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In this study, we aim to discover the importance of long non-coding RNA cyclin-dependent kinase 6 (CDK6)-AS1 in lipopolysaccharide (LPS)-induced HK-2 cells. We treated the HK-2 cells with LPS and knocked down CDK6-AS1 in HK-2 cells and then analyzed the effects of CDK6-AS1 on the viability of cell, cell apoptosis, the expression of cytokines via MTT, flow cytometry, enzyme-linked immunosorbent assay (ELISA), and qPCR. The results showed that silencing CDK6-AS1 alleviated LPS-induced inhibition of HK-2 cell proliferation, release of IL-1β, IL-8, IL-6, and TNF-α, cell apoptosis, and decrease in mitochondrial membrane potential. In addition, decreasing the level of CDK6-AS1 inhibited the reduction of Bcl-2 levels, the expression of Bax, cleaved caspade-9, and cleaved caspase-3, induced by LPS. In conclusion, lowering CDK6-AS1 level alleviates LPS-induced inflammatory damage in HK-2 cells.

Hypoxic preconditioning can protect against cerebral ischemia/reperfusion injury. However, the underlying mechanisms that mediate this effect are not completely clear. In this study, mice were pretreated with continuous, intermittent hypoxic preconditioning; 1 hour later, cerebral ischemia/reperfusion models were generated by middle cerebral artery occlusion and reperfusion. Compared with control mice, mice with cerebral ischemia/reperfusion injury showed increased Bederson neurological function scores, significantly increased cerebral infarction volume, obvious pathological damage to the hippocampus, significantly increased apoptosis; upregulated interleukin-1β, interleukin-6, and interleukin-8 levels in brain tissue; and increased expression levels of NOD-like receptor family pyrin domain containing 3 (NLRP3), NLRP inflammasome-related protein caspase-1, and gasdermin D. However, hypoxic preconditioning significantly inhibited the above phenomena. Taken together, these data suggest that hypoxic preconditioning mitigates cerebral ischemia/reperfusion injury in mice by reducing NLRP3 inflammasome expression. This study was approved by the Medical Ethics Committee of the Fourth Hospital of Baotou, China (approval No. DWLL2019001) in November 2019.

Background Glioma is one of the most aggressive malignant brain tumors that is characterized with inevitably infiltrative growth and poor prognosis. ARST is a novel lncRNA whose expression level is significantly decreased in the patients with glioblastoma multiforme. However, the exact mechanisms of ARST in gliomagenesis are largely unknown. Methods The expressions of ARST in the glioma samples and cell lines were analyzed by qRT-PCR. FISH was utilized to detect the distribution of ARST in the glioma cells. CCK-8, EdU and flow cytometry were used to examine cellular viability, proliferation and apoptosis. Transwell and wound-healing assays were performed to determine the migratory and invasive abilities of the cells. Intracranial tumorigenesis models were established to explore the roles of ARST in vivo. RNA pulldown assay was used to examine proteins that bound to ARST. The activities of key enzymes in the glycolysis and production of lactate acid were measured by colorimetry. In addition, RIP, Co-IP, western blot and immunofluorescence were used to investigate the interaction and regulation between ARST, F-actin, ALDOA and cofilin. Results In this study, we reported that ARST was downregulated in the gliomas. Overexpression of ARST in the glioma cells significantly suppressed various cellular vital abilities such as cell growth, proliferation, migration and invasion. The tumorigenic capacity of these cells in vivo was reduced as well. We further demonstrated that the tumor suppressive effects of ARST could be mediated by a direct binding to a glycolytic enzyme aldolase A (ALDOA), which together with cofilin, keeping the polymerization and depolymerization of actin filaments in an orderly dynamic equilibrium. Upregulation of ARST interrupted the interaction between ALDOA and actin cytoskeleton, which led to a rapid cofilin-dependent loss of F-actin stress fibers. Conclusions Taken together, it is concluded that ARST performs its function via a non-metabolic pathway associated with ALDOA, which otherwise modifies the morphology and invasive properties of the glioma cells. This has added new perspective to its role in tumorigenesis, thus providing potential target for glioma diagnosis, therapy, and prognosis.

Angiogenesis is a complex process, which involves the sprouting of new blood vessels from pre-existing vasculature. Pathological retinal angiogenesis can lead to vision loss and even blindness. Cyclin-dependent kinase 1 (CDK1) is involved in regulation of the cell cycle and is reported to contribute to tumor progression. However, the role of CDK1 in retinal angiogenesis is largely unknown. The purpose of the present study was to investigate the role of CDK1 in retinal angiogenesis. Western blotting, reverse transcription-quantitative PCR (RT-qPCR) analysis, immunofluorescence and immunohistochemistry were used to evaluate the expression of CDK1 in pathological angiogenesis using an oxygen-induced retinopathy (OIR) mouse model. Small interfering (si)RNA sequences against CDK1 were synthesized and incubated with retinal cells. The efficiency of knockdown was confirmed by western blot and RT-qPCR assays. The effect of CDK1 siRNAs on angiogenesis in vitro was investigated using EdU cell proliferation, cell migration and tube formation assays. Subsequently, flow cytometry was used to assess the effects of CDK1 siRNAs on cell cycle distribution and on the induction of apoptosis. The expression levels of cell cycle- and apoptosis-related genes were detected using western blotting. CDK1 was overexpressed in pathological retinal angiogenesis. CDK1 siRNAs inhibited human umbilical vein endothelial cell proliferation, migration and tube formation. The possible mechanisms involved the induction of cell cycle arrest at the G2/M phase and the induction of apoptosis via an increase in the expression levels of p21 and p53. In conclusion, the data indicated that CDK1 was overexpressed in the OIR model and that silencing of CDK1 inhibited angiogenesis in vitro. CDK1 may be a novel therapeutic target for pathological retinal angiogenesis.

Synaptojanin 2 (SYNJ2) regulates cell proliferation and apoptosis via dephosphorylating plasma membrane phosphoinositides. Aim of this study is to first seek the full-scale expression levels and potential emerging roles of SYNJ2 in hepatocellular carcinoma (HCC). We systematically analyzed SYNJ2 mRNA expression and protein levels in HCC tissues based on large-scale data and in-house immunohistochemistry (IHC). The clinical significance and risk factors for SYNJ2-related HCC cases were identified. A nomogram of prognosis was created and its performance was validated by concordance index (C-index) and shown in calibration plots. Based on the identified differentially coexpressed genes (DCGs) of SYNJ2, enriched annotations and potential pathways were predicted, and the protein interacting networks were mapped. Upregulated SYNJ2 in 3,728 HCC and 3,203 non-HCC tissues were verified and in-house IHC showed higher protein levels of SYNJ2 in HCC tissues. Pathologic T stage was identified as a risk factor. Upregulated mRNA levels and mutated SYNJ2 might cause a poorer outcome. The C-index of the nomogram model constructed by SYNJ2 level, age, gender, TNM classification, grade, and stage was evaluated as 0.643 (95%CI = 0.619–0.668) with well-calibrated plots. A total of 2,533 DCGs were extracted and mainly functioned together with SYNJ2 in metabolic pathways. Possible transcriptional axis of CTCF/POLR2A-SYNJ2/INPP5B (transcription factor-target) in metabolic pathways was discovered based on ChIP-seq datasets. In summary, transcriptional regulatory axis CTCF/POLR2A-SYNJ2 might influence SYNJ2 expression levels. Increased SYNJ2 expression level could be utilized for predicting HCC prognosis and potentially accelerates the occurrence and development of HCC via metabolic perturbations pathways., Graphical abstract

A growing number of studies have focused on the involvement of non-coding RNAs (ncRNAs) in the internal ribosome entry site (IRES)-mediated translation in tumorigenesis; however, the underlying mechanisms in colorectal cancer (CRC) remain elusive. In this study, we show that LINC00473 (LNC473) exerted its functions as a tumor suppressor in promoting apoptotic protease-activating factor 1 (APAF1) IRES activity through competitively sponging miR574-5p and miR15b-5p in CRC initiation and pathogenesis. Specifically, LNC473 and its downstream target APAF1 were significantly downregulated accompanied by upregulated miR574-5p and miR15b-5p in CRC cells and tissues, which had a significant prognostic impact on clinical outcomes in our CRC cohort (n = 157). Furthermore, ectopic LNC473 significantly sponged endogenous miR574-5p or miR15b-5p and thereby inhibited cell proliferation and colony formation capacity, and it accelerated cell apoptosis through activating the APAF1-CASP9-CASP3 pathway. Notably, LNC473 overexpression resulted in dramatic promotion of APAF1 IRES activity and translation, whereas rescue experiments confirmed the recovery by the existence of LNC473 and miR574/15b-5p. Mechanistically, LNC473 overexpression promoted IRES binding domain exposure and removed the constraints controlling from miR574-5p and miR15b-5p, and subsequently enhanced IRES-mediated APAF1 expression in vitro and in vivo. Therefore, our results uncover a novel LNC473-miR574/miR15b-APAF1 signaling axis, which provides new targets and crosstalk regulation mechanism for CRC prevention and treatment., Graphical Abstract, The authors show that LNC473 is critically involved in the tumorigenesis, progression, and prognosis in CRC by competitively sponging hsa-miR574/miR15b-5p, yet it regulates APAF1 IRES-mediated translation initiation. The novel signaling axis will provide strong support for finding efficient molecular biomarkers and pathways for CRC diagnosis and treatment.

Hepatocellular carcinoma (HCC) has high morbidity and mortality. MicroRNAs (miRNAs), which could be regulated by cancer-derived exosomes, play critical regulatory roles in the initiation and development of cancer. However, the expressions, effects, and mechanisms of abundant miRNAs regulated by HCC cancer-derived exosomes in HCC remain largely unclear. Exosomes of HepG2 cells under heat shock, TGF-β1, doxorubicin, acid and hypoxia/reoxygenation (H/R) conditions, and exosomes were successfully identified by transmission electron microscopy and Western blot analysis. The identified exosomes were then applied to evaluate the miRNA expression profiles by RNA sequencing. Mechanically, we discovered that doxorubicin was upregulated, TGF-β1 downregulated the expressions of Vps4A, Rab27A, Alix, and Hrs in HepG2 cells and exosomes, and Vps4A and Rab27A, as target genes for miR-4454, could also be downregulated by miR-4454. Functionally, we revealed that miR-4454 inhibitor and miR-4454 inhibitor-mediated exosomes could markedly suppress proliferation, migration, invasion, and vascularization and accelerate cycle arrest, apoptosis, and ROS of HepG2 cells. This study provided many potential HCC cancer-derived exosome-mediated miRNAs in HCC under 5 different stimulus conditions. Meanwhile, we certified that miR-4454 in exosomes could provide a novel and effective mechanism for HCC function.

BackgroundThe establishment of porcine pluripotent stem cells (piPSCs) is still a critical topic and challenging issue. However, all piPSCs are extremely sensitive to changes of the culture conditions.In addition, the side effect of inhibitors in culture medium confine the pluripotency and practicability. This study aimed to investigate the roles of AXIN in piPSCs and further explore the mechanism. Here, porcine AXIN1 gene and AXIN2 were knockdown, cloned, and overexpressed in piPSCs. Digital RNA-seq was performed to explore the mechanism of cell proliferation and anti-apoptosis. ResultsHere, we found (1): overexpression of the porcine AXIN2 gene significantly reduce the survivability of piPSCs, meanwhile wreck the pluripotency of piPSCs; (2): The Digital RNA-seq analysis reveals that AXIN2, as a negative effector of the WNT signaling pathway, whom after knockdown enhances the expression of genes involved in cell cycle such as CCND1, and reduced the expression of genes related to cell differentiation, cell death, and cell apoptosis. ConclusionAXIN2 could reduce the pluripotency and survival of piPSCs and also provided a potential to simplify the cultrue medium.

Long non-coding RNAs contribute to the development of human cancers. We compared the long non-coding RNA levels in gastric cancer (GC) and para-cancerous tissues in the Gene Expression Omnibus, and found that small nucleolar RNA host gene 12 (SNHG12) was upregulated in GC tissues. Fluorescence in situ hybridization confirmed that SNHG12 is overexpressed in GC tissues. We then used data from The Cancer Genome Atlas to assess the association of SNHG12 expression with the clinicopathological characteristics and prognosis of GC patients and found that higher SNHG12 expression was associated with a greater tumor invasion depth and poorer survival. In vitro, silencing SNHG12 suppressed GC cell proliferation, migration and invasion, but induced apoptosis and cell cycle arrest. Overexpressing SNHG12 had the opposite effects. In xenografted mice, knocking down SNHG12 reduced GC tumor growth. Taken together, cancer pathway microarray and bioinformatics analyses, RNA pulldown assays, Western blotting and immunohistochemistry revealed that SNHG12 induces GC tumorigenesis by activating the phosphatidylinositol 3-kinase/AKT pathway. SNHG12 may thus be a useful marker for predicting poor survival in GC patients.

Background: Hypoxia is associated with abnormal cell apoptosis in trophoblast cells, which causes fetal growth restriction and related placental pathologies. Few effective methods for the prevention and treatment of placenta-related diseases exist. Natural products and functional foods have always been a rich source of potential anti-apoptotic drugs. Nobiletin (NOB), a hexamethoxyflavonoid derived from the citrus pomace, shows an anti-apoptotic activity, which is a non-toxic constituent of dietary phytochemicals approved by the Food and Drug Administration. However, their effects on hypoxia-induced human trophoblast cells have not been fully studied. Objective: The aim of this study was to investigate the protective effects of NOB on hypoxia-induced apoptosis of human trophoblast JEG-3 and BeWo cells, and their underlying mechanisms. Design: First, the protective effect of NOB on hypoxia-induced apoptosis of JEG-3 and BeWo cells was studied. Cell viability and membrane integrity were determined by CCK-8 assay and lactate dehydrogenase activity, respectively. Real Time Quantitative PCR (RT-qPCR) and Western blot analysis were used to detect the mRNA and protein levels of HIF1α. Propidium iodide (PI)-labeled flow cytometry was used to detect cell cycle distribution. Cell apoptosis was detected by flow cytometry with Annexin V-FITC and PI double staining, and the expression of apoptosis marker protein cl-PARP was detected by Western blot analysis. Then, the molecular mechanism of NOB against apoptosis was investigated. Computer molecular docking and dynamics were used to simulate the interaction between NOB and p53 protein, and this interaction was verified in vitro by Ultraviolet and visible spectrum (UV-visible spectroscopy), fluorescence spectroscopy and circular dichroism. Furthermore, the changes in the expression of p53 signaling pathway genes and proteins were detected by RT-qPCR and Western blot analysis, respectively. Results: Hypoxia treatment resulted in a decreased cell viability and cell membrane integrity in JEG-3 and BeWo cell lines, and an increased expression of HIF1α, cell cycle arrest in the G1 phase, and massive cell apoptosis, which were alleviated after NOB treatment. Molecular docking and dynamics simulations found that NOB spontaneously bonded to human p53 protein, leading to the change of protein conformation. The intermolecular interaction between NOB and human p53 protein was further confirmed by UV-visible spectroscopy, fluorescence spectroscopy and circular dichroism. After the treatment of 100 μM NOB, a down-regulation of mRNA and protein levels of p53 and p21 and an up-regulation of BCL2/BAX mRNA and protein ratio were observed in JEG-3 cells; however, there was also a down-regulation of mRNA and protein levels observed for p53 and p21 in BeWo cells after the treatment of NOB. The BCL2/BAX ratio of BeWo cells did not change after the treatment of 100 μM NOB. Conclusion: NOB attenuated hypoxia-induced apoptosis in JEG-3 and BeWo cell lines and might be a potential functional ingredient to prevent pregnancy-related diseases caused by hypoxia-induced apoptosis. These findings would also suggest the exploration and utilization of citrus resources, and the development of citrus industry.

Cervical cancer is one of the leading causes of cancer-associated mortality in gynecological diseases and ranks third among female cancers worldwide. Although early detection and vaccination have reduced incidence rates, cancer recurrence and metastasis lead to high mortality due to the lack of effective medicines. The present study aimed to identify novel drug candidates to treat cervical cancer. In the present study, lanatoside C, an FDA-approved cardiac glycoside used for the treatment of heart failure, was demonstrated to have anti-proliferative and cytotoxic effects on cervical cancer cells, with abrogation of cell migration in a dose-dependent manner. Lanatoside C also triggered cell apoptosis by enhancing reactive oxygen species production and reducing the mitochondrial membrane potential, which induced cell cycle arrest at the S and G(2)/M phases. Furthermore, lanatoside C inhibited the phosphorylation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 6 (STAT6), while inducing the expression of suppressor of cytokine signaling 2, a negative regulator of JAK2-STAT6 signaling. Taken together, the results of the present study suggest that lanatoside C suppresses cell proliferation and induces cell apoptosis by inhibiting JAK2-STAT6 signaling, indicating that lanatoside C is a promising agent for the treatment of cervical cancer.

The present study aimed to investigate the influence of circular RNA nuclear receptor‑interacting protein 1 (circNRIP1) on the chemotherapeutic effect of 5‑fluorouracil (5‑FU) in colorectal cancer (CRC) and reveal its potential molecular mechanisms. The effects of circNRIP1 on cell proliferation, migration and invasion, and apoptosis were evaluated using Cell Counting Kit‑8, Transwell and flow cytometric assays, respectively. A dual‑luciferase reporter assay was performed to verify the potential interaction between circNRIP1 and microRNA (miR)‑532‑3p. The results of the present study indicated that circNRIP1 was upregulated in CRC and its increased expression was associated with CRC progression. Furthermore, overexpression of circNRIP1 promoted CRC cell proliferation, invasion and migration, while it inhibited apoptosis. Knockdown of circNRIP1 significantly enhanced the 5‑FU‑induced inhibition of the viability of HCT116 and SW480 cells. Bioinformatics analysis predicted that miR‑532‑3p was a direct target of circNRIP1, which was further confirmed by a dual‑luciferase reporter assay. miR‑532‑3p silencing reversed the effects of circNRIP1 knockdown on the sensitivity of 5‑FU in the chemotherapy of CRC. The results suggested that circNRIP1 and miR‑532‑3p may be utilized to improve the diagnosis of CRC and serve as diagnostic markers. In conclusion, overexpression of circNRIP1 promoted the progression of CRC, while circNRIP1 silencing sensitized CRC cells to 5‑FU via sponging miR‑532‑3p.

This study aimed to investigate the protective effects of dietary algae-derived polysaccharides (ADP) from Enteromorpha prolifera against heat stress (HS)-induced bursa of Fabricius injure in broilers, and to elucidate the molecular mechanisms underlying the protective effect. A total of 144 8-week-old male yellow-feathered broilers were randomly allocated into 3 treatments of 6 replicates each (8 broilers per replicate): thermoneutral zone group (TN, fed basal diet); heat stress group (HS, fed basal diet); heat stress + ADP group (HSA, basal diet supplemented with 1,000 mg/kg ADP). Broilers in TN group were raised at 23.6 ± 1.8°C during the whole study. Broilers in HS and HSA groups were exposed to 33.2 ± 1.5°C for 10 h/day. The experimental period lasted for four weeks. The results showed that HS and dietary ADP had no significant effects on bursa of Fabricius index (P > 0.05). HS exposure increased the apoptosis rate of bursa of Fabricius (P < 0.05), and the apoptosis rate was reduced by dietary ADP (P < 0.05). Besides, broilers in HS and HSA groups had a lower glutathione-S transferase (GST) activity and total anti-oxidation capacity (T-AOC), whereas had a higher malondialdehyde (MDA) levels of bursa of Fabricius than those in TN group (P < 0.05). HS exposure elevated the concentration of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-4, and IL-6, while decreased the concentration of interferon-γ (INF-γ) and IL-2 (P < 0.05), and dietary inclusion of ADP reduced the IL-1β level and increased the IL-2 level of bursa of Fabricius (P < 0.05). Compared with TN group, broilers in HS and HSA groups had lower relative mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and GSTT1 in bursa of Fabricius (P < 0.05). Additionally, HS exposure down-regulated the mRNA expression of inhibitor kappa B alpha (IκBα), IFN-γ, and IL-2, while up-regulated the mRNA expression of nuclear factor-kappa B (NF-κB) p65, TNF-α, IL-1β, and IL-6 in bursa of Fabricius (P < 0.05). However, dietary inclusion of ADP up-regulated the mRNA expression of IκBα and down-regulated the mRNA expression of NF-κB p65, TNF-α, and IL-6 in bursa of Fabricius (P < 0.05). Furthermore, HS exposure increased the relative protein expression levels of total and nuclear NF-κB p65 (P < 0.05), but dietary ADP supplementation reduced the relative protein expression levels of total and nuclear NF-κB p65 in bursa of Fabricius (P < 0.05). Collectively, dietary ADP ameliorated the impairment of histology, cell apoptosis and immune balance in bursa of Fabricius of heat stressed broilers, which is involved in modulation of NF-κB signaling pathway.

Excessive lung inflammation caused by endotoxins, including lipopolysaccharide (LPS), mediates the detrimental effects of acute lung injury (ALI), as evidenced by severe alveolar epithelial cell injury. CD40, a member of the tumor necrosis factor receptor superfamily, serves as a central activator in triggering and transducing a series of severe inflammatory events during the pathological processes of ALI. Ginkgolide C (GC) is an efficient and specific inhibitor of CD40. Therefore, the present study aimed to investigate whether GC alleviated LPS‑induced ALI, as well as the potential underlying mechanisms. LPS‑injured wild‑type and CD40 gene conditional knockout mice, and primary cultured alveolar epithelial cells isolated from these mice served as in vivo and in vitro ALI models, respectively. In the present study, histopathological assessment, polymorphonuclear neutrophil (PMN) infiltration, lung injury score, myeloperoxidase activity, wet‑to‑dry (W/D) weight ratio and hydroxyproline (Hyp) activity were assessed to evaluate lung injury. In addition, immunohistochemistry was performed to evaluate intracellular adhesion molecule‑1, vascular cell adhesion molecule‑1 and inducible nitric oxide synthase expression levels, and TNF‑α, IL‑1β, IL‑6 ELISAs and western blotting were conducted to elucidate the signaling pathway. The results demonstrated that GC alleviated LPS‑induced lung injury, as evidenced by improvements in ultrastructural characteristics and histopathological alterations of lung tissue, inhibited PMN infiltration, as well as reduced lung injury score, W/D weight ratio and hydroxyproline content. In LPS‑injured alveolar epithelial cells, GC significantly reduced IκBα phosphorylation, IKKβ activity and NF‑κB p65 subunit translocation via downregulating CD40, leading to a significant decrease in downstream inflammatory cytokine levels and protein expression levels. In conclusion, the results of the present study demonstrated that GC displayed a protective effect against LPS‑induced ALI via inhibition of the CD40/NF‑κB signaling pathway; therefore, the present study suggested that the CD40/NF‑κB signaling pathway might serve as a potential therapeutic target for ALI.

Purpose: Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is a common disease after long-term or high-dose glucocorticoid use. The pathogenesis of GIONFH is still controversial, and abnormal bone metabolism caused by glucocorticoids may be one of the important factors. Exosomes, owing to their positive effect on bone repair, show promising therapeutic effects on bone-related diseases. In this study, we hypothesised that exosomes reduce osteocyte apoptosis in rat GIONFH via the miR-21-PTEN-AKT signalling pathway. Methods: To evaluate the effects of exosomes in GIONFH, a dexamethasone-treated or exosome-treated in vitro cell model and a methylprednisolone-treated in vivo rat model were set up. In vitro, a CCK-8 assay and 5-ethynyl-2'-deoxyuridine staining were performed to evaluate the proliferation of osteocytes. Further, a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, annexin V-fluorescein isothiocyanate-propidium iodide staining, and western blotting were conducted to evaluate the apoptosis of osteocytes. In vivo, we used micro-computed tomography and histological and immunohistochemical analyses to assess the effects of exosomes. Moreover, the mechanism of exosome action on osteocyte apoptosis through the miR-21-PTEN-AKT pathway was investigated by high-throughput RNA sequencing, fluorescence in situ hybridisation, luciferase reporter assays, and western blotting. Results: High-throughput RNA sequencing results showed that the AKT signalling pathway was up-regulated in the exosome group. Quantitative PCR and western blotting confirmed that the relative expression of genes in the AKT pathway was up-regulated. Western blotting revealed that AKT activated by exosomes inhibited osteocyte apoptosis. RNA fluorescence in situ hybridisation and luciferase reporter assays were performed to confirm the interaction between miR-21 and PTEN. According to the experiment in vivo, exosomes prevented GIONFH in a rat model as evidenced by micro-computed tomography scanning and histological and immunohistochemical analyses. Conclusions: Exosomes are effective at inhibiting osteocyte apoptosis (in MLO-Y4 cells) and at preventing rat GIONFH. These beneficial effects are mediated by the miR-21-PTEN-AKT signalling pathway.

BACKGROUND Sorafenib, a multiple-target-point kinase inhibitor, has been used as a standard treatment for advanced liver cancer and has shown therapeutic benefits. However, resistance often occurs, prompting the need for identification of synergizing agents. Celastrol is a major active ingredient of Tripterygium wilfordii, which can increase the antitumor effect of traditional antitumor drugs. This work focused on the sensitization of liver cancers in use of celastrol combined with sorafenib. MATERIAL AND METHODS The IC50 values of sorafenib and celastrol on cancer cells were determined through MTT assays. The effects of sorafenib on AKT signaling and VEGF levels in sorafenib-treated cancer cells were analyzed by Western blotting and ELISA, respectively. After combined treatment with celastrol and sorafenib, the survival rate of tumor cells was determined by MTT and clonogenic assays, and the apoptosis rate was also determined by flow cytometry. In addition, the in vivo antitumor activity of celastrol combined with sorafenib was evaluated in Hepa1-6 tumor-bearing mice. RESULTS Sorafenib treatment induced the compensatory activation of the AKT pathway and autocrine VEGF in hepatoma cells, which could be reversed by celastrol. Furthermore, celastrol enhanced the growth inhibition and apoptosis induction of cancer cells by sorafenib both in vitro and in vivo and reduced the dosage of sorafenib needed. CONCLUSIONS Celastrol enhances the antitumor activity of sorafenib in HCC tumor cells by suppressing the AKT pathway and VEGF autocrine system.

BACKGROUND Pancreatic cancer has high incidence and low survival rates around the globe, mainly due to late diagnosis and unavailability of efficient chemotherapeutic agents. In the present study, the anticancer potential of glychionide-A was examined against PANC-1 pancreatic cancer cells. MATERIAL AND METHODS CellTiter-Glo Luminescent Cell Viability Assay Kits were used for assessment of cell viability. Electron microscopy and DAPI staining were used for the detection of apoptosis and autophagy, respectively. Annexin V/PI staining was used for determination of apoptotic cell percentage. Cell cycle distribution and ROS and MMP levels were determined by flow cytometry. Protein expression was examined by Western blot analysis. RESULTS The results revealed that glychionide-A significantly inhibited the proliferation of the PANC-1 pancreatic cancer cells (IC₅₀, 14 µM). However, minimal toxicity was observed against the normal hTRET-HPNE pancreatic cells (IC₅₀ 100 µM). The anticancer activity of glychionide-A against the PANC-1 cells was found to be due to induction of autophagy and apoptosis. Glychionide-A prompted apoptosis and autophagy and was also associated with alteration in apoptosis- (Bax, Caspase 9 and Bcl-2) and autophagy- (LC3I, II, Beclin 1 and p62) related protein expression. Glychionide-A also caused the arrest of PANC-1 cells in the G2/M phase of the cell cycle. The percentage of PANC-1 cells in G₂ phase increased from 19.5% to 49.4% upon treatment with glychionide-A. Finally, glychionide-A caused an increase in the level of ROS and decline in MMP levels of the PANC-1 pancreatic cancer cells. CONCLUSIONS In brief, these results reveal that glychionide-A significantly inhibits the growth of pancreatic cancer cells via inducing apoptosis and autophagy, and could prove valuable in the chemotherapeutic treatment of pancreatic cancer. Therefore, further research is needed, especially more advanced in vivo experiments.

Objective: Glioma is one of the leading causes of death worldwide with high incidence, recurrence, and mortality. Interleukin-10 (IL-10) is a cytokine with dual function in many types of tumors. Although IL-10 is overexpressed and promotes tumor progression in human primary brain tumor, the mechanisms are largely unknown. Materials and Methods: Glioma cells were treated with different dosages of IL-10. The cell growth was detected by CCK-8, and the invasion was measured by Transwell. The relative expression of messenger RNAs was detected by quantitative real-time polymerase chain reaction. Results: We found that IL-10 treatment significantly enhanced glioma cell growth and invasion. Moreover, KPNA2 was significantly upregulated after treatment with IL-10. By performing knockdown experiments, we found that the glioma cell growth and invasion were significantly declined. Conclusions: The results indicated that knockdown of KPNA2 significantly inhibited the growth and invasion of glioma cells. Moreover, IL-10 promotes glioma progression via upregulation of KPNA2. This study will be of important significance and provides a potential target for treatment of patients with glioma.

BACKGROUND Breast cancer (BC) is the leading cause of death in women worldwide. Golgi membrane protein 1 (GOLM1) has been identified as novel regulator in carcinogenesis, but its function in BC is unclear. MATERIAL AND METHODS The expression of GOLM1 in BC tissues and cell lines was detected by using qRT-PCR assay. CCK-8 and colony-formation assays were used to evaluate BC cell growth in vivo. Wound-healing and Transwell assays were used to detect cell migration and invasion. To investigate GOLM1 functions in vivo, we established a xenograft mice model and a lung metastasis model. The level of epithelial-to-mesenchymal transition (EMT)-related markers was analyzed by immunofluorescent staining. RESULTS GOLM1 was overexpressed in BC cell lines and tissues. Overexpression of GOLM1 induced EMT and promoted proliferation, migration, and invasion of BC cells. Furthermore, overexpressing of GOLM1 markedly promoted the tumorigenicity and metastasis of BC cells in vivo, whereas knock-down of GOLM1 caused the opposite outcomes. Furthermore, we proved that GOLM1 promoted BC cell aggressiveness by regulating matrix metalloproteinase-13 (MMP13). CONCLUSIONS Our results prove that GOLM1 facilitates the growth and metastasis of breast cancer cells.

BACKGROUND Recently, long noncoding RNAs (lncRNAs) have received wide attention in the area of tumor progression. Dysregulation of lncRNAs has been shown to participated in colon cancer, a known malignant tumor. This study aimed to identify the way lncRNA PVT1 affects the progression of colon cancer. MATERIAL AND METHODS Both human colon cancer tissues and 30 paired adjacent normal tissue samples, as well as the colon cancer cells, were collected. Then quantitative real-time (qRT-PCR) was performed to detect the expression of lncRNA PVT1 and miR-26b. Furthermore, the role of PVT1 was determined by function assays such as cell proliferation assay, invasion assay, and wound healing assay. The mechanism was studied using western blot assay and luciferase assay. RESULTS We demonstrate that the expression of PVT1 was significantly higher in tumor tissue compared with the adjacent normal tissue with a lower expression of miR-26b. Moreover, PVT1 promoted tumor growth, migration, and invasion in vitro. In addition, further experiments revealed that miR-26b was a direct target of PVT1 and could inhibit cell migration, invasion, and proliferation in colon cancer. CONCLUSIONS Our results suggest that PVT1 could promote metastasis and proliferation of colon cancer via endogenous sponging and inhibiting the expression of miR-26b, which may highlight the significance of lncRNA PVT1 in colon cancer tumorigenesis.

Chemo-PTT, which combines chemotherapy with photothermal therapy, offers a viable approach for the complete tumor eradication but would likely fail in drug-resistant situations if conventional chemotherapeutic agents are used. Here we show that a type of copper (Cu)-palladium (Pd) alloy tetrapod nanoparticles (TNP-1) presents an ideal solution to the chemo-PTT challenges. TNP-1 exhibit superior near-infrared photothermal conversion efficiency, thanks to their special sharp-tip structure, and induce pro-survival autophagy in a shape- and composition-dependent manner. Inhibition of autophagy with 3-methyl adenine or chloroquine has a remarkable synergistic effect on TNP-1-mediated PTT in triple-negative (4T1), drug-resistant (MCF7/MDR) and patient-derived breast cancer models, achieving a level of efficacy unattainable with TNP-2, the identically-shaped CuPd nanoparticles that have a higher photothermal conversion efficiency but no autophagy-inducing activity. Our results provide a proof-of-concept for a chemo-PTT strategy, which utilizes autophagy inhibitors instead of traditional chemotherapeutic agents and is particularly useful for eradicating drug-resistant cancer., “Conventional chemotherapy-photothermal therapy combination has limited efficacy in drug resistant cancers. Here they develop Copper-palladium tetrapod nanoparticles to overcome these challenges and show them to work in synergy with autophagy inhibitors to treat drug resistant cancers”

Background/Aims: Sonodynamic therapy (SDT), based on the synergistic effect of low-intensity ultrasound and sonosensitizer, is a potential approach for non-invasive treatment of cancers. In SDT, mitochondria played a crucial role in cell fate determination. However, mitochondrial activities and their response to SDT remain elusive. The purpose of this study was to examine the response of mitochondria to SDT in tumor cells. Methods: A human breast adenocarcinoma cell line - MCF-7 cells were subjected to 5-aminolevulinic acid (ALA)-SDT, with an average ultrasonic intensity of 0.25W/cm2. Mitochondrial dynamics and redox balance were examined by confocal immunofluorescence microscopy and western blot. The occurrence of mitophagy was determined by confocal immunofluorescence microscopy. Results: Our results showed that ALA-SDT could induce mitochondrial dysfunction through mitochondrial depolarization and fragmentation and lead to mitophagy. The Parkin-dependent signaling pathway was involved and promoted resistance to ALA-SDT induced cell death. Finally, excessive production of ROS was found to be necessary for the initiation of mitophagy. Conclusion: Taken together, we conclude that ROS produced by 5-ALA-SDT could initiate PINK1/Parkin-mediated mitophagy which may exert a protective effect against 5-ALA-SDT-induced cell death in MCF-7 cells.

Objectives. Most patients with systemic lupus erythematosus (SLE) develop lupus nephritis (LN) with severe kidney manifestations. Renal fibrosis can be primarily attributed to overproliferation of mesangial cells (MCs), which are subject to drug treatment. Nevertheless, the detailed mechanisms remain elusive. We sought to identify the effect of cyclophosphamide (CTX), a drug commonly used for LN treatment, on MC proliferation and explore its underlying mechanisms. Material/Methods. Cell proliferation and fibrosis in mouse kidney tissues were determined by histopathology staining techniques. Flow cytometry was used for cell cycle analysis. Cell cycle regulators were examined in vitro following treatment of immortalized human MCs with platelet-derived growth factor subunit B (PDGF-B). Quantitative real-time PCR and western blot analyses were used to measure the mRNA and protein levels of candidate cell cycle regulators, respectively. Results. CTX inhibited cell overproliferation induced by platelet-derived growth factor subunit B in vitro and in vivo. CTX (40 mg/l) was sufficient to induce G0/G1 phase cell cycle arrest. CTX treatment downregulated many critical cell cycle regulators including cyclins and cyclin-dependent kinases but upregulated cyclin-dependent kinase inhibitors. Additionally, CTX-treated samples showed significantly reduced fibrosis, as indicated by lower expression of interleukin-1β and α-smooth muscle actin. Conclusion. CTX inhibits proliferation of MCs by modulating cell cycle regulator and therefore arresting them at G1 phase. CTX treatment significantly alleviates the severity of renal fibrosis. These findings provide novel insights into the mechanisms by which CTX affects LN.

The mechanism of action of synovitis, as the vital pathological process of rheumatoid arthritis and osteoarthritis, remains to be elucidated. The effects and the mechanism of icariin (ICA), which is a promising therapeutic agent in synovitis, was investigated in the present study. In addition, ferroptosis, a vital cell process involved in several diseases, was also studied in synovitis for the first time. Lipopolysaccharide (LPS)-induced synoviocytes served as a synovitis cell model. The cells were divided into control, LPS and experimental groups and were treated with different concentrations of ICA. Cell viability was determined by Cell Counting Kit-8 assay and cell death was determined by flow cytometry. The expression levels of proteins (GPX4, SLC7A11, SLC3A2L, TRF, Nrf2 and NCOA4) were measured by western blotting. Quantification of malondialdehyde (MDA), iron and glutathione peroxidase 4 (GPX4) activity levels were performed via using corresponding assay kits. Cell death was increased, and cell viability was decreased in LPS-induced synoviocytes. Furthermore, MDA levels and iron content were elevated and GPX levels was reduced in LPS-induced synoviocytes. Transferrin receptor protein 1 and nuclear receptor coactivator 4 were upregulated and proteins of the Xc-/GPX4 axis, as well as nuclear factor erythroid 2-related factor 2, were decreased by LPS treatment. All aforementioned LPS affects were alleviated by ICA via a concentration-dependent manner. ICA counteracted the effects of RSL3, a ferroptosis activator, on cell viability, lipid peroxidation, iron content and relative protein expression of ferroptosis in synoviocytes. ICA protects the cells from death in synoviocytes induced by LPS, via the inhibition of ferroptosis by activating the Xc-/GPX4 axis, which can be exploited as a new therapeutic strategy for synovitis.

Tripartite motif 8 (TRIM8) is a member of the TRIM protein family that has been found to be implicated in cardiovascular disease. However, the role of TRIM8 in myocardial ischemia/reperfusion (I/R) has not been investigated. We aimed to explore the effect of TRIM8 on cardiomyocyte H9c2 cells exposed to hypoxia/reoxygenation (H/R). We found that TRIM8 expression was markedly upregulated in H9c2 cells after stimulation with H/R. Gain- and loss-of-function assays proved that TRIM8 knockdown improved cell viability of H/R-stimulated H9c2 cells. In addition, TRIM8 knockdown suppressed reactive oxygen species production and elevated the levels of superoxide dismutase and glutathione peroxidase. Knockdown of TRIM8 suppressed the caspase-3 activity, as well as caused significant increase in bcl-2 expression and decrease in bax expression. Furthermore, TRIM8 overexpression exhibited apposite effects with knockdown of TRIM8. Finally, knockdown of TRIM8 enhanced the activation of PI3K/Akt signaling pathway in H/R-stimulated H9c2 cells. Inhibition of PI3K/Akt by LY294002 reversed the effects of TRIM8 knockdown on cell viability, oxidative stress, and apoptosis of H9c2 cells. These present findings defined TRIM8 as a therapeutic target for attenuating and preventing myocardial I/R injury.

Ganoderma lucidum is a popular traditional Chinese medicine used in China to improve health. Previous researches have revealed that the polysaccharide from G. lucidum could exert diversity activities, including immunomodulation, antioxidant, and antitumor effects. However, the effect of enzymatically hydrolyzed G. lucidum polysaccharide (EGLP) in colorectal cancer (CRC) progression remains unknown. The present research aimed to investigate the antitumor mechanism of EGLP in human colon cancer cells. For this purpose, the cytotoxic effects of EGLP were measured by the (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method. The apoptosis was evoked upon EGLP treatment, which was assayed using flow cytometry. The results indicated that EGLP may induce apoptosis in human colon cancer cell (HCT-116) cells via the upregulation of BCL-2 associated X protein (Bax), phospho-extracellular regulated protein kinases (P-ERK), and cleaved caspase-3 expression and downregulation of B-cell lymphoma-2 (Bcl-2), phospho-serine/threonine kinase 1 (p-Akt1), and cyclo-oxygen-ase (COX-2) expression. The obtained findings indicated EGLP as a new therapeutic agent in fighting CRC.

Post-cardiac arrest myocardial dysfunction significantly contributes to early mortality after the return of spontaneous circulation. However, no effective therapy is available now. Aldehyde dehydrogenase 2 (ALDH2) enzyme has been shown to protect the heart from aldehyde toxicity such as 4-hydroxy-2-nonenal (4-HNE) and oxidative stress. In this study, we evaluated the effect of enhanced activity or expression of ALDH2 on post-cardiac arrest myocardial dysfunction and survival in a rat cardiac arrest model. Furthermore, we elucidated the underlying mechanisms with a focus on mitochondrial reactive oxygen species (ROS) production in a cell hypoxia/reoxygenation model. A total of 126 rats were used for the ALDH2 activation or cardiac overexpression of ALDH2 studies. Randomization was done 10 min before the respective agonist injection or in vivo gene delivery. We showed that enhanced activity or expression of ALDH2 significantly improved contractile function of the left ventricle and survival rate in rats subjected to cardiac arrest-cardiopulmonary resuscitation procedure. Moreover, ALDH2 prevented cardiac arrest-induced cardiomyocyte death from apoptosis and mitochondrial damage. Mechanistically, 4-HNE, a representative substrate of ALDH2, was dominantly increased in the hypoxia/reoxygenation-exposed cardiomyocytes. Direct addition of 4-HNE led to significantly augmented succinate accumulation and mitochondrial ROS production. Through metabolizing 4-HNE, ALDH2 significantly inhibited mitochondrial ROS production. Our findings provide compelling evidence of the cardioprotective effects of ALDH2 and therapeutic targeting this enzyme would provide an important approach for treating post-cardiac arrest myocardial dysfunction.