Knockdown of has_circ_0010452 Promotes Proliferation and Osteogenic Differentiation via Up-Regulating miR-543 in Human Bone Marrow Mesenchymal Stem Cells

Objective: The aim of this study was to explore the role of has_circ_0010452 in the progression of osteoporosis (OP) targeting miR-543, as well as their functions in regulating proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Methods: The expression levels of circ_0010452 and miR-543 in hBMSCs at different time points of osteogenic differentiation were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). After transfection of circ_0010452 siRNA or miR-543 inhibitor in hBMSCs, the relative expression levels of osteogenic marker proteins, including oat spelt xylan (OSX), osteocalcin (OCN) and collagen I (Col-1), were determined by western blot. Cell proliferation of hBMSCs was valued by Cell Counting Kit 8 (CCK-8) assay. Dual-Luciferase reporter gene assay was performed to verify the relationship between circ_0010452 and miR-543. Subsequently, the regulatory effects of circ_0010452 and miR-543 on osteogenic differentiation and the capability of mineralization were evaluated by alkaline phosphatase (ALP) determination and alizarin red staining, respectively. Results: The expression of circ_0010452 decreased gradually and miR-543 increased in hBMSCs with the prolongation of osteogenic differentiation. circ_0010452 could bind to miR-543, which was negatively regulated by miR-543 in hBMSCs. Moreover, knockdown of circ_0010452 inhibited proliferation and osteogenic differentiation by upregulating miR-543, as well as upregulating expressions of OSX, OCN and Col-1. Furthermore, knockdown of circ_0010452 markedly promoted the capability of mineralization of hBMSCs, which was further reversed by transfection of miR-543 inhibitor. The knockdown of miR-543 partially reversed the inhibitory effect of circ_0010452 on the osteogenesis of hBMSCs. Conclusions: Silence of circ_0010452 promotes the development of OP via binding to miR-543 regulating proliferation and osteogenic differentiation of hBMSCs, thus promoting the progression of osteoporosis.

Hydroxycamptothecin Impedes the Mesenchymal Stem Cells (MSCs)-Triggered Migrative Features of Breast Cancer Cells via Suppressing the Protein Kinase B/Mitogen-Activated Protein Kinase (AKT/MAPK) Activation in Bone Marrowmesenchymal Stem Cells

The current study aimed to dissect the impacts and mechanisms of hydroxycamptothecin on breast cancer. Collect conditioned medium from MSCs cells to apply it into the co-culture system of breast cancer cells, which were pre-treated with hydroxycamptothecin. The cell counting kit was employed to measure the proliferation potential of cells, while the phosphorylation degrees of AKT/MAPKrelated proteins were examined via Western blotting. Then the cellular migration was test by transwell. Finally, the transcriptional and translational levels of IL-6 and RANTES in cells were detected by real-time PCR and enzyme-linked immunosorbent assay. HC could remarkably influence the interplay between MSC and breast malignant cells, reduce the MSC-activated migrative behavior of breast malignant cells and impede the capability of MSC to maintain the migration of cancer cells. RANTES and IL-6 exerted a synergistic induction in the migrative feature of breast cancer cells. HC could retard the migrating activities of breast cancer cells via diminishing the RANTES and IL-6 levels. Hydroxycamptothecin could impede the proliferative and migrative activities of MSC, of which the impediment was accompanied by an inhibitory impact on the secretory production of two growth factors IL-6 and RANTES from MSC, thereby enhancing the migration of breast malignant cells.

Effects of Lycium barbarum polysaccharide on the photoinduced autophagy of retinal pigment epithelium cells

AIM: To investigate the relationship between autophagy and apoptosis in photoinduced injuries in retinal pigment epithelium (RPE) cells and how Lycium barbarum polysaccharide (LBP) contributes to the increased of RPE cells to photoinduced autophagy. METHODS: In vitro cultures of human RPE strains (ARPE-19) were prepared and randomly divided into the blank control, model, low-dose LBP, middle-dose LBP, high-dose LBP, and 3-methyladenine (3MA) groups. The viability of the RPE cells and apoptosis levels in each group were tested through cell counting kit-8 (CCK8) method with a flow cytometer (Annexin V/PI double staining technique). The expression levels of LC3II, LC3I, and P62 proteins were detected with the immunofluorescence method. The expression levels of beclin1, LC3, P62, PI3K, P-mTOR, mTOR, P-Akt, and Akt proteins were tested through Western blot. RESULTS: LBP considerably strengthens cell viability and inhibits the apoptosis of RPE cells after photoinduction. The PI3K/Akt/mTOR signal pathway is activated because of the upregulation of the phosphorylation levels of Akt and mTOR proteins, and thus autophagy is inhibited. CONCLUSION: LBP can inhibit the excessive autophagy in RPE cells by activating the PI3K/Akt/mTOR signaling pathways and thereby protect RPE cells from photoinduced injuries.

Heparanase-1 is downregulated in chemoradiotherapy orbital rhabdomyosarcoma and relates with tumor growth as well as angiogenesis

AIM: To determine the role of heparanase-1 (HPSE-1) in orbital rhabdomyosarcoma (RMS), and to investigate the feasibility of HPSE-1 targeted therapy for RMS. METHODS: Immunohistochemistry was performed to analyze HPSE-1 expression in 51 cases of orbital RMS patients (including 28 cases of embryonal RMS and 23 cases of alveolar RMS), among whom there were 27 treated and 24 untreated with preoperative chemoradiotherapy. In vitro, studies were conducted to examine the effect of HPSE-1 silencing on RMS cell proliferation and tube formation of human umbilical vein endothelial cells (HUVECs). RD cells (an RMS cell line) and HUVECs were infected with HPSE-1 shRNA lentivirus at a multiplicity of infection (MOI) of 10 and 30 separately. Real-time PCR and Western blot were applied to detect the mRNA and protein expression levels of HPSE-1. Cell viability of treated or control RD cells was evaluated by cell counting kit-8 (CCK-8) assay. Matrigel tube formation assay was used to evaluate the effect of HPSE-1 RNAi on the tube formation of HUVECs. RESULTS: Immunohistochemistry showed that the expression rate of HPSE-1 protein was 92.9% in orbital embryonal RMS and 91.3% in orbital alveolar RMS. Tissue from alveolar orbital RMS did not show relatively stronger staining than that from the embryonal orbital RMS. However, despite the types of RMS, comparing the cases treated chemoradiotherapy with those untreated, we have observed that chemoradiotherapy resulted in weaker staining in patients' tissues. The expression levels of HPSE-1 declined significantly in both the mRNA and protein levels in HPSE-1 shRNA transfected RD cells. The CCK-8 assay showed that lentivirus-mediated HPSE-1 silencing resulted in significantly reduced RD cells viability in vitro. Silencing HPSE-1 expression also inhibited VEGF-induced tube formation of HUVECs in Matrigel. CONCLUSION: HPSE-1 silencing may be a promising therapy for the inhibition of orbital RMS progression.

TLR4-Dependent DUOX2 Activation Triggered Oxidative Stress and Promoted HMGB1 Release in Dry Eye

Dry eye disease (DED) is one of the most common ocular surface diseases worldwide. DED has been characterized by excessive accumulation of reactive oxygen species (ROS), following significant corneal epithelial cell death and ocular surface inflammation. However, the key regulatory factor remains unclear. In this study, we tended to explore whether DUOX2 contributed to DED development and the underlying mechanism. Human corneal epithelial (HCE) cells were treated with hyperosmolarity, C57BL/6 mice were injected of subcutaneous scopolamine to imitate DED. Expression of mRNA was investigated by RNA sequencing (RNA-seq) and quantitative real-time PCR (qPCR). Protein changes and distribution of DUOX2, high mobility group box 1 (HMGB1), Toll-like receptor 4 (TLR4), and 4-hydroxynonenal (4-HNE) were evaluated by western blot assays and immunofluorescence. Cell death was assessed by Cell Counting Kit-8 (CCK8), lactate dehydrogenase (LDH) release, and propidium iodide (PI) staining. Cellular ROS levels and mitochondrial membrane potential (MMP) were analyzed by flow cytometry. RNA-seq and western blot assay indicated a significant increase of DUOX2 dependent of TLR4 activation in DED both in vitro and in vivo. Immunofluorescence revealed significant translocation of HMGB1 within corneal epithelial cells under hyperosmolar stress. Interestingly, after ablated DUOX2 expression by siRNA, we found a remarkable decrease of ROS level and recovered MMP in HCE cells. Moreover, knockdown of DUOX2 greatly inhibited HMGB1 release, protected cell viability and abolished inflammatory activation. Taken together, our data here suggest that upregulation of DUOX2 plays a crucial role in ROS production, thereafter, induce HMGB1 release and cell death, which triggers ocular surface inflammation in DED.

Downregulation of the LncRNA MEG3 Promotes Osteogenic Differentiation of BMSCs and Bone Repairing by Activating Wnt/β-Catenin Signaling Pathway

Background: Previous studies have demonstrated that long non-coding RNA maternally expressed gene 3 (MEG3) emerged as a key regulator in development and tumorigenesis. This study aims to investigate the function and mechanism of MEG3 in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and explores the use of MEG3 in skull defects bone repairing. Methods: Endogenous expression of MEG3 during BMSCs osteogenic differentiation was detected by quantitative real-time polymerase chain reaction (qPCR). MEG3 was knockdown in BMSCs by lentiviral transduction. The proliferation, osteogenic-related genes and proteins expression of MEG3 knockdown BMSCs were assessed by Cell Counting Kit-8 (CCK-8) assay, qPCR, alizarin red and alkaline phosphatase staining. Western blot was used to detect β-catenin expression in MEG3 knockdown BMSCs. Dickkopf 1 (DKK1) was used to block wnt/β-catenin pathway. The osteogenic-related genes and proteins expression of MEG3 knockdown BMSCs after wnt/β-catenin inhibition were assessed by qPCR, alizarin red and alkaline phosphatase staining. MEG3 knockdown BMSCs scaffold with PHMG were implanted in a critical-sized skull defects of rat model. Micro-computed tomography(micro-CT), hematoxylin and eosin staining and immunohistochemistry were performed to evaluate the bone repairing. Results: Endogenous expression of MEG3 was increased during osteogenic differentiation of BMSCs. Downregulation of MEG3 could promote osteogenic differentiation of BMSCs in vitro. Notably, a further mechanism study revealed that MEG3 knockdown could activate Wnt/β-catenin signaling pathway in BMSCs. Wnt/β-catenin inhibition would impair MEG3-induced osteogenic differentiation of BMSCs. By using poly (3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHHx)-mesoporous bioactive glass (PHMG) scaffold with MEG3 knockdown BMSCs, we found that downregulation of MEG3 in BMSCs could accelerate bone repairing in a critical-sized skull defects rat model. Conclusions: Our study reveals the important role of MEG3 during osteogenic differentiation and bone regeneration. Thus, MEG3 engineered BMSCs may be effective potential therapeutic targets for skull defects.

LncRNA HOXB-AS3 Promotes Proliferation, Migration, Invasion, and Epithelial-Mesenchymal Transition of Gallbladder Cancer Cells by Activating the MEK/ERK Pathway

OBJECTIVE: Long non-coding RNA HOXB-AS3 has been implicated in tumor progression in a variety of carcinomas. However, its biological role in gallbladder cancer (GBC) is unknown. The biological function and underlying mechanism of the lncRNA HOXB-AS3 for GBC were investigated in this study.MATERIALS AND METHODS: To investigate the function of lncRNA HOXB-AS3 in GBC, the level of lncRNA HOXB-AS3 in GBC cells was detected by quantitative reverse-transcription polymerase chain reaction. The cell viability was tested by cell counting kit-8 assay and colony formation assay. Flow cytometry was performed to investigate cell apoptosis and cell cycle. In addition, cell migration ability was assessed by wound healing assay and cell invasion ability by transwell invasion assay. RESULTS: It was found that HOXB-AS3 was obviously elevated in GBC tissues and cells. However, inhibition of HOXB-AS3 could depress NOZ and GBC-SD cell viability as well as induce cell apoptosis. Also, the gallbladder cancer cell cycle was blocked in the G1 phase. Meanwhile, NOZ and GBC-SD cell migration, invasion, and epithelial-mesenchymal transition were obviously suppressed by knockdown of HOXB-AS3. What is more, we found that HOXB-AS3 might promote gallbladder progress by activating the MEK/ERK pathway.CONCLUSION: The results show that lncRNA HOXB-AS3 serves as a key regulator in GBC progression, which provides a new treatment strategy for GBC.

lncRNA NEAT1-let 7b-P21 axis mediates the proliferation of neural stem cells cultured in vitro promoted by radial extracorporeal shock wave

In previous studies, we found radial extracorporeal shock wave (rESW), can promote the proliferation of neural stem cells（NSCs）. Emerging evidence suggests that lncRNA NEAT1 can regulate NSCs proliferation. Whether lncRNA NEAT1 plays a role in the proliferation of NSC induced by shock waves is unclear. Cell Counting Kit-8（CCK 8） method was used to detect the proliferation of NSCs, and the relative protein and mRNA expression of related genes of Nestin, Cyclin D1 and P21 were detected by Western Blot and Quantitative real-time PCR（RT-qPCR）respectively. Immunofluorescence staining was used to observe the changes in the number of BrdU/nestin positive cells. Overexpression of NEAT1 and let 7b in cells were used to explore whether rESW can rescue the decreased number of NSCs.We found that the optimal dose of R15 transmitter promoting NSCs proliferation is 1.5 bar, 500 pulse, 2 Hz. 1.2-1.5bar showed a dose-dependent effect on the proliferation of NSCs, but it was negatively correlated with the proliferation effect of NSC when it was more than 1.5bar. We revealed that let 7b-P21 axis was involved in regulating the inhibition of NSC proliferation which was activated by NEAT1 in NSCs. In addition, we demonstrated that rESW treatment resulted in the decrease of NEAT1 expression, which was accompanied by the improved biological function including proliferation.Our results confirm that low-intensity rESW（1.5bar，500pulse，2Hz） can promote the proliferation of NSCs through NEAT1-let 7b-P21 axis.

Collaborative workflow between pathologists and deep learning for evaluation of tumor cellularity in lung adenocarcinoma

Owing to the high demand for molecular testing, the reporting of tumor cellularity in cancer samples has become a mandatory task for pathologists. However, the pathological estimation of tumor cellularity is often inaccurate. We developed a collaborative workflow between pathologists and artificial intelligence (AI) models to evaluate tumor cellularity in lung cancer samples and prospectively applied it to routine practice. We also developed a quantitative model that we validated and tested on retrospectively analyzed cases and ran the model prospectively in a collaborative workflow where pathologists could access the AI results and apply adjustments (Adjusted-Score). The Adjusted-Scores were validated by comparing them with the ground truth established by manual annotation of hematoxylin-eosin slides with reference to immunostains with thyroid transcription factor-1 and napsin A. For training, validation, retrospective testing, and prospective application of the model, we used 40, 10, 50, and 151 whole slide images, respectively. The sensitivity and specificity of tumor segmentation were 97% and 87%, and the accuracy of nuclei recognition was 99%. Pathologists altered the initial scores in 87% of the cases after referring to the AI results and found that the scores became more precise after collaborating with AI. For validation of Adjusted-Score, we found the Adjusted-Score was significantly closer to the ground truth than non-AI-aided estimates (p<0.05). Thus, an AI-based model was successfully implemented into the routine practice of pathological investigations. The proposed model for tumor cell counting efficiently supported the pathologists to improve the prediction of tumor cellularity for genetic tests.

Cell-Free Fat Extract Attenuate Osteoarthritis via Chondrocytes Regeneration and Macrophages Immunomodulation

Background: The prevalence of osteoarthritis (OA) is increasing, yet clinically effective and economical treatments are unavailable. We have previously proposed a cell-free fat extract (CEFFE) containing multiple cytokines, which possessed anti-apoptotic, anti-oxidative, and proliferation promotion functions, as a “cell-free” strategy. In this study, we aimed to evaluate the therapeutic effect of CEFFE in vivo and in vitro . Methods: In vivo study, sodium iodoacetate-induced OA rats were treated with CEFFE by intra-articular injections for 8 weeks. Behavioral experiments were performed every two weeks. Histological analyses, anti-type II collagen, and toluidine staining provided structural evaluation. Macrophage infiltration was assessed by anti-CD68 and anti-CD206 staining. In vitro study, the effect of CEFFE on macrophage polarization and secretory factors was evaluated by flow cytometry, immunofluorescence, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The effect of CEFFE on cartilage regeneration was accessed by cell counting kit-8 assay and qRT-PCR. The generation of reactive oxygen species (ROS) and levels of ROS-related enzymes were investigated by qRT-PCR and western blotting. Results: In rat models with sodium iodoacetate (MIA)-induced OA, CEFFE increased claw retraction pressure while decreasing bipedal pressure in a dose-depend manner. Moreover, CEFFE promoted cartilage structure restoration and increased the proportion of CD206 + macrophages in the synovium. In vitro , CEFFE decreased the proportion of CD86 + cells and reduced the expression of pro-inflammatory factors in LPS + IFN-γ induced Raw 264.7. In addition, CEFFE decreased the expression of interleukin-6 and ADAMTs-5 and promoted the expression of SOX-9 in mouse primary chondrocytes. Besides, CEFFE reduced the intracellular levels of reactive oxygen species in both in vitro models through regulating ROS-related enzymes. Conclusions: CEFFE inhibits the progression of OA by promoting cartilage regeneration and limiting low-grade joint inflammation.