Remobilization causes site-specific cyst formation in immobilization-induced knee cartilage degeneration in an immobilized rat model.

An understanding of the articular cartilage degenerative process is necessary for the prevention and treatment of joint disease. The present study aimed to examine how long-term immobilization-induced cartilage degeneration is aggravated by remobilization. Sixty 8-week-old male Wistar rats were used in this study. The unilateral knee joint was immobilized using an external fixator for 8 weeks. The rats were killed at 0 and 3 days, and at 1, 2, 4 and 8 weeks after removing the fixator. After the rats were killed, the maximum knee extension angles were measured. Histological sections at the medial mid-condylar region (non-contact, transitional and contact regions of the femur and tibia) were prepared and scored. The cartilage thickness and number of chondrocytes were measured, and CD44 and Col2-3/4c expression levels were assessed immunohistochemically. The histological assessment revealed progressive aggravation of cartilage degeneration in the transitional region, with a decreased number of chondrocytes and CD44-positive chondrocytes as well as poor scoring over time, particularly in the tibia. Cyst formation was confirmed in the transitional region of the tibia at 8 weeks post-remobilization. The cartilage thickness in the transitional region was thicker than that in the contact region, particularly in the tibia. Col2-3/4c expression was observed in the non-contact and transitional regions, and the knee extension angle was recovered. In conclusion, immobilization-induced cartilage degeneration was aggravated by remobilization over time in the transitional region, followed by observations of a decreased number of chondrocytes and morphological disparity between different cartilage regions.

Alteration of cartilage surface collagen fibers differs locally after immobilization of knee joints in rats.

The purpose of this study was to examine the ultrastructural changes of surface cartilage collagen fibers, which differ by region and the length of the experimental period in an immobilization model of rat. Male Wistar rats were randomly divided into histological or macroscopic and ultrastructural assessment groups. The left knees of all the animals were surgically immobilized by external fixation for 1, 2, 4, 8 or 16 weeks (n = 5/time point). Sagittal histological sections of the medial mid-condylar region of the knee were obtained and assessed in four specific regions (contact and peripheral regions of the femur and tibia) and two zones (superficial and deep). To semi-quantify the staining intensity of the collagen fibers in the cartilage, picrosirius red staining was used. The cartilage surface changes of all the assessed regions were investigated by scanning electron microscopy (SEM). From histological and SEM observations, the fibrillation and irregular changes of the cartilage surface were more severe in the peripheral region than in the contact region. Interestingly, at 16 weeks post-immobilization, we observed non-fibrous structures at both the contact and peripheral regions. The collagen fiber staining intensity decreased in the contact region compared with the peripheral region. In conclusion, the alteration of surface collagen fiber ultrastructure and collagen staining intensity differed by the specific cartilage regions after immobilization. These results demonstrate that the progressive degeneration of cartilage is region specific, and depends on the length of the immobilization period.

Effect of microfabricated microgroove-surface devices on the morphology of mesenchymal stem cells.

The surface of a material that is in contact with cells is known to affect cell morphology and function. To develop an appropriate surface for tendon engineering, we used zigzag microgroove surfaces, which are similar to the tenocyte microenvironment. The purpose of this study was to investigate the effect of microgroove surfaces with different ridge angles (RAs), ridge lengths (RLs), ridge widths (RWs), and groove widths (GWs) on human bone marrow-derived mesenchymal stem cell (MSC) shape. Dishes with microgroove surfaces were fabricated using cyclic olefin polymer by injection-compression molding. The other parameters were fixed, and effects of different RAs (180 - 30 °), RLs (5 - 500 µm), RWs (5 - 500 µm), and GWs (5 - 500 µm) were examined. Changes in the zigzag shape of the cell due to different RAs, RLs, RWs, and GWs were observed by optical microscopy and scanning electron microscopy. Cytoskeletal changes were investigated using Phalloidin immunofluorescence staining. As observed by optical microscopy, MSCs changed to a zigzag shape in response to microgroove surfaces with different ridge and groove properties. . As observed by scanning electron microscopy, the cell shape changed at turns in the microgroove surface. Phalloidin immunofluorescence staining indicated that F-actin, not only in cell filopodia but also inside the cell body, changed orientation to conform to the microgrooves. In conclusion, the use of zigzag microgroove surfaces microfabricated by injection-compression molding demonstrated the property of MSCs to alter their shapes to fit the surface.

Evaluation of reference genes for human chondrocytes cultured in several different thermal environments.

PURPOSE: The purpose of this study was to identify reference genes showing stable expression in chondrocytes cultured under several different thermal environments and in different culture systems. MATERIALS AND METHODS: Human articular chondrocytes were cultured by monolayer or pellet culture system at 32 °C, 37 °C, and 41 °C for 3 days. Thereafter, the total RNA was extracted, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed. The qRT-PCR data was analysed using three different algorithms (geNorm, NormFinder, and BestKeeper) to identify reference genes exhibiting stable expression from among the seven candidate reference genes (B2M, ACTB, GAPDH, HSPCB, RPL13a, YWHAZ, and 18S). RESULTS: The candidate reference genes, except for HSPCB and YWHAZ, showed systematic variations in expression. In the monolayer culture, RPL13a was the most stable gene identified using NormFinder and BestKeeper; on using geNorm, ACTB and GAPDH showed the highest expression stability. In the pellet culture, ACTB was the most stable gene identified using NormFinder and BestKeeper, whereas GAPDH and RPL13a were the most stable reference genes as determined using geNorm. In the combined group, B2M and GAPDH were the most stable genes identified using geNorm, whereas RPL13a and YWHAZ were the most stable as per NormFinder and BestKeeper, respectively. The best combination of two candidate reference genes among all the groups determined using NormFinder was RPL13a and YWHAZ. CONCLUSION: The combination of RPL13a and YWHAZ might be suitable as reference genes for human chondrocytes cultured at 32 °C, 37 °C, and 41 °C in monolayer, pellet, or combined cultures.

Optimum temperature for extracellular matrix production by articular chondrocytes.

PURPOSE: The purpose of this study was to investigate the influence of temperature on the ability of the chondrocytes to produce extracellular matrix (ECM). MATERIALS AND METHODS: Articular chondrocytes were isolated from porcine knee joints. The chondrocytes were cultured at three different temperatures: 32 °C, 37 °C, and 41 °C. The ability to produce ECM was assessed by gene expression analysis, histological, and biochemical evaluation in a pellet culture system. RESULTS: Wet weight of the pellets generated after 21 days, was significantly heavier when cultured at lower temperatures. Picrosirius red staining, employed to evaluate collagen production, was higher at lower temperatures, and safranin-O staining, used to evaluate sulphated glycosaminoglycan (GAG), was lower at 32 °C than at 37 °C and 41 °C. Collagen type IIA1 mRNA expression was markedly up-regulated at 41 °C. However, picrosirius red staining was inhibited at 41 °C. GAG and DNA content were measured by 1,9-dimethylmethylene blue (DMMB) assay and PicoGreen® assay, respectively. The GAG content per pellet was significantly low at 41 °C compared to that at 32 °C and 37 °C. The DNA content per pellet was larger at lower temperatures. The GAG content normalised with the DNA content per pellet was significantly lower at 32 °C compared to that at 37 °C and 41 °C. CONCLUSION: Our results suggest that a culture temperature of approximately 41 °C inhibits ECM production by decreasing DNA content and perhaps by collagen misfolding. Taken together, the optimum temperature for ECM production in articular chondrocytes may be between 32 °C and 37 °C.

Contributions of biarticular myogenic components to the limitation of the range of motion after immobilization of rat knee joint.

BACKGROUND: Muscle atrophy caused by immobilization in the shortened position is characterized by a decrease in the size or cross-sectional area (CSA) of myofibers and decreased muscle length. Few studies have addressed the relationship between limitation of the range of motion (ROM) and the changes in CSA specifically in biarticular muscles after atrophy because of immobilization. We aimed to determine the contribution of 2 distinct muscle groups, the biarticular muscles of the post thigh (PT) and those of the post leg (PL), to the limitation of ROM as well as changes in the myofiber CSAs after joint immobilization surgery. METHODS: Male Wistar rats (n = 40) were randomly divided into experimental and control groups. In the experimental group, the left knee was surgically immobilized by external fixation for 1, 2, 4, 8, or 16 weeks (n = 5 each) and sham surgery was performed on the right knee. The rats in the control groups (n = 3 per time point) did not undergo surgery. After the indicated immobilization periods, myotomy of the PT or PL biarticular muscles was performed and the ROM was measured. The hamstrings and gastrocnemius muscles from the animals operated for 1 or 16 weeks were subjected to morphological analysis. RESULTS: In immobilized knees, the relative contribution of the PT biarticular myogenic components to the total restriction reached 80% throughout the first 4 weeks and decreased thereafter. The relative contribution of the PL biarticular myogenic components remained <20% throughout the immobilization period. The ratio of the myofiber CSA of the immobilized to that of the sham-operated knees was significantly lower at 16 weeks after surgery than at 1 week after surgery only in the hamstrings. CONCLUSIONS: The relative contribution of the PT and PL components to myogenic contracture did not significantly change during the experimental period. However, the ratio of hamstrings CSAs to the sham side was larger than the ratio of medial gastrocnemius CSAs to the sham side after complete atrophy because of immobilization.

Immature articular cartilage and subchondral bone covered by menisci are potentially susceptive to mechanical load.

BACKGROUND: The differences of mechanical and histological properties between cartilage covered by menisci and uncovered by menisci may contribute to the osteoarthritis after meniscectomy and these differences are not fully understood. The purpose of this study is to investigate potential differences in the mechanical and histological properties, and in particular the collagen architecture, of the superficial cartilage layer and subchondral bone between regions covered and uncovered by menisci using immature knee. METHODS: Osteochondral plugs were obtained from porcine tibial cartilage that was either covered or uncovered by menisci. Investigation of the thickness, mechanical properties, histology, and water content of the cartilage as well as micro-computed tomography analysis of the subchondral bone was performed to compare these regions. Collagen architecture was also assessed by using scanning electron microscopy. RESULTS: Compared to the cartilage uncovered by menisci, that covered by menisci was thinner and showed a higher deformity to compression loading and higher water content. In the superficial layer of cartilage in the uncovered regions, collagen fibers showed high density, whereas they showed low density in covered regions. Furthermore, subchondral bone architecture varied between the 2 regions, and showed low bone density in covered regions. CONCLUSIONS: Cartilage covered by menisci differed from that uncovered in both its mechanical and histological properties, especially with regards to the density of the superficial collagen layer. These regional differences may be related to local mechanical environment in normal condition and indicate that cartilage covered by menisci is tightly guarded by menisci from extreme mechanical loading. Our results indicate that immature cartilage degeneration and subchondral microfracture may occur easily to extreme direct mechanical loading in covered region after meniscectomy.

Stabilization of EREG via STT3B-mediated N-glycosylation is critical for PDL1 upregulation and immune evasion in head and neck squamous cell carcinoma.

Dysregulated Epiregulin (EREG) can activate epidermal growth factor receptor (EGFR) and promote tumor progression in head and neck squamous cell carcinoma (HNSCC). However, the mechanisms underlying EREG dysregulation remain largely unknown. Here, we showed that dysregulated EREG was highly associated with enhanced PDL1 in HNSCC tissues. Treatment of HNSCC cells with EREG resulted in upregulated PDL1 via the c-myc pathway. Of note, we found that N-glycosylation of EREG was essential for its stability, membrane location, biological function, and upregulation of its downstream target PDL1 in HNSCC. EREG was glycosylated at N47 via STT3B glycosyltransferases, whereas mutations at N47 site abrogated N-glycosylation and destabilized EREG. Consistently, knockdown of STT3B suppressed glycosylated EREG and inhibited PDL1 in HNSCC cells. Moreover, treatment of HNSCC cells with NGI-1, an inhibitor of STT3B, blocked STT3B-mediated glycosylation of EREG, leading to its degradation and suppression of PDL1. Finally, combination of NGI-1 treatment with anti-PDLl therapy synergistically enhanced the efficacy of immunotherapy of HNSCC in vivo. Taken together, STT3B-mediated N-glycosylation is essential for stabilization of EREG, which mediates PDL1 upregulation and immune evasion in HNSCC.

The clinical and genetic features in patients coexisting primary breast and thyroid cancers.

Background: We attempted to examine the clinical characteristics in patients with breast cancer (BC) and thyroid cancer (TC); explore the potential mechanisms of tumorigenesis and progression. Methods: Using the Surveillance, Epidemiology, and End Result Program-9 (SEER-9) database, a retrospective study (1975-2017) was conducted on patients with BC and TC. We identified the common differentially expressed genes involved in BC and TC using the Gene Expression Omnibus database (GEO). Immunohistochemical staining (IHC) was performed to verify the expression of the hit gene in patients with co-occurrence of BC and TC. Using The Cancer Genome Atlas (TCGA) database, the relationship between gene expression and clinicopathological characters was determined. Gene set enrichment analysis (GSEA) was used to identify the pathways enriched in BC and TC. Results: BC patients had a higher predisposition to develop TC (standardized incidence ratio, SIR: 1.29) and vice-versa (SIR: 1.12). Most of these patients were differentiated thyroid carcinoma (DTC) and hormone receptor (HR) - positive BC. The mRNA expression of COMP (Cartilage oligomeric matrix protein) was significantly overexpressed in BC and TC by analyzing the GEO database. The protein expression of COMP was increased in both BC and TC tissues obtained from the same patients validated by IHC. COMP was correlated with worse OS in BC (stage II-IV) and TC; it was the independent factor for prognosis of BC. GSEA indicated that the estrogen response and epithelial-mesenchymal transition (EMT) pathways were significantly enriched in both TC- and BC- COMP overexpressed groups. Conclusion: The co-occurrence risk of BC and TC in the same individual is higher than in the general population. Overexpression of COMP could promote oncogenesis and progression in patients with BC and TC through estrogen signaling and EMT pathways.

Multifunctionalized carbon-fiber-reinforced polyetheretherketone implant for rapid osseointegration under infected environment.

Carbon fiber reinforced polyetheretherketone (CFRPEEK) possesses a similar elastic modulus to that of human cortical bone and is considered as a promising candidate to replace metallic implants. However, the bioinertness and deficiency of antibacterial activities impede its application in orthopedic and dentistry. In this work, titanium plasma immersion ion implantation (Ti-PIII) is applied to modify CFRPEEK, achieving unique multi-hierarchical nanostructures and active sites on the surface. Then, hybrid polydopamine (PDA)@ZnO-EDN1 nanoparticles (NPs) are introduced to construct versatile surfaces with improved osteogenic and angiogenic properties and excellent antibacterial properties. Our study established that the modified CFRPEEK presented favorable stability and cytocompatibility. Compared with bare CFRPEEK, improved osteogenic differentiation of rat mesenchymal stem cells (BMSCs) and vascularization of human umbilical vein endothelial cells (HUVECs) are found on the functionalized surface due to the zinc ions and EDN1 releasing. In vitro bacteriostasis assay confirms that hybrid PDA@ZnO NPs on the functionalized surface provided an effective antibacterial effect. Moreover, the rat infected model corroborates the enhanced antibiosis and osteointegration of the functionalized CFRPEEK. Our findings indicate that the multilevel nanostructured PDA@ZnO-EDN1 coated CFRPEEK with enhanced antibacterial, angiogenic, and osteogenic capacity has great potential as an orthopedic/dental implant material for clinical application.

Effect of inoculating thermophilic bacterial consortia on compost efficiency and quality.

The objective of this study was to investigate the potential effects of thermophilic bacterial consortia on compost efficiency and quality. The application of bacterial consortia resulted in an earlier onset of the thermophilic period (THP), an increased upper temperature limit, and an extended duration of the THP by 3-5 days compared to the control group (CK). Microbial inoculation significantly improved the efficiency of organic matter degradation, as well as the content of water-soluble nitrogen (WSN) and humic acid-carbon (HAC). In the case of consortium Ⅱ inoculation (T2), the activities of cellobiohydrolase, ß-glucosidase, and protease were increased by 81.81 %, 70.13 %, and 74.09 % at the THP respectively compared to CK. During the maturation stage, T2 also exhibited the highest PV, n/PIII, n value (1.33) and HAC content (39.53 mg·g-1), indicating that inoculation of consortium Ⅱ effectively promoted substrate maturity and product quality. Moreover, this inoculation effectively optimized the bacterial communities, particularly the growth of Planococcus, Chelatococcus, and Chelativorans during the composting, which were involved in carbon and nitrogen conversion or HAC synthesis. Carbohydrate and amino acid metabolism, and membrane transport were predominant in the consortia-inoculated samples, with an increased gene abundance, suggesting that inoculation contributed to promoting the biodegradation of lignocellulose and the exchange of favorable factors. In conclusion, this study demonstrates that inoculating thermophilic bacterial consortia has a positive impact on enhancing the resource utilization efficiency of agricultural waste and improving the quality of compost products.

The functions of potential intermediates and fungal communities involved in the humus formation of different materials at the thermophilic phase.

Humus is the final product of humus precursors (HPS) during the humification process, while the associated mechanisms of humus formation have not been clarified. Here, the HPS degradation intermediate and core fungal function for wheat straw and chicken manure compost (SCM), cow dung compost (CD), Chinese traditional medicine residue compost (CTM) and mushroom dreg and chicken manure compost (MCM) was investigated during the thermophilic phase. The results showed SCM and MCM were rich in proteins, lipids, cellulose, low-molecular-weight organic acids, while CD and CTM contained abundant carbohydrates, aliphatic compounds, easily biodegradable aromatic structures, and intermediates from the lignocellulose degradation. In particular, the HPS degrading intermediates including O-alkyl-C and aromatic C compounds were the critical factors, and Scedosporium, Hypsizygus and Remersonia were the core fungal genera for the humification. Furthermore, the potential fungal functional genes involved in carbohydrate and lignin degradation might be the key factors to drive the humification process.

Deciphering the effect of exogenous lignocellulases addition on the composting efficiency and microbial communities.

This study aimed to reveal the potential effects of exogenous lignocellulases addition on the composting efficiency and microbial communities. The lignocellulases addition at the mesophilic phase (MEP) greatly expedited the substrate conversion and the rise of temperature at the initial stage, driving the early arrival of thermophilic phase (THP), caused by the positive effects of Sphingobacterium and Brevundimonas. When being added at the THP, the potential functions and interactions of microbial communities were stimulated, especially for Thermobispora and Mycothermus, which prolonged the duration of the THP and expedited the humic acid formation. Simultaneous addition (MEP and THP) significantly altered the microbial community succession and activated the microbes that contributed to the lignocellulases secretion, exhibiting the highest cellobiohydrolase (36.19 ± 3.25 U· g-1 dw) and xylanase (47.51 ± 3.32 U·g-1 dw) activity at the THP. These findings provide new strategies that can be effectively utilized to improve the efficiency and quality of composting.

Trace Element-Augmented Titanium Implant With Targeted Angiogenesis and Enhanced Osseointegration in Osteoporotic Rats.

Deteriorated bone quality in osteoporosis challenges the success of implants, which are in urgent need for better early osseointegration as well as antibacterial property for long-term stability. As osteoporotic bone formation tangles with angiogenic clues, the relationship between osteogenesis and angiogenesis has been a novel therapy target for osteoporosis. However, few designs of implant coatings take the compromised osteoporotic angiogenic microenvironment into consideration. Here, we investigated the angiogenic effects of bioactive strontium ions of different doses in HUVECs only and in a co-culture system with BMSCs. A proper dose of strontium ions (0.2-1 mM) could enhance the secretion of VEGFA and Ang-1 in HUVECs as well as in the co-culture system with BMSCs, exhibiting potential to create an angiogenic microenvironment in the early stage that would be beneficial to osteogenesis. Based on the dose screening, we fabricated a bioactive titanium surface doped with zinc and different doses of strontium by plasma electrolytic oxidation (PEO), for the establishment of a microenvironment favoring osseointegration for osteoporosis. The dual bioactive elements augmented titanium surfaces induced robust osteogenic differentiation, and enhanced antimicrobial properties. Augmented titanium implant surfaces exhibited improved bone formation and bone-implant contact under comprehensive assessment of an in vivo bone-implant interface. In conclusion, zinc- and strontium-augmented titanium surface benefits the osseointegration in osteoporosis via promoting osteogenic differentiation, exerting antibacterial efficacy, and stimulating early angiogenesis.

Bidirectional differentiation of BMSCs induced by a biomimetic procallus based on a gelatin-reduced graphene oxide reinforced hydrogel for rapid bone regeneration.

Developmental engineering strategy needs the biomimetic composites that can integrate the progenitor cells, biomaterial matrices and bioactive signals to mimic the natural bone healing process for faster healing and reconstruction of segmental bone defects. We prepared the gelatin-reduced graphene oxide (GOG) and constructed the composites that mimicked the procallus by combining the GOG with the photo-crosslinked gelatin hydrogel. The biological effects of the GOG-reinforced composites could induce the bi-differentiation of bone marrow stromal cells (BMSCs) for rapid bone repair. The proper ratio of GOG in the composites regulated the composites' mechanical properties to a suitable range for the adhesion and proliferation of BMSCs. Besides, the GOG-mediated bidirectional differentiation of BMSCs, including osteogenesis and angiogenesis, could be activated through Erk1/2 and AKT pathway. The methyl vanillate (MV) delivered by GOG also contributed to the bioactive signals of the biomimetic procallus through priming the osteogenesis of BMSCs. During the repair of the calvarial defect in vivo, the initial hypoxic condition due to GOG in the composites gradually transformed into a well-vasculature robust situation with the bi-differentiation of BMSCs, which mimicked the process of bone healing resulting in the rapid bone regeneration. As an inorganic constituent, GOG reinforced the organic photo-crosslinked gelatin hydrogel to form a double-phase biomimetic procallus, which provided the porous extracellular matrix microenvironment and bioactive signals for the bi-directional differentiation of BMSCs. These show a promised application of the bio-reduced graphene oxide in biomedicine with a developmental engineering strategy.

Targeting LSD1 suppresses stem cell-like properties and sensitizes head and neck squamous cell carcinoma to PD-1 blockade.

Head and neck squamous cell carcinoma (HNSCC) is a highly aggressive tumor with poor clinical outcomes due to recurrence, metastasis, and treatment resistance. Cancer stem cells (CSCs), a small population among tumor cells, are proposed to be responsible for tumor initiation, progression, metastasis, drug resistance, and recurrence. Here we show that high LSD1 expression was a predictor of poor prognosis for HNSCC patients. We found that high expression of LSD1 is essential for the maintenance of the CSC properties by regulating Bmi-1 expression. Moreover, tumor LSD1 ablation suppresses CSC-like characteristics in vitro and inhibits tumorigenicity in vivo in immune-deficient xenografts. However, this deletion induces the upregulation of PDL1 levels, which compromises antitumor immunity and reduces antitumor efficacy in an immune-competent mouse model. Functionally, the combination of LSD1 inhibitor and anti-PD-1 monoclonal antibody can overcome tumor immune evasion and greatly inhibit tumor growth, which was associated with reduced Ki-67 level and augmented CD8+ T cell infiltration in immunocompetent tumor-bearing mouse models. In summary, these findings provide a novel and promising combined strategy for the treatment of HNSCC using a combination of LSD1 inhibition and PD-1 blockade.

Magnesium Ammonium Phosphate Composite Cell-Laden Hydrogel Promotes Osteogenesis and Angiogenesis In Vitro.

Injectable hydrogels provide an effective strategy for minimally invasive treatment on irregular bony defects in the maxillofacial region. To improve the osteoinduction of gelatin methacrylate (GelMA), we fabricated a three-dimensional (3D) culture system based on the incorporation of magnesium ammonium phosphate hexahydrate (struvite) into GelMA. The optimal concentration of struvite was investigated using the struvite extracts, and 500 µg mL-1 was found to be the most suitable concentration for the osteogenesis of dental pulp stem cells (DPSCs) and angiogenesis of human umbilical vein endothelial cells (HUVECs). We prepared the GelMA composite (MgP) with 500 µg mL-1 struvite. Struvite did not affect the cross-linking of GelMA and released Mg2+ during degradation. The cell delivery system using MgP improved the laden-cell viability, upregulated the expression of osteogenic and angiogenic-differentiation-related genes, and promoted cell migration. Overall, the modifications made to the GelMA in this study improved osteoinduction and demonstrated great potential for application in vascularized bone tissue regeneration.

Long non-coding RNA EGFR-AS1 sponges micorRNA-381 to upregulate ROCK2 in bladder cancer.

The present study aimed to investigate the role of the long non-coding RNA EGFR-AS1 in bladder cancer (BC). In this study gene expression of both BC and non-tumor tissues from BC patients were measured by quantitative PCR. Cell transfections were performed to analyze gene interactions in HT-1197 cells. Transwell assays were performed to analyze cell invasion and migration of HT-1197 cells. It was revealed that epidermal growth factor receptor-antisense RNA 1 (EGFR-AS1) was upregulated in BC and positively associated with rho associated coiled-coil containing protein kinase 2 (ROCK2). Analysis of data collected in follow-ups indicated that EGFR-AS1 expression was significantly associated with poorer overall survival of patients with BC. Moreover, in bladder cancer cells, EGFR-AS1 overexpression mediated the upregulation of ROCK2, while microRNA (miR)-381 mediated the downregulation of ROCK2. However, EGFR-AS1 and ROCK2 failed to affect each other. Bioinformatics analysis indicated that miR-381 binds EGFR-AS1. In addition, EGFR-AS1 and ROCK2 overexpression resulted in the promotion of cell invasiveness and migration of HT-1197 BC cells. Conversely, miR-381 was revealed to partially reverse the effect of EGFR-AS1 overexpression. Therefore, EGFR-AS1 may sponge miR-381 to upregulate ROCK2 in BC, thereby promoting cell invasion and migration.

Targeting EGFR Enriches Stem Cell-Like Properties in Salivary Adenoid Cystic Carcinoma by Activating the Notch1 Pathway.

BACKGROUND: Salivary adenoid cystic carcinoma (SACC), a rare cancer arising in the salivary glands, is characterized by high rates of relapse and distant metastasis. Epidermal growth factor receptor (EGFR) has been implicated in SACC carcinogenesis. However, prospective trials of EGFR-targeting therapies in SACC are limited, and the optimum regimen is unclear. METHODS: The effects of erlotinib on cell proliferation, colony formation, ALDH enzymatic activity and tumorsphere formation were investigated in SACC cells. Expression of the cancer stem cell markers Bmi-1 and Oct4 was evaluated using Western blotting. RESULTS: We found that while it robustly inhibited cell growth, targeting EGFR with erlotinib enriched the ALDH+ cell population and elevated the clonogenicity of SACC cells, suggesting an increase in stem cell-like potential. In addition, we found that suppression of EGFR kinase activity with erlotinib led to the activation of Notch1 signaling, leading to an increase in stem cell-like properties. Moreover, the γ-secretase inhibitor GSI treatment eliminated the erlotinib-induced increase in stem cell-like properties by decreasing Notch activity. CONCLUSION: Our results provide an explanation for the worsened survival observed in some studies of erlotinib therapy in SACC and provide potential therapeutic strategies by combined blockade of the EGFR and Notch1 pathways.

Wntless (Wls): A Prognostic Index for Progression and Patient Survival of Breast Cancer.

BACKGROUND: Wntless (Wls) is an essential protein that is necessary for the secretion of Wnt proteins. While numerous researches have demonstrated that aberrations in Wnt/ß-catenin expression lead to tumorigenesis and progression in many cancer types, the effects of Wls in breast cancer (BC) are less studied. METHODS: The mRNA and protein expression of Wls in BC cell lines were detected by RT-qPCR and Western blot; the protein expression of patient samples was detected by immunohistochemistry (IHC). The associations between Wls expression and clinicopathological factors as well as survival time, including overall survival (OS) and disease-free survival (DFS) were analyzed. Bioinformatics analysis was used to reveal the correlation between Wls genes and associated genes or pathways. RESULTS: Wls was overexpressed in BC cell lines and tissues. The expression level of Wls was significantly correlated with tumor size, estrogen receptor (ER), progesterone receptor (PR), Ki-67, molecular classification, and follow-up status. Spearman correlation analysis showed that Wls protein expression was negatively correlated with ER and PR, which was confirmed by bioinformatics analysis in mRNA level. However, there were positive relationships with MBNG (modified Black's nuclear grade), tumor size, Ki-67, molecular classification, follow-up, and vital status. Univariate and multivariate analysis showed that Wls was an independent prognostic factor for OS and DFS in BC patients. Moreover, Wls was a significant prognostic indicator of OS and DFS in a hormone receptor-positive (HR+) subgroup. GSEA showed that estrogen and androgen response, as well as epithelial-mesenchymal transition pathways, were up-regulated in the Wls high-expression group. CONCLUSION: Overexpression of Wls is a significant marker of worse prognosis in BC and might play a crucial role in the HR+ subgroup.