METTL3 promotes IL‐1β–induced degeneration of endplate chondrocytes by driving m6A‐dependent maturation of miR‐126‐5p

METTL3 is an important regulatory molecule in the process of RNA biosynthesis. It mainly regulates mRNA translation, alternative splicing and microRNA maturation by mediating m6A‐dependent methylation. Interleukin 1β (IL‐1β) is an important inducer of cartilage degeneration that can induce an inflammatory cascade reaction in chondrocytes and inhibit the normal biological function of cells. However, it is unclear whether IL‐1β is related to METTL3 expression or plays a regulatory role in endplate cartilage degeneration. In this study, we found that the expression level of METTL3 and methylation level of m6A in human endplate cartilage with different degrees of degeneration were significantly different, indicating that the methylation modification of m6A mediated by METTL3 was closely related to the degeneration of human endplate cartilage. Next, through a series of functional experiments, we found that miR‐126‐5p can play a significant role in IL‐1β–induced degeneration of endplate chondrocytes. Moreover, we found that miR‐126‐5p can inhibit the PI3K/Akt signalling pathway by targeting PIK3R2 gene, leading to the disorder of cell vitality and functional metabolism. To further determine whether METTL3 could regulate miR‐126‐5p maturation, we first confirmed that METTL3 can bind the key protein underlying pri‐miRNA processing, DGCR8. Additionally, when METTL3 expression was inhibited, the miR‐126‐5p maturation process was blocked. Therefore, we hypothesized that METTL3 can promote cleavage of pri‐miR‐126‐5p and form mature miR‐126‐5p by combining with DGCR8.     

CircHIPK3 prevents chondrocyte apoptosis and cartilage degradation by sponging miR‐30a‐3p and promoting PON2

Osteoarthritis (OA) is a common joint disease featured by the deterioration of articular cartilage and chondrocyte death. Emerging evidence has indicated that circular RNAs (circRNAs) play an essential role in OA progress. Here, we found that the expression of circHIPK3 was significantly decreased in human and mouse OA cartilage. Knocking down circHIPK3 increased apoptosis and intracellular ROS level in HC‐a chondrocytes. We performed proteomic studies and identified that circHIPK3 regulated chondrocyte apoptosis through the mitochondrial pathway. Results of JC‐1 staining and western blot further confirmed that mitochondrial outer membrane permeabilization was promoted in HC‐a chondrocytes transfected by circHIPK3 siRNA. In terms of mechanism, we showed that PON2 functioned as a potential target of circHIPK3 to regulate chondrocyte apoptosis. Moreover, we revealed that circHIPK3 interacted with miR‐30a‐3p to regulate PON2 expression in chondrocytes. Taken together, our findings suggested that circHIPK3 regulated chondrocyte apoptosis by mitochondrial pathway, and targeting the circHIPK3/miR‐30a‐3p/PON2 axis might be a potential strategy for OA treatment.

The current study revealed the important role of circHIPK3 in regulating chondrocyte apoptosis and maintaining extracellular matrix (ECM) homeostasis. Mechanistically, circHIIPK3 might serve as a sponge of miR‐30a‐3p to regulate PON2 expression. The downregulation of circHIIPK3 resulted in the increased expression of miR‐30a‐3p and decreased expression of PON2, thus leading to mitochondrial pathway apoptosis and ECM destruction.     

KDM6A promotes chondrogenic differentiation of periodontal ligament stem cells by demethylation of SOX9

Objectives

KDM6A has been demonstrated critical in the regulation of cell fates. However, whether KDM6A is involved in cartilage formation remains unclear. In this study, we investigated the role of KDM6A in chondrogenic differentiation of PDLSCs, as well as the underlying epigenetic mechanisms.

Methods

KDM6A shRNA was transfected into PDLSCs by lentivirus. The chondrogenic differentiation potential of PDLSCs was assessed by Alcian blue staining. Immunofluorescence was performed to demonstrate H3K27me3 and H3K4me3 levels during chondrogenesis. SOX9, Col2a1, ACAN and miRNAs (miR‐29a, miR‐204, miR‐211) were detected by real‐time RT‐PCR. Western blot was performed to evaluate SOX9, H3K27me3 and H3K4me3.

Results

The production of proteoglycans in PDLSCs was decreased after knockdown of KDM6A. Depletion of KDM6A inhibited the expression of SOX9, Col2a1, ACAN and resulted in increased H3K27me3 and decreased H3K4me3 levels. EZH2 inhibitor rescued the chondrogenic potential of PDLSCs after knockdown of KDM6A by regulating H3K27me3. Additionally, miR‐29a, miR‐204 and miR‐211 were also involved in the process of PDLSCs chondrogenesis.

Conclusions

KDM6A is required in chondrogenic differentiation of PDLSCs by demethylation of H3K27me3, and EZH2 inhibitor could rescue chondrogenesis of PDLSCs after knockdown of KDM6A. It could be inferred that upregulation of KDM6A or application of EZH2 inhibitor might improve mesenchymal stem cell mediated cartilage regeneration in inflammatory tissue destruction such as osteoarthritis.

     

CircSLC7A2 protects against osteoarthritis through inhibition of the miR‐4498/TIMP3 axis

ObjectivesCircular RNAs (circRNAs) are noncoding RNAs that compete against other endogenous RNA species, such as microRNAs, and have been implicated in many diseases. In this study, we investigated the role of a new circRNA (circSLC7A2) in osteoarthritis (OA).Materials and MethodsThe relative expression of circSLC7A2 was significantly lower in OA tissues than it was in matched controls, as shown by real‐time quantitative polymerase chain reaction (RT‐qPCR). Western blotting, RT‐qPCR and immunofluorescence experiments were employed to evaluate the roles of circSLC7A2, miR‐4498 and TIMP3. The in vivo role and mechanism of circSLC7A2 were also conformed in a mouse model.ResultscircSLC7A2 was decreased in OA model and the circularization of circSLC7A2 was regulated by FUS. Loss of circSLC7A2 reduced the sponge of miR‐4498 and further inhibited the expression of TIMP3, subsequently leading to an inflammatory response. We further determined that miR‐4498 inhibitor reversed circSLC7A2‐knockdown‐induced OA phenotypes. Intra‐articular injection of circSLC7A2 alleviated in vivo OA progression in a mouse model of anterior cruciate ligament transection (ACLT).ConclusionsThe circSLC7A2/miR‐4498/TIMP3 axis of chondrocytes catabolism and anabolism plays a critical role in OA development. Our results suggest that circSLC7A2 may serve as a new therapeutic target for osteoarthritis.     

Bipolar‐associated miR‐499‐5p controls neuroplasticity by downregulating the Cav1.2 subunit CACNB2

Bipolar disorder (BD) is a chronic mood disorder characterized by manic and depressive episodes. Dysregulation of neuroplasticity and calcium homeostasis are frequently observed in BD patients, but the underlying molecular mechanisms are largely unknown. Here, we show that miR‐499‐5p regulates dendritogenesis and cognitive function by downregulating the BD risk gene CACNB2. miR‐499‐5p expression is increased in peripheral blood of BD patients, as well as in the hippocampus of rats which underwent juvenile social isolation. In rat hippocampal neurons, miR‐499‐5p impairs dendritogenesis and reduces surface expression and activity of the L‐type calcium channel Cav1.2. We further identified CACNB2, which encodes a regulatory β‐subunit of Cav1.2, as a direct functional target of miR‐499‐5p in neurons. miR‐499‐5p overexpression in the hippocampus in vivo induces short‐term memory impairments selectively in rats haploinsufficient for the Cav1.2 pore forming subunit Cacna1c. In humans, miR‐499‐5p expression is negatively associated with gray matter volumes of the left superior temporal gyrus, a region implicated in auditory and emotional processing. We propose that stress‐induced miR‐499‐5p overexpression contributes to dendritic impairments, deregulated calcium homeostasis, and neurocognitive dysfunction in BD.     

JUNB‐FBXO21‐ERK axis promotes cartilage degeneration in osteoarthritis by inhibiting autophagy

Osteoarthritis (OA) is a heterogeneous disease that is extremely hard to cure owing to its complex regulation network of pathogenesis, especially cartilage degeneration. FBXO21 is a subunit of ubiquitin E3 ligases that degrades P‐glycoprotein and EID1 by ubiquitination and activates the JNK and p38 pathways; however, its role in OA remains unknown. Here, the main objective of this study was to evaluate the potential effects and mechanism of FBXO21 in OA degeneration, we revealed that FBXO21 is upregulated in the cartilage of patients with OA, aging, and monosodium iodoacetate‐induced OA rats, and chondrocytes treated with interleukin‐1β, tumor necrosis factor‐α, and lipopolysaccharide. Moreover, the in vivo and in vitro knockdown of FBXO21 suppressed OA‐related cartilage degeneration, as evidenced by activated autophagy, upregulated anabolism, alleviated apoptosis, and downregulated catabolism. In contrast, its overexpression promoted OA‐related cartilage degeneration. In addition, using mass spectrometry and co‐immunoprecipitation assay, we demonstrated that the downstream mechanism of FBXO21 inhibits autophagy by interacting with and phosphorylating ERK. Furthermore, FBXO21 alleviated anabolism and enhanced apoptosis and catabolism by inhibiting autophagy in rat chondrocytes. Interestingly, for its upstream mechanism, JUNB promoted FBXO21 expression by directly targeting the FBXO21 promoter, thus further accelerating cartilage degeneration in SW1353 cells and rat chondrocytes. Overall, our findings reveal that the JUNB‐FBXO21‐ERK axis regulates OA apoptosis and cartilage matrix metabolism by inhibiting autophagy. Therefore, FBXO21 is an attractive target for regulating OA pathogenesis, and its knockdown may provide a novel targeted therapy for OA.     

Bone marrow macrophage‐derived exosomal miR‐143‐5p contributes to insulin resistance in hepatocytes by repressing MKP5

ObjectiveIn this study, we aim to explore the role of bone marrow macrophage‐derived exosomes in hepatic insulin resistance, investigate the substance in exosomes that regulates hepatic insulin signalling pathways, reveal the specific molecular mechanisms involved in hepatic insulin resistance and further explore the role of exosomes in type 2 diabetes.Materials and methodsHigh‐fat diet (HFD)‐fed mice were used as obesity‐induced hepatic insulin resistance model, exosomes were isolated from BMMs which were extracted from HFD‐fed mice by ultracentrifugation. Exosomes were analysed the spectral changes of microRNA expression using a microRNA array. The activation of the insulin signalling pathway and the level of glycogenesis were examined in hepatocytes after transfected with miR‐143‐5p mimics. Luciferase assay and western blot were used to assess the target of miR‐143‐5p.ResultsBMMs from HFD‐fed mice were polarized towards M1, and miR‐143‐5p was significantly upregulated in exosomes of BMMs from HFD‐fed mice. Overexpression of miR‐143‐5p in Hep1‐6 cells led to decreased phosphorylation of AKT and GSK and glycogen synthesis. Dual‐luciferase reporter assay and western blot demonstrated that mitogen‐activated protein kinase phosphatase‐5 (Mkp5, also known as Dusp10) was the target gene of miR‐143‐5p. Moreover, the overexpression of MKP5 could rescue the insulin resistance induced by transfection miR‐143‐5p mimics in Hep1‐6.ConclusionBone marrow macrophage‐derived exosomal miR‐143‐5p induces insulin resistance in hepatocytes through repressing MKP5.     

MicroRNA‐148a‐3p suppresses epithelial‐to‐mesenchymal transition and stemness properties via Wnt1‐mediated Wnt/β‐catenin pathway in pancreatic cancer

Although miR‐148a‐3p has been reported to function as a tumour suppressor in various cancers, the molecular mechanism of miR‐148a‐3p in regulating epithelial‐to‐mesenchymal transition (EMT) and stemness properties of pancreatic cancer (PC) cells remains to be elucidated. In the present study, we demonstrated that miR‐148a‐3p expression was remarkably down‐regulated in PC tissues and cell lines. Moreover, low expression of miR‐148a‐3p was associated with poorer overall survival (OS) in patients with PC. In vitro, gain‐of‐function and loss‐of‐function experiments showed that miR‐148a‐3p suppressed EMT and stemness properties as well as the proliferation, migration and invasion of PC cells. A dual‐luciferase reporter assay demonstrated that Wnt1 was a direct target of miR‐148a‐3p, and its expression was inversely associated with miR‐148a‐3p in PC tissues. Furthermore, miR‐148a‐3p suppressed the Wnt/β‐catenin pathway via down‐regulation of Wnt1. The effects of ectopic miR‐148a‐3p were rescued by Wnt1 overexpression. These biological functions of miR‐148a‐3p in PC were also confirmed in a nude mouse xenograft model. Taken together, these findings suggest that miR‐148a‐3p suppresses PC cell proliferation, invasion, EMT and stemness properties via inhibiting Wnt1‐mediated Wnt/β‐catenin pathway and could be a potential prognostic biomarker as well as a therapeutic target in PC.     

PTH‐induced EndMT via miR‐29a‐5p/GSAP/Notch1 pathway contributed to valvular calcification in rats with CKD

BackgroundEndothelial‐to‐mesenchymal transition (EndMT) is a common pathophysiology in valvular calcification (VC) among non‐chronic kidney disease (CKD) patients. However, few studies were investigated in CKD‐induced VC. Parathyroid hormone (PTH) was considered to be an important component of EndMT in CKD‐induced cardiovascular diseases. Therefore, determining whether PTH could induce valvular EndMT and elucidating corresponding mechanism involved further study.MethodsPerforming a 5/6 nephrectomy with a high phosphorus diet was done to construct VC models in rats with CKD. miRNA sequencing was used to ascertain changes in microRNA in human umbilical vein endothelial cells (HUVECs) intervened by PTH. VC was observed by Von Kossa staining and scanning electron microscope.ResultsPTH induced valvular EndMT in VC. Global microRNA expression profiling of HUVECs was examined in PTH versus the control in vitro, in which miR‐29a‐5p was most notably decreased and was resumed by PTHrP(7‐34) (PTH‐receptor1 inhibitor). Overexpression of miR‐29a‐5p could inhibit PTH‐induced EndMT in vitro and valvular EndMT in vivo. The dual‐luciferase assay verified that γ‐secretase‐activating protein (GASP) served as the target of miR‐29a‐5p. miR‐29a‐5p‐mimics, si‐GSAP and DAPT (γ‐secretase inhibitor) inhibited PTH‐induced γ‐secretase activation, thus blocking Notch1 pathway activation to inhibit EndMT in vitro. Moreover, Notch1 pathway activation was observed in VC. Blocking Notch1 pathway activation via AAV‐miR‐29a and DAPT inhibited valvular EndMT. In addition, blocking Notch1 pathway activation was also shown to alleviate VC.ConclusionPTH activates valvular EndMT via miR‐29a‐5p/GSAP/Notch1 pathway, which can contribute to VC in CKD rats.     

Growth differentiation factor 5 in cartilage and osteoarthritis: A possible therapeutic candidate

Growth differentiation factor 5 (GDF‐5) is essential for cartilage development and homeostasis. The expression and function of GDF‐5 are highly associated with the pathogenesis of osteoarthritis (OA). OA, characterized by progressive degeneration of joint, particularly in cartilage, causes severe social burden. However, there is no effective approach to reverse the progression of this disease. Over the past decades, extensive studies have demonstrated the protective effects of GDF‐5 against cartilage degeneration and defects. Here, we summarize the current literature describing the role of GDF‐5 in development of cartilage and joints, and the association between the GDF‐5 gene polymorphisms and OA susceptibility. We also shed light on the protective effects of GDF‐5 against OA in terms of direct GDF‐5 supplementation and modulation of the GDF‐5‐related signalling. Finally, we discuss the current limitations in the application of GDF‐5 for the clinical treatment of OA. This review provides a comprehensive insight into the role of GDF‐5 in cartilage and emphasizes GDF‐5 as a potential therapeutic candidate in OA.     

miR‐4286 functions in osteogenesis and angiogenesis via targeting histone deacetylase 3 and alleviates alcohol‐induced bone loss in mice

ObjectivesAlcohol consumption is one of the leading factors contributing to premature osteopenia. MicroRNA (miRNA) coordinates a cascade of anabolic and catabolic processes in bone homeostasis and dynamic vascularization. The aim was to investigate the protective role of miR‐4286 in alcohol‐induced bone loss and its mechanism.Materials and MethodsThe effect of miR‐4286 and alcohol on bone mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) was explored via multiple in vitro assays, including cell proliferation, QPCR, Western blot, osteogenesis, angiogenesis etc miR‐4286 directly regulated HDAC3 was investigated by luciferase reporter assay, and the function of HDAC3 was also explored in vitro. Moreover, alcohol‐induced bone loss in mice was established to reveal the preventive effect of miR‐4286 by radiographical and histopathological assays.ResultsIn vitro, ethanol dramatically inhibited the proliferation and osteogenesis of BMSCs, and substantially impaired the proliferation and vasculogenesis of HUVECs. However, a forced overexpression of miR‐4286 within BMSCs and HUVECs could largely abolish inhibitory effects by alcohol. Furthermore, alcohol‐induced inhibition on osteogenic and vasculogenic functions was mediated by histone deacetylase 3 (HDAC3), and dual‐luciferase reporter assay showed that HDAC3 was the direct binding target of miR‐4286. In vivo, micro‐CT scanning and histology assessment revealed that miR‐4286 could prevent alcohol‐induced bone loss.ConclusionsWe firstly demonstrated that miR‐4286 might function via intimate osteogenesis‐angiogenesis pathway to alleviate alcohol‐induced osteopenia via targeting HDAC3.     

MicroRNA‐411‐3p inhibits bleomycin‐induced skin fibrosis by regulating transforming growth factor‐β/Smad ubiquitin regulatory factor‐2 signalling

Skin fibrosis, which is characterized by fibroblast proliferation and increased extracellular matrix, has no effective treatment. An increasing number of studies have shown that microRNAs (miRNAs/miRs) participate in the mechanism of skin fibrosis, such as in limited cutaneous systemic sclerosis and pathological scarring. The objective of the present study was to determine the role of miR‐411‐3p in bleomycin (BLM)‐induced skin fibrosis and skin fibroblast transformation. Using Western blot analysis and real‐time quantitative polymerase chain reaction assess the expression levels of miR‐411‐3p, collagen (COLI) and transforming growth factor (TGF)‐β/Smad ubiquitin regulatory factor (Smurf)‐2/Smad signalling factors both in vitro and in vivo with or without BLM. To explore the regulatory relationship between miR‐411‐3p and Smurf2, we used the luciferase reporter assay. Furthermore, miR‐411‐3p overexpression was identified in vitro and in vivo via transfection with Lipofectamine 2000 reagent and injection. Finally, we tested the dermal layer of the skin using haematoxylin and eosin and Van Gieson''s staining. We found that miR‐411‐3p expression was decreased in bleomycin (BLM)‐induced skin fibrosis and fibroblasts. However, BLM accelerated transforming growth factor (TGF)‐β signalling and collagen production. Overexpression of miR‐411‐3p inhibited the expression of collagen, F‐actin and the TGF‐β/Smad signalling pathway factors in BLM‐induced skin fibrosis and fibroblasts. In addition, miR‐411‐3p inhibited the target Smad ubiquitin regulatory factor (Smurf)‐2. Furthermore, Smurf2 was silenced, which attenuated the expression of collagen via suppression of the TGF‐β/Smad signalling pathway. We demonstrated that miR‐411‐3p exerts antifibrotic effects by inhibiting the TGF‐β/Smad signalling pathway via targeting of Smurf2 in skin fibrosis.     

METTL3‐mediated maturation of miR‐589‐5p promotes the malignant development of liver cancer

MiR‐589‐5p could promote liver cancer, but the specific mechanisms are largely unknown. This study examined the role and mechanisms of miR‐589‐5p in liver cancer. The expressions of miR‐589‐5p, METTL3 and m6A in liver cancers were determined by RT‐qPCR. The relationship between miR‐589‐5p and METTL3‐mediated m6A methylation was examined by m6A RNA immunoprecipitation. After transfection, the viability, migration, invasion and expressions of METTL3 and miR‐589‐5p in liver cancer cells were detected by CCK‐8, wound‐healing, transwell and RT‐qPCR. After the xenograft tumour was established in mice, the tumour volume was determined and the expressions of METTL3, miR‐589‐5p, MMP‐2, TIMP‐2, E‐cadherin, N‐cadherin and Vimentin in tumour tissue were detected by RT‐qPCR and Western blotting. In vitro study showed that miR‐589‐5p and METTL3 were highly expressed in liver cancer. METTL3 was positively correlated with miR‐589‐5p. METTL3 up‐regulated the expression of miR‐589‐5p and promoted the maturation of miR‐589‐5p. Overexpressed miR‐589‐5p and METTL3 promoted the viability, migration and invasion of liver cancer cells, while the effects of silencing miR‐589‐5p and METTL3 on the cells were the opposite. The effects of METTL3 overexpression and silencing were reversed by miR‐589‐5p inhibitor and mimic, respectively. In vivo study showed that METLL3 silencing inhibited the growth of xenograft tumour and the expressions of METTL3, MMP‐2, N‐cadherin and Vimentin, promoted the expressions of TIMP‐2 and E‐cadherin, while miR‐589‐5p mimic caused the opposite results and further reversed the effects of METLL3 silencing. In summary, this study found that METTL3‐mediated maturation of miR‐589‐5p promoted the malignant development of liver cancer.     

Down‐regulated HDAC3 elevates microRNA‐495‐3p to restrain epithelial‐mesenchymal transition and oncogenicity of melanoma cells via reducing TRAF5

MicroRNAs (miRNAs) are emerging biomarkers in biological processes and the role of miR‐495‐3p has been identified in melanoma, while the detailed molecular mechanisms remain to be further explored. We aim to explore the effect of histone deacetylase 3 (HDAC3) and miR‐495‐3p on epithelial‐mesenchymal transition (EMT) and oncogenicity of melanoma cells by regulating tumour necrosis factor receptor‐associated factor 5 (TRAF5). Levels of HDAC3, miR‐495‐3p and TRAF5 in melanoma tissues and pigmented nevus tissues were determined, and the predictive roles of HDAC3 and miR‐495‐3p in prognosis of melanoma patients were measured. The melanoma cells were screened and transfected with relative oligonucleotides and plasmids, and the expression of HDAC3, miR‐495‐3p and TRAF5, and phenotypes of melanoma cells were gauged by a series of assays. The relations between HDAC3 and miR‐495‐3p, and between miR‐495‐3p and TRAF5 were confirmed. HDAC3 and TRAF5 were increased while miR‐495‐3p was decreased in melanoma cells and tissues, and the low expression of miR‐495‐3p as well as high expression of HDAC3 indicated a poor prognosis of melanoma patients. Inhibited HDAC3 elevated miR‐495‐3p to suppress EMT and oncogenicity of melanoma cells by reducing TRAF5. HDAC3 particularly bound to miR‐495‐3p and TRAF5 was the target gene of miR‐495‐3p. Our results revealed that down‐regulated HDAC3 elevates miR‐495‐3p to suppress malignant phenotypes of melanoma cells by inhibiting TRAF5, thereby repressing EMT progression of melanoma cells. This study may provide novel targets for melanoma treatment.     

METTL3/N6‐methyladenosine/ miR‐21‐5p promotes obstructive renal fibrosis by regulating inflammation through SPRY1/ERK/NF‐κB pathway activation

Renal fibrosis induced by urinary tract obstruction is a common clinical occurrence; however, effective treatment is lacking, and a deeper understanding of the mechanism of renal fibrosis is needed. Previous studies have revealed that miR‐21 impacts liver and lung fibrosis progression by activating the SPRY1/ERK/NF‐kB signalling pathway. However, whether miR‐21 mediates obstructive renal fibrosis through the same signalling pathway has not been determined. Additionally, studies have shown that N6‐methyladenosine (m⁶A) modification‐dependent primary microRNA (pri‐microRNA) processing is essential for maturation of microRNAs, but its role in the maturation of miR‐21 in obstructive renal fibrosis has not yet been investigated in detail. To address these issues, we employed a mouse model of unilateral ureteral obstruction (UUO) in which the left ureters were ligated for 3, 7 and 14 days to simulate the fibrotic process. In vitro, human renal proximal tubular epithelial (HK‐2) cells were transfected with plasmids containing the corresponding sequence of METTL3, miR‐21‐5p mimic or miR‐21‐5p inhibitor. We found that the levels of miR‐21‐5p and m⁶A modification in the UUO model groups increased significantly, and as predicted, the SPRY1/ERK/NF‐kB pathway was activated by miR‐21‐5p, confirming that miR‐21‐5p plays an important role in obstructive renal fibrosis by enhancing inflammation. METTL3 was found to play a major catalytic role in m⁶A modification in UUO mice and drove obstructive renal fibrosis development by promoting miR‐21‐5p maturation. Our research is the first to demonstrate the role of the METTL3‐m⁶A‐miR‐21‐5p‐SPRY1/ERK/NF‐kB axis in obstructive renal fibrosis and provides a deeper understanding of renal fibrosis.     

MiR‐26a‐tetrahedral framework nucleic acids mediated osteogenesis of adipose‐derived mesenchymal stem cells

ObjectivesDelivery systems that provide time and space control have a good application prospect in tissue regeneration applications, as they can effectively improve the process of wound healing and tissue repair. In our experiments, we constructed a novel micro‐RNA delivery system by linking framework nucleic acid nanomaterials to micro‐RNAs to promote osteogenic differentiation of mesenchymal stem cells.Materials and MethodsTo verify the successful preparation of tFNAs–miR‐26a, the size of tFNAs–miR‐26a were observed by non‐denaturing polyacrylamide gel electrophoresis and dynamic light scattering techniques. The expression of osteogenic differentiation‐related genes and proteins was investigated by confocal microscope, PCR and western blot to detect the impact of tFNAs–miR‐26a on ADSCs. And finally, Wnt/β‐catenin signaling pathway related proteins and genes were detected by confocal microscope, PCR and western blot to study the relevant mechanism.ResultsBy adding this novel complex, the osteogenic differentiation ability of mesenchymal stem cells was significantly improved, and the expression of alkaline phosphatase (ALP) on the surface of the cell membrane and the formation of calcium nodules in mesenchymal stem cells were significantly increased on days 7 and 14 of induction of osteogenic differentiation, respectively. Gene and protein expression levels of ALP (an early marker associated with osteogenic differentiation), RUNX2 (a metaphase marker), and OPN (a late marker) were significantly increased. We also studied the relevant mechanism of action and found that the novel nucleic acid complex promoted osteogenic differentiation of mesenchymal stem cells by activating the canonical Wnt signaling pathway.ConclusionsThis study may provide a new research direction for the application of novel nucleic acid nanomaterials in bone tissue regeneration.

MiR‐26a‐tetrahedral framework nucleic acids mediated osteogenesis of adipose‐derived mesenchymal stem cells.     

circ‐AKT3 aggravates renal ischaemia‐reperfusion injury via regulating miR‐144‐5p /Wnt/β‐catenin pathway and oxidative stress

Renal ischaemia‐reperfusion (RI/R) injury is one major pathological state of acute kidney injury (AKI) with a mortality rate ranking 50% to 80%. MiR‐144‐5p acts as a molecular trigger in various diseases. We presumed that miR‐144‐5p might be involved RI/R injury progression. We found that RI/R injury decreased miR‐144‐5p expression in rat models. MiR‐144‐5p downregulation promoted cell apoptosis rate and activated Wnt/β‐catenin signal in RI/R injury rats. By performing bioinformatic analysis, RIP, RNA pull‐down, luciferase reporter experiments, we found that circ‐AKT3 sponged to miR‐144‐5p and decreased its expression in RI/R injury rats. Moreover, we found that circ‐AKT3 promoted cell apoptosis rate and activated Wnt/β‐catenin signal, and miR‐144‐5p mimic reversed the promotive effect of circ‐AKT3 in rat models. We also found that circ‐AKT3 increased the oxidative stress level in rat models. In conclusion, our study suggests that the circAKT3 is involved RI/R injury progression through regulating miR‐144‐5p/Wnt/β‐catenin pathway and oxidative stress.     

Effect of MiR‐100‐5p on proliferation and apoptosis of goat endometrial stromal cell in vitro and embryo implantation in vivo

The growth of endometrial stromal cells (ESCs) at implantation sites may be a potential factor affecting the success rate of embryo implantation. Incremental proofs demonstrated that ncRNAs (e.g. miRNAs, lncRNAs and circRNAs) were involved in various biological procedures, including proliferation and apoptosis. In this study, the role of miR‐100‐5p on proliferation and apoptosis of goat ESCs in vitro and embryo implantation in vivo was determined. The mRNA expression of miR‐100‐5p was significantly inhibited in the receptive phase (RE) rather than in the pre‐receptive phase (PE). Overexpression of miR‐100‐5p suppressed ESCs proliferation and induced apoptosis. The molecular target of MiR‐100‐5p, HOXA1, was confirmed by 3′‐UTR assays. Meanwhile, the product of HOXA1 mRNA RT‐PCR increased in the RE more than that in the PE. The HOXA1‐siRNA exerted significant negative effects on growth arrest. Instead, incubation of ESCs with miR‐100‐5p inhibitor or overexpressed HOXA1 promoted the cell proliferation. In addition, Circ‐9110 which acted as a sponge for miR‐100‐5p reversed the relevant biological effects of miR‐100‐5p. The intrinsic apoptosis pathway was suppressed in ESCs, revealing a crosstalk between Circ‐9110/miR‐100‐5p/HOXA1 axis, PI3K/AKT/mTOR, and ERK1/2 pathways. To further evaluate the progress in study on embryo implantation regulating mechanism of miR‐100‐5p in vivo, the pinopodes of two phases were observed and analysed, suggesting that, as similar as in situ, miR‐100‐5p was involved in significantly regulating embryo implantation in vivo. Mechanistically, miR‐100‐5p performed its embryo implantation function through regulation of PI3K/AKT/mTOR and ERK1/2 pathways by targeting Circ‐9110/miR‐100‐5p/HOXA1 axis in vivo.