway, which maintains the stemness and pluripotency of MEPM cells. Using transmission electron microscopy (TEM), Western blot analysis, quantitative reverse transcriptase polymerase chain reaction (RT-qPCR), messenger ribonucleic acid (mRNA) microarray, dual-luciferase reporter system, and exosomes, we found that atRA blocks autophagy and osteogenic differentiation of MEPM cells through micro-ribonucleic acid (miR)-106a-5p by targeting the PTEN/Akt/mTOR autophagic pathway. ResultsIn vitro purified MEPM cells expressed cell surface markers similar to those of mouse bone marrow stem cells. Additionally, in vitro MEPM cells were ectomesenchymal and expressed the neural-crest marker human natural killer-1 (HNK-1), the mesodermal marker vimentin, and the ectodermal marker nestin. They were also positive for in vitro MEPM markers, including platelet-derived growth factor alpha (PDGFR), ephrin B1 (Efnb1), odd-skipped related 2 (Osr2), and Meox2, as well as for stemness markers including POU class 5 homeobox 4 (Oct4), Nanog, and sex-determining region Y-related HMG box 2 (Sox2). MEPM cell pluripotency was retained through activation of the PTEN/Akt/mTOR autophagic signaling pathway. We found that atRA blocked MEPM cell pluripotency to inhibit osteogenic differentiation via miR-106a-5p targeting of PTEN mRNA and subsequent suppression of the PTEN/Akt/mTOR autophagic pathway. ConclusionsIn vitro cultured MEPM cells are ectomesenchymal stem cells that have strong osteogenic differentiation potential, and MEPM pluripotency is regulated by autophagy via the PTEN/AKT/mTOR signaling pathway. atRA disrupts MEPM cell pluripotency through PTEN/AKT/mTOR signaling inactivation where miR-106a-5p targets PTEN mRNA to reduce osteogenic differentiation of MEPM cells and results in the development of cleft palates. Our findings provide new insight into the mechanism underlying the development of cleft palate, and miR-106a-5p may act as a prenatal screening biomarker for cleft palate as well as a new diagnostic and therapeutic target.

developmental biology↗

bioRxiv2020

MiRNA-223 Inhibits MouseGallstone Formation by Targeting Key Transporters in Hepatobiliary Cholesterol Secretion Pathway

BackgroudMiRNA-223 has previously been reported to play an essential role in hepatic cholesterol homeostasis by suppressing cholesterol synthesis, attenuating cholesterol uptake by hepatocyte from and promoting cholesterol efflux into the blood. However, its role in regulation of biliary cholesterol secretion and gallstone formation remains unknown. MethodsMice with conventional knockout (KO), hepatocyte-specific knockout ({Delta}Hepa) / knockdown (KD) or gain expression of miRNA-223 were included in the study and were subjected to lithogenic diet (LD) for various weeks. The gallbladders were harvested and subjected to cholesterol crystal imaging and gallstone mass measurement. Liver tissues were collected for western blotting, RT-qPCR, and immunohistochemistry staining. Levels of cholesterol, bile salt, phospholipids, and triglyceride were determined in serum, liver tissues, and bile by enzyme color reactive assays. 3 UTR reporter gene assays were used to verify the direct target genes for miRNA-223. ResultsLD-induced gallstone formation was remarkably accelerated in miRNA-223 KO, {Delta}Hepa, and KD mice with concurrent enhancement in total cholesterol levels in liver tissue and bile. Key biliary cholesterol transporters ABCG5 and ABCG8 were identified as direct targets of miRNA-223. Reversely, AAV-mediated hepatocyte-specific miRNA-223 overexpression prevented gallstone progression with reduced targets protein expression. ConclusionThe present study demonstrates a novel role of miRNA-223 in the regulation of hepatic bile cholesterol secretion pathway and gallstone formation by targeting ABCG5 and ABCG8 expression. Therefore, elevating miRNA-223 would be a potentially novel approach to overcome the sternness of cholesterol gallstone disease. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/344580v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1c418bforg.highwire.dtl.DTLVardef@134e20dorg.highwire.dtl.DTLVardef@e4e0d2org.highwire.dtl.DTLVardef@641b40_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗