K235 acetylation couples with PSPC1 to regulate the m6A demethylation activity of ALKBH5 and tumorigenesis.

N6-methyladenosine (m6A) modification plays important roles in bioprocesses and diseases. AlkB homolog 5 (ALKBH5) is one of two m6A demethylases. Here, we reveal that ALKBH5 is acetylated at lysine 235 (K235) by lysine acetyltransferase 8 and deacetylated by histone deacetylase 7. K235 acetylation strengthens the m6A demethylation activity of ALKBH5 by increasing its recognition of m6A on mRNA. RNA-binding protein paraspeckle component 1 (PSCP1) is a regulatory subunit of ALKBH5 and preferentially interacts with K235-acetylated ALKBH5 to recruit and facilitate the recognition of m6A mRNA by ALKBH5, thereby promoting m6A erasure. Mitogenic signals promote ALKBH5 K235 acetylation. K235 acetylation of ALKBH5 is upregulated in cancers and promotes tumorigenesis. Thus, our findings reveal that the m6A demethylation activity of ALKBH5 is orchestrated by its K235 acetylation and regulatory subunit PSPC1 and that K235 acetylation is necessary for the m6A demethylase activity and oncogenic roles of ALKBH5.

miRNA-19a exerts an anti-apoptotic effect in spinal cord injured rats via the PTEN pathway.

Introduction: Contusion spinal cord injury is involved in a number of cellular, biochemical and molecular changes. We studied the overall expression pattern of miRNAs on day 1 and 3 after spinal cord injury and the involved pathways. Material and methods: A spinal cord injury model was developed by contusion injury in rats. Microarray analysis and qRT-PCR were done for expression of miRs. The Basso, Beattie and Bresnahan (BBB) locomotor score was determined after spinal injury. Lesions at the injured site were analyzed by cresyl staining. Western blot analysis was carried out to analyze protein levels. Immunohistochemical staining was done to evaluate immunoreactivity. TUNEL assay was performed to determine the number of apoptotic cells. Results: The microarray analysis data suggested that about eight miRs were upregulated whereas four were downregulated in rats subjected to spinal cord injury on day 1. On comparing sham operated rats from the day 3 group two miRs were overexpressed and four were downregulated. miR-19a was the most deregulated. miR-19a antagomir was used as an inhibitor, which aggravated the functional deficit, decreased the protection of spinal cord tissue and elevated the number of apoptotic cells. The treatment of miR-19a antagomir increased the expression of FasL along with PTEN, but it failed to affect the levels of PDCD4. Conclusions: The results suggested that miR-19a plays a potential role in halting the neuronal cell death spinal cord injury and that the protective role of miR-19a may be due to its regulatory effect on pro-apoptotic genes.

LncRNA MEG3 inhibits renal fibrinoid necrosis of diabetic nephropathy via the MEG3/miR-21/ORAI1 axis.

INTRODUCTION: Diabetic nephropathy (DN) is one of the most common and lethal diabetic complications worldwide and is associated with a high risk of mortality. However, the exact mechanism behind its development is unknown. The mesangial cells (MCs) and non-coding RNAs are critical for DN, but it is unknown whether a MEG3/miR-21/ORAI1 regulatory axis exists in MCs. Hence, in this study, we aimed to understand whether the MEG3/miR-21/ORAI1 regulatory axis has a role in the pathophysiology of DN. RESULTS: We demonstrated that high-glucose stimuli downregulated MEG3 and ORAI1 expression while enhancing miR-21 expression. Exogenous miR-21 mimics inhibited ORAI1 expression, which was partially salvaged or reversed by MEG3 overexpression. Furthermore, RIP assay demonstrated that the beads labeled with AGO2 antibody could enrich more miR-21 and MEG3 than those labeled with control IgG antibody; both of them formed the RNA-induced silencing complex. Further, the biochemical indicators of db/db mice significantly improved, and renal fibrinoid necrosis was ameliorated using a miR-21 inhibitor. CONCLUSION: The MEG3/miR-21/ORAI1 axis regulates the manifestation of DN in diabetic mice and MCs, and the miR-21 inhibitor can be a potential therapeutic strategy to alleviate DN, once the presence of such an axis is found in humans.

The lncRNA NEAT1 promotes the epithelial-mesenchymal transition and metastasis of osteosarcoma cells by sponging miR-483 to upregulate STAT3 expression.

BACKGROUND: Osteosarcoma is one of the most prevalent primary bone tumours in adolescents. Accumulating evidence shows that aberrant expression of the long non-coding RNA (lncRNA) NEAT1 and microRNA-483 (miR-483) contribute to the epithelial-mesenchymal transition (EMT), invasion and metastasis of tumour cells. However, the potential regulatory effects of NEAT1 and miR-483 on the EMT of osteosarcoma remain elusive. METHODS: The expression of the NEAT1, miR-483, signal transducer and activator of transcription-1 (STAT1), STAT3, and EMT-associated markers was measured using qRT-PCR or western blotting. NEAT1 overexpression or knockdown was induced by lentivirus-mediated transfection. A luciferase reporter assay was employed to confirm the association between NEAT1/miR-483 and miR-483/STAT3. RNA immunoprecipitation (RIP) was also performed to verify the NEAT1 and miR-483 interaction. Wound healing and transwell assays were implemented to assess the migration and invasion of U2OS cells. Unilateral subcutaneous injection of U2OS into nude mice was performed to investigate tumour metastasis in vivo. RESULTS: The expression of miR-483 was downregulated in both osteosarcoma cell lines and osteosarcoma tissues. The overexpression of miR-483 suppressed the migration, invasion, and expression of EMT-associated proteins in U2OS cells, while simultaneous overexpression of STAT3 partially relieved this suppression. Mechanistically, miR-483 specifically targeted the 3' untranslated region (3'UTR) of STAT3 and repressed its expression. However, NEAT1 sponged miR-438, increased STAT3 expression, and repressed STAT1 expression, subsequently increasing the EMT of osteosarcoma cells. The knockdown of NEAT1 in transplanted U2OS cells impaired the liver and lung metastases of osteosarcoma in nude mice. Moreover, NEAT1 silencing inhibited the mesenchymal- epithelial transition (MET) of osteosarcoma at metastasis sites. CONCLUSIONS: The lncRNA NEAT1/miR-483/STAT3 axis plays a crucial role in regulating the metastasis of osteosarcoma and potentially represents one appealing therapeutic target for osteosarcoma treatment in the future.

Kaempferol promotes bone formation in part via the mTOR signaling pathway.

Previous research indicates that kaempferol (Kae) promotes osteogenesis, but its underlying mechanism of action remains unclear. The present study hypothesized that the osteogenic effects of Kae were mediated through mammalian target of rapamycin (mTOR). To validate this hypothesis, bone marrow mesenchymal stem cells (BMSCs) from ovariectomized (OVX) rats were differentiated into osteoblasts. The bone mineral density and bone microarchitecture of the OVX rats was measured in vivo, while osteogenesis was evaluated in vitro via Alizarin Red S staining and alkaline phosphatase activity measurements in cultured BMSCs. The levels of phosphorylated eukaryotic translation initiation factor 4E­binding protein 1 (p­4E/BP1) and phosphorylated ribosomal protein S6 kinase B1 (p­S6K), and the expression of Runt­related transcription factor 2 and Osterix, were concurrently quantified by western blot analysis. The data suggested that Kae prevented OVX­induced osteoporosis in rats by promoting osteoblastogenesis. Furthermore, treatment with Kae in rat BMSCs enhanced mineralization, elevated ALP activity, increased the expression levels of Runx­2 and Osterix and increased the levels of p­S6K and decreased the levels of p­4E/BP1 and, consistent with its ability to promote osteoblast differentiation. In contrast, treatment with rapamycin, an mTOR inhibitor, produced the opposite phenotype. Taken together, these data suggested that the protective effects of Kae in BMSCs and in the OVX rat model resulted from the induction of osteogenesis via mTOR signaling, or at least partially via the regulation of downstream effectors of the mTOR pathway.

The Clinical Effect of Arthroscopic Rotator Cuff Repair techniques: A Network Meta-Analysis and Systematic Review.

Rotator cuff tears are common and are associated with shoulder pain, disability, and dysfunction. Previous studies that have reported different arthroscopic techniques are controversial. A network meta-analysis with indirect and direct evidence was performed to compare the effectiveness of arthroscopic techniques for the treatment of rotator cuff tears. PUBMED, the Cochrane Register of Controlled Trials, EMBASE and Scopus were searched based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement, and related studies that evaluated arthroscopic techniques for the treatment of rotator cuff tears were identified in May 2018. The primary outcome measure was the retear rate. The secondary outcome measures included the constant score and the range of motion (forward flexion and external rotation). Twenty-one trials comprising 1815 shoulders were included in the study. This study indicated that single-row (SR) repair resulted in a higher retear rate than suture bridge (SB) and double-row (DR) repairs. Moreover, the SR and DR repairs resulted in higher incidences of retear than SB repair. The ranking of the treatments based on the constant score and external rotation was SB repair, SR repair and DR repair, whereas the treatment ranking according to forward flexion was SB repair, DR repair and SR repair. In summary, this network meta-analysis provides evidence that SB repairs might be the best choice to improve the postoperative recovery of function and decrease the retear rate.

Inhibition of miRNA-21 attenuates the proliferation and metastasis of human osteosarcoma by upregulating PTEN.

The present study aimed to investigate the expression of micro (mi)RNA-21 in osteosarcoma cells, and its role in inhibiting the invasion and metastasis of osteosarcoma. Human osteosarcoma MG-63 cells and osteoblast hFOB1.19 cells were used to compare the expression of miRNA-21 using reverse transcription-quantitative polymerase chain reaction analysis. A miRNA-21 mimic or inhibitor were transfected into the MG-63 cells to upregulate and downregulate the expression of miRNA-21, respectively. The present study investigated the proliferation and invasion of transfected MG-63 cells using MTT and Transwell assays. Western blot analyses were used to investigate the regulation of important proteins in the phosphatase and tensin homolog/phosphoinositide 3-kinase/RAC-α serine/threonine-protein kinase (PTEN/PI3K/AKT) signaling pathway. Compared with hFOB1.19 cells, miRNA-21 expression was significantly upregulated in the MG-63 cells (P<0.01), which lead to increased proliferation. Downregulating miRNA-21 expression reduced the proliferation of MG-63 cells compared with hFOB1.19 cells. Invasion assays and western blot analyses revealed that the overexpression of miRNA-21 significantly enhanced the invasion ability of MG-63 cells and the expression of phosphorylated (p-)AKT, while downregulation of miRNA-21 expression reduced the protein level of AKT and p-AKT. In the MG-63 cells, miRNA-21 upregulation significantly inhibited the protein level of PTEN, resulting in significantly increased AKT and PI3K protein levels (P<0.01). In conclusion, the results of the present study indicate that the expression of miRNA-21, PI3K and AKT is increased in the osteosarcoma cell line MG-63, which results in reduced expression of PTEN and increased expression of proteins in the PI3K/AKT signaling pathway, and thus increases the aggressiveness of osteosarcoma cells.