Objectives Carnosine (beta-alanyl-l-histidine) is a naturally occurring dipeptide that selectively inhibits cancer cell growth, possibly by influencing glucose metabolism. As its precise mode of action and its primary targets are unknown, we analysed carnosine's effect on metabolites and pathways in glioblastoma cells. Materials and methods Glioblastoma cells, U87, T98G and LN229, were treated with carnosine, and metabolites were analysed by gas chromatography coupled with mass spectrometry. Furthermore, mitochondrial ATP production was determined by extracellular flux analysis and reaction products of carnosine were investigated using mass spectrometry. Results Carnosine decreased the intracellular abundance of several metabolites indicating a reduced activity of the pentose phosphate pathway, the malate-aspartate shuttle and the glycerol phosphate shuttle. Mitochondrial respiration was reduced in U87 and T98G but not in LN229 cells, independent of whether glucose or pyruvate was used as substrate. Finally, we demonstrate non-enzymatic reaction of carnosine with dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. However, glycolytic flux from glucose to l-lactate appeared not to be affected by the reaction of carnosine with the metabolites. Conclusions Carnosine reacts non-enzymatically with glycolytic intermediates reducing the activity of the pentose phosphate pathway which is required for cell proliferation. Although the activity of the malate-aspartate and the glycerol phosphate shuttle appear to be affected, reduced mitochondrial ATP production under the influence of the dipeptide is cell-specific and appears to be independent of the effect on the shuttles.
Objectives The osteogenesis differentiation of human bone marrow stem cells (BMSCs) is essential for bone formation and bone homeostasis. In this study, we aim to elucidate novel molecular targets for bone metabolism diseases. Materials and methods The dataset GSE80614 which includes mRNA expression profile during BMSCs osteogenic differentiation was obtained from the GEO database (). The osteogenic differentiation of BMSCs was measured by ALP staining, AR staining and expression of osteogenic markers in vitro. For in vivo assay, we seeded BMSCs onto beta-tricalcium phosphate (beta-TCP) and transplanted them into muscle pockets of nude mice. Luciferase assay, co-immunoprecipitation assay and in vitro ubiquitination assay were carried out to investigate the molecular mechanism. Results We found that alpha-B-crystallin (CRYAB) expression was elevated during the process of BMSCs osteogenic differentiation. Further studies showed that upregulation of CRYAB significantly enhanced the osteogenic differentiation, while downregulation of CRYAB suppressed it. CRYAB regulated BMSCs osteogenic differentiation mainly through the canonical Wnt/beta-catenin signalling. In addition, we found that CRYAB could physically interact with beta-catenin and protect it from ubiquitination and degradation, which stabilized beta-catenin and promoted the Wnt signalling. Conclusions The present study provides evidences that CRYAB is an important regulator of BMSCs osteogenic differentiation by protecting beta-catenin from ubiquitination and degradation and promoting the Wnt signalling. It may serve as a potential therapeutic target for diseases related to bone metabolism.
Objective Withaferin A (WA) is a bioactive compound with a remarkable anti-cancer effect derived from Withania somnifera, commonly known as ashwagandha. However, the anti-cancer mechanisms of WA in glioblastoma multiforme (GBM) are still unclear. Materials and Methods Cell viability assays and xenografted nude mice were used to evaluate the effects of WA, along with flow cytometry to detect apoptosis and cell cycle of GBM. RNA-seq analysis, Western blotting, immunofluorescence staining, qRT-PCR and siRNA gene silencing were carried out to determine the signalling pathways affected by WA. Results Withaferin A significantly inhibited the growth of GBM in vitro and in vivo and triggered the intrinsic apoptosis of GBM cells by up-regulating expression of Bim and Bad. WA arrested GBM cells at the G2/M phase of the cell cycle through dephosphorylating Thr(161) of CDK1 by activating p53-independent p21 up-regulation. Knockdown of p21 restored cell cycle progression and cell viability by down-regulating the expression of Bad rather than Bim. We demonstrated that endoplasmic reticulum (ER) stress induced by WA through the ATF4-ATF3-CHOP axis, initiated apoptosis and G2/M arrest in GBM cells. Conclusion We revealed a novel pathway that elucidated WA activation of apoptosis and G2/M arrest in GBM cells through the ATF4-ATF3-CHOP axis. This discovery is important for optimization of WA-based regimens for prevention and/or treatment of GBM.
ObjectivesDue to the instability of microRNAs, the applications of microRNA are currently limited. Thus, we utilized tetrahedral framework nucleic acids and a targeted microRNAs to form a stable nanocomposite to explore whether this nanocomposite can promote apoptosis of tumour cells. Materials and methodsIn our study, the survivin gene, which is expressed only in tumour cells and embryonic cells, was selected as the target gene; miRNA-214-3p, which can reduce the expression of survivin, was modified onto tetrahedral framework nucleic acid, thereby producing a reduction in the expression of survivin upon intracellular delivery and eventually leading to tumour cell apoptosis. ResultsBy comparing the stability of microRNAs with that of microRNA-tetrahedral framework nucleic acid, we proved the superiority of this carrier system. The results of flow cytometry showed that after treated with this complex, the ratio of A549 cells in both late and early period of apoptosis in miRNA-214-3p-tetrahedral framework nucleic acid group had doubled and the cell cycle in the G2-M phase had declined. The decrease in the expression of anti-apoptotic protein and the increase in the expression of pro-apoptotic protein indicate that the ability of this complex to function in cells also makes it attractive as a new targeted therapy for cancer. ConclusionThe unique expression of survivin in tumour cells and embryonic cells makes microRNA-tetrahedral framework nucleic acid a new targeted therapy. In addition, due to the functional diversity of microRNAs, this delivery system approach can be applied to a wide variety of fields, such as targeted therapy and tissue regeneration.
Objectives Aconitine, the natural product extracted from Aconitum species, is widely used for the treatment of various diseases, including rheumatism, arthritis, bruises, fractures and pains. However, many studies have reported cardiotoxicity and neurotoxicity caused by aconitine, but the detailed mechanism underlying aconitine's effect on these processes remains unclear. Materials and methods The effects of aconitine on the inflammation, apoptosis and viability of H9c2 rat cardiomyocytes were evaluated by flow cytometry, Western blot, RNA sequencing and bioinformatics analysis. Results Aconitine suppressed cardiomyocyte proliferation and induced inflammation and apoptosis in a dose- and time-dependent manner. These inflammatory damages could be reversed by a TNF alpha inhibitor and BNIP3-mediated mitophagy. Consistent with the in vitro results, overexpression of BNIP3 in heart tissue partially suppressed the cardiotoxicity of aconitine by inhibiting apoptosis and the NLRP3 inflammasome. Conclusions Our findings lay a foundation for the application of a TNF alpha inhibitor and BNIP3 to aconitine-induced cardiac toxicity prevention and therapy, thereby demonstrating potential for further investigation.
Objectives Long non-coding RNA (lncRNA) MATN1-AS1 is a newfound lncRNA that has been rarely explored in cancers. Herein, we would like to investigate its role in glioma. Materials and methods qRT-PCR was conducted to examine gene expression in glioma. Then, MTT assay, colony formation assay and flow cytometry analysis were applied to evaluate the function of MATN1-AS1 on glioma cells. Western blot was performed to measure the protein levels of genes. Besides, the luciferase reporter assay, RNA pull-down assay, RIP assay and Spearman's correlation analysis were also performed as needed. Results Firstly, a data from TCGA showed that MATN1-AS1 might be largely implicated in glioma. Meanwhile, MATN1-AS1 upregulation confirmed in glioma predicted poor clinical outcomes. Functionally, MATN1-AS1 knockdown restrained cell proliferation but stimulated apoptosis in vitro and repressed tumour growth in vivo. Mechanistic investigations validated that MATN1-AS1 functioned as a ceRNA for miR-200b/c/429 to upregulate CHD1 which was also verified to exert a growth-promoting role in glioma cells here. Importantly, both CHD1 overexpression and miR-200b/c/429 inhibition could rescue the obstructive role of MATN1-AS1 silence in glioma cells. Conclusions MATN1-AS1 promotes glioma progression through regulating miR-200b/c/429-CHD1 axis, suggesting MATN1-AS1 as a probable target for glioma treatment.
Objectives DISHEVELLED, EGL-10, PLECKSTRIN (DEP) domain-containing 1B (DEPDC1B) promotes dismantling of focal adhesions and coordinates detachment events during cell cycle progression. DEPDC1B is overexpressed in several cancers with expression inversely correlated with patient survival. Here, we analysed the role of DEPDC1B in the regulation of murine and human skeletal myogenesis. Materials and methods Expression dynamics of DEPDC1B were examined in murine and human myoblasts and rhabdomyosarcoma cells in vitro by RT-qPCR and/or immunolabelling. DEPDC1B function was mainly tested via siRNA-mediated gene knockdown. Results DEPDC1B was expressed in proliferating murine and human myoblasts, with expression then decreasing markedly during myogenic differentiation. SiRNA-mediated knockdown of DEPDC1B reduced myoblast proliferation and induced entry into myogenic differentiation, with deregulation of key cell cycle regulators (cyclins, CDK, CDKi). DEPDC1B and beta-catenin co-knockdown was unable to rescue proliferation in myoblasts, suggesting that DEPDC1B functions independently of canonical WNT signalling during myogenesis. DEPDC1B can also suppress RHOA activity in some cell types, but DEPDC1B and RHOA co-knockdown actually had an additive effect by both further reducing proliferation and enhancing myogenic differentiation. DEPDC1B was expressed in human Rh30 rhabdomyosarcoma cells, where DEPDC1B or RHOA knockdown promoted myogenic differentiation, but without influencing proliferation. Conclusion DEPDC1B plays a central role in myoblasts by driving proliferation and preventing precocious myogenic differentiation during skeletal myogenesis in both mouse and human.
Scoliosis, a complex three-dimensional deformity of the spine with the Cobb angle (a measure of the spinal lateral curvature) >10 degree, encompasses a spectrum of pathologies, including congenital, idiopathic, syndromic and neuromuscular aetiologies. The pathogenesis is multifactorial involving both environmental and genetic factors but the exact cellular and molecular mechanisms of disease development remain largely unknown. Emerging evidence showed that non-coding RNAs (ncRNAs), namely microRNAs, long ncRNAs and circular RNAs, are deregulated in many orthopaedic diseases, including scoliosis. Importantly, these deregulated ncRNAs functionally participate in the initiation and progression of scoliosis. Here, we review recent progress in ncRNA research on scoliosis.
Objective The long non-coding RNA zinc finger E-box-binding homeobox 1 antisense 1 (ZEB1-AS1) acts as an oncogenic regulator in many human tumours. In the present study, we identify the role and potential molecular biological mechanisms of ZEB1-AS1 in colon adenocarcinoma (COAD). Methods QRT-PCR was used to detect the expression of ZEB1-AS1, miR-455-3p and p21-activated kinases 2 (PAK2) in COAD tissues. CCK8 assay, EdU assay, transwell assay and scratch wound assay were used to explore the biological function of ZEB1-AS1 in COAD cells. Bioinformatics, luciferase reporter assays and an RNA pull-down assay were used to demonstrate the mechanism of ZEB1-AS1. We further explore the role of ZEB1-AS1 in vivo though xenograft tumour assay. Results We found that ZEB1-AS1 expression was significantly up-regulated in COAD tissues, and high ZEB1-AS1 level was correlated with the poor prognosis of COAD patients. MiR-455-3p plays an anti-cancer role in COAD by targeting PAK2. We confirmed that ZEB1-AS1 promotes PAK2 expression by sponging miR-455-3p, thus facilitating COAD cell growth and metastasis. Conclusions To sum up, this result illustrates the novel molecular mechanism of ZEB1-AS1 in COAD and provides a new target for the diagnosis and treatment of COAD patients.
Objectives Many cancer cells depend on G2 checkpoint mechanism regulated by WEE family kinases to maintain genomic integrity. The PKMYT1 gene, as a member of WEE family kinases, participates in G2 checkpoint surveillance and probably links with tumorigenesis, but its role in breast cancer remains largely unclear. Materials and Methods In this study, we used a set of bioinformatic tools to jointly analyse the expression of WEE family kinases and investigate the prognostic value of PKMYT1 in breast cancer. Results The results indicated that PKMYT1 is the only frequently overexpressed member of WEE family kinases in breast cancer. KM plotter data suggests that abnormally high expression of PKMYT1 predicts poor prognosis, especially for some subtypes, such as luminal A/B and triple-negative (TNBC) types. Moreover, the up-regulation of PKMYT1 was associated with HER2-positive (HER2+), basal-like (Basal-like), TNBC statuses and increased classifications of Scarff, Bloom and Richardson (SBR). Co-expression analysis showed PKMYT1 has a strong positive correlation with Polo-like kinase 1 (PLK1), implying they may cooperate in regulating cancer cell proliferation by synchronizing rapid cell cycle with high quality of genome maintenance. Conclusions Collectively, this study demonstrates that overexpression of PKMYT1 is always found in breast cancer and predicts unfavourable prognosis, implicating it as an appealing therapeutic target for breast carcinoma.
Objectives Mixed lineage leukaemia protein-1 (MLL1) mediates histone 3 lysine 4 (H3K4) trimethylation (me3) and plays vital roles during early embryonic development and hematopoiesis. In our previous study, we found its expression was positively correlated with embryonic myogenic ability in pigs, indicating its potential roles in mammalian muscle development. The present work aimed to explore the roles and regulation mechanisms of MLL1 in myogenesis. Materials and methods The expression of MLL1 in C2C12 cells was experimentally manipulated using small interfering RNAs (siRNA). 5-ethynyl-2 '-deoxyuridine (EdU) assay, cell cycle assay, immunofluorescence, qRT-PCR and Western blot were performed to assess myoblast proliferation and differentiation. Chromatin immunoprecipitation assay was conducted to detect H3K4me3 enrichment on myogenic factor 5 (Myf5) promoter. A cardiotoxin (CTX)-mediated muscle regeneration model was used to investigate the effects of MLL1 on myogenesis in vivo. Results MLL1 was highly expressed in proliferating C2C12 cells, and expression decreased after differentiation. Knocking down MLL1 suppressed myoblast proliferation and impaired myoblast differentiation. Furthermore, knockdown of MLL1 resulted in the arrest of cell cycle in G1 phase, with decreased expressions of Myf5 and Cyclin D1. Mechanically, MLL1 transcriptionally regulated Myf5 by mediating H3K4me3 on its promoter. In vivo data implied that MLL1 was required for Pax7-positive satellite cell proliferation and muscle repair. Conclusion MLL1 facilitates proliferation of myoblasts and Pax7-positive satellite cells by epigenetically regulating Myf5 via mediating H3K4me3 on its promoter.
Objectives Carbon dots (CDs), as a fascinating class of fluorescent carbon nanomaterials, have been proven to be powerful tools in the field of bioimaging and biosensing due to their small size, suitable photostability and favourable biocompatibility. However, the cellular uptake of free CDs lacks selectivity and the same negative charges as cell membranes may cause inefficient cell internalization. In this study, an efficient detecting and targeting nanosystem was developed based on the DNA aptamer AS1411 modified CDs with polyethyleneimine (PEI) as connecting bridge. Materials and methods Hydrothermally prepared CDs were assembled with positive-charged PEI, followed by conjugation with AS1411 through electrostatic interaction to form CDs-PEI-AS1411 nanocomplexes. The CDs, CDs-PEI and CDs-PEI-AS1411 were characterized by transmission electron microscopy (TEM), fourier transform infrared (FTIR) spectra, UV-vis spectra, zeta potential measurements and capillary electrophoresis characterizations. The cytotoxicity investigation of the CDs-PEI-AS1411 and CDs-PEI in both MCF-7 and L929 cells was carried out by the CCK-8 assay. The cellular uptake of the CDs-PEI-AS1411 was studied with confocal microscopy and flow cytometry. Results The as-prepared nanosystem possessed good photostability and no obvious cytotoxicity. On the basis of the confocal laser scanning microscope observation and the flow cytometry studies, the cellular uptake of CDs-PEI-AS1411 nanosystem in MCF-7 cells was significantly higher than that of L929 cells, which revealed the highly selective detection ability of nucleolin-positive cells. Conclusions The results of this study indicated that the CDs-PEI-AS1411 nanosystem had a potential value in cancer cell targeted imaging.
Objectives Osteoclasts (OC) are unique terminally differentiated cells whose primary function is bone resorption. We previously showed that osteoprotegerin (OPG) inhibits OC differentiation in vitro by enhancing autophagy via the adenosine monophosphate-activated protein kinase (AMPK)/mTOR/p70S6K signalling pathway in vitro. Here, we aimed to elucidate the mechanism of AMPK mediated autophagy to regulate OPG-mediated inhibition of OC differentiation and identify potential therapeutic targets associated with bone loss. Materials and Methods We used the AMPK activator AICAR to determine the relationship between AMPK activation and OC differentiation, and studied the role of AMPK-mediated autophagy in OPG-mediated inhibition of OC differentiation by using autophagy inhibitors or AMPK knockdown. Results AMP-activated protein kinase activation caused LC3II accumulation and weakened OC differentiation activity. In contrast, inactivation of autophagy by 3-methyladenine or Bafilomycin A1 could attenuate OPG-mediated inhibition of OC differentiation via the AMPK/mTOR/p70S6K signalling pathway. Furthermore, the AMPK inhibitor compound C and knockdown of AMPK impaired OPG-mediated inhibition of OC differentiation by inducing autophagy. Conclusions These results demonstrated that the AMPK signalling pathway functions as a critical regulator in the OPG-mediated inhibition of OC differentiation, by inducing autophagy. Our results provide a basis for future bone-related studies on the AMPK signalling pathway.
Objectives miR-92b has been reported to play critical roles in several carcinomas; however, our understanding of the mechanisms by which miR-92b stimulates gastric cancer (GC) is incomplete. The aim of this study was to investigate the clinical significance and functional relevance of miR-92b in GC. Materials and methods Expression of miR-92b in GC and peritumoural tissues was determined using qRT-PCR, in situ hybridization and bioinformatics. CCK-8, colony formation and fluorescence-activated cell sorting assays were utilized to explore the effect of miR-92b on GC cells. A luciferase reporter assay and Western blotting were employed to verify miR-92b targeting of DAB2IP. Furthermore, Western blotting was used to evaluate the levels of DAB2IP and PI3K/Akt signalling pathway-related proteins. Results In this study, we found that miR-92b was upregulated in GC tissues compared with peritumoural tissues. Overexpression of miR-92b promoted cell proliferation, colony formation, and G(0)/G(1) transition and decreased apoptosis. Our results indicated that miR-92b repressed the expression of DAB2IP and that loss of DAB2IP activated the PI3K/AKT signalling pathway. Overexpression of DAB2IP rescued the effects of miR-92b in GC cells. Finally, our results demonstrated a significant correlation between miR-92b expression and DAB2IP expression in GC tissues. Conclusions Our results suggest that miR-92b promotes GC cell proliferation by activating the DAB2IP-mediated PI3K/AKT signalling pathway. The miR-92b/DAB2IP/PI3K/AKT signalling axis may be a potential therapeutic target to prevent GC progression.
Objectives miR-21 can promote osteoblast differentiation of periodontal ligament stem cells. However, the effect of miR-21 on bone remodelling in the midpalatal suture is unclear. This study aimed to elucidate the effects of miR-21 on the midpalatal suture bone remodelling by expanding the palatal sutures. Materials and methods miR-21 deficient (miR-21(-/-)) and wild-type (WT) mice were used to establish animal models by expanding the palatal sutures. Micro-CT, haematoxylin-eosin (HE) staining, tartrate-resistant acid phosphatase (TRAP) staining, fluorescence labelling and immunohistochemistry were used to investigate the function of miR-21 in midpalatal suture bone remodelling. Besides, bone mesenchymal stem cells (BMSCs) derived from both miR-21(-/-) and WT mice were cultured. The MTT, CCK8, EdU analysis, transwell and wound healing test were used to assess the effects of miR-21 on the characteristics of cells. Results The expression of ALP was suppressed in miR-21(-/-) mice after expansion except 28 days. The expression of Ocn in WT mice was much higher than that of miR-21(-/-) mice. Besides, with mechanical force, miR-21 deficiency downregulated the expression of Opg, upregulated the expression of Rankl, and induced more osteoclasts as TRAP staining showed. After injecting agomir-21 to miR-21(-/-) mice, the expression of Alp, Ocn and Opg/Rankl were rescued. In vitro, the experiments suggested that miR-21 deficiency reduced proliferation and migration ability of BMSCs. Conclusions The results showed that miR-21 deficiency reduced the rate of bone formation and prolonged the process of bone formation. miR-21 regulated the bone resorption and osteoclastogenesis by affecting the cell abilities of proliferation and migration.