Showing papers in "Cell Proliferation in 2019"

Role of pyroptosis in cardiovascular disease

Abstract: Cardiac function is determined by the dynamic equilibrium of various cell types and the extracellular matrix that composes the heart. Cardiovascular diseases (CVDs), especially atherosclerosis and myocardial infarction, are often accompanied by cell death and acute/chronic inflammatory reactions. Caspase-dependent pyroptosis is characterized by the activation of pathways leading to the activation of NOD-like receptors, especially the NLRP3 inflammasome and its downstream effector inflammatory factors interleukin (IL)-1β and IL-18. Many studies in the past decade have investigated the role of pyroptosis in CVDs. The findings of these studies have led to the development of therapeutic approaches based on the regulation of pyroptosis, and some of these approaches are in clinical trials. This review summarizes the molecular mechanisms, regulation and cellular effects of pyroptosis briefly and then discusses the current pyroptosis studies in CVD research.

Exosomes derived from miR-375-overexpressing human adipose mesenchymal stem cells promote bone regeneration

Abstract: Objectives The present study aimed to investigate whether exosomes derived from miR-375-overexpressing human adipose mesenchymal stem cells (hASCs) could enhance bone regeneration. Materials and methods Exosomes enriched with miR-375 (Exo [miR-375]) were generated from hASCs stably overexpressing miR-375 after lentiviral transfection and identified with transmission electron microscopy, nanosight and western blotting. The construction efficiency of Exo (miR-375) was evaluated with qRT-PCR and incubated with human bone marrow mesenchymal stem cells (hBMSCs) to optimize the effective dosage. Then, the osteogenic capability of Exo (miR-375) was investigated with ALP and ARS assays. Furthermore, dual-luciferase reporter assay and western blotting were conducted to reveal the underlying mechanism of miR-375 in osteogenic regulation. Finally, Exo (miR-375) were embedded with hydrogel and applied to a rat model of calvarial defect, and μ-CT analysis and histological examination were conducted to evaluate the therapeutic effects of Exo (miR-375) in bone regeneration. Results miR-375 could be enriched in exosomes by overexpressing in the parent cells. Administration of Exo (miR-375) at 50 μg/mL improved the osteogenic differentiation of hBMSCs. With miR-375 absorbed by hBMSCs, insulin-like growth factor binding protein 3 (IGFBP3) was inhibited by binding to its 3'UTR, and recombinant IGFBP3 protein reduced the osteogenic effects triggered by Exo (miR-375). After incorporated with hydrogel, Exo (miR-375) displayed a slow and controlled release, and further in vivo analysis demonstrated that Exo (miR-375) enhanced the bone regenerative capacity in a rat model of calvarial defect. Conclusions Taken together, our study demonstrated that exosomes derived from miR-375-overexpressing hASCs promoted bone regeneration.

Exosomes from human umbilical cord mesenchymal stem cells enhance fracture healing through HIF-1α-mediated promotion of angiogenesis in a rat model of stabilized fracture.

Abstract: Objectives Exosomes, as important players in intercellular communication due to their ability to transfer certain molecules to target cells, are believed to take similar effects in promoting bone regeneration with their derived stem cells. Studies have suggested that umbilical cord mesenchymal stem cells (uMSCs) could promote angiogenesis. This study investigated whether exosomes derived from uMSCs (uMSC-Exos) could enhance fracture healing as primary factors by promoting angiogenesis. Materials and methods uMSCs were obtained to isolate uMSC-Exos by ultrafiltration, with exosomes from human embryonic kidney 293 cells (HEK293) and phosphate-buffered saline (PBS) being used as control groups. NanoSight, laser light scattering spectrometer, transmission electron microscopy and Western blotting were used to identify exosomes. Next, uMSC-Exos combined with hydrogel were transplanted into the fracture site in a rat model of femoral fracture. Bone healing processes were monitored and evaluated by radiographic methods on days 7, 14, 21 and 31 after surgery; angiogenesis of the fracture sites was assessed by radiographic and histological strategies on post-operative day 14. In vitro, the expression levels of osteogenesis- or angiogenesis-related genes after being cultured with uMSC-Exos were identified by qRT-PCR. The internalization ability of exosomes was determined using the PKH67 assay. Cell cycle analysis, EdU incorporation and immunofluorescence staining, scratch wound assay and tube formation analysis were also used to determine the altered abilities of human umbilical vein endothelial cells (HUVECs) administered with uMSC-Exos in proliferation, migration and angiogenesis. Finally, to further explore the underlying molecular mechanisms, specific RNA inhibitors or siRNAs were used, and the subsequent effects were observed. Results uMSC-Exos had a diameter of approximately 100 nm, were spherical, meanwhile expressing CD9, CD63 and CD81. Transplantation of uMSC-Exos markedly enhanced angiogenesis and bone healing processes in a rat model of femoral fracture. In vitro, other than enhancing osteogenic differentiation, uMSC-Exos increased the expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1α (HIF-1α). uMSC-Exos were taken up by HUVECs and enhanced their proliferation, migration and tube formation. Finally, by using specific RNA inhibitors or siRNAs, it has been confirmed that HIF-1α played an important role in the uMSC-Exos-induced VEGF expression, pro-angiogenesis and enhanced fracture repair, which may be one of the underlying mechanisms. Conclusions These results revealed a novel role of exosomes in uMSC-mediated therapy and suggested that implanted uMSC-Exos may represent a crucial clinical strategy to accelerate fracture healing via the promotion of angiogenesis. HIF-1α played an important role in this process.

LncRNA XIST mediates bovine mammary epithelial cell inflammatory response via NF-κB/NLRP3 inflammasome pathway

Abstract: Objectives The correlations between long non-coding RNAs (lncRNAs) and diverse mammal diseases have been clarified by many researches, but the cognition about bovine mastitis-related lncRNAs remains limited. This study aimed to investigate the potential role of lncRNA X-inactive specific transcript (XIST) in the inflammatory response of bovine mammary epithelial cells. Materials and methods Two inflammatory bovine mammary alveolar cell-T (MAC-T) models were established by infecting the cells with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The expressions of pro-inflammatory cytokines were measured, and the proliferation, viability and apoptosis of the inflammatory cells were evaluated after XIST was knocked down by an siRNA. The relationship among XIST, NF-κB pathway and NOD-like receptor protein 3 (NLRP3) inflammasome was investigated using an inhibitor of NF-κB signal pathway. Results The expression of XIST was abnormally increased in bovine mastitic tissues and inflammatory MAC-T cells. Silencing of XIST significantly increased the expression of E. coli or S. aureus-induced pro-inflammatory cytokines. Additionally, knockdown of XIST could inhibit cell proliferation, suppress cell viability and promote cell apoptosis under inflammatory conditions. Furthermore, XIST inhibited E. coli or S. aureus-induced NF-κB phosphorylation and the production of NLRP3 inflammasome. Conclusions The expression of XIST was promoted by activated NF-κB pathway and, in turn, XIST generated a negative feedback loop to regulate NF-κB/NLRP3 inflammasome pathway for mediating the process of inflammation.

MicroRNA-130a controls bone marrow mesenchymal stem cell differentiation towards the osteoblastic and adipogenic fate.

Abstract: Objectives With age, bone marrow mesenchymal stem cells (BMSC) have reduced ability of differentiating into osteoblasts but have increased ability of differentiating into adipocytes which leads to age-related bone loss. MicroRNAs (miRNAs) play major roles in regulating BMSC differentiation. This paper explored the role of miRNAs in regulating BMSC differentiation swift fate in age-related osteoporosis. Material and methods Mice and human BMSC were isolated from bone marrow, whose miR-130a level was measured. The abilities of BMSC differentiate into osteoblast or fat cell under the transfected with agomiR-130a or antagomiR-130a were analysed by the level of ALP, osteocalcin, Runx2, osterix or peroxisome proliferator-activated receptorγ (PPARγ), Fabp4. Related mechanism was verified via qT-PCR, Western blotting (WB) and siRNA transfection. Animal phenotype intravenous injection with agomiR-130a or agomiR-NC was explored by Micro-CT, immunochemistry and calcein double-labelling. Results MiR-130a was dramatically decreased in BMSC of advanced subjects. Overexpression of miR-130a increased osteogenic differentiation of BMSC and attenuated adipogenic differentiation in BMSC, conversely, Inhibition of miR-130a reduced osteogenic differentiation and facilitated lipid droplet formation. Consistently, overexpression of miR-130a in elderly mice dropped off the bone loss. Furthermore, the protein levels of Smad regulatory factors 2 (Smurf2) and PPARγ were regulated by miR-130a with an negative effect through directly combining the 3'UTR of Smurf2 and PPARγ. Conclusions The results indicated that miR-130a promotes osteoblastic differentiation of BMSC by negatively regulating Smurf2 expression and suppresses adipogenic differentiation of BMSC by targeting the PPARγ, and supply a new target for clinical therapy of age-related bone loss.

Emerging roles of long non‐coding RNAs in neuropathic pain

Abstract: Neuropathic pain, a type of chronic and potentially disabling pain resulting from primary injury/dysfunction of the somatosensory nervous system and spinal cord injury, is one of the most intense types of chronic pain, which incurs a significant economic and public health burden. However, our understanding of its cellular and molecular pathogenesis is still far from complete. Long non-coding RNAs (lncRNAs) are important regulators of gene expression and have recently been characterized as key modulators of neuronal functions. Emerging evidence suggested that lncRNAs are deregulated and play pivotal roles in the development of neuropathic pain. This review summarizes the current knowledge about the roles of deregulated lncRNAs (eg, KCNA2-AS, uc.48+, NONRATT021972, MRAK009713, XIST, CCAT1) in the development of neuropathic pain. These studies suggested that specific regulation of lncRNAs or their downstream targets might provide novel therapeutic avenues for this refractory disease.

Aspirin inhibits inflammation and scar formation in the injury tendon healing through regulating JNK/STAT-3 signalling pathway.

Abstract: Objectively Tendinopathy is a common problem in sports medicine which can lead to severe morbidity. Aspirin, as the classical representative of non-steroidal anti-inflammatory drugs (NSAIDs) for its anti-inflammatory and analgesic actions, has been commonly used in treating tendinopathy. While its treatment effects on injury tendon healing are lacking, illuminating the underlying mechanism may provide scientific basis for clinical treatment. Materials and methods Firstly, we used immunohistochemistry and qRT-PCR to detect changes in CD14, CD206, iNOS, IL-6, IL-10, MMP-3, TIMP-3, Col-1a1, biglycan, Comp, Fibronectin, TGF-β1，ACAN，EGR-1 and FMOD. Next, Western blot was used to measure the protein levels (IL-6, IL-10, TGF-β1, COMP, TIMP-3, STAT-3/P-STAT-3 and JNK/P-JNK) in TSCs. Then, migration and proliferation of TSCs were measured through wound healing test and BrdU staining. Finally, the mechanical properties of injury tendon were detected. Results After aspirin treatment, the inflammation and scar formation in injury tendon were significantly inhibited by aspirin. Still, tendon's ECM was positively balanced. Increasing migration and proliferation ability of TSCs induced by IL-1β were significantly reversed. JNK/STAT-3 signalling pathway participated in the process above. In addition, biomechanical properties of injury tendon were significantly improved. Conclusions Taken together, the findings suggested that aspirin inhibited inflammation and scar formation via regulation of JNK/STAT-3 signalling and decreased rerupture risk of injury tendon. Aspirin could be an ideal therapeutic strategy in tendon injury healing.

PD-1/PD-L1 blockade rescue exhausted CD8+ T cells in gastrointestinal stromal tumours via the PI3K/Akt/mTOR signalling pathway.

Abstract: Objectives Although targeted therapy has revolutionized the treatment of gastrointestinal stromal tumours (GIST), it is almost never curative in GIST, and resistance commonly develops. One potential strategy is to combine targeted therapy with immunotherapy. Materials and methods We first studied Programmed cell death 1 ligand 1 (PD-L1) expression and tumour-infiltrating T cells (TILs) in GIST. IFN-γ was used to induce the upregulation of PD-L1 expression in GIST-882 cells, a well-known GIST cell line. CD8+ T-cell apoptosis was determined by flow cytometry. The PI3K/Akt/mTOR levels in CD8+ T cells were examined by Western blotting. Results PD-L1 expression was an independent factor of poor prognosis in GIST and resulted in exhausted T cells in the TILs population or the blood. Then, we found that PD-L1 blockade alone could not increase tumour cell apoptosis in GIST. The apoptosis rate of CD8+ T cells was higher when T cells were cultured with PD-L1+ GIST-882 cells (GIST-882 cells with high PD-L1 expression) than when T cells were cultured with control GIST-882 cells. However, when the PD-L1 blockade was used, the apoptosis rates of the CD8+ T cells in the two groups became similar. Then, Western blotting showed the PI3K/Akt/mTOR levels of the CD8+ T cells rescued by the PD-1/PD-L1 blockade were higher than those of the CD8+ T cells not treated with the PD-1/PD-L1 blockade. Conclusions PD-L1 expression was an independent poor prognosis factor in GIST. PD-1/PD-L1 blockade rescued exhausted CD8+ T cells in GIST via the PI3K/Akt/mTOR signalling pathway. In GIST, PD-1/PD-L1 not only function as predictive biomarkers but also improve current therapies as treatment targets.

Circ_0078767 suppresses non-small-cell lung cancer by protecting RASSF1A expression via sponging miR-330-3p.

Abstract: Objectives This study was designed to investigate the role of circ_0078767/miR-330-3p/RASSF1A in non-small-cell lung cancer (NSCLC). Bioinformatic analysis was performed to screen for the differentially expressed genes in NSCLC tissues from adjacent lung tissues. Materials and methods qRT-PCR was used to detect the RNA expression of genes in cells and tissues, and Western blot was conducted to determine the protein levels of RASSF1A in tissues and cells. A miRanda algorithm was used to predict the targeted relationship among RNAs. A dual-luciferase reporter gene assay was conducted to verify the targeted relationship. Flow cytometry was performed to investigate the effects of circ_0078767/miR-330-3p/RASSF1A on cell cycle progression and apoptosis. A CCK-8 assay was conducted to explore the effects of circ_0078767/miR-330-3p/RASSF1A on cell proliferation. A transwell invasion assay was completed to study the effects of circ_0078767/miR-330-3p/RASSF1A on cell invasion. Lastly, an in vivo assay was conducted to investigate the effects of circ_0078767/miR-330-3p/RASSF1A on tumour development. Results Circ_0078767 and RASSF1A were downregulated, while miR-330-3p was upregulated in NSCLC tissues than that in adjacent tissues. miR-330-3p had a binding relationship with circ_0078767 and RASSF1A. The overexpression of circ_0078767 and RASSF1A or the underexpression of miR-330-3p significantly suppressed NSCLC cell viability, cell cycle progression and invasion while also significantly promoting cell apoptosis. Additionally, these modulations significantly suppressed in vivo tumour growth. Conclusions Circ_0078767 could suppress NSCLC progression by inhibiting miR-330-3p, which thereby increased RASSF1 levels.

Blockage of SLC31A1-dependent copper absorption increases pancreatic cancer cell autophagy to resist cell death.

Abstract: Objectives Clinical observations have demonstrated that copper levels elevate in several cancer types, and copper deprivation is shown to inhibit tumour angiogenesis and growth in both animal models and preclinical trials. However, the content of copper in pancreatic duct adenocarcinoma (PDAC) and whether it is a potential therapy target is still unknown. Materials and methods The levels of copper in PDAC specimens were detected by ICP-MS assays. Copper depletion in Panc-1 or MiaPaCa-2 cells was conducted via copper transporter 1 (SLC31A1) interference and copper chelator tetrathiomolybdate (TM) treatment. The effects of copper deprivation on cancer cells were evaluated by cell proliferation, migration, invasion, colony formation and cell apoptosis. The mechanism of copper deprivation-caused cancer cell quiescence was resolved through mitochondrial dysfunction tests and autophagy studies. The tumour-suppression experiments under the condition of copper block and/or autophagy inhibition were performed both in vitro and in xenografted mice. Results SLC31A1-dependent copper levels are correlated with the malignant degree of pancreatic cancer. Blocking copper absorption could inhibit pancreatic cancer progression but did not increase cell death. We found that copper deprivation increased mitochondrial ROS level and decreased ATP level, which rendered cancer cells in a dormant state. Strikingly, copper deprivation caused an increase in autophagy to resist death of pancreatic cancer cells. Simultaneous treatment with TM and autophagy inhibitor CQ increased cell death of cancer cells in vitro and retarded cancer growth in vivo. Conclusions These findings reveal that copper deprivation-caused cell dormancy and the increase in autophagy is a reason for the poor clinical outcome obtained from copper depletion therapies for cancers. Therefore, the combination of autophagy inhibition and copper depletion is potentially a novel strategy for the treatment of pancreatic cancer and other copper-dependent malignant tumours.

LncRNA NEAT1 promotes the tumorigenesis of colorectal cancer by sponging miR-193a-3p.

Abstract: Objectives LncRNA nuclear-enriched abundant transcript 1 (NEAT1) participates in the development and progression of multiple malignancies. However, the molecular mechanism by which NEAT1 contributes to colorectal cancer (CRC) remains unclear. Methods The association between lncRNA NEAT1 expression and clinicopathological characteristics and prognosis in patients with CRC was analysed by TCGA RNA-sequencing data. MTT, colony formation, flow cytometry, transwell assays and a xenograft tumour model were used to assess the functions of NEAT1. Bioinformatics and spearman correlation analysis were used to identify the NEAT1-specific binding with miRNAs, and luciferase gene report and RIP assays were performed to confirm the interaction between miR-193a-3p (miR-193a) and NEAT1 in CRC cells. Results Upregulation of NEAT1 expression was significantly correlated with TNM stage, poor survival and tumour recurrence in patients with CRC, and acted as an independent prognostic factor for tumour recurrence. Knockdown of NEAT1 suppressed cell proliferation, colony formation abilities and invasive potential and induced cell apoptosis, but overexpression of NEAT1 reversed these effects. Furthermore, NEAT1 was confirmed to act as a sponge of miR-193a, and knockdown of NEAT1 attenuated miR-193a inhibitor-induced tumour promoting effects and L17RD expression in CRC cells. miR-193a harboured negative correlation with NEAT1 and IL17RD expression in CRC specimens. In vivo experiment further validated the inhibitory effects of NEAT1 knockdown on xenograft tumour growth. Conclusion Our findings demonstrate that lncRNA NEAT1 acts as an oncogenic role in CRC cells by sponging miR-193a and may represent a potential marker for CRC patients.

Ginsenoside Rb3 provides protective effects against cisplatin-induced nephrotoxicity via regulation of AMPK-/mTOR-mediated autophagy and inhibition of apoptosis in vitro and in vivo.

Abstract: Objectives: Based on previous reports that ginsenosides have been shown to exert better preventive effects on cisplatin‐induced kidney injury, the present work aims to evaluate the protective effects of ginsenoside Rb3 (G‐Rb3) on cisplatin‐induced renal damage and underlying mechanisms in vivo and in vitro. Materials and methods: The protective effect of G‐Rb3 on cisplatin‐induced acute renal failure in ICR mouse model and HEK293 cell model was investigated, and the underlying possible mechanisms were also explored. For animal experiment, renal function, kidney histology, inflammation, oxidative stress, relative protein molecules involved in apoptosis and autophagy signalling pathways were assessed. In addition, rapamycin (a specific inhibitor of mTOR), compound C (a specific inhibitor of AMPK) and acetylcysteine (NAC, a specific ROS scavenger) were employed to testify the effects of AMPK/mTOR signal pathway on the protective effects of G‐Rb3 in HEK293 cells. Results: Pre‐treatment with G‐Rb3 at doses of 10 and 20 mg/kg for ten days significantly reversed the increases in serum creatinine (CRE), blood urea nitrogen (BUN) and malondialdehyde (MDA), and decrease in glutathione (GSH) content and superoxide dismutase (SOD) activity. Histopathological examination further revealed that G‐Rb3 inhibited cisplatin‐induced nephrotoxicity. G‐Rb3 diminished cisplatin‐induced increase in protein expression levels of p62, Atg3, Atg5 and Atg7, and decrease in protein expression level of p‐mTOR and the ratio of LC3‐I/LC3‐II, indicating that G‐Rb3 suppressed cisplatin‐induced activation of autophagy. Inhibition of autophagy induced inactivation of apoptosis, which suggested that autophagy played an adverse effect on cisplatin‐evoked renal damage. Further, we found that G‐Rb3 might potentially modulate the expressions of AMPK‐related signal pathways. Conclusions: These findings clearly suggested that G‐Rb3‐mediated alleviation of cisplatin‐induced nephrotoxicity was in part due to regulation of AMPK‐/mTOR‐mediated autophagy and inhibition of apoptosis in vitro and in vivo.

Resveratrol enhances the chemotherapeutic response and reverses the stemness induced by gemcitabine in pancreatic cancer cells via targeting SREBP1.

Abstract: Objectives Gemcitabine is a standard treatment for advanced pancreatic cancer patients but can cause chemoresistance during treatment. The chemoresistant cells have features of cancer stem cells (CSCs). Resveratrol has been reported to overcome the resistance induced by gemcitabine. However, the mechanism by which resveratrol enhances chemosensitivity remains elusive. Here, we explored the mechanism by which resveratrol enhanced chemosensitivity and the role of sterol regulatory element binding protein 1 (SREBP1) in gemcitabine-induced stemness. Materials and methods The pancreatic cancer cell lines MiaPaCa-2 and Panc-1 were treated under different conditions. Methyl thiazolyl tetrazolium and colony formation assays were performed to evaluate effects on proliferation. Flow cytometry was conducted to detect apoptosis. Oil red O staining was performed to examine lipid synthesis. The sphere formation assay was applied to investigate the stemness of cancer cells. Immunohistochemistry was performed on tumour tissue obtained from treated KPC mice. Results Resveratrol enhanced the sensitivity of gemcitabine and inhibited lipid synthesis via SREBP1. Knockdown of SREBP1 limited the sphere formation ability and suppressed the expression of CSC markers. Furthermore, suppression of SREBP1 induced by resveratrol reversed the gemcitabine-induced stemness. These results were validated in a KPC mouse model. Conclusions Our data provide evidence that resveratrol reverses the stemness induced by gemcitabine by targeting SREBP1 both in vitro and in vivo. Thus, resveratrol can be an effective chemotherapy sensitizer, and SREBP1 may be a rational therapeutic target.

Circular RNA DOCK1 promotes bladder carcinoma progression via modulating circDOCK1/hsa-miR-132-3p/Sox5 signalling pathway.

Abstract: Objectives To reveal the role of circular RNA (circRNA) DOCK1 (circDOCK1) as a potential biomarker and therapeutic target and its competing endogenous RNA mechanism in bladder carcinoma (BC). Methods The next-generation sequencing (NGS) technology was introduced to screen the circRNA expression profiles of BC using microarray. qPCR and Western blots assay were employed to measure the gene expression in different groups. Cell counting kit-8, EdU and transwell assays were applied to detect the cell viability, proliferation and migration potential, respectively. Luciferase reporter assay was used to test the binds between hsa-miR-132-3p/Sox5. Xenografted tumour growth of nude mice was performed to test the role of circDOCK1 in vivo. Results CircDOCK1 was upregulated in BC tissues and cell lines. Repression of circDOCK1 reduced cell viability, inhibited cell proliferation and curbed the cell migration potential of BC cell. CircDOCK1 played its role via regulation of circDOCK1/hsa-miR-132-3p/Sox5 pathway in BC cells. Suppression circDOCK1 inhibited the tumour growth in vivo. Conclusion In this study, we revealed that circDOCK1 affected the progression of BC via modulation of circDOCK1/hsa-miR-132-3p/Sox5 pathway both in vitro and in vivo and providing a potential biomarker and therapeutic targets for BC.

DANCR promotes HCC progression and regulates EMT by sponging miR-27a-3p via ROCK1/LIMK1/COFILIN1 pathway.

Abstract: Objectives This research aims to verify that the long non-coding RNA differentiation antagonizing nonprotein coding RNA (LncRNA DANCR) could modulate the proliferation and metastasis of hepatocellular carcinoma (HCC), and it thus may work as a novel biomarker to render new orientation for early diagnosis and clinical therapy of HCC. Materials and methods Firstly, qRT-PCR was used to detect the expression of genes including LncRNA DANCR and miR-27a-3p. Next, MTT assay, Ethynyldeoxyuridine (EdU) analysis and clone formation assay were used for investigating cell growth and proliferation. Meanwhile, transwell assay and wound healing assay were applied to evaluate the capacity of cell metastasis and motility, respectively. In addition, bioinformatic analysis and dual-luciferase reporter assay were applied to analyse molecular interaction. Next, we conducted immunofluorescence and Western blot for mechanic investigation. Last but not the least, xenograft tumours in nude mice were built by subcutaneously injecting Hep3B cells stably transfected with sh-NC and sh-DANCR to detect proliferation and SMMC-7721 cells stably transfected with sh-NC and sh-DANCR to investigate metastasis. Results The results of qRT-PCR and bioinformatic analysis revealed the high expression of DANCR in HCC. DANCR accelerated proliferation and metastasis of HCC cells and the knockdown of DANCR had the opposite effect. Meanwhile, xenograft tumours in sh-DANCR group grow slower and have smaller volumes compared with negative control group. Next, the antineoplastic effect of miR-27a-3p on cell growth and motility of HCC was confirmed. In addition, we clarified that DANCR acted as a ceRNA to decoy miR-27a-3p via mediating ROCK1/LIMK1/COFILIN1 pathway. In the end, we validated that DANCR/miR-27a-3p axis regulates EMT progression by cell immunofluorescence and Western blot. Conclusions In a word, DANCR promotes HCC development and induces EMT by decoying miR-27a-3p to regulate ROCK1/LIMK1/COFILIN1 pathway.

Circular RNAs in nucleus pulposus cell function and intervertebral disc degeneration.

Abstract: Intervertebral disc degeneration (IDD) is a common cause of low back pain, which inflicts more global disability than any other condition. Although IDD was deemed to be a natural process that comes with ageing, a growing body of evidence suggested that both genetic and environmental factors could modify the development of IDD. In this connection, aberrant function of nucleus pulposus cells has been implicated in IDD pathogenesis. Circular RNAs are a novel class of endogenous non-coding RNAs that play crucial regulatory roles in diverse cellular processes. Recently, deregulation of circRNAs in nucleus pulposus cells was found to functionally participate in IDD development. In this review, we summarize the current knowledge regarding the deregulation of circRNAs in IDD in relation to their actions on nucleus pulposus cell functions, including cell proliferation, apoptosis and extracellular matrix synthesis/degradation. The potential clinical utilities of circRNAs as therapeutic targets for the management of IDD are also discussed.

Circ-SERPINE2 promotes the development of gastric carcinoma by sponging miR-375 and modulating YWHAZ.

Abstract: Objectives Circular RNAs (circRNAs) exist extensively in the eukaryotic genome. The study aimed to identify the role of hsa_circ_0008365 (Circ-SERPINE2) in gastric carcinoma (GC) cells and its downstream mechanisms. Materials and methods Gene Expression Omnibus (GEO) database was applied to screen differentially expressed circRNAs. CircInteractome, TargetScan and miRecords websites were used to predict target relationships. qRT-PCR and RNase R treatment were utilised to detect molecule expression and confirm the existence of circ-SERPINE2. RNA pull-down assay and dual-luciferase reporter assay were performed for interaction between circRNA and miRNA or mRNA. EdU assay, colony formation assay, and flow cytometry for apoptosis and cell cycle detections were utilised to assess cell function. Western blot and immunohistochemistry (IHC) assays were applied for detection of proteins in tissues or cells. Results Circ-SERPINE2 and YWHAZ were upregulated, and miR-375 was downregulated in GC tissues and cells. Circ-SERPINE2 and YWHAZ targetedly bound to miR-375. Circ-SERPINE2 promoted cell proliferation and cell cycle progress and inhibited cell apoptosis by sponging miR-375 and regulating YWHAZ expression in vitro. Circ-SERPINE2 repressed solid tumour growth through enhancing miR-375 expression and reducing YWHAZ expression in vivo. Conclusions Circ-SERPINE2 is a novel proliferative promoter through the regulation of miR-375/YWHAZ. Circ-SERPINE2/miR-375/YWHAZ axis might provide a novel therapeutic target of GC.

δ-Tocotrienol induces apoptosis, involving endoplasmic reticulum stress and autophagy, and paraptosis in prostate cancer cells.

Abstract: Objectives Prostate cancer, after the phase of androgen dependence, may progress to the castration-resistant prostate cancer (CRPC) stage, with resistance to standard therapies. Vitamin E-derived tocotrienols (TTs) possess a significant antitumour activity. Here, we evaluated the anti-cancer properties of δ-TT in CRPC cells (PC3 and DU145) and the related mechanisms of action. Materials and methods MTT, Trypan blue and colony formation assays were used to assess cell viability/cell death/cytotoxicity. Western blot, immunofluorescence and MTT analyses were utilized to investigate apoptosis, ER stress and autophagy. Morphological changes were investigated by light and transmission electron microscopy. Results We demonstrated that δ-TT exerts a cytotoxic/proapoptotic activity in CRPC cells. We found that in PC3 cells: (a) δ-TT triggers both the endoplasmic reticulum (ER) stress and autophagy pathways; (b) autophagy induction is related to the ER stress, and this ER stress/autophagy axis is involved in the antitumour activity of δ-TT; in autophagy-defective DU145 cells, only the ER stress pathway is involved in the proapoptotic effects of δ-TT; (c) in both CRPC cell lines, δ-TT also induces an intense vacuolation prevented by the ER stress inhibitor salubrinal and the protein synthesis inhibitor cycloheximide, together with increased levels of phosphorylated JNK and p38, supporting the induction of paraptosis by δ-TT. Conclusions These data demonstrate that apoptosis, involving ER stress and autophagy (in autophagy positive PC3 cells), and paraptosis are involved in the anti-cancer activity of δ-TT in CRPC cells.

Long non-coding RNA HCG11 modulates glioma progression through cooperating with miR-496/CPEB3 axis.

Abstract: Objectives It has been widely reported that long non-coding RNAs (lncRNAs) can participate in multiple biological processes of human cancers. lncRNA HLA complex group 11 (HCG11) has been reported in human cancers as a tumour suppressor. This study focused on investigating the function and mechanism of HCG11 in glioma. Materials and methods Based on The Cancer Genome Atlas (TCGA) data set and qRT-PCR analysis, the expression pattern of HCG11 was identified in glioma samples. The mechanism associated with HCG11 downregulation was determined by mechanism experiments. Gain-of-function assays were conducted for the identification of HCG11 function in glioma progression. Mechanism investigation based on the luciferase reporter assay, RIP assay and pull-down assay was used to explore the downstream molecular mechanism of HCG11. The role of molecular pathway in the progression of glioma was analysed in accordance with the rescue assays. Results HCG11 was expressed at low level in glioma samples compared with normal samples. FOXP1 could bind with HCG11 and transcriptionally inactivated HCG11. Overexpression of HCG11 efficiently suppressed cell proliferation, induced cell cycle arrest and promoted cell apoptosis. HCG11 was predominantly enriched in the cytoplasm of glioma cells and acted as a competing endogenous RNAs (ceRNAs) by sponging micro-496 to upregulate cytoplasmic polyadenylation element binding protein 3 (CPEB3). CEPB3 and miR-496 involved in HCG11-mediated glioma progression. Conclusions HCG11 inhibited glioma progression by regulating miR-496/CPEB3 axis.

Overexpression of matrix metalloproteinase-9 in breast cancer cell lines remarkably increases the cell malignancy largely via activation of transforming growth factor beta/SMAD signalling.

Abstract: Objectives Matrix metalloproteinase 9 (MMP-9) has been frequently noticed in the breast cancers. In this study, we aim to investigate the associations of MMP-9 with the activation of transforming growth factor beta (TGF-β)/SMAD signalling and the malignancy of breast malignant tumour cells. Materials and methods The distributions of MMP-9 and TGF-β in the tissues of canine breast cancers were screened by immunohistochemical assays. A recombinant plasmid expressing mouse MMP-9 was generated and transiently transfected into three different breast cancer cell lines. Cell Counting Kit-8 and colony formation assay were used to study cell viability. Migration and invasion ability were analysed by wound assay and transwell filters. Western blot and quantitative real-time PCR were used to determine the protein and mRNA expression. Result Remarkable strong MMP-9 and TGF-β signals were observed in the malignant tissues of canine breast cancers. In the cultured three cell lines receiving recombinant plasmid expressing mouse MMP-9, the cell malignancy was markedly increased, including the cell colony formation, migration and epithelial-mesenchymal transition. The levels of activated TGF-β, as well as SMAD4, SMAD2/3 and phosphorylation of SMAD2, were increased, reflecting an activation of TGF-β/SMAD signalling. We also demonstrated that the inhibitors specific for MMP-9 and TGF-β sufficiently blocked the overexpressing MMP-9 induced the activation of SMAD signalling and enhancement on invasion in the tested breast cancer cell lines. Conclusion Overexpression of MMP-9 increases the malignancy of breast cancer cell lines, largely via activation of the TGF-β/SMAD signalling.

STAT3-induced upregulation of lncRNA ABHD11-AS1 promotes tumour progression in papillary thyroid carcinoma by regulating miR-1301-3p/STAT3 axis and PI3K/AKT signalling pathway.

Abstract: Objectives Emerging evidences indicated the importance of long non-coding RNAs (lncRNAs) in the tumorigenesis and deterioration of malignant tumours. To our knowledge, the study about lncRNAs in papillary thyroid carcinoma (PTC) is still inadequate. ABHD11-AS1 was highly expressed in the PTC samples of The Cancer Genome Atlas database. This study focused on the biological function and mechanism of lncRNA ABHD11-AS1 in PTC. Materials and methods qRT-PCR analysis was used to examine the expression of ABHD11-AS1 in PTC tissues and cell lines. The prognostic significance of ABHD11-AS1 for the patients with PTC was analysed with Kaplan-Meier analysis. The effects of ABHD11-AS1 knockdown on the cell proliferation and metastasis were evaluated by in vitro functional assays and in vivo experiments. The molecular mechanism which contributed to the oncogenic role of ABHD11-AS1 in PTC was explored by conducting mechanism experiments. Rescue assays were carried out for final demonstration. Results High expression of ABHD11-AS1 predicted poor prognosis for patients with PTC and promoted cell proliferation and metastasis in vitro and in vivo. ABHD11-AS1 was activated by the transcription factor STAT3. ABHD11-AS1 positively regulated PI3K/AKT signalling pathway. ABHD11-AS1 acted as a competitive endogenous (ce) RNA to upregulate STAT3 by sponging miR-1301-3p. Conclusions STAT3-induced lncRNA ABHD11-AS1 promoted PTC progression by regulating PI3K/AKT signalling pathway and miR-1301-3p/STAT3 axis.

Elevated levels of hsa_circ_006100 in gastric cancer promote cell growth and metastasis via miR-195/GPRC5A signalling.

Abstract: Objectives Circular RNAs (circRNAs) are non-coding RNAs, some of which are thought to be involved in gastric cancer development. Here, we examined the functions of circRNA hsa_circ_006100 in gastric cancer cells and an animal model of gastric cancer. Materials and methods The expression of hsa_circ_006100, miR-195 and various functional genes was determined by quantitative RT-PCR. Cell viability, clone formation, apoptosis and cell migration/invasion abilities were analysed by the CCK-8 assay, crystal violet staining, Hoechst staining and Transwell assay, respectively. A tumour model was established by subcutaneously injecting tumour cells into nude mice. Levels of protein expression were analysed by Western blotting and immunohistochemistry. Results A bioinformatics analysis showed that miR-195 was negatively co-expressed with hsa_circ_006100. Patients with a high hsa_circ_006100 level or low miR-195 level had tumours with a high TNM stage, poor cellular differentiation and lymph node metastasis. miR-195 was targeted and inhibited by hsa_circ_006100. Overexpression of hsa_circ_006100 enhanced cellular viability and proliferation, while miR-195 suppressed hsa_circ_006100-enhanced cell growth and induced apoptosis in MGC-803 and AGS cells. Forced hsa_circ_006100 expression promoted the migration and invasion of MGC-803 and AGS cells, while those activities were inhibited by miR-195. Mechanistically, GPRC5A was predicted as a target of miR-195 and was upregulated in gastric cancer. A miR-195 inhibitor restored cell viability, proliferation, migration and invasion, and repressed apoptosis via GPRC5A. In vivo studies showed that knockdown of hsa_circ_006100 delayed tumour growth, reduced PCNA expression and upregulated miR-195 and BCL-2 expression which was restored by miR-195 inhibition due to GPRC5A/EGFR signalling, and changed the EMT phenotype in vivo. Conclusions Hsa_circ_006100 functions as an oncogene in gastric cancer and exerts its effects via miR-195/GPRC5A signalling.

Mechanisms of Panax ginseng action as an antidepressant

Abstract: Objectives Panax ginseng, a well-known traditional Chinese medicine with multiple pharmacological activities, plays a crucial role in modulating mood disorders. Several recent studies have identified an underlying role of Panax ginseng in the prevention and treatment of depression. However, the cellular and molecular mechanisms remain unclear. Materials and methods In this review, we summarized the recent progress of antidepressant effects and underlying mechanisms of Panax ginseng and its representative herbal formulae. Results The molecular and cellular mechanisms of Panax ginseng and its herbal formulae include modulating monoamine neurotransmitter system, upregulating the expression of neurotrophic factors, regulating the function of HPA axis, and anti-inflammatory action. Conclusions Therefore, this review may provide theoretical bases and clinical applications for the treatment of depression by Panax ginseng and its representative herbal formulae.

QingBai decoction regulates intestinal permeability of dextran sulphate sodium-induced colitis through the modulation of notch and NF-κB signalling

Abstract: Objective Chinese Herb QingBai decoction (QBD) has been approved affective in the treatment of IBD patients in clinic. However, the underlying mechanism remains unknown. We aim to investigate the effect of QBD on the mouse model of ulcerative colitis and its possible mechanism. Methods C57/bL mice were given 5% DSS to induce colitis and were divided as QBD and mesalazine group. Weight, faeces and mental status were recorded each day and the histopathological changes (goblet cells etc) of the colon were observed after sacrificed. Fluorescein isothiocyanate-dextran 4000 was measured to reflect the intestinal mucosal permeability. In addition, cell junction-related proteins and possible signal pathways were investigated. Results QingBai decoction could significantly alleviate the inflammation and the protection effect of colitis is comparable as those in mesalazine enema group. It was found that the permeability reduced significantly with QBD treatment vs the control group, while no significant difference between the mesalazine and QBD groups. QBD treatment could upregulate the expression of tight junction complex（ZO-1, claudin-1 and occludin）and muc-2 expression. It significantly reduced the production and secretion of serials proinflammatory cytokines (IL-1β, IL-6, Kc and TNF-α) compared with the control group. Meanwhile, NF-κB and Notch pathways were regulated. Conclusion QingBai decoction can effectively alleviate intestinal inflammation and mucosal barrier function in colitis mice, and the mechanism may be related to the inhibition of inflammatory cascade as well as enhanced mucus layer barrier and mechanical barrier function by NF-κB and Notch signalling.

Inhibition of CREB-mediated ZO-1 and activation of NF-κB-induced IL-6 by colonic epithelial MCT4 destroys intestinal barrier function.

Abstract: Objective Inflammatory bowel disease (IBD) is a disorder intestinal inflammation and impaired barrier function, associated with increased epithelial expression of monocarboxylate transporter 4 (MCT4). However, the specific non-metabolic function and clinical relevance of MCT4 in IBD remain to be fully elucidated. Methods Lentivirus-mediated overexpression of MCT4 was used to assess the role of MCT4 in transcriptionally regulating ZO-1 and IL-6 expression by luciferase assays, WB and ChIP. IP was used to analyse the effect of MCT4 on the interaction NF-κB-CBP or CREB-CBP, and these MCT4-mediated effects were confirmed in vivo assay. Results We showed that ectopic expression of MCT4 inhibited ZO-1 expression, while increased pro-inflammatory factors expression, leading to destroy intestinal epithelial barrier function in vitro and in vivo. Mechanistically, MCT4 contributed NF-κB p65 nuclear translocation and increased the binding of NF-κB p65 to the promoter of IL-6, which is attributed to MCT4 enhanced NF-κB-CBP interaction and dissolved CREB-CBP complex, resulting in reduction of CREB activity and CREB-mediated ZO-1 expression. In addition, treatment of experimental colitis with MCT4 inhibitor α-cyano-4-hydroxycinnamate (CHC) ameliorated mucosal intestinal barrier function, which was due to attenuation of pro-inflammation factors expression and enhancement of ZO-1 expression. Conclusion These findings suggested a novel role of MCT4 in controlling development of IBD and provided evidence for potential targets of IBD.

LncRNA LOXL1-AS1 facilitates the tumorigenesis and stemness of gastric carcinoma via regulation of miR-708-5p/USF1 pathway.

Abstract: Objectives As one of the most life-threatening malignancies, gastric cancer is the third contributor of cancer mortalities globally. Increasing studies have proven the regulatory roles of lncRNAs in the development of diverse malignant tumours. But little is known about its function and molecular mechanism in gastric carcinoma. Materials and methods RT-qPCR was performed to measure the expression pattern of LOXL1-AS1 in gastric cancer. To ascertain its definite role, CCK-8, EdU, Western blot, transwell and sphere formation assays were adopted. RNA pull-down, RIP, ChIP and luciferase reporter assays were carried out to investigate the molecular mechanism of LOXL1-AS1 in gastric carcinoma. Results LOXL1-AS1 was highly expressed in tissues and cells of gastric cancer. The upregulation of LOXL1-AS1 predicted poor prognosis in gastric carcinoma. Our findings demonstrated that LOXL1-AS1 accelerated the deterioration of gastric cancer by inducing cell proliferation, migration, EMT and stemness. Moreover, the expression of USF1 in gastric cancer was higher than in normal control and LOXL1-AS1 negatively modulated USF1. Functionally, LOXL1-AS1 acted as a ceRNA to upregulate USF1 via sponging miR-708-5p. Besides, we confirmed USF1 promoted the transcription of stemness marker SOX2. Rescue experiments testified the stimulative role of LOXL1-AS1/miR-708-5p/USF1 pathway in gastric cancer progression. It was also validated that LOXL1-AS1 facilitated cell growth of gastric carcinoma in vivo. Conclusions Our study unravelled that LOXL1-AS1/miR-708-5p/USF1 pathway contributed to the development of gastric cancer.

MALAT1 accelerates the development and progression of renal cell carcinoma by decreasing the expression of miR-203 and promoting the expression of BIRC5.

Abstract: Objective We aimed to investigate the roles of the lncRNA MALAT1 in renal cell carcinoma (RCC) progression. Methods qRT-PCR was used for the assessment of BIRC5, miRNA-203 and MALAT1 expression. Furthermore, the targeted relationships between miR-203 and BIRC5, as well as MALAT1 and miR-203, were predicted by the miRanda/starBase database and verified by dual-luciferase reporter gene assay. The effects of MALAT1, miRNA-203 and BIRC5 on cell proliferation, cell cycle, cell apoptosis, cell invasion and cell migration were studied by using CCK-8, flow cytometry, transwell and wound healing assays, respectively. In addition, the effects of MALAT1 on RCC tumorigenesis were evaluated in vivo by nude mouse tumorigenesis. Results The expression levels of BIRC5 and MALAT1 were higher in RCC tissues and cell lines than in adjacent normal tissues and a normal renal cortex proximal tubule epithelial cell line. In contrast, the expression of miRNA-203 in RCC tissues and cell lines was higher than that in adjacent normal tissues and a normal renal cortex proximal tubule epithelial cell line. BIRC5 and MALAT1 promoted cell proliferation yet decreased the percentage of RCC cells at G0/G1 phase. Conclusions Our study demonstrated that MALAT1 functions as a miR-203 decoy to increase BIRC5 expression in RCC.

Long non-coding RNA MYOSLID functions as a competing endogenous RNA to regulate MCL-1 expression by sponging miR-29c-3p in gastric cancer.

Abstract: Objective Long non-coding RNA (lncRNA) has become an important regulator of many human malignancies. However, the biological role and clinical significance of most lncRNA in gastric cancer (GC) remain unclear. Methods We investigate the biological function, mechanism of action and clinical expression of lncRNA MYOSLID in GC. First, we analysed the differential expression of lncRNA MYOSLID in GC tissues and non-cancerous tissues by analysing the sequencing data obtained from The Cancer Genome Atlas. Subsequently, we verified that lncRNA MYOSLID regulates the proliferation and apoptosis of GC cells by acting as a ceRNA against miR-29c-3p. The nude mouse xenograft was used to further confirm the functional significance of lncRNA MYOSLID in vivo. Results We found for the first time that the expression of lncRNA MYOSLID was significantly up-regulated in GC tissues, and the up-regulation of lncRNA MYOSLID in GC was correlated with tumour size, AJCC stage, depth of invasion and survival time. In addition, apoptosis and growth arrest can be induced in vitro after knockdown of lncRNA MYOSLID, which inhibits tumorigenesis in mouse xenografts in vivo. Further in-depth studies revealed that lncRNA MYOSLID acts as a ceRNA of miR-29c-3p, resulting in de-repression of its downstream target gene MCL-1. Conclusion The lncRNA MYOSLID-miR-29c-3p-MCL-1 axis plays a key role in the development of GC. Our findings may provide potential new targets for the diagnosis and treatment of human GC.

Aptamer-targeted DNA nanostructures with doxorubicin to treat protein tyrosine kinase 7-positive tumours

Abstract: Objectives Aptamer sgc8c is a short DNA sequence that can target protein tyrosine kinase 7 (PTK7), which was overexpressed on many tumour cells. This study aimed to fabricate a novelty DNA nanostructure drug delivery system target on PTK7-positive cells-CCRF-CEM (human T-cell ALL). Methods Aptamer-modified tetrahedron DNA was synthesized through one-step thermal annealing process. The sgc8c-TDNs (s-TDNs) loading DOX complexes were applied to investigate the effect to PTK7-negative and -positive cells. Results When s-TDN:DOX acted on PTK7-positive and -negative cells respectively, the complexes exhibited specific toxic effect on PTK7-positive cells but not on PTK7-negative Ramos cells in vitro research. Conclusions In this work, we successfully constructed a PTK7-targeting aptamer-guided DNA tetrahedral nanostructure (s-TDN) as a drug delivery system via a facile one-pot synthesis method. The results showed that s-TDN:DOX exhibited enhanced cytotoxicity against PTK7-positive CCRF-CEM cells, with a minor effect against PTK7-negative Ramos cells. Hence, this functionalized TDNs drug delivery system displayed its potential application in targeting PTK7-positive tumour T-cell acute lymphoblastic leukaemia.

Induction of neutrophil extracellular traps during tissue injury: Involvement of STING and Toll-like receptor 9 pathways.

Abstract: Objectives Neutrophils are thought to release neutrophil extracellular traps (NETs) to form in response to exogenous bacteria, viruses and other pathogens. However, the mechanisms underlying NET formation during sterile inflammation are still unclear. In this study, we would like to identify neutrophil extracellular traps formation during sterile inflammation and tissue injury and associated pathways and its mechanism. Materials and methods We identified different injuries such as chemical-induced and trauma-induced formation of NETs and investigated mechanism of the formation of NETs in vitro and in vivo during the treatment of mtDNA. Results Here, we find the release of mitochondrial DNA (mtDNA) and oxidized mtDNA in acute peripheral tissue trauma models or other chemically induced lung injury, and moreover, endogenous mtDNA and oxidized mtDNA induce the formation of NETs and sterile inflammation. Oxidized mtDNA is a more potent inducer of NETs. Mitochondrial DNA activates neutrophils via cyclic GMP-AMP synthase (cGAS)-STING and the Toll-like receptor 9 (TLR9) pathways and increases the production of neutrophil elastase and extracellular neutrophil-derived DNA in NETs. Mitochondrial DNA also increases the production of reactive oxygen species (ROS) and expression of the NET-associated proteins Rac 2 and peptidylarginine deiminase 4 (PAD4). Conclusions Altogether, these findings highlight that endogenous mitochondrial DNA inducted NETs formation and subsequent sterile inflammation and the mechanism associated with NET formation.