Facilitate Cell Migration Research Articles

Corneal stromal filler injection of gelatin-based photocurable hydrogels for maintaining the corneal thickness and reconstruction of corneal stroma

Refractive errors manifest as thinning of the central cornea, which can seriously impair vision. The corneal stromal filler is an innovative concept for treating refractive problems. However, corneal stromal regeneration poses a challenge for the tissue-engineered cornea. Hence, materials for corneal reconstruction with high transparency and biocompatibility are urged to be developed. This study aimed to establish a novel therapeutic approach by injecting gelatin-based photocurable hydrogel fillers into the corneal stroma to maintain corneal thickness and promote corneal stromal regeneration for refractive error. The hydrogel possesses high transparency and suitable mechanical qualities, which degrade slowly in the cornea. Furthermore, the hydrogel presents high biocompatibility for cellular adhesion and proliferation. The hydrogel can also provide space for cells to develop and facilitate cell migration when cells are infused into it. In vivo experiments showed that the hydrogel could effectively fortify corneal thickness and induce corneal stromal regeneration. The photocurable hydrogel showed potential in terms of corneal reconstruction.

Revolutionizing wound healing: Ultrashort pulse electric fields in seconds for highly aligned extracellular matrix and efficient cell migration

Wound healing is a complicated process for maintaining skin integrity after injury, for which electrical stimulations (ES) have been attributed to promote wound healing by facilitating cell migration. Reducing the duration of the stimulation treatment from hours to minutes to promote efficient wound healing while avoiding cell damage poses a considerable challenge. Here, a promising technology of ultrashort pulse electric field (PEF), microsecond PEF at higher voltage, is proposed and realized to promote wound healing under a much short time (seconds) for the total treatment. We revealed that microsecond PEF regulated actin cytoskeleton reorganization and focal adhesion turnover, promoting fibroblasts migration in 2D cell cultures under the pulse stimulation. This accelerated fibroblast migration was accompanied by the mutual promotion with extracellular matrix (ECM) alignment in 3D microenvironments, which cooperatively benefit the eventual wound healing. These findings were further corroborated by the significant 2.5-fold enhancement observed in the healing of skin wounds in the classical mouse model by day 6 after electrical stimulation. Additionally, we coined an actin- and collagen-dependent mechanism of microsecond PEF-mediated wound healing. The quantitative mechanism proposed here for our novel microsecond pulse electric filed (μsPEF) methodology orients the new practical electric treatment toward a wide range of biomedical applications, such as wound healing, regenerative medicine and tissue engineering.

Efficacy of collagen-only scaffolds compared to polymer-associated collagen and nanomaterials in skin wound repair – a review

Wound healing remains a clinical problem, with cases of atrophic, hypertrophic, or keloid scars. Three-dimensional scaffolds have been used to restore skin function, facilitating cell migration, adhesion, and proliferation. Collagen is the most common, presenting low antigenicity, decreased inflammation, and replacement by autologous tissue. It is used as sheets/films, sponges, membranes, sprays, and hydrogels of various origins. This integrative literature review aimed to evaluate the application of unassociated collagen scaffolds for skin wound healing and compare them to collagen associations with nanomaterials and polymers. Properties such as applications in humans and other unconventional models cause burns, partial and full-thickness wounds, and others. Scaffold, biomaterials, collagen, wound, injury, repair, and healing were among the descriptors. We found 3,098 articles published between 1995 and 2022 (Mendeley platform), including clinical/experimental trials. After exclusion, 26 studies were identified and analyzed. Autologous and heterologous collagens are the most used in the clinic and favor wound closure by improving re-epithelialization and reducing inflammation but may present challenges in aesthetic acceptance and loss of repair function in the wound site. Furthermore, collagen integration with other nanomaterials improved wound repair and experimental models.

Adoptive Treg therapy with metabolic intervention via perforated microneedles ameliorates psoriasis syndrome.

Regulatory T (Treg) cells underlie multiple autoimmune disorders and potentialize an anti-inflammation treatment with adoptive cell therapy. However, systemic delivery of cellular therapeutics often lacks tissue targeting and accumulation for localized autoimmune diseases. Besides, the instability and plasticity of Treg cells also induce phenotype transition and functional loss, impeding clinical translation. Here, we developed a perforated microneedle (PMN) with favorable mechanical performance and a spacious encapsulation cavity to support cell survival, as well as tunable channels to facilitate cell migration for local Treg therapy of psoriasis. In addition, the enzyme-degradable microneedle matrix could release fatty acid in the hyperinflammatory area of psoriasis, enhancing the Treg suppressive functions via the fatty acid oxidation (FAO)-mediated metabolic intervention. Treg cells administered through PMN substantially ameliorated psoriasis syndrome with the assistance of fatty acid-mediated metabolic intervention in a psoriasis mouse model. This tailorable PMN could offer a transformative platform for local cell therapy to treat a variety of diseases.

Atlas of the anatomical localization of atypical chemokine receptors in healthy mice.

Atypical chemokine receptors (ACKRs) scavenge chemokines and can contribute to gradient formation by binding, internalizing, and delivering chemokines for lysosomal degradation. ACKRs do not couple to G-proteins and fail to induce typical signaling induced by chemokine receptors. ACKR3, which binds and scavenges CXCL12 and CXCL11, is known to be expressed in vascular endothelium, where it has immediate access to circulating chemokines. ACKR4, which binds and scavenges CCL19, CCL20, CCL21, CCL22, and CCL25, has also been detected in lymphatic and blood vessels of secondary lymphoid organs, where it clears chemokines to facilitate cell migration. Recently, GPR182, a novel ACKR-like scavenger receptor, has been identified and partially deorphanized. Multiple studies point towards the potential coexpression of these 3 ACKRs, which all interact with homeostatic chemokines, in defined cellular microenvironments of several organs. However, an extensive map of ACKR3, ACKR4, and GPR182 expression in mice has been missing. In order to reliably detect ACKR expression and coexpression, in the absence of specific anti-ACKR antibodies, we generated fluorescent reporter mice, ACKR3GFP/+, ACKR4GFP/+, GPR182mCherry/+, and engineered fluorescently labeled ACKR-selective chimeric chemokines for in vivo uptake. Our study on young healthy mice revealed unique and common expression patterns of ACKRs in primary and secondary lymphoid organs, small intestine, colon, liver, and kidney. Furthermore, using chimeric chemokines, we were able to detect distinct zonal expression and activity of ACKR4 and GPR182 in the liver, which suggests their cooperative relationship. This study provides a broad comparative view and a solid stepping stone for future functional explorations of ACKRs based on the microanatomical localization and distinct and cooperative roles of these powerful chemokine scavengers.

Aquaporin 1 (AQP1)/ GSK3β interactions control pulmonary arterial smooth muscle cell (PASMC) function

Pulmonary hypertension (PH) is a complex, incurable, devastating condition, due to pathology of the pulmonary circulation. PH can be induced by hypoxia and is notable for severe wall thickening in the distal vasculature, characterized by enhanced PASMC migration and proliferation. Previously our lab discovered AQP1, a water channel, was highly expressed in PASMCs and mediated hypoxia-induced migration and proliferation. Moreover, increasing AQP1 protein was sufficient to induce PASMC migration and proliferation without hypoxia. We showed the C-terminal tail was required for regulating cell function via accumulation of β-catenin independent of water transport.β-catenin protein is tightly controlled by glycogen synthase kinase 3 β (GSK3β). Binding to GSK3β results in β-catenin degradation; when stabilized, β-catenin mediates transcription of pro-growth genes. Through in silico analysis, we identified a putative binding site for GSK3β in the AQP1 C-terminal tail, allowing us to hypothesize a potential mechanism whereby AQP1 binds GSK3β, releasing β-catenin from degradation, and thereby increases PASMC migration and proliferation.First, we used BioID, a proximity-based assay, to identify GSK3β and AQP1 interactions. We designed BioID constructs with wild-type AQP1 (BioID-AQP1) and AQP1 with mutations in the putative GSK3β binding site (BioID-AQP1M) fused to biotin ligase and compared results to wild-type AQP1 (AQP1), as a control for transfection. We used HEK293 cells which do not express endogenous AQP1. In cells transfected with AQP1 and incubated with cell permeable biotin, following biotin-affinity capture of streptavidin, no biotinylated GSK3b was measured. In cells transfected with BioID-AQP1, a significant amount of GSK3β protein was biotinylated, suggesting AQP1/GSK3β protein-protein interactions. When the putative GSK3b binding site was mutated, very low levels of GSK3b were biotinylated.Next, primary cultured rat distal PASMCs were infected with adenoviral constructs expressing wild-type AQP1 (AdAQP1), AQP1 with the putative GSK3β binding site mutated (AdAQP1M), or green fluorescent protein (AdGFP; control for infection). Compared to AdGFP, infection with AdAQP1 and AdAQP1M increased PASMC total and surface AQP1 protein levels. Using calcein self-quenching, the rate change of water influx was similar for both AQP1 constructs, indicating the mutation did not impair function as a water channel. Infection with AdAQP1 significantly increased both PASMC migration and proliferation, measured by BrdU incorporation and transwell assays, respectively, whereas infection with AdAQP1M had no effect.Our results suggest AQP1 controls PASMC migration and proliferation via a mechanism that requires GSK3β interactions. We speculate high levels of AQP1, as occur during PH, act as a sink for GSK3β, allowing β-catenin to escape from the destruction complex and facilitate cell migration and initiate gene transcription involved in cell proliferation. HL126514 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The activation of gastric inhibitory peptide/gastric inhibitory peptide receptor axis via sonic hedgehog signaling promotes the bridging of gapped nerves in sciatic nerve injury.

Schwann cells play an essential role in peripheral nerve regeneration by generating a favorable microenvironment. Gastric inhibitory peptide/gastric inhibitory peptide receptor (GIP/GIPR) axis deficiency leads to failure of sciatic nerve repair. However, the underlying mechanism remains elusive. In this study, we surprisingly found that GIP treatment significantly enhances the migration of Schwann cells and the formation of Schwann cell cords during recovery from sciatic nerve injury in rats. We further revealed that GIP and GIPR levels in Schwann cells were low under normal conditions, and significantly increased after injury demonstrated by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Wound healing and Transwell assays showed that GIP stimulation and GIPR silencing could affect Schwann cell migration. In vitro and in vivo mechanistic studies based on interference experiment revealed that GIP/GIPR might promote mechanistic target of rapamycin complex 2 (mTORC2) activity, thus facilitating cell migration; Rap1 activation might be involved in this process. Finally, we retrieved the stimulatory factors responsible for GIPR induction after injury. The results indicate that sonic hedgehog (SHH) is a potential candidate whose expression increased upon injury. Luciferase and chromatin immunoprecipitation (ChIP) assays showed that Gli3, the target transcription factor of the SHH pathway, dramatically augmented GIPR expression. Additionally, in vivo inhibition of SHH could effectively reduce GIPR expression after sciatic nerve injury. Collectively, our study reveals the importance of GIP/GIPR signaling in Schwann cell migration, providing a therapeutic avenue toward peripheral nerve injury.

High-Performance Polarization Microscopy Reveals Structural Remodeling in Rat Calcaneal Tendons Cultivated In Vitro.

Collagenous tissues exhibit anisotropic optical properties such as birefringence and linear dichroism (LD) as a result of their structurally oriented supraorganization from the nanometer level to the collagen bundle scale. Changes in macromolecular order and in aggregational states can be evaluated in tendon collagen bundles using polarization microscopy. Because there are no reports on the status of the macromolecular organization in tendon explants, the objective of this work was to evaluate the birefringence and LD characteristics of collagen bundles in rat calcaneal tendons cultivated in vitro on substrates that differ in their mechanical stiffness (plastic vs. glass) while accompanying the expected occurrence of cell migration from these structures. Tendon explants from adult male Wistar rats were cultivated for 8 and 12 days on borosilicate glass coverslips (n = 3) and on nonpyrogenic polystyrene plastic dishes (n = 4) and were compared with tendons not cultivated in vitro (n = 3). Birefringence was investigated in unstained tendon sections using high-performance polarization microscopy and image analysis. LD was studied under polarized light in tendon sections stained with the dichroic dyes Ponceau SS and toluidine blue at pH 4.0 to evaluate the orientation of proteins and acid glycosaminoglycans (GAG) macromolecules, respectively. Structural remodeling characterized by the reduction in the macromolecular orientation, aggregation and alignment of collagen bundles, based on decreased average gray values concerned with birefringence intensity, LD and morphological changes, was detected especially in the tendon explants cultivated on the plastic substrate. These changes may have facilitated cell migration from the lateral regions of the explants to the substrates, an event that was observed earlier and more intensely upon tissue cultivation on the plastic substrate. The axial alignment of the migrating cells relative to the explant, which occurred with increased cultivation times, may be due to the mechanosensitive nature of the tenocytes. Collagen fibers possibly played a role as a signal source to cells, a hypothesis that requires further investigation, including studies on the dynamics of cell membrane receptors and cytoskeletal organization, and collagen shearing electrical properties.

Simple Technique for Microscopic Evaluation of Active Cellular Invasion into 3D Hydrogel Constructs.

Materials that are evaluated for bioengineering purposes are carefully tested to evaluate cellular interactions with respect to biocompatibility and in some cases cell differentiation. A key perspective that is often considered is the ability for decellularized synthetic or natural based matrices to facilitate cell migration or tissue ingrowth. Current methods of measuring cell migration range from simple scratch assays to Boyden chamber inserts and fluorescent imaging of seeded spheroids. Many of these methods require tissue processing for histological analysis and fixing and staining for imaging, which can be difficult and dependent on the stability of the hydrogel subject. Herein we present a simple platform that can be manufactured using 3D printing and easily applied to in vitro cell culture, allowing the researcher to image live cellular migration into a cellular materials. We found this to be an adaptable, cheap, and replicable technique to evaluate cellular interaction that has applications in the research and development of hydrogels for tissue engineering purposes.

CEBPB regulates the migration, invasion and EMT of breast cancer cells by inhibiting THBS2 expression and O-fucosylation.

Breast cancer (bc) is the second most common type of human malignancies with highest morbidity and mortality in the female population. Therefore, it is essential to develop novel and effective therapies for bc treatment. The main aim of the current study is to investigate the functions of CEBPB and THBS2 in bc and the underlying mechanism. Reverse transcription-quantitative real-time polymerase chain reaction and western blot were performed for the measurement of ribonucleic acids and proteins. Function and mechanism assays were, respectively, conducted for the evaluation of bc biological behaviors and exploration of the potential correlation of genes. According to bioinformatics analyses and experimental results, THBS2, up-regulated in bc tissues and cell lines, could facilitate cell migration, invasion and EMT in bc. CEBPB was validated to facilitate miR-29a-3p transcription, thus negatively modulating THBS2 expression. The results of rescue experiments reflected that CEBPB could regulate the malignant behaviors of bc cells via THBS2. Furthermore, CEBPB was ascertained to inhibit the transcription of B3GALTL to affect THBS2 protein O-fucosylation and secretion. The interaction between THBS2 and ITGB1 was confirmed, and THBS2 was found to activate the PI3K/AKT signaling pathway. To conclude, CEBPB could restrain bc cell migration, invasion and EMT via inhibition on THBS2 expression and O-fucosylation.

The combination of positive anti‑WDR1 antibodies with negative anti‑CFL1 antibodies in serum is a poor prognostic factor for patients with esophageal carcinoma.

WD repeat-containing protein 1 (WDR1) regulates the cofilin 1 (CFL1) activity, promotes cytoskeleton remodeling, and thus, facilitates cell migration and invasion. A previous study reported that autoantibodies against CFL1 and β-actin were useful biomarkers for diagnosing and predicting the prognosis of patients with esophageal carcinoma. Therefore, the present study aimed to evaluate the serum levels of anti-WDR1 antibodies (s-WDR1-Abs) combined with serum levels of anti-CFL1 antibodies (s-CFL1-Abs) in patients with esophageal carcinoma. Serum samples obtained from 192 patients with esophageal carcinoma and other solid cancers. And s-WDR1-Ab and s-CFL1-Ab titers were analyzed using the amplified luminescent proximity homogeneous assay-linked immunosorbent assay. Compared with those of healthy donors, the s-WDR1-Ab levels were significantly higher in the 192 patients with esophageal, whereas these were not significantly higher in the samples from patients with gastric, colorectal, lung, or breast cancer. In 91 patients treated with surgery, sex, tumor depth, lymph node metastasis, stage and C-reactive protein levels were significantly associated with overall survival, as determined using the log-rank test, whereas the squamous cell carcinoma antigen, p53 antibody and s-WDR1-Ab levels tended to be associated with a worse prognosis. Although no significant difference was observed in the survival between the positive and negative groups of s-WDR1-Abs or s-CFL1-Abs alone in the Kaplan-Meier test, the patients in the s-WDR1-Ab-positive and s-CFL1-Ab-negative groups exhibited a significantly poorer prognosis in the overall survival analysis. On the whole, the present study demonstrates that the combination of positive anti-WDR1 antibodies with negative anti-CFL1 antibodies in serum may be a poor prognostic factor for patients with esophageal carcinoma.

The Actin Crosslinker Fascin Regulates Cell Chirality.

The left-right (L-R) asymmetry of the cells, or cell chirality, is a well-known intrinsic property derived from the dynamic organization of the actin cytoskeleton. Cell chirality can be regulated by actin-binding proteins such as α-actinin-1 and can also be mediated by certain signaling pathways, such as protein kinase C (PKC) signaling. Fascin, an actin crosslinker known to mediate parallel bundling of actin filaments, appears as a prominent candidate in cell chirality regulation, given its role in facilitating cell migration as an important PKC substrate. Here, it is shown that the chirality of NIH/3T3 cells can be altered by PKC activation and fascin manipulation. With either small-molecule drug inhibition or genetic knockdown of fascin, the chirality of 3T3 cells is reversed from a clockwise (CW) bias to a counterclockwise (CCW) bias on ring-shaped micropatterns, accompanied by the reversal in cell directional migration. The Ser-39 fascin-actin binding sites are further explored in cell chirality regulation. The findings of this study reveal the critical role of fascin as an important intermediator in cell chirality, shedding novel insights into the mechanisms of L-R asymmetric cell migration and multicellular morphogenesis.

Expression and function of myelin expression factor 2 in hepatocellular carcinoma

IntroductionHepatocellular carcinoma (HCC) is one of the most common malignant tumours in the world and has a high mortality rate. However, the pathogenesis of HCC remains unclear. This study aimed to investigate the potential biomarkers of HCC.MethodsONCOMINE, HCCDB and THE HUMAN PROTEIN ATLAS were used to identify myelin expression factor 2 (MYEF2) as a potential biomarker for HCC. The Cancer Genome Atlas database was used to further validate and analyse the value of MYEF2. Kaplan–Meier Plotter was used for the prognostic analysis. The COX regression model and Kaplan–Meier method were used to investigate the clinical value of MYEF2 in the prognosis of HCC by reviewing the survival status of patients. Fluorescent quantitative polymerase chain reaction (qPCR) and immunohistochemistry were used to detect the expressions of the MYEF2 mRNA and protein in HCC tissues and cell lines. qPCR and Western blotting were used to validate the efficiency of MYEF2 knockout and overexpression in HCC cells. The invasion and migration abilities regulated by MYEF2 were detected by performing transwell and wound healing assays.ResultsMYEF2 is significantly upregulated in HCC and is mainly located in the nucleus of HCC cells. MYEF2 expression is significantly associated with the tumour stage, histological grade and TNM stage. High MYEF2 expression is an independent prognostic factor for patients with HCC. Functionally, elevated MYEF2 facilitated cell migration and invasion in vitro. In contrast, decreased MYEF2 inhibited cell migration and invasion.ConclusionsMYEF2 may be a novel biomarker with potential diagnosis and prognosis values and as a potential therapeutic target for HCC.

The scaffold RhoGAP protein ARHGAP8/BPGAP1 synchronizes Rac and Rho signaling to facilitate cell migration.

Rho GTPases regulate cell morphogenesis and motility under the tight control of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). However, the underlying mechanism(s) that coordinate their spatiotemporal activities, whether separately or together, remain unclear. We show that a prometastatic RhoGAP, ARHGAP8/BPGAP1, binds to inactive Rac1 and localizes to lamellipodia. BPGAP1 recruits the RacGEF Vav1 under epidermal growth factor (EGF) stimulation and activates Rac1, leading to polarized cell motility, spreading, invadopodium formation, and cell extravasation and promotes cancer cell migration. Importantly, BPGAP1 down-regulates local RhoA activity, which influences Rac1 binding to BPGAP1 and its subsequent activation by Vav1. Our results highlight the importance of BPGAP1 in recruiting Vav1 and Rac1 to promote Rac1 activation for cell motility. BPGAP1 also serves to control the timing of Rac1 activation with RhoA inactivation via its RhoGAP activity. BPGAP1, therefore, acts as a dual-function scaffold that recruits Vav1 to activate Rac1 while inactivating RhoA to synchronize both Rho and Rac signaling in cell motility. As epidermal growth factor receptor (EGFR), Vav1, RhoA, Rac1, and BPGAP1 are all associated with cancer metastasis, BPGAP1 could provide a crucial checkpoint for the EGFR-BPGAP1-Vav1-Rac1-RhoA signaling axis for cancer intervention.

Evaluation of the Lysyl Oxidase-Like 2 (LOXL2) inhibitory activity of pimaranes and their glycosyl derivatives

Lysyl oxidase (LOX) and LOX-like 1-4 (LOXL 1-4) enzymes catalyze the cross-linking of elastin and collagen in the extracellular matrix, facilitating cell migration and invasion. The inhibition of these enzymes, particularly LOXL2, has been suggested as a therapeutic strategy to prevent breast cancer metastasis. In this work, new natural LOXL2 inhibitors were searched from Aeollanthus rydingianus, a medicinal plant rich in bioactive products. Five pimarane diterpenoids, two isolated from the plant and three derivatives, were tested. These compounds have been described for their bioactive properties such as anti-tumor, anti-inflammatory, analgesic, and antibacterial activities. In this regard, we intended to explore the mechanisms of these compounds by studying their effects on LOXL2 activity. Two pimarane diterpenoids showed a mild LOXL2 inhibitory activity as evaluated by an Amplex Ultra Red-based technique. The cytotoxicity of the most active compound was analyzed by the MTT assay in the MDA-MB-231 cell line, representative of triple-negative breast cancer. This compound decreased cell viability as single agent and increased the cytotoxic effect of doxorubicin. Its glycoconjugate was considerably more toxic, likely due to a higher uptake by cancer cells. Keywords: breast cancer, lysyl oxidase-like 2, inhibitors, pimaranes, Aeollanthus rydingianus, MTT

Synthesis and characterization of mussel-inspired nanocomposites based on dopamine–chitosan–iron oxide for wound healing: In vitro study

There are many challenges faced the soft tissue adhesives in the medical application field. For example, there is a limited effective binding between the medical adhesive and different types of soft tissues. Chitosan (CS) and dopamine (DA) were used as structural units for synthesizing nanocomposites utilized as a wet tissue adhesive. To produce dopamine–chitosan–iron oxide nanocomposites (DA–CS–Fe3O4 NCs), DA was loaded onto chitosan–iron oxide nanocomposites. The nanocomposites have been prepared using ionic gelation method under vigorous homogenization and characterized by different techniques. Fourier-transform infrared spectroscopy (FTIR) have shown that DA–CS– Fe3O4 NCs could attach to the tissue through two possible functional groups, namely, the catechol and amine groups. The results of in vitro scratch wound-healing assay suggested that the prepared DA–CS– Fe3O4 NCs facilitate cell migration (the wound-closure percentage reached 96% at 72 h). All experimental results confirm that DA–CS– Fe3O4 NCs are strongly recommended for use as a soft medical tissue adhesive in wound healing and surgeries such as vascular surgery. In addition, the results of the whole blood clotting, antibacterial assessment, live and dead assay, cytotoxicity test, and wound-healing assay indicate that DA-CS–Fe3O4 NCs can be used as a multifunctional biomedical adhesive.

A short peptide encoded by long non-coding RNA small nucleolar RNA host gene 6 promotes cell migration and epithelial-mesenchymal transition by activating transforming growth factor-beta/SMAD signaling pathway in human endometrial cells.

Endometrial dysfunction is closely correlated with the development of multiple severe gynecological disorders including intrauterine adhesion. Accumulating evidence supports that some long non-coding RNAs (lncRNAs) have peptide-coding potential. In this text, the peptide-coding ability of lncRNA SNHG6 was examined. Also, the effects of an SNHG6-encoded peptide on the viability and migration of human endometrial stromal cells (hESCs) and human endometrial epithelial cells (hEECs) and related molecular mechanisms were explored. The peptide-encoding potential of SNHG6 was predicted by FuncPEP and getorf databases and validated by western blot assay. Cell viability was tested by cell counting kit-8 assay. Cell migratory ability was examined by wound healing and transwell migration assays. Protein levels of genes were measured by western blot assay. Prediction analysis suggested that SNHG6 had the potential peptide-coding ability and multiple open-reading frames (ORFs). Western blot validated that SNHG6 ORF#1 and ORF#2 could translate into short peptides. SNHG6 ORF#2 overexpression facilitated cell migration and epithelial-mesenchymal transition (EMT) in hESCs and hEECs, while these effects were abrogated by transforming growth factor-beta (TGF-β)/SMAD signaling inhibitor GW788388. Moreover, GW788388 inhibited the increase of p-SMAD2 and p-SMAD3 levels induced by SNHG6 ORF#2 in hESCs. SNHG6 ORF#2-encoded peptide did not influence endometrial stromal and epithelial cell viability. LncRNA SNHG6 ORF#1 and ORF#2 could translate into small peptides and SNHG6 ORF#2 overexpression promoted cell migration and EMT by activating the TGF-β/SMAD pathway in hESCs and hEECs, suggesting the potential roles of SNHG6-encoded peptides in the development of endometrial stromal and epithelial cells and related gynecological diseases.

Integrin Signaling Pathway Is Crucial for the Recently Divided Fraction of CLL Cells and Facilitates Cell Migration in a Disease Progressive Manner

Chronic lymphocytic leukemia (CLL) cells can be subdivided into fractions based on the time since cell birth/division. The fraction of CLL B cells bearing a CXCR4DimCD5Bright phenotype divided recently (proliferative fraction, PF), while those cells that divided earlier and are quiescent bear a CXCR4BrightCD5Dim phenotype (resting fraction, RF). Prior studies suggest that cells of the PF are recent emigrants of solid tissue, whereas cells of the RF did so earlier and are most adept at homing to lymphoid niches. Here we report gene expression and functional analyses of the PF and RF that address the signaling pathway most relevant for the PF and the relative abilities of the PF and RF to migrate using the pathway. When analyzing genes significantly differentially expressed (DEGs) between PF and RF of 26 CLL patients (13 U-CLL and 13 M-CLL), we found 198 genes upregulated in the PF and 88 in the RF. Using Ingenuity Pathway Analysis (IPA), the DEGs of the PF overlapped best with the integrin signaling pathway (Z-score = 2.887). Moreover, of the other identified pathways with Z scores > 2.0, each impacted integrin expression and function (upregulated: Actin Nucleating by ARP-WASP Complex and Regulation of Actin-based Motility by Rho; downregulated: RhoGDI signaling). To validate the transcriptomic data, mRNA and protein levels of various upregulated integrin subunits were confirmed by Q-PCR and cell surface staining by flow cytometry. When comparing the DEGs between the PF and the RF in U-CLL vs M-CLL patients, a core of 144 genes were shared by the two and 358 were unique to U-CLL and 35 to M-CLL. Notably, IPA indicated that the shared genes overlapped with the integrin signaling pathway and the U-CLL unique genes resembled pathways used by integrins to carry out their functions, such as migration. When the 3 sets of DEGs were subjected to Upstream Regulator analysis using IPA, multiple cytokines were identified as potential regulators of the DEGs shared by U-CLL and M-CLL (Z scores > 2.0) clones, whereas for the DEGs unique to the PF of U-CLL, only IL-4 was identified (Z scores = 2.6). To corroborate the above, we cultured CLL B cells for 3 days with the of putative upstream regulators of the shared and U-CLL unique DEGs. For the former, we found that [1] IFNα upregulated CD11c in all CLL, [2] IL-1 and IL-2 upregulated CD11a in all CLL, and [3] IL-1, IL-2, IFNα and IFNγ upregulated CD29 in M-CLL only. When testing a regulatory role for IL-4 in the U-CLL unique genes, we found that IL-4 upregulated CD11a, CD11c, and CD18 in U-CLL (P < 0.05), but not in M-CLL. Finally, we evaluated several functions mediated by integrin molecules. First, we tested the binding of the PF and RF to fibronectin, an important step in cell migration. This indicated that the PF bound better to fibronectin than RF (P < 0.01), and led to extended survival (P < 0.05). Consistent with this, co-culture with anti-CD49d antibody significantly blocked CLL cell adhesion to fibronectin (P < 0.01). Next, we addressed CLL cell migration. In vitro migration studies indicated that PF cells migrated significantly better than the RF with (P < 0.0001) or without (P < 0.001) CXCL12/SDF-1. In line with this, 24-hours after transferring PF and RF into NSG mice, significantly more PF than RF cells were found in the spleen (P < 0.05). Notably, the ability to more rapidly enter solid tissue associated with the rate of disease progression of individual patients (P < 0.001). Since these were unexpected findings, we questioned if sorting PF and RF altered their ability to migrate in vitro. Based on the analysis of 16 patients (U-CLL = 8, M-CLL = 8), simultaneous preincubation of CLL B cells with mAbs to CD5 plus CXCR4 did not change spontaneous nor CXCL12/SDF-1-mediated migration. Collectively, our findings indicate that integrins and the integrin signaling pathway are key elements in the biology of the recently divided fraction of CLL cells. Moreover, those DEGS that are shared by U-CLL and M-CLL cells and those unique to U-CLL are regulated by distinct upstream influences, with IL-4 standing out as a crucial cytokine for the development of the U-CLL PF. Finally, in vitro and in vivo studies suggest a key role for the integrin pathway in CLL migration, and pinpoint, surprisingly, the PF, not the RF, as the better migrater and document that the rate of migration correlates with disease activity.

SPP1 facilitates cell migration and invasion by targeting COL11A1 in lung adenocarcinoma

BackgroundSecreted phosphoprotein 1 (SPP1), an extracellular secreted glycol phosphoprotein, is closely related to tumor biologies, such as proliferation, migration, and invasion. However, the role and biological function of SPP1 in lung adenocarcinoma (LUAD) was still ambiguous.MethodsSPP1 expression in LUAD tissues and its associations with clinical features and prognosis was investigated using meta-analysis, immunohistochemistry (IHC) staining methods, and quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, the potential mechanism related to SPP1 was identified by using the Gene Set Enrichment Analysis (GSEA) method. A series of function assays were conducted to determine the biological role of SPP1 in LUAD cell migration and invasion in vitro and vivo. The co-expressed genes of SPP1 were obtained and verified by western blot assays. The influence of SPP1 on Collagen type XI alpha 1 (COL11A1) expression and epithelial-mesenchymal transition (EMT) markers was analyzed using western blot assays.ResultsThe expression of SPP1 in LUAD tissues and cells was significantly higher than that in normal tissues and cells. And positively associations of SPP1 expression with TNM stage, lymph node metastasis, and invasion depth were observed. Patients with high SPP1 expression had unfavorable survival. The multivariable Cox regression analysis revealed that SPP1 expression was an independent prognostic factor of LUAD patients. Furthermore, downregulation of SPP1 could inhibit cell migration and invasion both in vitro and vivo, reduce the expression of epithelial marker (E-cadherin), and increase the expression of mesenchymal markers (N-cadherin and vimentin). Using bioinformatics and western blot assays, we confirmed that COL11A1 acted as the downstream of SPP1, and SPP1 knockdown could significantly downregulate the COL11A1 expression. Importantly, suppression of cell migration and invasion and the expression changes of EMT markers induced by SPP1 downregulation could be reversed by COL11A1 overexpression.ConclusionsSPP1 facilitates cell migration and invasion by upregulating COL11A1 expression and that acts as a potential biomarker of metastasis and prognosis for LUAD.

Apelin Promotes Prostate Cancer Metastasis by Downregulating TIMP2 via Increases in miR-106a-5p Expression.

Prostate cancer commonly affects the urinary tract of men and metastatic prostate cancer has a very low survival rate. Apelin belongs to the family of adipokines and is associated with cancer development and metastasis. However, the effects of apelin in prostate cancer metastasis is undetermined. Analysis of the database revealed a positive correlation between apelin level with the progression and metastasis of prostate cancer patients. Apelin treatment facilitates cell migration and invasion through inhibiting tissue inhibitor of metalloproteinase 2 (TIMP2) expression. The increasing miR-106a-5p synthesis via c-Src/PI3K/Akt signaling pathway is controlled in apelin-regulated TIMP2 production and cell motility. Importantly, apelin blockade inhibits prostate cancer metastasis in the orthotopic mouse model. Thus, apelin is a promising therapeutic target for curing metastatic prostate cancer.