Elabela, a Novel Peptide, Exerts Neuroprotective Effects Against Ischemic Stroke Through the APJ/miR-124-3p/CTDSP1/AKT Pathway.

Elabela (ELA), which is the second endogenous peptide ligand of the apelin receptor (APJ) to be discovered, has been widely studied for potential use as a therapeutic peptide. However, its role in ischemic stroke (IS), which is a leading cause of disability and death worldwide and has limited therapeutic options, is uncertain. The aim of the present study was to investigate the beneficial effects of ELA on neuron survival after ischemia and the underlying molecular mechanisms. Primary cortical neurons were isolated from the cerebral cortex of pregnant C57BL/6J mice. Flow cytometry and immunofluorescence showed that ELA inhibited oxygen-glucose deprivation (OGD) -induced apoptosis and axonal damage in vitro. Additionally, analysis of the Gene Expression Omnibus database revealed that the expression of microRNA-124-3p (miR-124-3p) was decreased in blood samples from patients with IS, while the expression of C-terminal domain small phosphatase 1 (CTDSP1) was increased. These results indicated that miR-124-3p and CTDSP1 were related to ischemic stroke, and there might be a negative regulatory relationship between them. Then, we found that ELA significantly elevated miR-124-3p expression, suppressed CTDSP1 expression, and increased p-AKT expression by binding to the APJ receptor under OGD in vitro. A dual-luciferase reporter assay confirmed that CTDSP1 was a direct target of miR-124-3p. Furthermore, adenovirus-mediated overexpression of CTDSP1 exacerbated neuronal apoptosis and axonal damage and suppressed AKT phosphorylation, while treatment with ELA or miR-124-3p mimics reversed these effects. In conclusion, these results indicated that ELA could alleviate neuronal apoptosis and axonal damage by upregulating miR-124-3p and activating the CTDSP1/AKT signaling pathway. This study, for the first time, verified the protective effect of ELA against neuronal injury after ischemia and revealed the underlying mechanisms. We demonstrated the potential for the use of ELA as a therapeutic agent in the treatment of ischemic stroke.

Corrigendum: CircAFF1 aggravates vascular endothelial cell dysfunction mediated by miR-516b/SAV1/YAP1 axis.

[This corrects the article DOI: 10.3389/fphys.2020.00899.].

Bioinformatics identification of the candidate microRNAs and construction of a competing endogenous RNA regulatory network in lacrimal gland adenoid cystic carcinoma high-grade transformation.

Adenoid cystic carcinoma of the lacrimal gland (LACC) is a major orbital malignancy. The recurrence rate and mortality rate are higher in LACC high-grade transformation (LACC-HGT) compared with in LACC. The present study aimed to identify the candidate microRNAs (miRNAs/miRs) and construct a competing endogenous RNA (ceRNA) regulatory network for LACC-HGT. A miRNA microarray on paraffin-embedded tissues was performed to identify the differentially expressed miRNAs (DEMs) of LACC-HGT. The overlap with the salivary adenoid cystic carcinoma miRNA/RNA sequencing dataset in the Gene Expression Omnibus was used to identify candidate miRNAs. In order to construct a ceRNA regulatory network of LACC-HGT, a microarray of mRNA and circRNA in primary cell lines was performed. The circRNAs and genes with high expression in LACC-HGT were predicted as targeting miRNAs, and the circRNA-miRNA-mRNA regulatory network was constructed. miR-140-3p was identified as part of the ceRNA network and as a candidate miRNA, therefore this was further analyzed using reverse transcription-quantitative (RT-q)PCR. Overall, the Agilent Human microarray analysis identified a total of 16 DEMs from the LACC-HGT paraffin-embedded tissues. A total of 653 DECs and 9,566 DEGs of LACC-HGT primary cell lines were screened via the microarray of mRNA and circRNA. The ceRNA regulatory network was constructed using the cross-binding of circRNA-miRNA, miRNA-mRNA and the downregulated miRNAs in LACC-HGT to clearly demonstrate the circRNA-miRNA-mRNA interaction relationship. RT-qPCR results confirmed that miR-140-3p was downregulated in LACC-HGT tissues and primary cell lines compared with LACC. Target genes CD200 and parathyroid hormone-related protein were significantly upregulated in LACC-HGT primary cell lines. miR-140-3p and its target genes may play an important role in LACC-HGT pathogenesis. In conclusion, the current bioinformatics study constructed a ceRNA network based on a microarray, which may help identify novel miRNA therapeutic targets for LACC-HGT.

circAFF1 Aggravates Vascular Endothelial Cell Dysfunction Mediated by miR-516b/SAV1/YAP1 Axis.

Pathological vascular endothelial damage caused by hypoxia is the basis of many vascular-related diseases. However, the role of circular RNA in hypoxic vascular injury is still poorly understood. Here, we found that hypoxia induced AFF1 circular RNA (circAFF1) can activate the SAV1/YAP1 and lead to the dysfunction of vascular endothelial cells. In HUV-EC-C and HBEC-5i cells, circAFF1 was upregulated under CoCl2 induced hypoxic conditions. The abnormal expression of circAFF1 inhibited the proliferation, tube formation, migration of vascular endothelial cells. The effect of circAFF1 is achieved by the adsorption of miR-516b to release SAV1, which in turn causes the phosphorylation of YAP1. Moreover, we found that the upregulation of circAFF1 in 235 Patients with subarachnoid hemorrhage. Taken together, we clarify the role of circAFF1/miR-516b/SAV1/YAP1 axis in vascular endothelial dysfunction and its potential early diagnostic value of disease caused by hypoxia injury in blood vessels.

Downregulation of circFASTKD1 ameliorates myocardial infarction by promoting angiogenesis.

Circular RNAs (circRNAs), a novel class of endogenous long non-coding RNAs, have attracted considerable attention due to their closed continuous loop structure and potential clinical value. In this study, we investigated the function of circFASTKD1 in vascular endothelial cells. CircFASTKD1 bound directly to miR-106a and relieved its inhibition of Large Tumor Suppressor Kinases 1 and 2, thereby suppressing the Yes-Associated Protein signaling pathway. Under both normal and hypoxic conditions, the ectopic expression of circFASTKD1 reduced the viability, migration, mobility and tube formation of vascular endothelial cells, whereas the downregulation of circFASTKD1 induced angiogenesis by promoting these processes. Moreover, downregulation of circFASTKD1 in mice improved cardiac function and repair after myocardial infarction. These findings indicate that circFASTKD1 is a potent inhibitor of angiogenesis after myocardial infarction and that silencing circFASTKD1 exerts therapeutic effects during hypoxia by stimulating angiogenesis in vitro and in vivo.

Carbon Monoxide Improves Neurologic Outcomes by Mitochondrial Biogenesis after Global Cerebral Ischemia Induced by Cardiac Arrest in Rats.

Mitochondrial dysfunction contributes to brain injury following global cerebral ischemia after cardiac arrest. Carbon monoxide treatment has shown potent cytoprotective effects in ischemia/reperfusion injury. This study aimed to investigate the effects of carbon monoxide-releasing molecules on brain mitochondrial dysfunction and brain injury following resuscitation after cardiac arrest in rats. A rat model of cardiac arrest was established by asphyxia. The animals were randomly divided into the following 3 groups: cardiac arrest and resuscitation group, cardiac arrest and resuscitation plus carbon monoxide intervention group, and sham control group (no cardiac arrest). After the return of spontaneous circulation, neurologic deficit scores (NDS) and S-100B levels were significantly decreased at 24, 48, and 72 h, but carbon monoxide treatment improved the NDS and S-100B levels at 24 h and the 3-day survival rates of the rats. This treatment also decreased the number of damaged neurons in the hippocampus CA1 area and increased the brain mitochondrial activity. In addition, it increased mitochondrial biogenesis by increasing the expression of biogenesis factors including peroxisome proliferator-activated receptor-γ coactivator-1α, nuclear respiratory factor-1, nuclear respiratory factor-2 and mitochondrial transcription factor A. Thus, this study showed that carbon monoxide treatment alleviated brain injury after cardiac arrest in rats by increased brain mitochondrial biogenesis.

[Expression, purification, antibody preparation and identification of tumor protein translationally-controlled 1 (TPT1)].

AIM: To express and purify the fusion protein of TPT1 (tumor protein translationally-controlled 1) in prokaryocytes and to prepare rabbit anti-TPT1 antibody. METHODS: The expression vector pRSETA(2)-TPT1 was reconstructed and transformed into BL21 (DE3). TPT1 fusion protein was induced by IPTG and the TPT1 fusion protein purified by Ni-NTA His Bind resin was used to immunize the rabbit. The titer of the polyclonal antibody was detected by ELISA and its specificity was analyzed by Western blot and IF (immunofluorescence). RESULTS: The TPT1 fusion protein was highly expressed in E.coli and specific polyclonal antibody was obtained after the immunization. CONCLUSION: The recombinant prokaryotic expression vector pRSETA(2)-TPT1 is successfully constructed and high expression of TPT1 is induced in E.coli, which may lay the basis for further study of the development and treatment of tumors and other diseases.

[Establishment of high-yield suspension cell line of Curcuma zedoaria (Berg.) Rosc and study on the volatile oil synthesis-controlling with precursors].

The condition for high-yield suspension cell line and the precursors of volatile oil synthesis of Curcuma zedoaria (Berg.) Rosc were studied. The results showed that the light yellow particle callus was suitable for establishment of the high-yield suspension cell line. The optimum conditions for cell growth were MS medium added 15-30 g/L glucose and 15-30 g/L sucrose (1:1) as carbon source, the total concentration of 80 mmol/L nitrogen source combined NH4+ with NO3- (1:3), hormones of 3.0-5.0 mg/L 6-BA, 1.0 mg/L 2,4-D and dark culture after 10-15 days light culture. The 229 g/L cell (FW) and 2.11% content of volatile oil were obtained in vitro. The addition of precursors of calcium pantothenate, ammonium acetate and potassium acetate during the middle period of the cell suspension culture enhanced the volatile oil content respectively, and ammonium acetate was most effective among them. The highest yield of volatile oil obtained was 3.11% and 8.27 g/L respectively , which was 1.25 and 1.2 times of the control group.