Loss of TP53I11 Enhances the Extracellular Matrix-independent Survival by Promoting Activation of AMPK.

Extracellular matrix (ECM)-independent survival is an essential prerequisite for tumor metastasis, and a hallmark of epithelial cancer stem cells and epithelial-mesenchymal transition (EMT). Here, we found that loss of TP53I11 enhanced, and overexpression of TP53I11 suppressed the ECM-independent survival, EMT, and migration in MCF10A cells. TP53I11 has long been considered as a transcriptional target of TP53. However, we found that TP53I11 regulated the ECM-independent survival by a TP53-independent way. As a metabolic sensor, AMPK promoted anoikis resistance by inhibiting AKT/m-TOR/p70S6K signaling pathway. It was recently revealed that the reciprocal inhibitory relationship between AKT and AMPK regulated adaptation of cells to ECM-detachment. Our results demonstrated that loss of TP53I11 promoted the activation of AKT/m-TOR pathway, increased PGC-1α expression and thereby enhanced OXPHOS in attach-cultured MCF10A cells, but promoted AMPK activation to inhibit AKT/m-TOR/p70S6K signaling pathway in detach-cultured MCF10A cells. This indicates that TP53I11 functions as a mediator to balance activation of AKT and AMPK to adapt cells to different cellular contexts such as ECM-attachment and -detachment. © 2018 IUBMB Life, 71(1):183-191, 2019.

A functionalized collagen-I scaffold delivers microRNA 21-loaded exosomes for spinal cord injury repair.

MicroRNA (miRNA)-based therapies have shown great potential in the repair of spinal cord injury (SCI). MicroRNA 21 (miR21) has been proven to have an essential protective effect on SCI. However, there are some challenges for miRNAs application due to their easy degradation and ineffective cell penetration. As natural vesicles, exosomes were considered ideal carriers for miRNAs delivery for their advantages of low immunogenicity, inherent stability and tissue/cell penetration. However, poor targeting and the low capacity of specific miRNAs impede their practical applications. This study aims to develop a type of genetically engineered miR21-loaded exosomes that can be entrapped in collagen-I (Col-I) scaffold to repair SCI. The collagen-binding domain (CBD)-fused lysosome-associated membrane glycoprotein 2b (Lamp2b) protein (CBD-LP) and miR21 were overexpressed in host HEK293T (293T) cells that were used to produce engineered miR21-loaded exosomes. The CBD peptide fused in Lamp2b on the exosome surface can stably tether exosomes to Col-I scaffold, facilitate the retention of miR21-loaded exosomes in lesion sites, promote the sustained release of miR21 to cells. Finally, a functionalized Col-I scaffold biomaterial enriched with miR21-loaded exosomes was developed and it could benefit the repair of SCI. STATEMENT OF SIGNIFICANCE: MiRNA-based therapeutics have promising potential in spinal cord injury (SCI) repair. However, easy degradation and ineffective cell penetration impede miRNAs application. Exosomes are natural vehicles for miRNAs delivery but face the challenge of diffusion in vivo. Here, the collagen-binding domain (CBD)-fused Lamp2b and miR21 were overexpressed in HEK293T cells to produce miR21-loaded and CBD-modified exosomes (CBD-LP-miR21-EXOs). The CBD modified on the exosome surface can stably tether exosomes to collagen-I scaffold to form functionalized CBD-LP-miR21-EXO-Col scaffold that can facilitate the retention of miR21-loaded exosomes, promote the sustained release of miR21 to cells and finally benefit SCI repair. Furthermore, this type of functionalized collagen-I materials can be widely applied for other tissue injury repairs by enriching the CBD-LP-EXOs loaded with appropriate miRNAs.

A functional extracellular matrix biomaterial enriched with VEGFA and bFGF as vehicle of human umbilical cord mesenchymal stem cells in skin wound healing.

The construction of microvascular network is one of the greatest challenges for tissue engineering and cell therapy. Endothelial cells are essential for the construction of network of blood vessels. However, their application meets challenges in clinic due to the limited resource of autologous endothelium. Mesenchymal stem cells can effectively promote the angiogenesis in ischemic tissues for their abilities of endothelial differentiation and paracrine, and abundant sources. Extracellular matrix (ECM) has been widely used as an ideal biomaterial to mimic cellular microenvironment for tissue engineering due to its merits of neutrality, good biocompatibility, degradability, and controllability. In this study, a functional cell derived ECM biomaterial enriched with VEGFA and bFGF by expressing the collagen-binding domain fused factor genes in host cells was prepared. This material could induce endothelial differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) and promote angiogenesis, which may improve the healing effect of skin injury. Our research not only provides a functional ECM material to inducing angiogenesis by inducing endothelial differentiation of hUCMSCs, but also shed light on the ubiquitous approaches to endow ECM materials different functions by enriching different factors. This study will benefit tissue engineering and regenerative medicine researches.

The Extracellular Matrix Enriched With Exosomes for the Treatment on Pulmonary Fibrosis in Mice.

Pulmonary fibrosis (PF) is a severe respiratory disease caused by lung microenvironment changes. TGF-ß/Smad3 signaling pathway plays a critical role in the fibrotic process. MicroRNA-29 (miR-29) has proved to alleviate the occurrence of PF by downregulating TGF-ß/Smad3 signaling pathway. The miRNA application encounters obstacles due to its low stability in body and no targeting to lesions. Exosomes can be used for therapeutic delivery of miRNA due to their favorable delivery properties. However, low efficiency of separation and production impedes the therapeutic application of exosomes. In this study, we developed a liquid natural extracellular matrix (ECM) enriched with miR-29-loaded exosomes for PF treatment. The collagen-binding domain (CBD)-fused Lamp2b (CBD-Lamp2b) and miR-29 were overexpressed in human foreskin fibroblast (HFF) host cells for the entrapment of miR-29-loaded exosomes in ECM of the cells. The repeated freeze-thaw method was performed to prepare the liquid ECM enriched with exosomes without destroying the exosomal membrane. In summary, this study developed a novel functional ECM biomaterial for therapy of PF, and also provided a promising gene therapy platform for different diseases by treatment with liquid ECM that is, enriched with exosomes loaded with different functional miRNAs.

PM2.5 collecting in a tire manufacturing plant affects epithelial differentiation of human umbilical cord derived mesenchymal stem cells by Wnt/ß-catenin pathway.

Mesenchymal stem cells (MSCs) can differentiate into pulmonary epithelial cells by Wnt/ß-catenin pathway and promote lung repair. However, whether fine particulate matter (PM2.5) could affect Wnt pathway and finally reduce the ability of MSCs to differentiate into epithelial cells is still unknown. This study aimed to investigate whether PM2.5 could inhibit the epithelial differentiation of human umbilical cord-derived MSCs cells (hUCMSCs) and the related underlying mechanism. hUCMSCs were incubated with different concentrations of PM2.5. Then, the cell viability, reactive oxygen species level, and single-cell sphere formation were assessed. The underlying mechanism of PM2.5 in epithelial differentiation of hUCMSCs was further evaluated by co-culturing hUCMSCs with A549 cells. Our results demonstrated that PM2.5 exposures could affect the expressions of ß-catenin and lung epithelial markers (zonula occludens-1 (ZO-1); cytokeratins 5 and 19) in the co-cultured hUCMSCs. The Wnt/ß-catenin pathway is involved in regulating the epithelial differentiation of MSCs. As expected, co-treatment with Wnt3a, which is the activator of the Wnt pathway, attenuated the downregulation of lung epithelial markers (ZO-1; cytokeratins 5 and 19) and paracrine factors (keratinocyte growth factor and hepatocyte growth factor) caused by PM2.5. Altogether, these results demonstrated that PM2.5 could affect the epithelial differentiation of hUCMSCs via the Wnt/ß-catenin pathway.

Single cell derived spheres of umbilical cord mesenchymal stem cells enhance cell stemness properties, survival ability and therapeutic potential on liver failure.

Umbilical cord mesenchymal stem cells (UCMSCs) have shown great potentials in regenerative medicine for their extensive sources, multilineage differentiation potential, low immunogenicity and self-renewal ability. However, the clinical application of UCMSCs still confronts many challenges including the requirement of large quantity of cells, low survival ability in vivo and the loss of main original characteristics due to two-dimensional (2D) culture. The traditional three-dimensional (3D)-spheroid culture can mimic in vivo conditions, but still has limitations in clinical application due to large size of spheroid against direct injection and inner cell death. Based on self-renewal tenet, we produced single cell derived sphere (SCDS) of UCMSCs through combining single cell pattern on chip with 3D culture. Compared with the 2D and traditional 3D culture, SCDS culture has many advantages to meet clinical requirements, including small size, higher abilities of survival and migration, and stronger hypoxia resistance and stemness maintenance. Furthermore, SCDS culture promotes angiogenesis in UCMSCs-xenografts and displays greater therapeutic potential on acute liver failure (ALF) in vivo. Our results suggest that SCDS culture may serve as a simple and effective strategy for UCMSCs optimization to meet clinical demand.

Comprehensive analysis of the lncRNA-associated ceRNA network identifies neuroinflammation biomarkers for Alzheimer's disease.

Accumulating evidence has highlighted the important roles of long non-coding RNAs (lncRNAs) acting as competing endogenous RNAs (ceRNAs) in Alzheimer's disease (AD). In this study, we constructed an AD-derived lncRNA-associated ceRNA network (LncACeNET) based on the ceRNA hypothesis and co-expressed correlation analysis of RNAs (miRNAs, mRNAs and lncRNAs) from AD patients. Based on this network, we preliminarily identified new potential AD biomarkers including hsa-miR-155-5p, CERS6-AS1, and CTB-89H12.4. The functional enrichment analysis demonstrated that these inferred biomarkers were significantly correlated with AD-related biological processes such as neuron projection development and neuron projection morphogenesis. Notably, lncRNA CTB-89H12.4 is significantly associated with "calcium ion-regulated exocytosis of neurotransmitter", "chemical synaptic transmission", "presynaptic membrane assembly", "receptor localization to synapse", and "learning". This indicates the important role of CTB-89H12.4 as a promising target for AD therapy. Subsequently, we used the computational pipeline DTINet and discovered 19 lines of probable therapeutic relationships between FDA-approved drugs and CTB-89H12.4, which offered a new avenue to repurpose existing FDA-approved drugs for AD indication. Our study provides a new landscape for LncACeNET in AD, and will benefit mechanism study and new drug development for AD.

TP53I11 suppresses epithelial-mesenchymal transition and metastasis of breast cancer cells.

Epithelial-mesenchymal transition (EMT) is widely-considered to be a modulating factor of anoikis and cancer metastasis. We found that, in MDA-MB-231 cells, TP53I11 (tumor protein P53 inducible protein 11) suppressed EMT and migration in vitro, and inhibited metastasis in vivo. Our findings showed that hypoxic treatment upregulated the expression of HIF1α, but reduced TP53I11 protein levels and TP53I11 overexpression reduced HIF1α expression under normal culture and hypoxicconditions, and in xenografts of MDA-MB-231 cells. Considering HIF1α is a master regulator of the hypoxic response and that hypoxia is a crucial trigger of cancer metastasis, our study suggests that TP53I11 may suppress EMT and metastasis by reducing HIF1α protein levels in breast cancer cells. [BMB Reports 2019; 52(6): 379-384].

Quantitative assessment of human uterine fibroid xenograft using sequential in vivo bioluminescence imaging.

Uterine fibroid is one of the most common solid tumors occurring in reproductive age women. Lack of accurate methods for In vivo quantitative assessment of uterine fibroid progression severely impedes the basic research and drug screen of this disease. To solve this problem, the correlation between bioluminescence imaging (BLI) and initial cell number used to form xenograft was investigated in this study. The results showed that both subcutaneous (SC) and intraperitoneal (IP) D-luciferin administration led to fast increase of bioluminescence signal (BLS) intensity and caused large variation of peak signal intensity of xenografts through the analysis of BLI kinetic curves. We found that a distinct linear stage appeared in xenograft BLI curve for each mouse subjected to IP-injection of D-luciferin. Moreover, a high positive correlation was found between linear slope and the initial number of human uterine fibroid smooth muscle cells (fSMCs) used for xenograft formation. Our research indicates that the slope of linear stage in BLI curve is more appropriate for in vivo quantitative assessment of human uterine fibroid xenograft.

The extracellular matrix enriched with membrane metalloendopeptidase and insulin-degrading enzyme suppresses the deposition of amyloid-beta peptide in Alzheimer's disease cell models.

Amyloid plaque is a typical feature of Alzheimer's disease (AD) and is one of the targets for AD therapy. Membrane metalloendopeptidase (MME) and insulin-degrading enzyme (IDE) are two types of proteases that could cleave beta-amyloid (Aß) peptides generated by neuron cells of AD patients. Extracellular matrix (ECM) plays a crucial role in regulating tissue-specific functions and is an ideal biomaterial for tissue repair. In this study, we extracted the liquid ECM enriched with collagen-binding-domain-fused IDE or MME from human foreskin fibroblast cells. We found that these ECM biomaterials reduced the aggregation of Aß peptides, prevented the formation of amyloid plaques, and also suppressed phosphorylation of Tau protein in AD cell models. Overall, our research provides a novel ECM biomaterial that can be potentially used for AD therapy.

Nuclear respiratory factor 1 promotes spheroid survival and mesenchymal transition in mammary epithelial cells.

Epithelial cells aggregate into spheroids when deprived of matrix, and the proclivity for spheroid formation and survival is a hallmark of normal and tumorigenic mammary stem cells. We show here that Nuclear Respiratory Factor 1 (NRF1) is a spheroid promoter by in silico identification of this transcription factor as highly connected to top shRNA-hits deduced from re-iterative selections for shRNAs enriched in MCF10A spheroids. NRF1-promoted spheroid survival is linked to its stimulation of mitochondrial OXPHOS, cell migration, invasion, and mesenchymal transition. Conversely, NRF1 knockdown in breast cancer MDA-MB-231 cells reduced spheroids, migration, invasion, and mesenchymal marker expression. NRF1 knockdown also reduced tumor burden in mammary fat pads and lungs of orthotopic- or tail vein-transplanted mice. With the Luminal A subtype of breast cancer, higher NRF1 expression is associated with lower survival. These results show that NRF1, an activator of mitochondrial metabolism, supports mammary spheroid survival and tumor development.

BCL11A expression in acute phase chronic myeloid leukemia.

Chronic myeloid leukemia (CML) has chronic and acute phases. In chronic phase myeloid differentiation is preserved whereas in acute phase myeloid differentiation is blocked. Acute phase CML resembles acute myeloid leukemia (AML). Chronic phase CML is caused by BCR-ABL1. What additional mutation(s) cause transition to acute phase is unknown and may differ in different persons with CML. BCL11A encodes a transcription factor and is aberrantly-expressed in several haematological and solid neoplasms. We analyzed BCL11A mRNA levels in subjects with chronic and acute phase CML. BCL11A transcript levels were increased in subjects with CML in acute phase compared with those in normals and in subjects in chronic phase including some subjects studied in both phases. BCL11A mRNA levels were correlated with percent bone marrow blasts and significantly higher in lymphoid versus myeloid blast crisis. Differentiation of K562 with butyric acid, a CML cell line, decreased BCL11A mRNA levels. Cytology and flow cytometry analyses showed that ectopic expression of BCL11A in K562 cells blocked differentiation. These data suggest BCL11A may operate in transformation of CML from chronic to acute phase in some persons.

BCL11A expression in acute myeloid leukemia.

BACKGROUND: BCL11A encodes a C2H2 type zinc-finger protein. During normal haematopoietic cell differentiation BCL11A expression is down-regulated. Data in mice suggest up-regulation of BCL11A is involved in the pathogenesis of myeloid leukaemias. BCL11A expression in persons with acute myeloid leukaemia (AML) is not systematically studied. OBJECTIVE: Interrogate associations between BCL11A expression at diagnosis and clinical and laboratory valuables and outcomes in newly-diagnosed persons with AML. METHODS: We determined BCL11A mRNA levels in bone marrow and blood mononuclear cells in 292 consecutive newly-diagnosed subjects with AML by reverse transcript and real-time polymerase chain reaction. Data were compared to mRNA levels in bone marrow cells of normals. RESULTS: Subjects with BCL11A transcript levels at diagnosis exceeding the median value of 2.434 (±3.423 SD; 25th-75th inter-quartile range, 1.33-4.29) had higher WBC levels, a greater proportion of bone marrow myeloblasts, were more likely to be FAB M0 subtype, less likely to be FAB M3 subtype, more likely to be in the intermediate cytogenetic risk cohort, less likely to have a complex karyotype and more likely to have DNMT3A(R882) and FLT3-ITD mutations than subjects with transcript levels below the median value. In 89 subjects receiving conventional induction chemotherapy the complete remission rate was 54% (95% confidence interval [CI]; 33, 75%) in the lower BCL11A cohort and 65% (45, 85%; P=0.26) in the higher BCL11A cohort. 3 year survival was 33% (2, 65%) in the lower BCL11A cohort and 15% (0, 39%; P=0.35) in the high BCL11A cohort. CONCLUSION: BCL11A transcript levels at diagnosis was significantly associated with several clinical and laboratory variables. There were also non-significant associations with complete remission rate and survival. These data suggest a possible role for BCL11A expression in AML biology.