Functionalized DNA-Origami-Protein Nanopores Generate Large Transmembrane Channels with Programmable Size-Selectivity.

The DNA-origami technique has enabled the engineering of transmembrane nanopores with programmable size and functionality, showing promise in building biosensors and synthetic cells. However, it remains challenging to build large (>10 nm), functionalizable nanopores that spontaneously perforate lipid membranes. Here, we take advantage of pneumolysin (PLY), a bacterial toxin that potently forms wide ring-like channels on cell membranes, to construct hybrid DNA-protein nanopores. This PLY-DNA-origami complex, in which a DNA-origami ring corrals up to 48 copies of PLY, targets the cholesterol-rich membranes of liposomes and red blood cells, readily forming uniformly sized pores with an average inner diameter of ∼22 nm. Such hybrid nanopores facilitate the exchange of macromolecules between perforated liposomes and their environment, with the exchange rate negatively correlating with the macromolecule size (diameters of gyration: 8-22 nm). Additionally, the DNA ring can be decorated with intrinsically disordered nucleoporins to further restrict the diffusion of traversing molecules, highlighting the programmability of the hybrid nanopores. PLY-DNA pores provide an enabling biophysical tool for studying the cross-membrane translocation of ultralarge molecules and open new opportunities for analytical chemistry, synthetic biology, and nanomedicine.

A cellular endolysosome-modulating pore-forming protein from a toad is negatively regulated by its paralog under oxidizing conditions.

Endolysosomes are key players in cell physiology, including molecular exchange, immunity, and environmental adaptation. They are the molecular targets of some pore-forming aerolysin-like proteins (ALPs) that are widely distributed in animals and plants and are functionally related to bacterial toxin aerolysins. ßγ-CAT is a complex of an ALP (BmALP1) and a trefoil factor (BmTFF3) in the firebelly toad (Bombina maxima). It is the first example of a secreted endogenous pore-forming protein that modulates the biochemical properties of endolysosomes by inducing pore formation in these intracellular vesicles. Here, using a large array of biochemical and cell biology methods, we report the identification of BmALP3, a paralog of BmALP1 that lacks membrane pore-forming capacity. We noted that both BmALP3 and BmALP1 contain a conserved cysteine in their C-terminal regions. BmALP3 was readily oxidized to a disulfide bond-linked homodimer, and this homodimer then oxidized BmALP1 via disulfide bond exchange, resulting in the dissociation of ßγ-CAT subunits and the elimination of biological activity. Consistent with its behavior in vitro, BmALP3 sensed environmental oxygen tension in vivo, leading to modulation of ßγ-CAT activity. Interestingly, we found that this C-terminal cysteine site is well conserved in numerous vertebrate ALPs. These findings uncover the existence of a regulatory ALP (BmALP3) that modulates the activity of an active ALP (BmALP1) in a redox-dependent manner, a property that differs from those of bacterial toxin aerolysins.

Site-specific cation release drives actin filament severing by vertebrate cofilin.

Actin polymerization powers the directed motility of eukaryotic cells. Sustained motility requires rapid filament turnover and subunit recycling. The essential regulatory protein cofilin accelerates network remodeling by severing actin filaments and increasing the concentration of ends available for elongation and subunit exchange. Although cofilin effects on actin filament assembly dynamics have been extensively studied, the molecular mechanism of cofilin-induced filament severing is not understood. Here we demonstrate that actin filament severing by vertebrate cofilin is driven by the linked dissociation of a single cation that controls filament structure and mechanical properties. Vertebrate cofilin only weakly severs Saccharomyces cerevisiae actin filaments lacking this "stiffness cation" unless a stiffness cation-binding site is engineered into the actin molecule. Moreover, vertebrate cofilin rescues the viability of a S. cerevisiae cofilin deletion mutant only when the stiffness cation site is simultaneously introduced into actin, demonstrating that filament severing is the essential function of cofilin in cells. This work reveals that site-specific interactions with cations serve a key regulatory function in actin filament fragmentation and dynamics.

Host-derived, pore-forming toxin-like protein and trefoil factor complex protects the host against microbial infection.

Aerolysins are virulence factors belonging to the bacterial ß-pore-forming toxin superfamily. Surprisingly, numerous aerolysin-like proteins exist in vertebrates, but their biological functions are unknown. ßγ-CAT, a complex of an aerolysin-like protein subunit (two ßγ-crystallin domains followed by an aerolysin pore-forming domain) and two trefoil factor subunits, has been identified in frogs (Bombina maxima) skin secretions. Here, we report the rich expression of this protein, in the frog blood and immune-related tissues, and the induction of its presence in peritoneal lavage by bacterial challenge. This phenomena raises the possibility of its involvement in antimicrobial infection. When ßγ-CAT was administrated in a peritoneal infection model, it greatly accelerated bacterial clearance and increased the survival rate of both frogs and mice. Meanwhile, accelerated Interleukin-1ß release and enhanced local leukocyte recruitments were determined, which may partially explain the robust and effective antimicrobial responses observed. The release of interleukin-1ß was potently triggered by ßγ-CAT from the frog peritoneal cells and murine macrophages in vitro. ßγ-CAT was rapidly endocytosed and translocated to lysosomes, where it formed high molecular mass SDS-stable oligomers (>170 kDa). Lysosomal destabilization and cathepsin B release were detected, which may explain the activation of caspase-1 inflammasome and subsequent interleukin-1ß maturation and release. To our knowledge, these results provide the first functional evidence of the ability of a host-derived aerolysin-like protein to counter microbial infection by eliciting rapid and effective host innate immune responses. The findings will also largely help to elucidate the possible involvement and action mechanisms of aerolysin-like proteins and/or trefoil factors widely existing in vertebrates in the host defense against pathogens.

High-performance quantum dot light-emitting diodes with hybrid hole transport layer via doping engineering.

Here, we report on the hybrid hole transport materials 4,4'-bis-(carbazole-9-yl)biphenyl (CBP) or poly-N-vinylcarbazole (PVK) doped into poly(4-butyl-phenyl-diphenyl-amine) (Poly-TPD) as the hybrid hole transport layer (HTL) to tailor the energy band alignment between hole injection layer (HIL) and quantum dot (QD) light emitting layer in order to realize efficient quantum dot light emitting diodes (QLEDs) in all solution-processed fabrication. Compared to the pristine Poly-TPD based device, it is found that the electroluminescence (EL) performance of QLEDs can be significantly improved by 1.5 fold via addition of CBP into Poly-TPD, which can be attributed to the lowered highest occupied molecular orbital (HOMO) level of Poly-TPD to reduce the energy barrier between HTL and valance band (VB) of QDs. Thus, after doping small molecules into polymer under optimized proportion (Poly-TPD:CBP = 2:1 by weight), the hole transport rate can be balanced, facilitating the carrier injection from HTL to QDs and enhancing the efficiency of QLEDs. As a result, a maximum luminance, a maximum current efficiency and a maximum power efficiency of 7600 cd/m2, 5.41 cd/A and 4.25 lm/W can be obtained based on this variety of hybrid HTL employed QLEDs.

Mitochonic Acid 5 Increases Ram Sperm Quality by Improving Mitochondrial Function during Storage at 4 °C.

Semen preservation involves lengthening sperm's fertile lifespan without any detrimental effects on its biochemical, functional, and ultrastructural properties. Liquid storage at 4 °C is a ram sperm preservation method. However, this method of storage causes irreversible damage due to cold shocks, osmotic stresses, oxidative stresses, and reductions in sperm metabolism. The present study aims to investigate whether the supplementation of mitochonic acid 5 (MA-5) in a sperm extender could improve chilled ram sperm quality and elucidate its mechanism of action. Ram sperm were diluted with a tris-citrate-glucose extender containing different concentrations of MA-5 (0, 0.1, 1, 10, and 100 nM) and stored at 4 °C for up to 48 h. Sperm motility, membrane integrity, acrosome integrity, mitochondrial membrane potential, reactive oxygen species (ROS) level, ATP content, and the expression of NADPH dehydrogenase subunits 1 (MT-ND1) and NADPH dehydrogenase subunits 6 (MT-ND6) were evaluated. It was observed that compared to the control, the 10 nM MA-5 treatment significantly (p < 0.05) increased total motility (82 ± 3.5% vs. 76 ± 5.9%), progressive motility (67.6 ± 8.2% vs. 51 ± 8.3%), and other parameters (straight-line velocity (VSL), average path velocity (VAP), and curvilinear velocity (VCL)). In addition, 10 nM MA-5 supplementation also improved ram sperm membrane integrity and acrosomal integrity as well increased mitochondrial membrane potential (51.1 ± 0.7% vs. 37.7 ± 1.3%), reduced ROS levels, and elevated adenosine triphosphate (ATP) contents. Furthermore, a Western blot analysis demonstrated that the addition of MA-5 significantly (p < 0.05) increased the expression of MT-ND1 and MT-ND6 proteins in ram sperm, with the 10 nM MA-5 treatment resulting in the highest expression level. These results suggest that MA-5 improves ram sperm quality by maintaining high sperm mitochondrial function during liquid storage at 4 °C.

Carboxylated ε-Poly-l-lysine Improves Post-Thaw Quality, Mitochondrial Functions and Antioxidant Defense of Goat Cryopreserved Sperm.

Carboxylated ε-poly-l-lysine (CPLL), a novel cryoprotectant, can protect the sperm membranes by inhibiting ice crystal formation during the cryopreservation process. The present study was conducted to investigate the consequence of CPLL supplementation on the post-thaw quality of cryopreserved goat sperm. For this, different doses (0, 0.5%, 1%, 1.5%, and 2%; v/v) of CPLL were added to the cryopreservation medium, and the motility, membrane and acrosome integrity, mitochondrial membrane potential (MMP), ATP level, ROS production, anti-oxidant defense system, malondialdehyde (MDA) level, and apoptosis in post-thaw sperm were evaluated. It was observed that the addition of 1% CPLL significantly (p < 0.05) increased the total motility, membrane integrity, acrosome integrity, and catalase (CAT) activity of post-thaw sperm compared to those of control and other CPLL doses. The ATP content was observed significantly (p < 0.05) higher in 0.5% and 1% CPLL, however, the SOD activity and progressive motility were significantly (p < 0.05) increased by adding CPLL at 1% and 1.5% level. Moreover, the addition of CPLL at 1% dose not only showed a lower percentage of apoptosis, but also significantly (p < 0.05) increased the MMP while reducing ROS production and MDA levels compared to those of other CPLL doses and/or control. Therefore, it is clear that the supplementation of 1% CPLL can remarkably improve the post-thaw goat sperm motility, membrane and acrosome integrity, antioxidant abundance, mitochondrial potentials, and ATP supply by protecting the sperm from cryodamage and undergoing apoptosis. These findings will provide novel insights into sperm cryobiology.

Exosome miR-4738-3p-mediated regulation of COL1A2 through the NF-κB and inflammation signaling pathway alleviates osteoarthritis low-grade inflammation symptoms.

This study aimed to elucidate the roles of microRNA (miR)-4738-3p and the collagen type I alpha 2 chain (COL1A2) gene in the pathogenesis of osteoarthritis (OA) through bioinformatics analysis and cellular assays. The GSE55235 dataset was analyzed using the weighted gene co-expression network analysis (WGCNA) method to identify gene modules associated with OA. Key overlapping genes were identified from these modules and the GSE55235-differential expressed genes (DEGs). The expression levels of selected genes were determined in C28/I2 cells using the quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between miR-4738-3p and COL1A2 was examined in the context of interleukin 1 beta (IL-1ß) induction. Exosome characterization was achieved through transmission electron microscopy (TEM), western blotting (WB), and other analyses. The study also investigated the functional relevance of miR-4738-3p in OA pathology through various molecular and cellular assays. Our findings revealed that the green module exhibited a strong correlation with the OA phenotype in the GSE55235 dataset, with COL1A2 emerging as a hub gene and miR-4738-3p as its key downstream target. IL-1ß induction suggested that COL1A2 is involved in inflammation and apoptosis, while miR-4738-3p appeared to play an antagonistic role. The analysis of exosomes underscored the significance of miR-4738-3p in cellular communication, with an enhanced level of exo-miR-4738-3p antagonizing IL-1ß-induced inflammation and promoting cell survival. Conversely, a reduction in exo-miR-4738-3p led to increased cell damage. This study established a clear regulatory relationship between miR-4738-3p and COL1A2, with the nuclear factor kappa B (NF-κB) signaling pathway playing a central role in this regulation. The miR-4738-3p significantly influences the OA-associated inflammation, primarily through modulation of COL1A2 and the NF-κB pathway. Therefore, targeting miR-4738-3p offers a potential therapeutic approach for OA, with exosome miR-4738-3p presenting a promising strategy.

Toll-like receptor 5 forms asymmetric dimers in the absence of flagellin.

The structure of full-length human TLR5 determined by electron microscopy single-particle image reconstruction at 26Å resolution shows that TLR5 forms an asymmetric homodimer via ectodomain interactions. The structure shows that like TLR9, TLR5 dimerizes in the absence of ligand. The asymmetry of the dimer suggests that TLR5 may recognize two flagellin molecules cooperatively to establish an optimal flagellin response threshold. A TLR5 homology model was generated and fitted into the electron microscopy structure. All seven predicted N-linked glycosylation sites are exposed on the molecular surface, away from the dimer interface. Glycosylation at the first five sites was confirmed by tandem mass spectrometry. Two aspartate residues proposed to interact with flagellin (Asp294 and Asp366) are sterically occluded by a glycan at position 342. In contrast, the central region of the ectodomains near the dimer interface is unobstructed by glycans. Ligand binding in this region would be consistent with the ligand binding sites of other TLRs.

Polyamines protect boar sperm from oxidative stress in vitro.

Sperm are susceptible to excessive reactive oxygen species (ROS). Spermine and spermidine are secreted in large amounts by the prostate and potent natural free radical scavengers and protect cells against redox disorder. Thus, we used boar sperm as a model to study the polyamines uptake and elucidate whether polyamines protected sperm from ROS stress. Seven mature and fertile Duroc boars (aged 15 to 30 mo) were used in this study. In experiment 1, spermine and spermidine (3.6 ± 0.3 and 3.3 ± 0.2 mmol/L, respectively) were abundant in seminal plasma, and the content of polyamine decreased (P < 0.05) after preservation at 17 °C for 7 d or incubation at 37 °C for 6 h. In experiment 2, using labeling of spermine or spermidine by conjugation with fluorescein isothiocyanate and ultra-high-performance liquid chromatography, we found that the accumulation of spermine or spermidine in sperm was inhibited by quinidine and dl-tetrahydropalmatine (THP, organic cation transporters [OCT] inhibitors, P < 0.05), but not mildronate and l-carnitine (organic cation/carnitine transporter [OCTN] inhibitors, P > 0.05). In experiment 3, the addition of spermine or spermidine (0.5 mmol/L) in the extender resulted in higher motility, plasma membrane and acrosome integrity, and lower ROS level after preservation in vitro at 17 °C for 7 d (P < 0.05). In experiment 4, in the condition of oxidative stress (treatment with H2O2 at 37 °C for 2 h), the addition of spermine (1 mmol/L) or spermidine (0.5 mmol/L) in extender increased activities of glutathione peroxidase, glutathione reductase, and glutathione S-transferase; reduced glutathione and oxidized glutathione ratio (P < 0.05); and alleviate oxidative stress-induced lipid peroxidation, DNA damage, mitochondrial membrane potential (ΔΨm) decline, adenosine triphosphate depletion, and intracellular calcium concentration ([Ca2+]i) overload (P < 0.05), thereby improving boar sperm motility, the integrity of plasma membrane and acrosome (P < 0.05) in vitro. These data suggest that spermine and spermidine alleviate oxidative stress via the antioxidant capacity, thereby improving the efficacy of boar semen preservation.

Boar semen preservation and artificial insemination are widely used in the pig industry. Although preservation in vitro prolongs sperm lifespan, reactive oxidative species (ROS) also accumulate in sperm with the increased preservation period. ROS over-accumulation would impair motility, the integrity of plasma membrane and acrosome, mitochondrial function, and eventually lead to infertility. Spermine and spermidine are secreted in large amounts by the prostate and are potent natural free radical scavengers. Thus, we used boar sperm as a model to study the polyamines uptake and elucidate whether polyamines protected sperm from ROS stress. We found for the first time that organic cation transporters mediated polyamines uptake in sperm cells, and that extracellular polyamines decreased during preservation in vitro. The addition of polyamines increased the activities of glutathione-related antioxidant enzymes and reduced glutathione and oxidized glutathione ratio, and alleviate oxidative stress-induced mitochondrial dysfunction, lipid peroxidation, and DNA damage, thereby maintaining sperm quality in vitro. These data suggest that spermine and spermidine alleviate oxidative stress, thereby improving the efficacy of boar semen preservation.

Effects of supplementing low-protein diets with sodium dichloroacetate and glucose on growth performance, carcass traits, and meat quality of growing-finishing pigs.

The objective of this study was to evaluate the effects of supplementing low-protein diets with sodium dichloroacetate (DCA) and glucose on growth performance, carcass traits, and meat quality of growing-finishing pigs. A total of 80 crossbred (Duroc × Landrace × Large White) growing barrows (27 ± 0.4 kg body weight) were allocated randomly to one of the five treatments during three successive 4-wk periods. There were five diets in each phase. Diet 1 was the control diet with normal protein levels (CON) where protein levels in the three phases were 18%, 16.5%, and 15.5%, respectively. The dietary protein levels of Diets 2, 3, 4, and 5 (the low-protein diets, LP) were decreased by 4.5% compared to Diet 1. Additionally, Diets 3 and 4 were supplemented with an extra 120 mg/kg DCA (LP + DCA) or 1.8% glucose (LP + GLUC), respectively. Diet 5 was further supplemented with an extra 120 mg/kg DCA and 1.8% glucose (LP + DCA + GLUC). The LP + DCA diet increased the average daily weight gain of pigs compared to the CON and LP diet in phase 3 and the overall experimental period (P < 0.001). The LP diet reduced the gain:feed ratios of the pigs compared to the CON, LP + DCA, and LP + DCA + GLUC diets in phase 1 and the overall experimental period (P < 0.001). Furthermore, gain:feed ratios in LP + DCA and LP + DCA + GLUC groups did not differ from that of the CON group (P > 0.10). Pigs fed the LP + DCA diet had higher pH values of meat at 24 h post-mortem than the CON group (P < 0.05). The LP + DCA + GLUC diet increased the total protein content in the longissimus dorsi (LD) muscle of pigs, compared to the other dietary treatments (P < 0.05), and increased the Arg and Leu contents in the LD muscle compared to the LP + DCA diet (P < 0.05). Moreover, the LP + DCA diet induced a higher C18:1n9t percentage in the LD muscle of pigs compared to other groups (P < 0.05). In conclusion, an LP diet reduced the feed efficiency in pigs and barely affected meat quality, whereas 120 mg/kg DCA supplementation in an LP diet improved the growth performance of growing-finishing pigs, showed modest effects on carcass traits, and improved the muscle protein content with the addition of glucose.

NAD+ and its possible role in gut microbiota: Insights on the mechanisms by which gut microbes influence host metabolism.

Nicotinamide adenine dinucleotide (NAD+) is an enzyme cofactor, co-substrate, and redox factor in all living cells and is necessary for maintaining cell metabolism. It has been shown that appropriate supplementation of NAD+ precursors or inhibition of NAD+-depleting enzymes can promote mitochondrial oxidative phosphorylation and improve host energy utilization efficiency. In addition, increasing evidence indicates that the gut microbiota plays a pivotal role in host metabolism. Theoretically, there should be a close correlation among NAD+, gut microbiota, and host metabolism; however, the information is limited. In this review, we summarize the metabolic process of NAD+ and its impact on host metabolism, the link between gut microbiota and host metabolism, as well as the potential effects of NAD+ on microbial metabolism, providing a new perspective on the interaction between gut microbiota and host metabolism.

Expression Profile Analysis of Long Non-coding RNA in OVX Models-Derived BMSCs for Postmenopausal Osteoporosis by RNA Sequencing and Bioinformatics.

Osteoporosis (OP) has the characteristics of a systematically impaired bone mass, strength, and microstructure. Long non-coding RNAs (lncRNAs) are longer than 200 nt, and their functions in osteoporosis is yet not completely understood. We first harvested the bone marrow mesenchymal stem cells (BMSCs) from ovariectomy (OVX) and sham mice. Then, we systematically analyzed the differential expressions of lncRNAs and messenger RNAs (mRNAs) and constructed lncRNA-mRNA coexpression network in order to identify the function of lncRNA in osteoporosis. Totally, we screened 743 lncRNAs (461 upregulated lncRNAs and 282 downregulated lncRNAs) and 240 mRNAs (128 upregulated and 112 downregulated) with significantly differential expressions in OP compared to normal. We conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analyses to investigate the functions and pathways of the differential expression of messenger RNAs (mRNAs), a coexpressed network of lncRNA/mRNA. Quantitative PCR (qPCR) validated that the expressions of NONMMUT096150.1, NONMMUT083450.1, and NONMMUT029743.2 were all downregulated, whereas NONMMUT026970.2, NONMMUT051734.2, NONMMUT003617.2, and NONMMUT034049.2 were all upregulated in the OVX group. NONMMUT096150.1, as a key lncRNA in OP, was identified to modulate the adipogenesis of BMSCs. Further analysis suggested that NONMMUT096150.1 might modulate the adipogenesis of BMSCs via the peroxisome proliferator-activated receptor (PPAR) signaling pathway, AMPK signaling pathway, and the lipolysis regulation in adipocyte and adipocytokine signaling pathway. Our study expands the understanding of lncRNA in the pathogenesis of OP.

Sorting sub-150-nm liposomes of distinct sizes by DNA-brick-assisted centrifugation.

In cells, myriad membrane-interacting proteins generate and maintain curved membrane domains with radii of curvature around or below 50 nm. To understand how such highly curved membranes modulate specific protein functions, and vice versa, it is imperative to use small liposomes with precisely defined attributes as model membranes. Here, we report a versatile and scalable sorting technique that uses cholesterol-modified DNA 'nanobricks' to differentiate hetero-sized liposomes by their buoyant densities. This method separates milligrams of liposomes, regardless of their origins and chemical compositions, into six to eight homogeneous populations with mean diameters of 30-130 nm. We show that these uniform, leak-resistant liposomes serve as ideal substrates to study, with an unprecedented resolution, how membrane curvature influences peripheral (ATG3) and integral (SNARE) membrane protein activities. Compared with conventional methods, our sorting technique represents a streamlined process to achieve superior liposome size uniformity, which benefits research in membrane biology and the development of liposomal drug-delivery systems.

Long Noncoding RNA HAGLROS Promotes Cell Invasion and Metastasis by Sponging miR-152 and Upregulating ROCK1 Expression in Osteosarcoma.

BACKGROUND: Long noncoding RNAs (lncRNAs) played a crucial role in a number of biological processes. lncRNA HAGLROS was demonstrated to facilitate cell proliferation and migration in various cancers. However, the functions and molecular mechanisms of HAGLROS in osteosarcoma remained to be elucidated. METHODS: qRT-PCR assay was used to detect the relative expression of HAGLROS in osteosarcoma tissue samples and cells. CCK-8 and Transwell assays were performed to assess the effects of HAGLROS on OS cells proliferation and invasion. Luciferase reporter assay verified the interaction between ROCK1 and miR-152. RESULTS: In our study, we found that the expression of HAGLROS increased osteosarcoma samples and cell lines compared with normal tissues and cells. HAGLROS knockdown inhibited certain functions of U2OS and SW1353 cells in vitro. Moreover, HAGLROS depletion inhibited tumor growth and metastasis in vivo. Mechanically, we found that HAGLROS sponged miR-152 to promote ROCK1 expression in U2OS and SW1353 cells. CONCLUSION: In summary, our study indicated that HAGLROS could promote osteosarcoma progression by sponging miR-152 to promote ROCK1 expression. The results showed HAGLROS/miR-152/ROCK1 axis might act as a novel therapeutic strategy for osteosarcoma.

Circular RNA circFAT1(e2) Promotes Osteosarcoma Progression and Metastasis by Sponging miR-181b and Regulating HK2 Expression.

As a subclass of noncoding RNAs, circular RNAs (circRNAs) have been demonstrated to play a critical role in regulating gene expression in eukaryotes. Recent studies have revealed the pivotal functions of circRNAs in cancer progression. Nevertheless, how circRNAs participate in osteosarcoma (OS) development and progression are not well understood. In the present study, we identified a circRNA circFAT1(e2) with an upregulated expression level in OS tissues. By functional experiments, we found that circFAT1(e2) depletion significantly suppressed the proliferation and reduced migration in OS. In terms of mechanism, we found that circFAT1(e2) inhibited miR-181b, while miR-181b targeted HK2. By releasing the inhibition of miR-181b on HK2 expression, leading to attenuated OS progression. Mechanistic investigations suggested that circFAT1(e2) served as a competing endogenous RNA (ceRNA) of miR-181b to enhance HK2 expression. On the whole, our study indicated that circFAT1(e2) exerted oncogenic roles in OS and suggested the circFAT1(e2)/miR-181b/HK2 axis might be a potential therapeutic target.

Hypoxia-induced let-7f-5p/TARBP2 feedback loop regulates osteosarcoma cell proliferation and invasion by inhibiting the Wnt signaling pathway.

Osteosarcoma (OS) is the most common bone tumor in children and adolescents and is characterized by high metastatic and recurrence rates. In the past, it has been shown that microRNAs may play critical roles in hypoxia-related OS proliferation and invasion. However, the mechanisms by which OS cells acquire this malignant phenotype have remained largely unknown. In the present study, we report that let-7f-5p and TARBP2 were expressed in lower amounts in human OS cell lines when compared with the hFOB normal human osteoblastic cell line; however, both types of cells were repressed by hypoxia. let-7f-5p and TARBP2 significantly inhibited the proliferation and invasion of OS cells. Furthermore, TARBP2 as a downstream and functional target of let-7f-5p regulated the expression of let-7f-5p, and there was a regulatory feedback loop between let-7f-5p and TARBP2. This loop reduced the expression of let-7f-5p and TARBP2 in OS cells to a very low level, which was induced by hypoxia. Furthermore, the hypoxia-induced let-7f-5p/TARBP2 feedback loop contributed to activation of the Wnt signaling pathway. Taken together, our data clearly showed that the feedback loop between let-7f-5p and TARBP2 induced by the hypoxia-promoted OS cell malignant phenotype increased with activation of the Wnt signaling pathway.

Downregulation of microRNA­143 promotes osteogenic differentiation of human adipose­derived mesenchymal stem cells through the k­Ras/MEK/ERK signaling pathway.

MicroRNAs (miRNAs) are known to have regulatory roles in the osteogenic differentiation of various mesenchymal stem cells (MSCs), although their regulatory role on human adipose­derived mesenchymal stem cells (hADSCs) remains unclear. The aim of the present study was to investigate the biological function and underlying molecular mechanism of miRNAs in regulating the osteogenic differentiation of hADSCs using microarray assay. hADSCs differentiated into osteoblasts under culture with osteogenic medium, with an increase observed in calcium deposits and alkaline phosphatase activity. The mRNA levels of bone sialoprotein, osteopontin and osteocalcin increased, whereas Runt­related transcription factor­2 expression decreased during osteogenic differentiation. In addition, miR­143 was markedly downregulated during osteogenic differentiation, while miR­143 overexpression inhibited and miR­143 knockdown enhanced this process. miR­143 overexpression also blocked extracellular signal­regulated kinase 1/2 (ERK1/2) pathway activation, while miR­143 inhibition enhanced it. The promoting effects of miR­143 knockdown on the osteogenic differentiation of hADSCs were partly diminished by the mitogen­activated protein kinase (MEK) inhibitors U0126 and PD98059. Bioinformatics analysis further revealed that miR­143 targets k­Ras and directly binds to the 3'­untranslated region of its mRNA. Inhibition of miR­143 enhanced the activation of the k­Ras/MEK/ERK pathway during osteogenic differentiation, whereas miR­143 overexpression had the opposite effect. Collectively, these results demonstrated that miR­143 negatively regulates the osteogenic differentiation of hADSCs through the k­Ras/MEK/ERK pathway, providing further insight into the underlying molecular mechanisms.

Circular RNA circHIPK3 Promotes Cell Metastasis through miR-637/STAT3 Axis in Osteosarcoma.

Recent studies have suggested that circular RNAs play an important role in the progression of various cancers. However, few studies have revealed the great value of circRNAs in the diagnosis and prognosis prediction of osteosarcoma (OS). In this study, we performed experiments with the human OS cell lines and the results showed that the expression of circHIPK3 in OS cell lines was significantly upregulated compared to that in the normal cell line. In addition, the results showed that circHIPK3 could promote the migration, invasion, and growth of OS cells. Furthermore, miR-637 was identified as a target of circHIPK3, while STAT3 was targeted by miR-637. circHIPK3 could promote STAT3 expression via interacting with miR-637 in OS cells. In conclusion, our research uncovered an important role of the circHIPK3/miR-637/STAT3 pathway in the migration and invasion of OS cells and suggested that circHIPK3 may be a prognostic marker and a promising therapeutic target for OS.

Identification of circRNA-associated ceRNA network in BMSCs of OVX models for postmenopausal osteoporosis.

Circular RNAs (circRNAs) serve as competing endogenous RNAs (ceRNAs) and indirectly regulate gene expression through shared microRNAs (miRNAs). However, the potential circRNAs functioning as ceRNAs in osteoporosis remain unclear. The bone marrow mesenchymal stem cells (BMSCs) were isolated from ovariectomy (OVX) mice and controls. We systematically analyzed RNA-seq and miRNA-microarray data, miRNA-target interactions, and prominently coexpressed gene pairs to identify aberrantly expressed circRNAs, miRNAs, and messenger RNAs (mRNAs) between the OVX mice and controls. A total of 45 circRNAs, 22 miRNAs, and 548 mRNAs were significantly dysregulated (fold change > 1.5; p < 0.05). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were conducted for differentially expressed mRNAs, and subsequently a circRNA-associated ceRNA network involved in osteoporosis was constructed. We identified two ceRNA regulatory pathways in this osteoporosis mouse model-novel circRNA 0020/miR-206-3p/Nnmt and circRNA 3832/miR-3473e/Runx3, which were validated by real-time PCR. This is the first study to elucidate the circRNA-associated ceRNA network in OVX and control mice using deep RNA-seq and RNA-microarray analysis. The data further expanded the understanding of circRNA-associated ceRNA networks, and the regulatory functions of circRNAs, miRNAs and mRNAs in the pathogenesis and pathology of osteoporosis.