YKL-40/CHI3L1 facilitates migration and invasion in HER2 overexpressing breast epithelial progenitor cells and generates a niche for capillary-like network formation Abstract Epithelial to mesenchymal transition (EMT) is a developmental event that is hijacked in some diseases such as fibrosis and cancer. In cancer, EMT has been linked to increased invasion and metastasis and is generally associated with a poor prognosis. In this study, we have compared phenotypic and functional differences between two isogenic cell lines with an EMT profile: D492M and D492HER2 that are both derived from D492, a breast epithelial cell line with stem cell properties. D492M is non-tumorigenic while D492HER2 is tumorigenic. Thus, the aim of this study was to analyze the expression profile of these cell lines, identify potential oncogenes, and evaluate their effects on cellular phenotype. We performed transcriptome and secretome analyses of D492M and D492HER2 and verified expression of selected genes at the RNA and protein level. One candidate, YKL-40 (also known as CHI3L1), was selected for further studies due to its differential expression between D492M and D492HER2, being considerably higher in D492HER2. YKL-40 has been linked to chronic inflammation diseases and cancer, yet its function is not fully understood. Knock-down experiments of YKL-40 in D492HER2 resulted in reduced migration and invasion as well as reduced ability to induce angiogenesis in an in vitro assay, plus changes in the EMT-phenotype. In summary, our data suggest that YKL-40 may provide D492HER2 with increased aggressiveness, supporting cancer progression and facilitating angiogenesis.

The effect of extracellular matrix protein binding and culture confluence status on the effect of ROCK on TNF-α- and IL-1-stimulated CXCL8 secretion by colonic epithelial cell Abstract Colonic and intestinal epithelial cells (EC) attach to a basement membrane of laminins, fibronectin, and collagen IV. Wounding of the epithelial layer can change the types of extracellular matrix (ECM) proteins to which the EC attach. In this study, we determined the effect of culturing Caco-2 cells on different ECM proteins on the capacity of EC to produce TNF-α- or IL-1-stimulated CXCL8. The effect of the ECM proteins was such that CXCL8 secretion by cells cultured on collagen I > collagen IV > fibronectin or laminin-111. However, suppression of ROCK activity resulted in a similar 75 to 85% suppression of CXCL8 secretion regardless of the ECM protein type. This suggests that EC can produce different levels of CXCL8 depending on the type of ECM proteins they attach to, but all cases result in a similar requirement for ROCK activity for optimal CXCL8 secretion. Furthermore, when confluent cells were compared to subconfluent cells, the level of TNF- or IL-1-stimulated CXCL8 secretion was greatly elevated with the subconfluent cells and inhibiting ROCK had no effect on CXCL8 secretion levels by the confluent cells. These experiments suggest that CXCL8 responses by confluent cells, which would model for intact, unwounded epithelial, do not involve ROCK activation. However, CXCL8 responses by subconfluent cells, which would model for cells attaching to and moving on ECM proteins in wounded epithelia, require ROCK activation for greatly elevated CXCL8 responses. These results provide a model to examine the important conditions which regulate chemokine production by EC in wounded epithelia.

C1QTNF6 as a novel biomarker regulates cellular behaviors in A549 cells and exacerbates the outcome of lung adenocarcinoma patients Abstract C1q/tumor necrosis factor-related protein 6 (C1QTNF6) is a member of the CTRP family and implicated to cardiovascular diseases, inflammatory reaction, and adipogenesis. However, the function of C1QTNF6 in lung adenocarcinoma remains unknown. We downloaded the expression profiles of C1QTNF6 from TCGA database and Oncomine dataset in order to analyze the relationship between C1QTNF6 expression level and tumorigenesis by bioinformatics methods, such as chi-square test, Kaplan-Meier, and Cox regression analysis. In addition, we performed experiments to investigate the biological function of C1QTNF6 on cancer cells in vitro. The siRNA strategy was conducted to decrease the C1QTNF6 expression and then Cell Counting Kit-8 (CCK8) assay and wound-healing and transwell assays were to determine the proliferation, migration, and invasion. Western blot and qRT-PCR were used to confirm the expression levels. Based on the TCGA database and Oncomine dataset, we found that C1QTNF6 was over expressed in lung adenocarcinoma. The statistical data also showed that the high-regulated C1QTNF6 was related to poor prognosis in patients with lung adenocarcinoma. Moreover, the capabilities of proliferation, migration, and invasion were inhibited owing to the knockdown of C1QTNF6 in lung adenocarcinoma cells. And the phosphorylation of MEK and ERK was blocked by treated si-C1QTNF6 compared with the GAPDH. In conclusion, aberrant C1QTNF6 expression was implicated in terrible prognosis accompanying with the damage of relevant cell potential in lung adenocarcinoma. C1QTNF6 might be an independent predictor of prognosis in lung adenocarcinoma.

S100A12 inhibits fibroblast migration via the receptor for advanced glycation end products and p38 MAPK signaling Abstract The migration of lung fibroblasts plays a pivotal role in wound repair and fibrotic processes in the lung. Although the receptor for advanced glycation end products (RAGE) has been implicated in the pathogenesis of lung diseases, its role in lung fibroblast migration is unclear. The current study examined the effect of three different RAGE ligands, namely, high mobility group box 1 (HMGB1), S100A12, and N-epsilon-(carboxymethyl) lysine (CML), on human fibronectin-directed human fetal lung fibroblast (HFL-1) migration. HMGB1 augmented, whereas S100A12 inhibited, HFL-1 migration in a concentration-dependent manner. CML did not affect HFL-1 migration. The effect of HMGB1 was not through RAGE. However, the effect of S100A12 was mediated by RAGE, but not Toll-like receptor 4. S100A12 did not exert a chemoattractant effect, but inhibited HFL-1 chemotaxis and/or chemokinesis. Moreover, S100A12 mediated HFL-1 migration through p38 mitogen-activated protein kinase (MAPK) but not through nuclear factor-kappa B, protein kinase A, phosphatase and tensin homolog deleted on chromosome 10, or cyclooxygenase. In addition, western blot analysis showed that S100A12 augmented p38 MAPK activity in the presence of human fibronectin. In conclusion, S100A12 inhibits lung fibroblast migration via RAGE-p38 MAPK signaling. This pathway could represent a therapeutic target for pulmonary conditions characterized by abnormal tissue repair and remodeling.

Site-specific integration of rotavirus VP6 gene in rabbit β-casein locus by CRISPR/Cas9 system Abstract Rotavirus (RV) is the leading cause of viral gastroenteritis in neonates and VP6 protein has been discussed as a potential candidate vaccine. CRISPR/Cas9 was the latest generation of gene editing tools that can mediate the site-specific knock-in of exogenous genes, providing strong support for the expression of recombinant proteins. Here, seeking to design a rotavirus vaccine that would be suitable for both mammary-gland-based production and milk-based administration, rabbit β-casein (CSN2) locus was chosen as the target site to integrate the VP6 gene. The efficiency of inducing mutations in different target sites of rabbit CSN2 locus was analyzed and g4 site seems to be the best one to generate mutations (g4 72.76 ± 0.32% vs g1 30.14 ± 1.93%, g2 38.53 ± 0.75%, g3 52.26 ± 1.16%, P < 0.05). We further compared the knock-in efficiency through cytoplasmic injection of two group mixtures (containing 100 ng/μL Cas9 mRNA or Cas9 protein, 20 ng/μL sgRNA4, and 100 ng/μL donor vector) in rabbit zygotes, though the Cas9 mRNA group induced an HDR efficiency as high as 20.0% ± 2.6% than Cas9 protein group (10.3% ± 3.1%), 37.5% of the knock-in events were partial integration in the target site, when Cas9 protein used in the CRISPR/Cas9 system, all of the positive blastocysts showed completely integrated, results showed that the use of Cas9 protein is better than Cas9 mRNA to integrate the correct exogenous gene into the target site. Moreover, the transgenic rabbit that harbored correct integration of VP6 gene was obtained using Cas9 protein group and was used to produce an experimental milk-based rotavirus vaccine. Our research provides a novel strategy to produce rotavirus subunit vaccine and make a foundation for building broader milk-based vaccine protection against other pathogens.

Ultrastructural morphology is distinct among primary progenitor cell isolates from normal, inflamed, and cryopreserved equine hoof tissue and CD105 + K14 + progenitor cells Abstract The equine hoof dermal-epidermal interface requires progenitor cells with distinct characteristics. This study was designed to provide accurate ultrastructural depictions of progenitor cells isolated from inflamed tissue and normal tissue before and after cryopreservation and following selection of cells expressing both keratin (K) 14 (ectodermal) and cluster of differentiation (CD) 105 (mesodermal). Passage 3 cell ultrastructure was assessed following 2D culture and after 3D culture on decellularized hoof tissue scaffolds. Outcome measures included light, transmission electron, and scanning electron microscopy, immunocytochemistry, and CD105+K14+ cell trilineage plasticity. Cells from normal tissue had typical progenitor cell characteristics. Those from inflamed tissue had organelles and morphology consistent with catabolic activities including lysosomes, irregular rough endoplasmic reticulum, and fewer vacuoles and early endosomes than those from normal tissue. Cryopreserved tissue cells appeared apoptotic with an irregular cell membrane covered by cytoplasmic protrusions closely associated with endocytic and exocytic vesicles, chromatin aggregated on the nuclear envelop, abundant, poorly organized rough endoplasmic reticulum, and plentiful lysosomes. Cells that were CD105+K14+ were distinguishable from heterogenous cells by infrequent microvilli on the cell surface, sparse endosomes and vesicles, and desmosomes between cells. Cells expressed ectodermal (K15) and mesodermal (CD105) proteins in 2D and 3D cultures. Inflamed and cryopreserved tissue isolates attached poorly to tissue scaffold while normal tissue cells attached well, but only CD105+K14+ cells produced extracellular matrix after 4 d. The CD105+K14+ cells exhibited osteoblastic, adipocytic, and neurocytic differentiation. Ultrastructural information provided by this study contributes to understanding of equine hoof progenitor cells to predict their potential contributions to tissue maintenance, healing, and damage as well post-implantation behavior.

Genome mutation after the introduction of the gene editing by electroporation of Cas9 protein (GEEP) system into bovine putative zygotes Abstract The present study was designed to investigate the effects of voltage strength on embryonic developmental rate and mutation efficiency in bovine putative zygotes during electroporation with the CRISPR/Cas9 system to target the MSTN gene at different time points after insemination. Results showed that there was no significant interaction between electroporation time and voltage strength on the embryonic cleavage and blastocyst formation rates. However, increasing the voltage strength to 20 V/mm to electroporate the zygotes at 10 h after the start of insemination yielded significantly lower blastocyst formation rates (P < 0.05) than those of the 10-V/mm electroporated zygotes. Mutation efficiency was then assessed in individual blastocysts by DNA sequence analysis of the target sites in the MSTN gene. A positive correlation between mutation rate and voltage strength was observed. The mutation efficiency in mutant blastocysts was significantly higher in the zygotes electroporated with 20 V/mm at 10 h after the start of insemination (P < 0.05) than in the zygotes electroporated at 15 h, irrespective of the voltage strength. We also noted that a certain number of blastocysts from zygotes that were electroporated with more than 15 V/mm at 10 h (4.8–16.7%) and 20 V/mm at 15 h (4.8%) were biallelic mutants. Our results suggest that the voltage strength during electroporation as well as electroporation time certainly have effects on the embryonic developmental rate and mutation efficiency in bovine putative zygotes.

Protective effects of noradrenaline on benzo[a]pyrene-induced oxidative stress responses in brain tumor cell lines Abstract Benzo[a]pyrene (B[a]P) is an ubiquitous environmental pollutant that is generated during combustion of fossil fuels. We examine the effect of noradrenaline (NA) on B[a]P-induced neurotoxicity in brain tumor cell lines like neuroblastoma (Neuro2a) and glioma (C6). We pre-treated tumor cells with NA for 6 h, followed by addition of B[a]P for additional 24 h. Cell viability was measured using trypan blue dye-exclusion assay and comet assay was performed to measure DNA damage. Cell cycle status was analyzed using flow cytometry and oxidative DNA damage (8-oxodG) production was examined by immunostaining. The intracellular Ca2+ concentration was analyzed using Fura-2AM. Our results showed viability of Neuro2a and C6 cells declined (24% and 20%) in B[a]P-treated groups. However, pre-treating with NA increased viability of cells by reducing percentage of cell death in both. Furthermore, B[a]P-induced deregulation of cell cycle (G2/M and S phase cell arrest) was significantly restored by pre-treatment with NA in Neuro2a cells as compared to C6 cells. We further observed increased 8-oxodG production in B[a]P-treated cells; however, NA pre-treatment significantly (p < 0.05) reduced the 8-oxodG production in Neuro2a, while C6 cells were less affected possibly due to better protective machinery. B[a]P-induced intracellular Ca2+ influx was significantly reduced in both the cell lines due to co-treatment of NA possibly by reducing Ca2+ influx. NA protects brain tumor cells against B[a]P-induced neurotoxicity may be by decreasing percentage of G2 cell arrest, oxidative DNA damage, and reducing intracellular Ca2+ influx. These findings suggested that NA may be considered as a natural potential protective agent against B[a]P-induced neurotoxicity. Graphical abstract Graphical abstract showing differential protective mechanism of NA against B[a]P-induced toxicity through antioxidant mechanism maintaining homeostasis for oxidative stress in Neuro2a and C6 cell lines. The schematic graph showed the biological significance of the NA that regulates the induction of metabolic processes of cell cycle after exposure to the environmental pollutants. B[a]P increases the intracellular levels of Ca2+, but also induces damage to cellular molecules including DNA causing cell cycle arrest. The B[a]P-induced DNA damage due to base lesions generated in the genome, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is one of the most abundant because of guanine’s lowest redox potential among DNA bases through intracellular calcium homoeostasis

ULK1 affects cell viability of goat Sertoli cell by modulating both autophagy and apoptosis Abstract Sertoli cells (SCs) are necessary for proper germ cell development and viability. Unc-51 like autophagy activating kinase (ULK1) protein kinase is an important regulator of autophagy activation. This study aims to investigate the role of autophagy promoter ULK1 on cell viability of goat SCs. Our results showed that ULK1 knockdown in goat SCs decreased autophagy activation, which was confirmed by decreased expression of autophagy-related markers including LC3, Beclin1, Atg5, and Atg7 (P < 0.05). Meanwhile, lower ULK1 levels resulted in decreased expressions of goat SC marker genes ABP, AMH, FASL, and GATA4. However, a reverse trend of these parameters occurred when the goat SCs were transfected with ULK1 overexpression construct; higher ULK1 levels in goat SCs also decreased the ratio of Bax/Bcl-2. Moreover, ULK1 overexpression in goat SCs activated the autophagy levels when cells were exposed to an environmental contaminant bisphenol A (BPA). The above results indicated that ULK1 gene might play important roles in goat SC function by regulating cell viability.

Long noncoding RNA FGD5-AS1 promotes colorectal cancer cell proliferation, migration, and invasion through upregulating CDCA7 via sponging miR-302e Abstract The biologic function as well as the mechanism of long noncoding RNAs (lncRNAs) in colorectal cancer (CRC) still remain largely unknown. Long noncoding RNA FGD5 antisense RNA 1 (FGD5-AS1) has been reported to have a promotive effect on other human cancers, but its function in CRC still remains unknown. The expression levels of long noncoding RNA FGD5-AS1, CDCA7 mRNA, and miR-302e were assessed by RT-qPCR. The protein levels of CDCA7 were assessed by Western blot. The function of FGD5-AS1 was detected using cell viability assay, 5-ethynyl-2′-deoxyuridine (EdU) assay, transwell, and caspase-3 activity assay. Additionally, the microRNAs (miRNAs) sponge potential of FGD5-AS1 was examined by RNA immunoprecipitation assay, RNA pull-down assay, and luciferase reporter assay. FGD5-AS1 was increased in colorectal cancer cell lines compared to normal cell lines. Inhibition of FGD5-AS1 suppressed cell proliferation, migration, invasion, and accelerated cell apoptosis in CRC. FGD5-AS1 competitively bound with miR-302e to modulate CDCA7. The inhibiting effects of FGD5-AS1 knockdown on CRC cell proliferation, migration, and invasion, and the promoting effects on CRC cell apoptosis could be revived by miR-302e suppression or CDCA7 upregulation. LncRNA FGD5-AS1 could promote CRC progression through sponging miR-302e and upregulating CDCA7. FGD5-AS1 might serve as a potential therapeutic target for CRC.