Platinum-induced upregulation of ITGA6 promotes chemoresistance and spreading in ovarian cancer.

Platinum (PT)-resistant Epithelial Ovarian Cancer (EOC) grows as a metastatic disease, disseminating in the abdomen and pelvis. Very few options are available for PT-resistant EOC patients, and little is known about how the acquisition of PT-resistance mediates the increased spreading capabilities of EOC. Here, using isogenic PT-resistant cells, genetic and pharmacological approaches, and patient-derived models, we report that Integrin α6 (ITGA6) is overexpressed by PT-resistant cells and is necessary to sustain EOC metastatic ability and adhesion-dependent PT-resistance. Using in vitro approaches, we showed that PT induces a positive loop that, by stimulating ITGA6 transcription and secretion, contributes to the formation of a pre-metastatic niche enabling EOC cells to disseminate. At molecular level, ITGA6 engagement regulates the production and availability of insulin-like growth factors (IGFs), over-stimulating the IGF1R pathway and upregulating Snail expression. In vitro data were recapitulated using in vivo models in which the targeting of ITGA6 prevents PT-resistant EOC dissemination and improves PT-activity, supporting ITGA6 as a promising druggable target for EOC patients.

HtrA4 is required for human trophoblast stem cell differentiation into syncytiotrophoblast.

INTRODUCTION: The syncytiotrophoblast (STB) of the human placenta facilitates vital maternal-fetal communication and is maintained by fusion (syncytialization) of cytotrophoblasts. Serine protease HtrA4 (high temperature requirement factor A4) is highly expressed only in the human placenta and was previously reported to be important for BeWo fusion. This study investigated whether HtrA4 is critical for differentiation of human trophoblast stem cells (TSCs) into STB. METHODS: Primary TSCs were isolated from first trimester placentas (n = 5) and validated by immunofluorescence (IF) for CD49f, CK7 and vimentin. TSCs were then differentiated into STB and the success of syncytialization was confirmed by RT-PCR, IF and ELISA of known markers. TSCs were next stably transfected with a HtrA4-targetting CRISPR/Cas9 plasmid, and cells with severe HtrA4 knockdown (HtrA4-KD) were analyzed to investigate the impact on STB differentiation. RESULTS: Primary TSCs were confirmed to be of high purity by staining positively for CD49f and CK7 but negatively for vimentin. These TSCs readily syncytialized when stimulated for STB differentiation, significantly increasing ß-hCG and syncytin-1, substantially decreasing E-cadherin, and markedly losing cell borders. While TSCs produced very low levels of HtrA4, upon stimulation for STB differentiation the cells drastically upregulated HtrA4 expression; secretion of HtrA4 protein also increased sharply, correlating positively and significantly with that of ß-hCG. The HtrA4-KD TSCs, however, failed to show this surge of HtrA4 production upon stimulation, and ultimately remained primarily mononucleated with no significant STB differentiation. DISCUSSION: This study demonstrates that HtrA4 plays a critical role in TSC differentiation into syncytiotrophoblast.

Targeted m6A demethylation of ITGA6 mRNA by a multisite dCasRx-m6A editor inhibits bladder cancer development.

INTRODUCTION: N6-methyladenosine (m6A) modification contributes to the pathogenesis and development of various cancers, including bladder cancer (BCa). In particular, integrin α6 (ITGA6) promotes BCa progression by cooperatively regulating multisite m6A modification. However, the therapeutic effect of targeting ITGA6 multisite m6A modifications in BCa remains unknown. OBJECTIVES: We aim to develop a multisite dCasRx- m6A editor for assessing the effects of the multisite dCasRx-m6A editor targeted m6A demethylation of ITGA6 mRNA in BC growth and progression. METHODS: The multisite dCasRx- m6A editor was generated by cloning. m6A-methylated RNA immunoprecipitation (meRIP), luciferase reporter, a single-base T3 ligase-based qPCR-amplification, Polysome profiling and meRIP-seq experiments were performed to determine the targeting specificity of the multisite dCasRx-m6A editor. We performed cell phenotype analysis and used in vivo mouse xenograft models to assess the effects of the multisite dCasRx-m6A editor in BC growth and progression. RESULTS: We designed a targeted ITGA6 multi-locus guide (g)RNA and established a bidirectional deactivated RfxCas13d (dCasRx)-based m6A-editing platform, comprising a nucleus-localized dCasRx fused with the catalytic domains of methyltransferase-like 3 (METTL3-CD) or α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5-CD), to simultaneously manipulate the methylation of ITGA6 mRNA at four m6A sites. The results confirmed the dCasRx-m6A editor modified m6A at multiple sites in ITGA6 mRNA, with low off-target effects. Moreover, targeted m6A demethylation of ITGA6 mRNA by the multisite dCasRx-m6A editor significantly reduced BCa cell proliferation and migration in vitro and in vivo. Furthermore, the dCasRx-ALKBH5-CD and ITGA6 multi-site gRNA delivered to 5-week-old BALB/cJNju-Foxn1nu/Nju nude mice via adeno-associated viral vectors significantly inhibited BCa cell growth. CONCLUSION: Our study proposes a novel therapeutic tool for the treatment of BC by applying the multisite dCasRx-m6A editor while highlighting its potential efficacy for treating other diseases associated with abnormal m6A modifications.

Biophysical phenotype mixtures reveal advantages for tumor muscle invasion in vivo.

Bladder, colon, gastric, prostate, and uterine cancers originate in organs surrounded by laminin-coated smooth muscle. In human prostate cancer, tumors that are organ confined, without extracapsular extension through muscle, have an overall cancer survival rate of up to 97% compared with 32% for metastatic disease. Our previous work modeling extracapsular extension reported the blocking of tumor invasion by mutation of a laminin-binding integrin called α6ß1. Expression of the α6AA mutant resulted in a biophysical switch from cell-ECM (extracellular matrix) to cell-cell adhesion with drug sensitivity properties and an inability to invade muscle. Here we used different admixtures of α6AA and α6WT cells to test the cell heterogeneity requirements for muscle invasion. Time-lapse video microscopy revealed that tumor mixtures self-assembled into invasive networks in vitro, whereas α6AA cells assembled only as cohesive clusters. Invasion of α6AA cells into and through live muscle occurred using a 1:1 mixture of α6AA and α6WT cells. Electric cell-substrate impedance sensing measurements revealed that compared with α6AA cells, invasion-competent α6WT cells were 2.5-fold faster at closing a cell-ECM or cell-cell wound, respectively. Cell-ECM rebuilding kinetics show that an increased response occurred in mixtures since the response was eightfold greater compared with populations containing only one cell type. A synthetic cell adhesion cyclic peptide called MTI-101 completely blocked electric cell-substrate impedance sensing cell-ECM wound recovery that persisted in vitro up to 20 h after the wound. Treatment of tumor-bearing animals with 10 mg/kg MTI-101 weekly resulted in a fourfold decrease of muscle invasion by tumor and a decrease of the depth of invasion into muscle comparable to the α6AA cells. Taken together, these data suggest that mixed biophysical phenotypes of tumor cells within a population can provide functional advantages for tumor invasion into and through muscle that can be potentially inhibited by a synthetic cell adhesion molecule.

Aging alters the cell cycle control and mitogenic signaling responses of human hematopoietic stem cells.

Human hematopoietic stem cells (HSCs), like their counterparts in mice, comprise a functionally and molecularly heterogeneous population of cells throughout life that collectively maintain required outputs of mature blood cells under homeostatic conditions. In both species, an early developmental change in the HSC population involves a postnatal switch from a state in which most of these cells exist in a rapidly cycling state and maintain a high self-renewal potential to a state in which the majority of cells are in a quiescent state with an overall reduced self-renewal potential. However, despite the well-established growth factor dependence of HSC proliferation, whether and how this mechanism of HSC regulation might be affected by aging has remained poorly understood. To address this knowledge gap, we isolated highly HSC-enriched CD34+CD38-CD45RA-CD90+CD49f+ (CD49f+) cells from cord blood, adult bone marrow, and mobilized peripheral blood samples obtained from normal humans spanning 7 decades of age and then measured their functional and molecular responses to growth factor stimulation in vitro and their regenerative activity in vivo in mice that had undergone transplantation. Initial experiments revealed that advancing donor age was accompanied by a significant and progressively delayed proliferative response but not the altered mature cell outputs seen in normal older individuals. Importantly, subsequent dose-response analyses revealed an age-associated reduction in the growth factor-stimulated proliferation of CD49f+ cells mediated by reduced activation of AKT and altered cell cycle entry and progression. These findings identify a new intrinsic, pervasive, and progressive aging-related alteration in the biological and signaling mechanisms required to drive the proliferation of very primitive, normal human hematopoietic cells.

In vitro propagation of XXY human Klinefelter spermatogonial stem cells: A step towards new fertility opportunities.

Klinefelter Syndrome (KS) is characterized by a masculine phenotype, supernumerary sex chromosomes (47, XXY), and impaired fertility due to loss of spermatogonial stem cells (SSCs). Early testicular cryopreservation could be an option for future fertility treatments in these patients, including SSCs transplantation or in vitro spermatogenesis. It is critically essential to adapt current in vitro SSCs propagation systems as a fertility option for KS patients. KS human testicular samples (13,15- and 17-year-old non-mosaic KS boys) were donated by patients enrolled in an experimental testicular tissue banking program. Testicular cells were isolated from cryopreserved tissue and propagated in long-term culture for 110 days. Cell-specific gene expression confirmed the presence of all four main cell types found in testes: Spermatogonia, Sertoli, Leydig, and Peritubular cells. A population of ZBTB16+ undifferentiated spermatogonia was identified throughout the culture using digital PCR. Flow cytometric analysis also detected an HLA-/CD9+/CD49f+ population, indicating maintenance of a stem cell subpopulation among the spermatogonial cells. FISH staining for chromosomes X and Y showed most cells containing an XXY karyotype with a smaller number containing either XY or XX. Both XY and XX populations were able to be enriched by magnetic sorting for CD9 as a spermatogonia marker. Molecular karyotyping demonstrated genomic stability of the cultured cells, over time. Finally, single-cell RNAseq analysis confirmed transcription of ID4, TCN2, and NANOS 3 within a population of putative SSCs population. This is the first study showing successful isolation and long-term in vitro propagation of human KS testicular cells. These findings could inform the development of therapeutic fertility options for KS patients, either through in vitro spermatogenesis or transplantation of SSC, in vivo.

Midkine mediates dysfunction of liver sinusoidal endothelial cells through integrin α4 and α6.

Midkine (MK)2 is an important regulatory molecule that promotes pathological angiogenesis of hepatocellular carcinoma (HCC). Although some studies have shown that its expression is increased in chronic liver disease, its effect on sinusoidal vasculopathy are still unclear. In this study, we demonstrated that MK was mainly secreted by liver sinus endothelial cells (LSECs) during the stage of precancerous lesions. Increased expression of its receptor integrin was an important mechanism by which MK participated in sinusoidal vasculopathy through autocrine and positive feedback effects. LSECs with high expression of integrin α6 (Itgα6+) and integrin α4 (Itgα4+) were used to study the mechanism of MK, and it was found that the effect of MK on LSECs was closely related to the integrin subtypes. The activation of MK /integrin α6/Src/Shc signaling pathway promoted the expression of ET-1, TXA2 and reduced the production of NO, and then induced the capillary vascularization of liver sinusoids, while the activation of MK/integrin α4/NF-κB pathway mainly induced angiogenesis by promoting the production of VEGF and Ang2. In the three-dimensional co-culture system of hepatocytes (BRL-3A) and LSECs, MK significantly increased the production of reactive oxygen species (ROS) in the co-culture system of highly expressed integrin LSECs and decreased the expression of tumor suppressor gene P53 in hepatocytes. These results suggested that MK /integrin signaling pathway, especially MK /integrin α6, was an important mechanism leaded to persistent sinusoidal hepatic vasculopathy in chronic liver disease and induced HCC，while MK/integrin α 4 activation was more involved in pathological angiogenesis.

Immortalised canine buccal epithelial cells' CXCL8 secretion is affected by allergen extracts, Toll-like receptor ligands, IL-17A and calcitriol.

Epithelial cells are known to produce mediators which can influence the behaviour of neighbouring immune cells. Although the oral mucosa has gained increased interest as a route to induce allergy desensitisation and mucosal pathogen immunisation in dogs, there is only limited knowledge on the factors which impact mediator secretion by canine oral epithelial cells. The study's objective was to enlarge the knowledge on the stimuli that can influence the secretion of some pro- and anti-inflammatory cytokines and the chemokine CXCL8 by canine buccal epithelial cells. To investigate this, buccal epithelial cells were isolated from a biopsy of a dog and immortalised by lentiviral transduction of the SV40 large T antigen. The cells were stained with a CD49f and cytokeratin 3 antibody to confirm their epithelial origin. Cells were incubated with allergen extracts, Toll-like receptor ligands (TLRL), recombinant cytokines and vitamin A and D metabolites. Subsequently, the secretion of the cytokines interleukin (IL)-4, IL-6, IL-10, IL-17A, IFN-γ, TGF-ß1 and the chemokine CXCL8 was assayed by ELISA. Immortalised canine buccal epithelial cells stained positive for CD49f but not for cytokeratin 3. The cells produced detectable amounts of CXCL8 and TGF-ß1. A Dermatophagoides farinae extract, an Alternaria alternata extract, Pam3CSK4, heat-killed Listeria monocytogenes, FSL-1, flagellin and canine recombinant IL-17A significantly increased CXCL8 secretion, while the vitamin D metabolite calcitriol significantly suppressed the production of this chemokine. This study showed that certain allergens, TLRL, IL-17A and calcitriol modulate CXCL8 secretion in a cell line of canine buccal epithelial cells.

Role of Snai2 and Notch signaling in salivary gland myoepithelial cell fate.

Myoepithelial (ME) cells in exocrine glands exhibit both epithelial and mesenchymal features, contributing to fluid secretion through contraction. However, the regulation mechanism of behind this unique phenotype in salivary glands remains unclear. We established a flow cytometry-based purification method using cell surface molecules, epithelial cell adhesion molecule (EpCAM) and alpha 6 integrin (CD49f), to characterize ME cells. EpCAM+CD49fhigh cells showed relatively high expression of ME cell-marker genes, such as alpha-smooth muscle actin (α-SMA). For lineage tracing and strict isolation, tdTomato+EpCAM+CD49fhigh-ME cells were obtained from myosin heavy chain 11 (Myh11) -CreERT2/tdTomato mice. Transcriptome analysis revealed that expression of genes involved in the epithelial-mesenchymal transition, including Snai2, were upregulated in the ME cell-enriched subset. Snai2 suppression in stable ME cells decreased α-SMA and increased Krt14 expression, suggesting that ME cell features may be controlled by the epithelial-mesenchymal balance regulated by Snai2. In contrast, ME cells showed reduced ME properties and expressed the ductal markers Krt18/19 under sphere culture conditions. Notch signaling was activated under sphere culture conditions; excessive activation of Notch signaling accelerated Krt18/19 expression, but reduced α-SMA and Snai2 expression, suggesting that the behavior of Snai2-expressing ME cells may be controlled by Notch signaling.

TM4SF1 promotes esophageal squamous cell carcinoma metastasis by interacting with integrin α6.

Transmembrane-4 L-six family member-1 (TM4SF1) is a member of the L6 family and functions as a signal transducer to regulate tumor cell behaviors. However, the function and mechanism of TM4SF1 in esophageal squamous cell carcinoma (ESCC) metastasis remains unclear. Here, we find that TM4SF1 expression is increased and positively correlated with clinical TNM stage, N classification, differentiation, tumor size, and poor prognosis in ESCC patients. Interestingly, we demonstrate that TM4SF1 promotes ESCC cell adhesion, spreading, migration, and invasion, but not cell proliferation, in a laminin-dependent manner by interacting with integrin α6. Mechanistically, the TM4SF1/integrin α6/FAK axis signal pathway mediates cell migration under laminin-coating condition. Inhibiting FAK or knocking down TM4SF1 can attenuate TM4SF1-mediated cell migration and lung metastasis. Clinically, the TM4SF1/integrin α6/FAK axis positively correlates with ESCC. Altogether, these findings reveal a new mechanism of TM4SF1 in promoting ESCC metastasis via binding to integrin α6 and suggest that the cross-talk between TM4SF1 and integrin α6 may serve as a therapeutic target for ESCC.

Integrin alpha 6 is upregulated and drives hepatocellular carcinoma progression through integrin α6ß4 complex.

Integrin α6 (ITGA6) forms integrin receptors with either integrin ß1 (ITGB1) or integrin ß4 (ITGB4). How it functions to regulate hepatocellular carcinoma (HCC) progression is not well-elucidated. We found that ITGA6 RNA and protein expression levels are significantly elevated in human HCC tissues in comparison with paired adjacent nontumor tissues by RNA sequencing, RT-qPCR, Western blotting and immunofluorescence staining. Stable knockdown of ITGA6 with different ITGA6 shRNA expression lentivectors significantly inhibited proliferation, migration and anchorage-independent growth of HCC cell lines in vitro, and xenograft tumor growth in vivo. The inhibition of anchorage-dependent and -independent growth of HCC cell lines was also confirmed with anti-ITGA6 antibody. ITGA6 knockdown was shown to induce cell-cycle arrest at G0/G1 phase. Immunoprecipitation assay revealed apparent interaction of ITGA6 with ITGB4, but not ITGB1. Expression studies showed that ITGA6 positively regulates the expression of ITGB4 with no or negative regulation of ITGB1 expression. Finally, while high levels of ITGA6 and ITGB4 together were associated with significantly worse survival of HCC patients in TCGA data set, the association was not significant for high levels of ITGA6 and ITGB1. In conclusion, ITGA6 is upregulated in HCC tumors and has a malignant promoting role in HCC cells through integrin α6ß4 complex. Thus, integrin α6ß4 may be a therapeutic target for treating patients with HCC.

The miR-410-5p /ITGA6 axis participates in the pathogenesis of recurrent abortion by regulating the biological function of trophoblast.

The purpose of this study was to determine the regulation of the miR-410-5p /ITGA6 axis on the biological functions of trophoblast cells and the mechanism involved in recurrent spontaneous abortion（RSA）. We used qRT-PCR and Western blotting to quantify the expression levels of Mir-410-5p and ITGA6 in placenta of RSA and normal, and found that compared with normal placenta, the placenta of RSA patients showed higher miR-410-5p and lower ITGA6 expression. Dual luciferase reporter gene assay confirmed the binding of miR-410-5p to ITGA6. The expression of miR-410-5p and ITGA6, and proliferation, apoptosis, invasion and migration of trophoblast cells and the effect on the polarization of M2 macrophages were detected in the trophoblast derived cell lines HTR8/Svneo transfected with miR-410-5p mimic, sh-miR-410-5p and si-ITGA6 respectively. Meanwhile, the molecular mechanism of ITGA6 regulation on trophoblast cells was explored. Transfection with miR-410-5p mimic or si-ITGA6 attenuated the proliferation, migration and invasion and induced apoptosis of HTR-8/SVneo cells. Transfection of sh-miR-410-5p promoted proliferation, migration and invasion, and weakened apoptosis of HTR-8/SVneo cells. In addition, overexpression of miR-410-5p in trophoblast cells inhibited the polarization of M2 macrophages, while knockdown of miR-410-5p was beneficial to recruitment of trophoblast cell and promoted the polarization of M2 macrophages. ITGA6 may affect the biological functions of trophoblast cells by regulating PI3K/AKT and MAPK signaling pathways. In conclusion, miR-410-5p mediates trophoblast cell proliferation, apoptosis, invasion and migration through regulating ITGA6 expression.

Glycan Epitope and Integrin Expression Dynamics Characterize Neural Crest Epithelial-to-Mesenchymal Transition (EMT) in Human Pluripotent Stem Cell Differentiation.

The neural crest gives rise to progeny as diverse as peripheral neurons, myelinating cells, cranial muscle, bone and cartilage tissues, and melanocytes. Neural crest derivation encompasses complex morphological change, including epithelial-to-mesenchymal transition (EMT) and migration to the eventual target locations throughout the body. Neural crest cultures derived from stem cells provide an attractive source for developmental studies in human model systems, of immediate biomedical relevance for neurocristopathies, neural cancer biology and regenerative medicine, if only appropriate markers for lineage and cell type definition and quality control criteria were available. Implementing a defined, scalable protocol to generate neural crest cells from embryonic stem cells, we identify stage-defining cluster-of-differentiation (CD) surface markers during human neural crest development in vitro. Acquisition of increasingly mesenchymal phenotype was characterized by absence of neuroepithelial stemness markers (CD15, CD133, CD49f) and by decrease of CD57 and CD24. Increased per-cell-expression of CD29, CD44 and CD73 correlated with established EMT markers as determined by immunofluorescence and immunoblot analysis. The further development towards migratory neural crest was associated with decreased CD24, CD49f (ITGA6) and CD57 (HNK1) versus an enhanced CD49d (ITGA4), CD49e (ITGA5) and CD51/CD61 (ITGAV/ITGB3) expression. Notably, a shift from CD57 to CD51/CD61 was identified as a sensitive surrogate surface indicator of EMT in neural crest in vitro development. The reported changes in glycan epitope and integrin surface expression may prove useful for elucidating neural crest stemness, EMT progression and malignancies.

Differential gene expression and hallmarks of stemness in epithelial cells of the developing rat epididymis.

Epididymal development can be subdivided into three phases: undifferentiated, a period of differentiation, and expansion. The objectives of this study were (1) to assess gene expression profiles in epididymides, (2) predict signaling pathways, and (3) develop a novel 3D cell culture method to assess the regulation of epididymal development in vitro. Microarray analyses indicate that the largest changes in differential gene expression occurred between the 7- to 18-day period, in which 1452 genes were differentially expressed, while 671 differentially expressed genes were noted between days 18 and 28, and there were 560 differentially expressed genes between days 28 and 60. Multiple signaling pathways were predicted at different phases of development. Pathway associations indicated that in epididymides of 7- to 18-day old rats, there was a significant association of regulated genes implicated in stem cells, estrogens, thyroid hormones, and kidney development, while androgen- and estrogen-related pathways were enriched at other phases of development. Organoids were derived from CD49f + columnar cells from 7-day old rats, while no organoids developed from CD49f- cells. Cells cultured in an epididymal basal cell organoid medium versus a commercial kidney differentiation medium supplemented with DHT revealed that irrespective of the culture medium, cells within differentiating organoids expressed p63, AQP9, and V-ATPase after 14 days of culture. The commercial kidney medium resulted in an increase in the number of organoids positive for p63, AQP9, and V-ATPase. Together, these data indicate that columnar cells represent an epididymal stem/progenitor cell population.

Effect of different iodine levels on the DNA methylation of PRKAA2, ITGA6, THEM4 and PRL genes in PI3K-AKT signaling pathway and population-based validation from autoimmune thyroiditis patients.

PURPOSE: Autoimmune thyroiditis (AIT) is one of the most common autoimmune endocrine diseases. The currently recognized causes are genetic susceptibility, environmental factors and immune disorders. It is important to clarify the pathogenesis for the prevention, diagnosis, treatment of AIT and scientific iodine supplementation. This study analyzed the DNA methylation levels of PRKAA2, ITGA6, PRL and THEM4 genes related to PI3K-AKT signaling pathway, compared the DNA methylation levels between cases and controls from different water iodine levels in Shandong Province of China, and evaluated the contribution of PI3K-AKT signaling pathway-related genes in AIT. METHODS: A total of 176 adult AIT patients were included from three different water iodine areas, and 176 healthy controls were included according to gender, age and BMI. According to the results of the Illumina Methylation 850 K BeadChip in our previous research, the significant methylation differences of genes on the PI3K-AKT signaling pathway related to AIT were determined. The MethylTarget™ assay was used to detect the methylation levels of the target genes, and real-time PCR experiments were used to verify the mRNA expression levels. RESULTS: Compared with the control group, PRKAA2_3 and 15 CpG sites were hyper-methylated. ITGA6 gene and 2 CpG sites were hypo-methylated in AIT cases. The mRNA expression of ITGA6 gene was negatively correlated with the DNA methylation levels of ITGA6 gene and 2 CpG sites. Compared with cases and controls in areas with different water iodine levels, methylation differences were mainly in PRKAA2 and ITGA6 genes. The methylation levels of PRKAA2_1 and PRKAA2_3 were positively correlated with age. The methylation levels of PRL and THEM4 genes were negatively correlated with age. The methylation level of PRKAA2_3 was positively correlated with FT4. CONCLUSION: In summary, we identified aberrant DNA methylation levels of PRKAA2 and ITGA6 genes related to PI3K-AKT signaling pathway in the blood of AIT patients. Both iodine supplementation after long-term iodine deficiency and iodine excess can affect the DNA methylation levels of PRKAA2 and ITGA6 genes, and the former affects more obviously. In ITGA6 gene, this aberrant epigenetic modification is associated with the increased mRNA expression.

Protein Ligand Nanopattern Size Selects for Cellular Adhesion via Hemidesmosomes over Focal Adhesions.

Hemidesmosomes (HDs) are multiprotein complexes that firmly anchor epidermal cells to the basement membrane of skin through the interconnection of the cytoplasmic intermediate filaments with extracellular laminin 332 (Ln332). Considerably less attention has been paid to HDs compared to focal complexes/focal adhesions (FC/FAs) in mechanistic single-cell structures due to the lack of suitable in vitro model systems. Here nanopatterns of Ln332 (100-1000 nm) are created to direct and study the formation of HD in adherent HaCaT cells. It is observed that HaCaT cells at Ln 332 nanopatterns adhere via hemidesmosomes, in stark contrast to cells at homogeneous Ln332 surfaces that adhere via FC/FAs. Clustering of α6 integrin is observed at nanopatterned Ln332 of 300 nm patches and larger. Cells at 500 nm diameter patterns show strong colocalization of α6 integrin with ColXVII or pan-cytokeratin compared to 300 nm/1000 nm indicating a threshold for HD initiation >100 nm but a pattern size selection for maturation of HDs. It is demonstrated that the pattern of Ln332 can determine the cellular selection of adhesion types with a size-dependent initiation and maturation of HDs. The protein nanopatterning approach that is presented provides a new in vitro route to study the role of HDs in cell signaling and function.

Identification of Surface Antigens That Define Human Pluripotent Stem Cell-Derived PRRX1+Limb-Bud-like Mesenchymal Cells.

Stem cell-based therapies and experimental methods rely on efficient induction of human pluripotent stem cells (hPSCs). During limb development, the lateral plate mesoderm (LPM) produces limb-bud mesenchymal (LBM) cells that differentiate into osteochondroprogenitor cells and form cartilage tissues in the appendicular skeleton. Previously, we generated PRRX1-tdTomato reporter hPSCs to establish the protocol for inducing the hPSC-derived PRRX1+ LBM-like cells. However, surface antigens that assess the induction efficiency of hPSC-derived PRRX1+ LBM-like cells from LPM have not been identified. Here, we used PRRX1-tdTomato reporter hPSCs and found that high pluripotent cell density suppressed the expression of PRRX1 mRNA and tdTomato after LBM-like induction. RNA sequencing and flow cytometry suggested that PRRX1-tdTomato+ LBM-like cells are defined as CD44high CD140Bhigh CD49f-. Importantly, other hPSC lines, including four human induced pluripotent stem cell lines (414C2, 1383D2, HPS1042, HPS1043) and two human embryonic stem cell lines (SEES4, SEES7), showed the same results. Thus, an appropriate cell density of hPSCs before differentiation is a prerequisite for inducing the CD44high CD140Bhigh CD49f- PRRX1+ LBM-like cells.

Functional characterization of CD49f+ hepatic stem/progenitor cells in adult mice liver.

Hepatic Stem/progenitor cells (HSPCs) have gained a large amount of interest for treating acute liver disease. However, the isolation and identification of HSPCs are unclear due to the lack of cell-specific surface markers. To isolate adult HSPCs, we used cell surface-marking antibodies, including CD49f and Sca-1. Two subsets of putative HSPCs, Lin-CD45-Sca-1-CD49f+ (CD49f+) and Lin-CD45-Sca-1+CD49f- (Sca-1+) cells, were isolated from adult mice liver by flow cytometry. Robust proliferative activity and clonogenic activity were found in both CD49f+ and Sca-1+ cells through colony-forming tests and cell cycle analyses. Immunofluorescence staining revealed that CD49f+ cells expressed ALB and CK-19 while Sca-1+ cells expressed only ALB, indicating that CD49f+ cells were bipotential and capable of differentiating into hepatocyte and cholangiocyte. Consequently, PAS stain showed that differentiated CD49f+ and Sca-1+ cells synthesised glycogen, indicating they could differentiate into functional hepatocytes. mRNA expression profile indicated that both CD49f+ and Sca-1+ cells showed differential expression of genes that are associated with liver progenitor function such as Sox9 and EpCam. Moreover, two subsets of putative HSPCs were activated by DDC and we found that their abundance and proliferation increased with age. In summary, we hypothesized that CD49f+ cells were a type of potential HSPCs and may be utilised for clinical stem cell therapy.

Long non-coding RNA OIP5-AS1 suppresses microRNA-92a to augment proliferation and metastasis of ovarian cancer cells through upregulating ITGA6.

OBJECTIVE: Recently, long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been identified as essential biomarkers during development of malignancies. This study was performed to study the roles of lncRNA opa-interacting protein 5 antisense transcript 1 (OIP5-AS1) and miR-92a in ovarian cancer (OC). METHODS: OIP5-AS1, miR-92a and integrin alpha 6 (ITGA6) expression in OC tissues and cells was assessed. The screened OC cells were respectively with OIP5-AS1-, miR-92a- and ITGA6-related vectors or oligonucleotides . The viability, migration, invasion and apoptosis of the cells were determined and the levels of epithelial-mesenchymal transition (EMT)-related proteins were also measured. The interactions between OIP5-AS1 and miR-92a, and between miR-92a and ITGA6 were confirmed. RESULTS: OIP5-AS1 and ITGA6 were upregulated while miR-92a was downregulated in OC. Inhibited OIP5-AS1 or downregulated ITGA6 or elevated miR-92a repressed EMT, viability, migration and invasion, and promoted apoptosis of OC cells. OIP5-AS1 as a competing endogenous RNA interacted with miR-92a to regulate ITGA6. These effects that induced by silenced OIP5-AS1 could be reversed by miR-92a inhibition while those that induced by up-regulated miR-92a were reduced by restored ITGA6. CONCLUSION: OIP5-AS1 silencing promoted miR-92a to repress proliferation and metastasis of OC cells through inhibiting ITGA6.

Integrin α6 overexpression promotes lymphangiogenesis and lymphatic metastasis via activating the NF-κB signaling pathway in lung adenocarcinoma.

OBJECTIVE: It has been reported that tumor-associated lymphangiogenesis plays an important role in lymph node metastasis and contributes to the poor survival of lung adenocarcinoma (LUAD) patients. As yet, however, the molecular mechanism underlying LUAD-associated lymphangiogenesis has remained elusive. METHODS: Immunohistochemistry (IHC) was used to determine the expression of integrin subunit alpha 6 (ITGA6) and the lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1) in clinicopathologically characterized LUAD specimens. The effect of ITGA6 overexpression on lymphangiogenesis and lymphatic metastasis was examined by tube formation, scratch wound-healing, and cell migration assays in vitro and a popliteal lymph node metastasis model in vivo. Mechanistically, overexpression of ITGA6 and activation of NF-κB signaling were examined by real-time PCR, ubiquitination and dual-luciferase reporter assays. Finally, high ITGA6 expression in LUAD tissue samples was related to copy number variation (CNV) using the TCGA database. RESULTS: We found that ITGA6 overexpression correlated with microlymphatic vessel density in LUAD specimens (p < 0.01). Importantly, by using a popliteal lymph node metastasis model, we found that ITGA6 upregulation significantly enhanced lymphangiogenesis and lymphatic metastasis in vivo (p < 0.05). In addition, we found that ITGA6 overexpression enhanced the capability of A549 and H1299 LUAD cells to induce tube formation and migration in human lymphatic endothelial cells (HLECs). Mechanistically, we found that ITGA6 sustained NF-κB activity via binding and promoting K63 polyubiquitination of TNF receptor-associated factor 2 (TRAF2). Finally, CNV analysis revealed ITGA6 amplification of 27.5% in the LUAD tissue samples in the TCGA database. CONCLUSIONS: Taken together, our results uncover a plausible role for ITGA6 in mediating lymphangiogenesis and lymphatic metastasis and may provide a basis for targeting ITGA6 to treat LUAD lymphatic metastasis.