Signaling Pathway-related Factors Research Articles

A comprehensive transcriptomic analysis of alternate interferon signaling pathways in peripheral blood mononuclear cells in rheumatoid arthritis.

Interferon (IFN) signaling pathways play crucial roles in the pathogenesis of rheumatoid arthritis (RA). Prior studies have mainly studied mixed alterations in the IFN signaling pathway in RA, but these studies have not been sufficient to elucidate how imbalanced IFN signaling subtly influences immune cells. Single-cell RNA (scRNA) sequencing makes it possible to better understand the alternations in the interferon signaling pathways in RA. In the present study, we found that IFN signaling pathways were activated in natural killer (NK) cells, monocytes, T cells, B cells, and most immune cell subclasses in RA. We then explored and analyzed the connections between abnormal IFN signaling pathways and cellular functional changes in RA. Single-Cell rEgulatory Network Inference and Clustering (SCENIC) analysis and gene regulatory network (GRN) construction were also performed to identify key transcription factors in RA. Finally, we also investigated altered IFN signaling pathways in multiple RA peripheral blood samples, which indicated that abnormal IFN signaling pathways were universally observed in RA. Our study contributes to a better understanding of the delicate and precise regulation of IFN signaling in the immune system in RA. Furthermore, common alternations in IFN signaling pathway-related transcription factors could help to identify novel therapeutic targets for RA treatment.

Icariin inhibits oral squamous cell carcinoma cell proliferation and induces apoptosis via inhibiting the NF-κB and PI3K/AKT pathways.

Oral squamous cell carcinoma (OSCC), one of the most common types of human cancer, has a high mortality rate and a poor prognosis due to its high rates of recurrence and metastasis. In recent years, icariin (ICA) has been reported to play an important role in a variety of malignancies, such as gastric, colorectal, pancreatic and ovarian cancer. However, its role and mechanism in OSCC remains to be elucidated. The present study aimed to investigate the effect of ICA in OSCC cells and to reveal its underlying mechanisms. The OSCC cell lines SCC9 and Cal 27 were used to explore the effect of different concentrations of ICA on the biological behavior of OSCC cells. The effect of ICA on OSCC cell proliferation and apoptosis was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide and flow cytometric assays, respectively. Subsequently, the protein expression levels of caspase-3 and cleaved-caspase-3 were detected using western blot analysis. Additionally, the protein and mRNA expression levels of nuclear factor-κB (NF-κB) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway-related factors were determined using western blot analysis and reverse transcription-quantitative PCR, respectively. The results demonstrated that ICA inhibited OSCC cell proliferation and significantly increased the apoptosis rate in a dose-dependent manner. In addition, treatment of OSCC cells with ICA upregulated the protein expression of cleaved-caspase-3 and increased the cleaved-caspase-3/caspase-3 ratio. The protein expression levels of phosphorylated (p)-p65, p-PI3K and p-AKT were decreased in OSCC cells treated with ICA. The aforementioned findings revealed that ICA could attenuate the proliferation of OSCC cells and induce apoptosis via inhibiting the NF-κB and PI3K/AKT signaling pathways. Therefore, the current study provided a new insight into the clinical treatment of OSCC.

Silencing UHRF1 Enhances Radiosensitivity of Esophageal Squamous Cell Carcinoma by Inhibiting the PI3K/Akt/mTOR Signaling Pathway.

PurposeResistance to radiotherapy results in a high treatment failure rate for locally advanced esophageal squamous cell carcinoma (ESCC). Ubiquitin-like with plant homeodomain and ring-finger domains 1 (UHRF1), is associated with poor prognosis in ESCC. The present study aims to characterize the effect of UHRF1 silencing on the radiosensitivity of ESCC and its potential mechanism.MethodsBoth in vitro and in vivo experiments were conducted to observe the effects of UHRF1 silencing on the radiosensitivity of ESCC. The effects of UHRF1 silencing on the apoptosis of ESCC cells were assessed by flow cytometry. The expression of apoptosis-related factors (caspase-3 and Bcl-2), PI3K/Akt/mTOR signaling pathway-related factors (PTEN, p-Akt and Akt, p-mTOR and mTOR), and DNMT1 were measured via Western blot, and the status of PTEN methylation was detected by methylation-specific PCR. Immunohistochemistry was used to detect the expressions of PTEN, p-AKT, and p-mTOR in xenograft tumor tissues.ResultsIn vitro and in vivo experiments showed that UHRF1 knock-down inhibited ESCC cell growth and enhanced their radiosensitivity. shUHRF1 combined with radiation significantly increased ESCC cell apoptosis. Meanwhile, it activated the expression of caspase-3 and inhibited the expression of Bcl-2. shUHRF1 inhibited the expression of DNMT1 and reduced the methylation of PTEN, and then upregulated the expression of PTEN to inhibit the PI3K/Akt/mTOR signaling pathway. On the contrary, the PI3K/Akt/mTOR signaling pathway can be activated by upregulation of UHRF1.ConclusionOur findings provide a theoretical basis for UHRF1 as a target to improve the radiosensitivity of ESCC.

Effect of Zhuang Medicine Feilongzhangxue on the Expression of HMGB1-TLR4 / RANKL-NF-?B Signaling Pathway Related Factors in Osteoclasts

Objective: To study the regulatory effect of feilongzhangxue on the levels of HMGB1-TLR4 / RANKL-NF-?B signaling pathway related factors HMGB1, RANKL, rank, TRAF-6 and NF-?Bp65 in osteoclasts, so as to explore the mechanism of feilongzhangxue intervention in RA; Methods: The osteoclasts with good activity were randomly divided into blank group, methotrexate control group and Zhuang medicine feilongzhang blood containing serum treatment group, which were divided into OC + blank group, OC + methotrexate control group, OC + Zhuang medicine feilongzhang blood containing serum group; The expression of HMGB1, RANKL, rank, TRAF-6 and NF-?Bp65 mRNA was detected by RT-PCR; The protein expressions of HMGB1, RANKL, RANK, TRAF-6 and NF-?Bp65 were detected by immunofluorescence. Results: PCR results showed that: Compared with the blank group, feilongzhangxue could effectively inhibit the expression levels of HMGB1, RANKL, rank, TRAF-6 and NF-? B p65 mRNA in OC cells, and the inhibitory effect was stronger than methotrexate. Immunofluorescence test results showed that: Compared with the blank group and methotrexate group, feilongzhangxue could effectively inhibit the protein expression of HMGB1, RANKL, rank, TRAF-6 and NF - ? B p65 in OC cells. Conclusion: The effect of Zhuang medicine feilongzhangxue on hmgb1-tlr4 / rankl-nf - ? B signaling pathway of osteoclasts is through the regulation of related factors HMGB1, RANKL, rank, TRAF-6 and NF-?Bp65, which may be the key mechanism of Zhuang medicine feilongzhangxue on rheumatoid arthritis.

Mesenchymal stem cell-derived exosomes containing miR-145-5p reduce inflammation in spinal cord injury by regulating the TLR4/NF-κB signaling pathway

ABSTRACT EXs (Exosomes) secreted by mesenchymal stem cells (MSCs) have the potential to treat spinal cord injury (SCI), this study aimed to further explore the therapeutic effect of EXs on SCI. Firstly, EXs were extracted from MSCs and analyzed with a transmission electron microscope. Next, MSCs with or without the miR-145-5p plasmid were injected into the SCI rat model, and then rat damage was evaluated by BBB score, HE staining and Nissl staining. And then Luciferase experiment verified the targeting relationship between miR-145-5p and TLR4. Furthermore, LPS-induced PC12 cells were established and incubated with Dil-labeled MSC-EXs to explore their effects on cell viability, apoptosis and inflammation through MTT, flow cytometry and ELISA, respectively. In addition, expressions of TLR4/NF-κB signaling pathway related factors were measured by qRT-PCR and Western blot. The results showed that after MSCs were successfully isolated, the existence of EXs in MSCs was confirmed. Moreover, MSC-EXs containing miR-145-5p improved functional recovery and reduced histopathological injury and inflammation in SCI rats. And MSC-EXs promoted miR-145-5p expression in spinal cord tissue and inhibited TLR4/NF-κB pathway activation in SCI rats. MSC-EXs inhibited LPS-induced inflammatory response and activation of the TLR4/NF-κB pathway in PC12 cells. In addition, we also found that miR-145-5p specifically targeted TLR4. TLR4 overexpression significantly reversed the effect of EX-miR-145-5p on maintaining PC12 cell viability, inhibiting apoptosis and inflammatory response, and activating TLR4/NF- κB pathway. In conclusion, mesenchymal stem cell-derived EXs containing miR-145-5p reduce inflammation in spinal cord injury by regulating the TLR4/NF-κB signaling pathway.

Melittin inhibits lung metastasis of human osteosarcoma: Evidence of wnt/β-catenin signaling pathway participation

Melittin is a major active peptide component of bee venom that has been demonstrated to show anti-tumor effects. Osteosarcoma is a type of bone tumor with a high degree of malignancy, and metastasis is the main challenge of osteosarcoma therapy. This study aimed to investigate the role of melittin in the lung metastasis of osteosarcoma. 143 B cells were treated with different concentrations of melittin in vitro. Wound-healing and transwell assays were performed to determine the cell migration and invasion potential. Quantitative real-time PCR and Western blot experiments were performed to evaluate the expression levels of Wnt/β-catenin signaling pathway-related factors after treatment with melittin. The orthotopic implantation model and hematoxylin-eosin staining were used to investigate the effect of melittin treatment on tumor formation and lung metastasis. Immunohistochemical staining and Western blot experiments were performed to indicate the melittin-mediated expression changes in Wnt/β-catenin signaling pathway-related factors. The cell migration and invasion potential were observed to be inhibited in a dose-dependent manner upon treatment with melittin. Treatment with medium and high concentrations of melittin attenuated the mRNA and protein expression of LRP5, β-catenin, MMP-2, cyclin D, c-Myc, survivin, MMP-9, and VEGF genes in vitro. Melittin significantly inhibited the growth of tibia xenografts in nude mice and decreased the number of lung metastatic nodules. Consistent with the results observed in vitro, treatment with melittin at medium and high concentrations attenuated the expression of Wnt/β-catenin signaling pathway-related factors in vivo. In vitro, Wnt/β-catenin signaling pathway was involved in Melittin-mediated -migration and invasion potential of 143 B cells. Similarly, as observed in the in vivo experiments, Wnt/β-catenin signaling pathway was also associated with the role of melittin on lung metastasis of osteosarcomas.

Ginsenoside Rh2 alleviates ulcerative colitis by regulating the STAT3/miR-214 signaling pathway

Ethnopharmacological relevanceGinseng is a valuable medicinal herb used in China for the prevention and treatment of cancer, diabetes, cardiovascular diseases and other diseases. As the main active ingredient of ginseng, ginsenoside has a wide range of pharmacological effects. Ginsenoside Rh2, a protopanaxadiol saponin from ginseng, exhibits anti-inflammatory and anticancer effects. Aim of the studyThe potential biological mechanism of Rh2 in the treatment of ulcerative colitis (UC) has not been clarified clearly. In our research, we aimed to explore the therapeutic effects of Rh2 on dextran sodium sulfate (DSS)-induced colitis and elucidate the mechanism of Rh2 in treating UC. MethodsDSS-induced UC mice were established and randomly divided into the following four groups: control group, DSS group, Rh2 (50 mg/kg) group and sulfasalazine (SASP, 200 mg/kg) group. Except for the control group, 3% DSS drinking water was given to each group for 7 days, and the other two groups were intragastrically administered with Rh2 and SASP for 10 days. At the end of the experiment, colon samples were collected, and phenotypic and pathological analyses were performed in UC mice. Then, Western blot, immunohistochemistry and quantitative real-time PCR analyses were performed to determine the expression of signaling pathway-related factors. ResultsRh2 markedly alleviated DSS-induced body weight loss, intestinal damage, colon length shortening and disease activity index (DAI) scores. Furthermore, proinflammatory cytokines, such as TNF-α, IL-6 and IL-1β, were reduced by Rh2. Additionally, STAT3/miR-214 activation was also suppressed by Rh2 administration. In vitro, we demonstrated that Rh2 effectively inhibited IL-6-induced STAT3 phosphorylation and miR-214 expression in cultured normal colonic epithelial cells. ConclusionOur results suggested that Rh2 exhibits potential application value in the treatment of UC, and its mechanism is related to the downregulation of STAT3/miR-214 levels, which is expected to be applicable in the treatment of clinical UC.

Silymarin nanoliposomes attenuate renal injury on diabetic nephropathy rats via co-suppressing TGF-β/Smad and JAK2/STAT3/SOCS1 pathway

AimsTo investigate the improvement and mechanisms of silymarin on renal injury in mouse podocytes and streptozotocin (STZ)-induced diabetic nephropathy model (DN) rats. Main methodsFirstly, the effects of silymarin on the cell viability and cellular injury-related indicators of high-glucose incubated mouse podocytes MPC-5 were assessed by CCK-8 and western blotting (WB) methods, respectively. The STZ-induced diabetic rats with DN were treated with silymarin nanoliposomes at three doses for consecutive 8-week. General metabolic indicators, renal functions and lipid accumulation-related factors were all measured. The renal tissue sections were stained and observed via hematoxylin-eosin (H&E) staining method. Real-time RT-PCR and WB methods were utilized to measure the expression of JAK2/STAT3/SOCS1 and TGF-β/Smad signaling pathway related factors. Key findingsSilymarin significantly improve the high-glucose induced up-regulation of podoxin and nephrin, as well as the expression of inflammatory cytokines IL-6, ICAM-1 and TNF-α, and the cell survival rates were also significantly increased in a dose-dependent manner. Significant improvement on body weight/kidney ratio, renal functions and lipid profiles in renal tissues were observed in STZ-induced diabetic rats after chronic silymarin treatment. The H&E staining exhibited that the pathological damages in renal tissues were obviously improved. Moreover, silymarin nanoliposomes treatment notably suppressed expression levels of inflammation-related proteins as well as IL-6 and ICAM-1, and regulated JAK2/STAT3/SOCS1 and TGF-β/Smad signaling pathway, thereby exhibited protective effects on kidney of DN model rats. SignificanceSilymarin nanoliposomes ameliorate STZ-induced kidney injury by improving oxidative stress, renal fibrosis, and co-inhibiting JAK2/STAT3/SOCS1 and TGF-β/Smad signaling pathways in diabetic rats.

Polystyrene microplastics lead to pyroptosis and apoptosis of ovarian granulosa cells via NLRP3/Caspase-1 signaling pathway in rats

Microplastics (MPs) considered as a new persistent environmental pollutant could enter into the circulatory system and result in decrease of sperm quantity and quality in mice. However, the effects of Polystyrene MPs (PS MPs) on the ovary and its mechanism in rats remained unclear. In this present study, thirty-two healthy female Wistar rats were exposed to different concentrations of 0.5 µm PS MPs dispersed in deionized water for 90 days. Using hematoxylin-eosin (HE) staining, the number of growing follicles was decreased compared to the control group. In addition, the activity of glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) were decreased while the expression level of malondialdehyde (MDA) was increased in ovary tissue. Confirmed by immunohistochemistry, the integrated optical density of NLRP3 and Cleaved-Caspase-1 had been elevated by 13.9 and 14 in granulosa cells in the 1.5 mg/kg/d group. Furthermore, compared to the control group, the level of AMH had been decreased by 23.3 pg/ml while IL-1β and IL-18 had been increased by 32 and 18.5 pg/ml in the 1.5 mg/kg/d group using the enzyme-linked immune sorbent assay (ELISA). Besides, the apoptosis of granulosa cells was elevated measured by terminal deoxyribonucleotide transferase-mediated nick end labeling (TUNEL) staining and flow cytometry. Moreover, western blot assays showed that the expressions of NLRP3/Caspase-1 signaling pathway related factors and Cleaved-Caspase-3 were increased. These results demonstrated that PS MPs could induce pyroptosis and apoptosis of ovarian granulosa cells via the NLRP3/Caspase-1 signaling pathway maybe triggered by oxidative stress. The present study suggested that exposure to microplastics had adverse effects on ovary and could be a potential risk factor for female infertility, which provided new insights into the toxicity of MPs on female reproduction.

RETRACTED: Exosomal miR-22-3p from human umbilical cord blood-derived mesenchymal stem cells protects against lipopolysaccharid-induced acute lung injury

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. Concern was raised about the reliability of the Western blot results in Figs. 2D/H/L, and 7B/F, which appear to have the same eyebrow shaped phenotype as many other publications tabulated here (https://docs.google.com/spreadsheets/d/149EjFXVxpwkBXYJOnOHb6RhAqT4a2llhj9LM60MBffM/edit#gid=0). Concerns were also raised over the provenance of the flow cytometry plots in Fig. 1C. The journal requested the corresponding author comment on these concerns and provide the raw data. However the authors were not able to satisfactorily fulfil this request and therefore the Editor-in-Chief decided to retract the article.

Combination therapy with semaglutide and rosiglitazone as a synergistic treatment for diabetic retinopathy in rodent animals

ObjectiveTo investigate the protective efficacies and potent mechanisms of combination therapy with semaglutide and rosiglitazone (RSG) on the high-glucose incubated human ARPE-19 cells and diabetic retinopathy (DR) model rats. Main methodsThe CCK-8 methods were used to evaluate the protective effects of semaglutide and RSG alone or combination on the cell viability of high-glucose treated ARPE-19 cells. After the DR rat model was established, the effects of combined treatment on general indexes, retinal morphological changes, retinal Müller cells as well as PI3K/Akt/MTOR related factors of DR model rats were investigated. ResultsThe CCK-8 assay showed obviously enhanced protective efficacies of combination therapy with semaglutide and RSG on the ARPE-19 with oxidative stress induced by high-glucose with combination index all below 1.5 demonstrating obvious synergistic effects. Combined incubation could also effectively decrease the expression of inflammatory factors, including TNF-α, IL-1β, IL-6, and the increase of ROS content in ARPE cell culture supernatant induced by high-glucose. Combined use of the antioxidant, PI3K/Akt and mTOR inhibitors, we further demonstrated that combined incubation of semaglutide and RSG could effectively by reduce high glucose-induced inflammatory injury inhibiting ROS/PI3K/Akt/mTOR signaling. Furthermore, chronic combination treatment effectively improved the histopathological characteristics and down-regulated the GFAP expression in Müller cells as well as PI3K/Akt/MTOR signaling pathway-related factors in retina which was better than any monomer treatment group. ConclusionsCombined semaglutide with RSG exhibited synergistically protective efficacies on retinal cells by decreasing the GFAP expression, inhibiting oxidative stress and PI3K/Akt/MTOR signaling-transduction in DR model rats.

MiR-92a promotes proliferation and inhibits apoptosis of prostate cancer cells through the PTEN/Akt signaling pathway

ABSTRACT MicroRNAs (miRNAs) play an important role in the development of prostate cancer (PCa). Recent studies have shown that miR-92a expression is significantly increased in various cancers including PCa. However, its specific mechanism in PCa remains unknown. The goal of this study was to investigate the effect of miR-92a expression on the function and mechanism of PCa. PCa cell lines PC-3 and LNCap were transfected with miR-92a inhibitor to reduce the expression of miR-92a, respectively. The cell proliferation, cell viability, apoptosis, cell invasion and migration ability of PCa cells were examined by CCK8 assay, cell cloning, flow cytometry, Transwell assay and scratch assay, respectively. The effects of miR-92a on PTEN/Akt signaling pathway-related factors (PI3k, Akt, p-PI3k, p-Akt, PTEN) were also observed by RT-qPCR and Western blot. Compared with the control group and NC inhibitor group, the viability, cell migration and invasion ability of PC-3 and LNCap cells were decreased and apoptosis was significantly increased after interference with miR-92a expression. In addition, the mRNA and protein levels of PTEN in PC-3 and LNCap cells in the miR-92a inhibitor group were significantly increased, while the phosphorylation levels of PI3K and AKT were significantly decreased. MiR-92a might play a key role in regulating the proliferation, migration and invasion of PCa cells through the PTEN/Akt signaling pathway. Inhibition of miR-92a expression has practical value against PCa and provides ideas for further clinical treatment of patients with PCa.

Combination therapy with metformin and IL-12 to inhibit the growth of hepatic carcinoma by promoting apoptosis and autophagy in HepG2-bearing mice.

To investigate the effects and mechanism of metformin (Met) combined the interleukin-12 (IL-12) on inhibiting hepatoma HepG2 cell proliferation via in vitro and in vivo assays. MTT assay was used to detect inhibitory effects of Met, IL-12 alone or combination on HepG2 cells proliferation. Half inhibitory concentration (IC50) and combination index (CI) were also calculated. Anti-tumor effects of combination or monotherapy on the HepG2-bearing mice were investigated and protein expression levels of apoptosis, as well as the Akt/mTOR/STAT3 signaling pathway-related factors were detected by Western blot. MTT results showed that the inhibitory effect of Met combined with IL-12 on HepG2 cell proliferation was significantly enhanced (both p<0.01) compared with monomer therapy group with a significant synergistic effect (CI<1). The apoptosis rate of HepG2 cells treated with Met combined with IL-12 were 88.12±7.15% and significantly higher than the others (all p<0.01). Moreover, combination treatment significantly suppressed hepatoma growth and increased the survival rate of HepG2-bearing mice without evident body weight loss. Western blot analysis showed that Met combined with IL-12 significantly increased the expression of autophagy-related marker proteins, downregulated the protein expression levels of Bcl-2, p-Akt, p-mTOR, p-STAT3, upregulated the expression level of BAX in both HepG2 cells and tumor tissues. Met combined with IL-12 exhibited a synergistic antitumor effect on hepatoma HepG2 cells, and the mechanism may be related to its common inhibition of Akt/mTOR/STAT3 signaling pathway and increase of autophagy in HepG2-bearing mice.

Electroacupuncture improves learning and memory functions in a rat cerebral ischemia/reperfusion injury model through PI3K/Akt signaling pathway activation.

Electroacupuncture has been widely used to treat cognitive impairment after cerebral ischemia, but the underlying mechanism has not yet been fully elucidated. Studies have shown that autophagy plays an important role in the formation and development of cognitive impairment, and the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway plays an important role in autophagy regulation. To investigate the role played by the PI3K/Akt signaling pathway in the electroacupuncture treatment of cerebral ischemia/reperfusion rat models, we first established a rat model of cerebral ischemia/reperfusion through the occlusion of the middle cerebral artery using the suture method. Starting at 2 hours after modeling, electroacupuncture was delivered at the Shenting (GV24) and Baihui (GV20) acupoints, with a dilatational wave (1-20 Hz frequency, 2 mA intensity, 6 V peak voltage), for 30 minutes/day over 8 consecutive days. Our results showed that electroacupuncture reduced the infarct volume in a rat model of cerebral ischemia/reperfusion injury, increased the mRNA expression levels of the PI3K/Akt signaling pathway-related factors Beclin-1, mammalian target of rapamycin (mTOR), and PI3K, increased the protein expression levels of phosphorylated Akt, Beclin-1, PI3K, and mTOR in the ischemic cerebral cortex, and simultaneously reduced p53 mRNA and protein expression levels. In the Morris water maze test, the latency to find the hidden platform was significantly shortened among rats subjected to electroacupuncture stimulation compared with rats without electroacupuncture stimulation. In the spatial probe test, the number of times that a rat crossed the target quadrant was increased in rats subjected to electroacupuncture stimulation compared with rats without electroacupuncture stimulation. Electroacupuncture stimulation applied to the Shenting (GV24) and Baihui (GV20) acupoints activated the PI3K/Akt signaling pathway and improved rat learning and memory impairment. This study was approved by the Animal Ethics Committee of the First Affiliated Hospital of Henan University of Traditional Chinese Medicine, China (approval No. 8150150901) on March 10, 2016.

Osteoglycin silencing exerts inhibitory effects on myocardial fibrosis and epithelial/endothelial-mesenchymal transformation in a mouse model of myocarditis.

Osteoglycin (Ogn), a class III SLRP member with multiple glycosylation sites, has been proposed to be engaged in cardiac dysfunction and adverse remodeling in human heart failure following myocardial infarction. However, the underlying mechanism remains to be elucidated. Thus, we sought to define the role of Ogn in regulation of the Wnt pathway on myocardial fibrosis and epithelial/endothelial-mesenchymal transformation (EMT/EndMT) in mice with myocarditis. The pathological changes are observed, while hematoxylin-eosin staining and picric acid Sirius red staining were conducted in successfully constructed myocarditis mouse models. Immunohistochemistry and enzyme-linked immunosorbent assay were adopted to determine Ogn and β-catenin levels and serum procollagen propeptide concentrations in the mouse myocardial tissues, respectively. Expression of Ogn and Wnt signaling pathway-related factors were measured by reverse transcription quantitative polymerase chain reaction and western blot assay, cell viability by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cell cycle distribution and apoptosis by flow cytometry. We saw indicative pathological changes accompanied by many Ogn and β-catenin positive cells and increased serum procollagen propeptide, in the mouse myocardial tissues. Loss function assays showed reduced levels of Ogn, β-catenin, LRP6, TGF-β1, Twist, FSP-1, α-SMA and higher levels of E-cadherin and VE-cadherin, together with decreased proliferation rate, as well as increased apoptosis rate, indicating that the Wnt signaling pathway, proliferation were inhibited while apoptosis was enhanced with upon gene silencing. Coherently, depletion of Ogn inhibits myocardial fibroblasts proliferation and EMT/EndMT while facilitating myocardial fibroblasts apoptosis in myocarditis through the Wnt signaling pathway, thus serving as an intervention target for the molecular treatment of myocarditis.

A Temporal Examination of Cytoplasmic Ca2 + Levels, Sarcoplasmic Reticulum Ca2 + Levels, and Ca2 + -Handling-Related Proteins in Different Skeletal Muscles of Hibernating Daurian Ground Squirrels

To explore the possible mechanism of the sarcoplasmic reticulum (SR) in the maintenance of cytoplasmic calcium (Ca2+) homeostasis, we studied changes in cytoplasmic Ca2+, SR Ca2+, and Ca2+-handling proteins of slow-twitch muscle (soleus, SOL), fast-twitch muscle (extensor digitorum longus, EDL), and mixed muscle (gastrocnemius, GAS) in different stages in hibernating Daurian ground squirrels (Spermophilus dauricus). Results showed that the level of cytoplasmic Ca2+ increased and SR Ca2+ decreased in skeletal muscle fiber during late torpor (LT) and inter-bout arousal (IBA), but both returned to summer active levels when the animals aroused from and re-entered into torpor (early torpor, ET), suggesting that intracellular Ca2+ is dynamic during hibernation. The protein expression of ryanodine receptor1 (RyR1) increased in the LT, IBA, and ET groups, whereas the co-localization of calsequestrin1 (CSQ1) and RyR1 in GAS muscle decreased in the LT and ET groups, which may increase the possibility of RyR1 channel-mediated Ca2+ release. Furthermore, calcium pump (SR Ca2+-ATPase 1, SERCA1) protein expression increased in the LT, IBA, and ET groups, and the signaling pathway-related factors of SERCA activity [i.e., β-adrenergic receptor2 protein expression (in GAS), phosphorylation levels of phospholamban (in GAS), and calmodulin kinase2 (in SOL)] all increased, suggesting that these factors may be involved in the up-regulation of SERCA1 activity in different groups. The increased protein expression of Ca2+-binding proteins CSQ1 and calmodulin (CaM) indicated that intracellular free Ca2+-binding ability also increased in the LT, IBA, ET, and POST groups. In brief, changes in cytoplasmic and SR Ca2+ concentrations, SR RyR1 and SERCA1 protein expression levels, and major RyR1 and SERCA1 signaling pathway-related factors were unexpectedly active in the torpor stage when metabolic functions were highly inhibited.

Decorin deficiency promotes epithelial-mesenchymal transition and colon cancer metastasis

The tumor microenvironment encompasses a complex cellular network that includes cancer-associated fibroblasts, inflammatory cells, neo-vessels, and an extracellular matrix enriched in angiogenic growth factors. Decorin is one of the main components of the tumor stroma, but it is not expressed by cancer cells. Lack of this proteoglycan correlates with down-regulation of E-cadherin and induction of β-catenin signaling. In this study, we investigated the role of a decorin-deficient tumor microenvironment in colon carcinoma progression and metastasis. We utilized an established model of colitis-associated cancer by administering Azoxymethane/Dextran sodium sulfate to adult wild-type and Dcn−/- mice. We discovered that after 12 weeks, all the animals developed intestinal tumors independently of their genotype. However, the number of intestinal neoplasms was significantly higher in the Dcn−/− microenvironment vis-à-vis wild-type mice. Mechanistically, we found that under unchallenged basal conditions, the intestinal epithelium of the Dcn−/− mice showed a significant increase in the protein levels of epithelial-mesenchymal transition associated factors including Snail, Slug, Twist, and MMP2. In comparison, in the colitis-associated cancer evoked in the Dcn−/− mice, we found that intercellular adhesion molecule 1 (ICAM-1) was also significantly increased, in parallel with epithelial-mesenchymal transition signaling pathway-related factors. Furthermore, a combined Celecoxib/decorin treatment revealed a promising therapeutic efficacy in treating human colorectal cancer cells, in decorin-deficient animals. Collectively, our results shed light on colorectal cancer progression and provide a protein-based therapy, i.e., treatment using recombinant decorin, to target the tumor microenvironment.

Metformin inhibits the growth of ovarian cancer cells by promoting the Parkin-induced p53 ubiquitination.

Ovarian cancer is the most lethal diseases among women. The chemo-resistance has been a big challenge for the cancer treatment. It has been reported that metformin may inhibit ovarian cancer and is able to impede the development of drug resistance, but the molecular mechanisms remain elusive. In this study, we explored the molecular roles of metformin in Parkin expression and p53 ubiquitination in chemo-resistant ovarian cancer cells. Firstly, ovarian cancer and chemo-resistant ovarian cancer cells were selected for determining the expression of Parkin, p53, and p53 signaling pathway-related factors. Then the cell proliferation and viability after loss- and gain-of-function assays were measured. Besides, immunoprecipitation (IP) was used to determine the interactions between Parkin and p53, and the ubiquitination level of p53 was measured using in vitro ubiquitination assay. Finally, the degradation of p53 proteasome regulated by Parkin was monitored using the MG132 proteasome inhibitor. We found that metformin significantly inhibited the growth of ovarian cancer parental cells and chemo-resistant cells, and metformin promoted Parkin expression in chemo-resistant cells. Further, up-regulated Parkin expression promoted the ubiquitination and degradation of p53, and metformin inhibited the expression of p53 to suppress the proliferation of chemo-resistant ovarian cancer cells. Mechanistically, metformin could inhibit the growth of ovarian cancer cells by promoting the Parkin-induced p53 ubiquitination. Altogether, our study demonstrated an inhibitory role of metformin in the growth of chemo-resistant cancer cells through promoting the Parkin-induced p53 ubiquitination, which provides a novel mechanism of metformin for treating ovarian cancer.

Reduced LINC00467 Elevates microRNA-125a-3p to Suppress Cisplatin Resistance in Non-Small Cell Lung Cancer Through Inhibiting Sirtuin 6 and Inactivating the ERK1/2 Signaling Pathway

Objective: Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been identified as vital biomarkers in cancers. This study aimed to investigate effect of long intergenic non-protein coding RNA 467 (LINC00467)/miR-125a-3p/sirtuin 6 (SIRT6) axis on non-small cell lung cancer (NSCLC). Methods: Expression of LINC00467, miR-125a-3p, SIRT6 and the extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling pathway-related factors in NSCLC tissues and cells was determined. Parent cells A549 and H1299 were induced by cisplatin (DDP) to construct DDP resistant cell lines A549/DDP and H1299/DDP, and the drug resistance was verified. The DDP resistant cells were treated with relative plasmids to observe the impacts of altered LINC00467 and miR-125a-3p on biological processes and DDP resistance of cells. Results: LINC00467 and SIRT6 were upregulated, miR-125a-3p was suppressed and the ERK1/2 pathway was activated in NSCLC tissues and cells. Inhibited LINC00467 or elevated miR-125a-3p repressed malignant behaviors and DDP resistance of NSCLC cells. Depletion of miR-125a-3p reversed impacts of inhibited LINC00467 on NSCLC cells. Moreover, LINC00467 competitively bound to miR-125a-3p, and SIRT6 was targeted by miR-125a-3p. Conclusion: Reduced LINC00467 elevated miR-125a-3p to suppress DDP resistance in NSCLC cells by restricting SIRT6 and inactivating the ERK1/2 signaling pathway. This research may provide potential candidates for NSCLC chemotherapy. Funding Statement: The current work is funded by Research project of Health and Family Planning Commission of Heilongjiang Province.(Contract grant number: 2018268) and Research project of Health and Family Planning Commission of Heilongjiang Province. (Contract grant number: 2018416). Declaration of Interests: The authors declare that they have no conflicts of interest. Ethics Approval Statement: This study was approved and supervised by the animal ethics committee of The Affiliated Hospital of Guilin Medical University. The treatment of animals in all experiments conforms to the ethical standards of experimental animals. Written informed consents were acquired from all patients before this study. The protocol of this study was confirmed by the Ethic Committee of The Affiliated Hospital of Guilin Medical University and based on the ethical principles for medical research involving human subjects of the Helsinki Declaration.

MiR-21 protects neonatal rats from hypoxic-ischemic brain damage by targeting CCL3.

Hypoxic-ischemic brain damage (HIBD) represents one of the leading causes of neonatal mortality and permanent neurological disability worldwide. Compelling studies have identified implication of microRNAs (miRNAs) in HIBD. However, the molecular mechanism of miR-21 underlying the disease pathogenesis is unknown. The present study aims to explore the role of miR-21 in neonatal rats with HIBD. HIBD rat models were developed by carotid artery ligation and hypoxia treatment, and in vitro cell models were induced by oxygen-glucose deprivation. Through RT-qPCR and western blot analysis, high expression of CCL3 and poor expression of miR-21 were detected in brain tissues of rats with HIBD. Results of dual-luciferase reporter gene assay demonstrated that miR-21 could target and downregulate CCL3. The effect of miR-21 on the neurobehavioral ability of rats, the pathological characteristics of brain tissues, neuron apoptosis and as well as its impact on the NF-κB signaling pathway-related factors was examined by gain- and loss-of-function experiments. The obtained data suggested that upregulation of miR-21 resulted in significantly reduced cerebral infarct volume and degree of brain tissue damage, and improved neurobehavioral ability and memory ability in rats with HIBD through downregulation of CCL3. Besides, overexpression of miR-21 downregulated CCL3 to repress IKKα/β and p65 phosphorylation both in vivo and in vitro, hence disrupting the NF-κB signaling pathway. Taken together, the key findings of the current study underlie the cerebral protective effect of miR-21 against HIBD in neonatal rats through the inhibition of CCL3.