TTN-AS1 delivered by gastric cancer cell-derived exosome induces gastric cancer progression through in vivo and in vitro studies.

Extracellular communication within the tumor microenvironment exerts critical functions in tumor progression. Moreover, exosomes are capable of packaging into long non-coding RNAs (lncRNAs) to regulate extracellular communication. We tried to discuss the role of exosomal lncRNA TTN-AS1 and its molecular mechanism on gastric cancer (GC) progression. Bioinformatics analysis depicted increased TTN-AS1 in GC which shared correlation with poor prognosis. Clinical tissue and cellular experiments also confirmed the elevation of TTN-AS1 in GC tissues and cells. GC cell (AGS)-derived Exo could be uptake by NCI-N87 cells to induce malignant features of GC cells. Functionally, TTN-AS1 could upregulate ZEB1 expression by binding to miR-499a-5p. In addition, in vitro experiments demonstrated that ZEB1 targeted and activated CDX2 transcription and promoted CDX2 expression; silencing CDX2 inhibited malignant phenotypes of AGS and NCI-N87 cells. Furthermore, Exo-TTN-AS1 promoted GC cell growth and migration by promoting CDX2 expression. Exosomal TTN-AS1 from GC cells could also promote metastasis of GC in vivo. In conclusion, our findings provided evidence describing that exosomes derived from GC cells transferred TTN-AS1 to GC cells, which aggravate GC through the miR-499a-5p/ZEB1/CDX2 axis. 1. Exo derived from GC cells promotes the growth and metastasis of GC cells by carrying TTN-AS1. 2. TTN-AS1 acts as a ceRNA to adsorb miR-499a-5p to regulate the expression of ZEB1. 3. ZEB1 targets and activates CDX2 transcription. 4. GC cell-derived Exo-TTN-AS1 enhances the growth and metastasis of GC cell xenografts in vivo.

Upregulation of Reg IV and Hgf mRNAs by Intermittent Hypoxia via Downregulation of microRNA-499 in Cardiomyocytes.

Sleep apnea syndrome (SAS) is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]), and is a risk factor for cardiovascular disease (CVD) and insulin resistance/Type 2 diabetes. However, the mechanisms linking IH stress and CVD remain elusive. We exposed rat H9c2 and mouse P19.CL6 cardiomyocytes to experimental IH or normoxia for 24 h to analyze the mRNA expression of several cardiomyokines. We found that the mRNA levels of regenerating gene IV (Reg IV) and hepatocyte growth factor (Hgf) in H9c2 and P19.CL6 cardiomyocytes were significantly increased by IH, whereas the promoter activities of the genes were not increased. A target mRNA search of microRNA (miR)s revealed that rat and mouse mRNAs have a potential target sequence for miR-499. The miR-499 level of IH-treated cells was significantly decreased compared to normoxia-treated cells. MiR-499 mimic and non-specific control RNA (miR-499 mimic NC) were introduced into P19.CL6 cells, and the IH-induced upregulation of the genes was abolished by introduction of the miR-499 mimic, but not by the miR-499 mimic NC. These results indicate that IH stress downregulates the miR-499 in cardiomyocytes, resulting in increased levels of Reg IV and Hgf mRNAs, leading to the protection of cardiomyocytes in SAS patients.

Cyclic stretching boosts microRNA-499 to regulate Bcl-2 via microRNA-208a in atrial fibroblasts.

MicroRNAs that modulate transcription can regulate other microRNAs and are also up-regulated under pathological stress. MicroRNA-499 (miR-499), microRNA-208a (miR-208a) and B-cell lymphoma 2 (Bcl-2) play roles in cardiovascular diseases, such as direct reprogramming of cardiac fibroblast into cardiomyocyte and cardiomyocyte apoptosis. Whether miR208a, miR499 and Bcl-2 were critical regulators in cardiac fibroblast apoptosis under mechanical stretching conditions in human cardiac fibroblasts-adult atrial (HCF-aa) was investigated. Using negative pressure, HCF-aa grown on a flexible membrane base were cyclically stretched to 20% of their maximum elongation. In adult rats, an aortocaval shunt was used to create an in vivo model of volume overload. MiR208a was up-regulated early by stretching and returned to normal levels with longer stretching cycles, whereas the expression of miR499 and Bcl-2 was up-regulated by longer stretching times. Pre-treatment with antagomir-499 reversed the miR-208a down-regulation, whereas Bcl-2 expression could be suppressed by miR-208a overexpression. In the HCF-aa under stretching for 1 h, miR-499 overexpression decreased pri-miR-208a luciferase activity; this inhibition of pri-miR-208a luciferase activity with stretching was reversed when the miR-499-5p binding site in pri-miR-208a was mutated. The addition of antagomir-208a reversed the Bcl-2-3'UTR suppression from stretching for 1 h. Flow cytometric analysis revealed that pre-treatment with miR-499 or antagomir-208a inhibited cellular apoptosis in stretched HCF-aa. In hearts with volume overload, miR-499 overexpression inhibited myocardial miR-208a expression, whereas Bcl-2 expression could be suppressed by the addition of miR-208a. In conclusion, miR-208a mediated the regulation of miR-499 on Bcl-2 expression in stretched HCF-aa and hearts with volume overload.

Overexpression of long non-coding RNA XIST promotes IL-1ß-induced degeneration of nucleus pulposus cells through targeting miR-499a-5p.

BACKGROUND: Long non-coding RNA X-interactive specific transcript (XIST) is implicated in many diseases. However, its role and interaction with microRNA (miR)-499a-5p in intervertebral disc degeneration (IDD) remained unclear. METHODS: Nucleus pulposus (NP) tissue samples were collected and nucleus pulposus cells (NPCs) were isolated for Interleukin-1ß (IL-1ß) treatment and identification. XIST and miR-499a-5p expressions in the tissue were measured with quantitative real-time polymerase chain reaction (qRT-PCR). After IL-1ß treatment, NPC apoptosis was detected by flow cytometry. The potential binding sites of XIST and miR-499a-5p were predicted by starBase and confirmed by dual-luciferase reporter assay. Relative expressions of tissue inhibitor of metalloproteinases-3 (TIMP-3), Matrix metalloproteinases-3 (MMP-3), MMP-13, Collagen II, Aggrecan and apoptosis-related proteins (Bcl-2 associated X protein, Bax; B-cell lymphoma 2, Bcl-2; cleaved caspase-3) were measured by qRT-PCR and Western blot as needed. RESULTS: XIST expression was upregulated in the NP tissues of patients with IDD, and IL-1ß treatment resulted in a degradation of NPCs. Overexpressed XIST promoted the effects of IL-1ß on increasing NPC apoptosis and expressions of XIST, MMP-3, MMP-13, Bax and Cleaved caspase-3, but down-regulated TIMP-3, Collagen II, Aggrecan and Bcl-2 expressions. Silencing XIST, however, showed the opposite effects to its overexpression. MiR-499a-5p expression was downregulated in NP tissues of IDD patients and could bind with XIST, while its upregulation reversed the effects of overexpressed XIST in the IL-1ß-treated NPCs. CONCLUSION: Overexpressed XIST caused NPC degeneration through promoting apoptosis and extracellular matrix degradation of IL-1ß-treated NPCs through targeting miR-499a-5p, and therefore can serve as a potential treatment for IDD.

Retracted: Tetramethylpyrazine partially relieves hypoxia-caused damage of cardiomyocytes H9c2 by downregulation of miR-449a.

Inadequate oxygen supply is probably one of the most important pathophysiological mechanisms of cardiomyocyte damage in ischemic heart disease. Tetramethylpyrazine (TMP, also known as ligustrazine) is the main active ingredient isolated from the rhizome of Ligusticum chuanxiong Hort. A previous study reported that the TMP could exert cardioprotective activity. This study aimed to explore the molecular mechanism of the protective effects of TMP on cardiomyocyte damage caused by hypoxia. The viability and apoptosis of cardiomyocytes H9c2 were detected using cell counting kit-8 assay and annexin V-FITC/PI staining, respectively. Quantitative reverse transcription polymerase chain reaction was conducted to measure the expression level of microRNA-449a (miR-449a). Cell transfection was performed to upregulate the expression level of miR-449a or downregulate the expression level of sirtuin 1 (Sirt1). The protein expression levels of Sirt1 and key factors involved in cell apoptosis and phosphatidylinositol 3-kinase/protein kinase 3 (PI3K/AKT) pathway were evaluated using western blot analysis. We found that the hypoxia incubation inhibited H9c2 viability, induced cell apoptosis, and inactivated the PI3K/AKT pathway. TMP treatment partially relieved the hypoxia-caused H9c2 cell viability loss and apoptosis, as well as reversed the hypoxia-caused inactivation of the PI3K/AKT pathway. Moreover, TMP partially alleviated the upregulation of miR-449a in H9c2 cells caused by hypoxia. Overexpression of miR-449a weakened the effects of TMP on hypoxia-treated H9c2 cells. Furthermore, Sirt1 was a target gene of miR-449a. Knockdown of Sirt1 also weakened the effects of TMP on hypoxia-treated H9c2 cells. In conclusion, TMP partially relieved hypoxia-caused cardiomyocytes H9c2 viability loss and apoptosis at least through downregulating miR-499a, upregulating Sirt1, and then activating the PI3K/AKT pathway.

Multiplexed fluorometric determination for three microRNAs in acute myocardial infarction by using duplex-specific nuclease and MoS2 nanosheets.

Circulating microRNAs are of diagnostic value for acute myocardial infarction (AMI). This study describes a fluorometric assay for multiplexed detection of the AMI biomarkers microRNA-499, microRNA-133a and microRNA-1. The assay involves the following two steps: (a) duplex-specific nuclease (DSN)-mediated signal amplification using aptamer-based sensor; (b) MoS2 nanosheets-based multiplexed fluorometric signal detection, and fluorometric signals have excitation/emission maxima at 492/518 nm for microRNA-499, 565/580 nm for microRNA-133a and 649/663 nm for microRNA-1. The assay has detection limits of around 100 fM for all three microRNAs. The assay is highly specific and rapid. It demonstrates that the expressions of microRNA-499, microRNA-133a and microRNA-1 are significantly higher in AMI patients. The ROC curves allow a clear distinction to be made between AMI group and non-AMI group. Graphical abstractSchematic representation of a multiplexed fluorometric method based on duplex-specific nuclease (DSN)-mediated signal amplification and MoS2 nanosheets-based fluorometric signal detection (DSN-MoS2) for rapid, sensitive and specific detection of microRNAs in AMI.

Pre-micro RNA-499 Gene Polymorphism rs3746444 T/C is Associated with Susceptibility to Rheumatoid Arthritis in Egyptian Population.

Pre-miRNA-499 gene is associated with autoimmune disease. Mir-449 rs3746444 polymorphism is inconsistent for rheumatoid arthritis (RA). This study aimed to investigate association of mir-499 rs3746444 polymorphism with RA activity and severity in Egyptian population. The study population was conducted as case control study in 100 RA patients diagnosed according to the American College of Rheumatology classification criteria for RA, and the control group included 100 healthy subjects who were age-and sex-matched to the RA group. Different genotypes were assessed using polymerase chain reaction-restriction fragment length polymorphism. 95% Confidence interval and odds ratio were defined to assess the strength of association. Regarding patients, thirty-three patients carried TT genotype, fifty-three patients carried TC genotype and fourteen patients carried CC genotype. So the frequency of the minor C allele in RA patients was significantly higher than the control subjects (P = 0.037). TC, CC genotypes and C allele frequencies were significantly associated with disease severity as they had high rheumatoid factor (55.78 µIU/ml) and anti-cyclic citrullinated peptide (Anti-CCP) antibody (297.32 µIU/ml). Moreover, the heterozygote TC had more severe and more active form of the disease compared with homozygote CC or TT as they had high Anti-CCP antibody, and disease activity score 28 (score 5). Our work suggests that C allele of Pre-miRNA rs3746444 polymorphism contributes to heritability of susceptibility to RA compared to T allele. This polymorphism was associated with the activity and severity of the disease.

Effects of MicroRNA-499 On the Inflammatory Damage of Endothelial Cells During Coronary Artery Disease Via the Targeting of PDCD4 Through the NF-Κß/TNF-α Signaling Pathway.

BACKGROUND/AIMS: This study aimed to analyze the impact of microRNA-499 (miR-499) on the inflammatory damage of endothelial cells during coronary artery disease (CAD) via the targeting of PDCD4 through the NF-kB/ TNF-α signaling pathway. METHODS: A total of 216 CAD patients (CAD group) and 90 healthy people (normal group) were enrolled in our study. Endothelial cells were collected and assigned into normal, OX-LDL, negative control (NC), miR-499 inhibitor, miR-499 mimic, PDCD4 siRNA, and miR-499 inhibitor + PDCD4 siRNA groups. The qRT-PCR and western blotting were performed to detect the mRNA and protein expression levels of PDCD4 and miR-499. The MTT assay was performed to determine cell viability, ELISA was performed to determine the expression levels of inflammatory factors, and flow cytometry assay to evaluate cell apoptosis. RESULTS: Increased miR-499 expression and decreased PDCD4 expression in the plasma were observed in the CAD group compared with the normal group, demonstrating a negative correlation between miR-499 and PDCD4. Compared to the normal and miR-499 inhibitor groups, the survival rate of cells and PDCD4 expression were decreased; and the expressions of miR-499, IL-6, IL-8, IL-1ß, TNF-α, NF-kB, VCAM-1, ICAM-1 and MCP-1 and the apoptosis rate were all elevated in the OX-LDL, NC, miR-499 mimic, PDCD4 siRNA and miR-499 inhibitor + PDCD4 siRNA groups. Compared to the OX-LDL, NC and miR-499 inhibitor + PDCD4 siRNA groups, PDCD4 expression and the survival rate of cells were increased; and the IL-6, IL-8, IL-1ß, TNF-α, NF-κB, VCAM-1, ICAM-1 and MCP-1 expression levels and the apoptosis rate were all reduced in the miR-499 inhibitor group. In the PDCD4 siRNA group, PDCD4 expression and the survival rate of cells were lower, and the expression levels of IL-6, IL-8, IL-1ß, TNF-α, NF-κB, VCAM-1, ICAM-1 and MCP-1 and the apoptosis rate were all higher compared with the miR-499 mimic group. In the miR-499 inhibitor + PDCD4 siRNA group, PDCD4 expression and the survival rate of cells were higher, and the expression levels of IL-6, IL-8, IL-1ß, TNF-α, NF-κB, VCAM-1, ICAM-1, and MCP-1 and the apoptosis rate were all lower than those in the PDCD4 siRNA group. CONCLUSION: Down-regulated miR-499 expression increased PDCD4 expression and protected endothelial cells from inflammatory damage during CAD by inhibiting the NF-κB/TNF-α signaling pathway.

Rs3746444 T>C locus in miR-499 increases the susceptibility to hepatocellular carcinoma: A meta-analysis 14812 subjects.

BACKGROUND: Recently, many investigations have suggested that the rs3746444 T>C locus in the microRNA (miR)-499 gene may contribute to the occurrence of cancer. However, reports on the association between rs3746444 and hepatocellular carcinoma (HCC) are conflicting. AIM: To further understand and explore the potential correlation between the single-nucleotide polymorphism of rs3746444 and the incidence of HCC. METHODS: In this meta-analysis, we obtained electronic literature by searching the PubMed, Embase and Chinese BioMedical Disc databases (through May 20, 2022). All eligible case-control, prospective cohort or nested case-control studies with sufficient data for calculating the odds ratios with their 95% confidence intervals were included. RESULTS: Ultimately, a total of 17 independent studies were included. We identified that rs3746444 was associated with the development of HCC (C vs T: P = 0.019 and CC/CT vs TT: P = 0.016). In Asian individuals, rs3746444 was associated with susceptibility to HCC (C vs T: P = 0.013 and CC/CT vs TT: P = 0.016). In addition, this study identified that the miR-499 rs3746444 locus was associated with susceptibility to HCC in the normal/healthy control subgroup (C vs T: P = 0.034 and CC/CT vs TT: P = 0.024). CONCLUSION: In summary, this meta-analysis highlights that rs3746444 in the miR-499 gene is involved in the occurrence of HCC, especially in Asian individuals.

The additive effects of nicotinamide mononucleotide and melatonin on mitochondrial biogenesis and fission/fusion, autophagy, and microRNA-499 in the aged rat heart with reperfusion injury.

The prognosis of myocardial ischemia/reperfusion (I/R) injury is poor in elderly patients. Aging increases the susceptibility of the heart to cell death from I/R injury and prevents the optimal effectiveness of cardioprotective modalities. Since the interaction of aging with cardioprotection is multifactorial, combination therapy may overcome the above-mentioned burden through correcting various components of the injury. Here, we explored the effects of nicotinamide mononucleotide (NMN)/melatonin combination therapy on mitochondrial biogenesis and fission/fusion, autophagy, and microRNA-499 in the aged rat heart with reperfusion injury. Ex vivo model of myocardial I/R injury was established by coronary occlusion and re-opening in 30 aged male Wistar rats (400-450 g, 22-24 months old). NMN (100 mg/kg/48 h, intraperitoneally) was administered over 28 days before I/R, and melatonin (50 µM) was added to the perfusion solution at early reperfusion. CK-MB release and expression of mitochondrial biogenesis genes and proteins, mitochondrial fission/fusion proteins, autophagy genes, and microRNA-499 were assessed. NMN/melatonin combination therapy concomitantly decreased CK-MB release in aged reperfused hearts (P < .001). It also upregulated SIRT1/PGC-1α/Nrf1/TFAM profiles at both gene and protein levels, Mfn2 protein, and microRNA-499 expression, and downregulated Drp1 protein and Beclin1, LC3, and p62 genes (P < .05 to P < .001). The effect of combination therapy was greater than individual ones. Co-application of NMN/melatonin within the setting of I/R injury in the aged rat heart induced noticeable cardioprotection through modulation of a coordinated network including microRNA-499 expression along with mitochondrial biogenesis associated with SIRT1/PGC-1α/Nrf1/TFAM profiles, mitochondrial fission/fusion, and autophagy, therefore, appears to prevent the burden of myocardial I/R injury in elderly patients.

Combination of azithromycin and methylprednisolone alleviates Mycoplasma pneumoniae induced pneumonia by regulating miR-499a-5p/STAT3 axis.

Mycoplasma pneumoniae (M. pneumoniae) is a contributing factor to community-acquired pneumonia in children. The present study sought to explain the underlying mechanism of azithromycin (AZM) combined with methylprednisolone (MP) in the treatment of M. pneumoniae infection. Peripheral blood samples were obtained from patients with M. pneumoniae and healthy volunteers for analysis. A549 cells were infected with M. pneumoniae to construct an in vitro cell model with M. pneumoniae, followed by treatment with AZM and MP. Cell Counting Kit-8 and TUNEL assays were conducted to detect cell viability and apoptosis. RT-qPCR was employed to measure the expression levels of microRNA (miR)-499a-5p and STAT3. Western blotting was performed to measure the expression of STAT3 and apoptosis-related proteins. Luciferase report assay was performed to verify the binding site between miR-499a-5p and STAT3. The production of inflammatory cytokines was determined using ELISA kits. The results exhibited the downregulated miR-499a-5p and dysregulated inflammatory cytokines in peripheral blood of patients and M. pneumoniae-infected A549 cells. AZM and MP treatment alone or combined significantly inhibited inflammatory response, cell viability loss and promoted apoptosis in A549 cells infected with M. pneumoniae, which was partly reversed by inhibition of miR-499a-5p. Furthermore, miR-499a-5p could negatively regulate its direct target STAT3. In addition, STAT3 is also regulated by AZM and MP. Collectively, the present results suggested that combination treatment of AZM and MP could inhibit M. pneumoniae infection-induced inflammation, cell viability loss and promoted apoptosis partly by regulating miR-499a-5p/STAT3 axis.

The correlation of microRNA-499 rs3746444 T>C locus with the susceptibility of gastric cancer: from a case-control study to a meta-analysis.

The relationship between rs3746444 T>C single-nucleotide polymorphism (SNP) in microRNA (mir)-499 and risk of gastric cancer (GC) has been widely investigated. However, the association was still unconfirmed. Here, we first recruited 490 GC patients and 1476 controls, and conducted a case-control study. And we did not find any association between rs3746444 T>C SNP polymorphism and risk of GC. Subsequently, we conducted a meta-analysis to explore the association of mir-499 rs3746444 polymorphism with GC development. Two authors searched the PubMed and EMBASE databases up to October 15, 2019 independently. Finally, nine literatures involving 12 independent studies were included. In total, 3954 GC cases and 9745 controls were recruited for meta-analysis. The results suggested that allele model, homozygote model and recessive model could increase the risk of overall GC (P = 0.002, 0.009 and 0.013, respectively). When we excluded the studies violated HWE, this association was also found in allele model (P = 0.020) and dominant model (P= 0.044). In subgroup analyses, we identified that rs3746444 SNP in mir-499 increased the risk of GC in Asians and gastric cardiac adenocarcinoma (GCA) subgroups. No significant bias of selection was found (all P>0.1). Test of sensitivity analysis indicated that our findings were stable. Additionally, we found that the power value was 0.891 in the allele model, suggesting the reliability of our findings. In summary, our analysis confirmed the association between rs3746444 and the risk of GC, especially in Asians and in patients with GCA.

MicroRNA-499 serves as a sensitizer for lung cancer cells to radiotherapy by inhibition of CK2α-mediated phosphorylation of p65.

The present study aimed to define the tumor-suppressive role of microRNA-499 (miR-499) in lung cancer cells and its underlying mechanism. First, qRT-PCR analysis revealed poor expression of miR-499 in clinical samples and cell lines of lung cancer. Next, we performed loss- and gain-of-function experiments for the expression of miR-499 in lung cancer cells exposed to irradiation (IR) to determine the effect of miR-499 expression on cell viability and apoptosis as well as tumor growth. Results showed that overexpression of miR-499 inhibited cell viability, enhanced the radiosensitivity of lung cancer cells, and promoted cell apoptosis under IR. Furthermore, CK2α was verified to be a target of miR-499, and miR-499 was identified to repress p65 phosphorylation by downregulating CK2α expression, which ultimately diminished the survival rate of lung cancer cells under IR. Collectively, the key findings of the study illustrate the tumor-inhibiting function of miR-499 and confirmed that miR-499-mediated CK2α inhibition and altered p65 phosphorylation enhances the sensitivity of lung cancer cells to IR.

Restoring microRNA-499-5p Protects Sepsis-Induced Lung Injury Mice Via Targeting Sox6.

BACKGROUND: MicroRNAs (miRs) are known to participate in sepsis; hence, we aim to discuss the protective effect of miR-499-5p targeting sex-determining region Y-related high-mobility-group box 6 (Sox6) on sepsis-induced lung injury in mice. METHODS: The sepsis-induced lung injury model was established by cecal ligation and puncture. The wet/dry weight (W/D) ratio, miR-499-5p, Sox6, Caspase-3 and Caspase-9 expression in lung tissues of mice were tested. Lung injury score, collagen fibers and the degree of pulmonary fibrosis in lung tissues were determined. Further, the cell apoptosis in lung tissues was measured. The inflammatory factors contents and oxidative stress indices in bronchoalveolar lavage fluid (BALF) and lung tissues were detected via loss- and gain-of-function assays. The targeting relation between miR-499-5p and Sox6 was verified. RESULTS: W/D ratio and Sox6 were increased while miR-499-5p was decreased in lung tissues of sepsis-induced lung injury mice. Restored miR-499-5p or depleted Sox6 alleviated lung tissues pathology, reduced lung injury score, collagen fibers, the degree of pulmonary fibrosis, TUNEL positive cells, Caspase-3 and Caspase-9 protein expression and inflammatory factors contents in BALF and lung tissues as well as oxidative stress response in lung tissues of sepsis-induced lung injury mice. miR-499-5p targeted Sox6. CONCLUSION: High expression of miR-499-5p can attenuate cell apoptosis in lung tissues and inhibit inflammation of sepsis-induced lung injury mice via depleting Sox6, and it is a potential candidate marker and therapeutic target for sepsis-induced lung injury.

Long non-coding RNA LINC01018 inhibits the progression of acute myeloid leukemia by targeting miR-499a-5p to regulate PDCD4.

Acute myeloid leukemia (AML) is a highly heterogeneous disease with a very high mortality rate. In recent years, an increasing number of studies have proven that long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) may serve as useful biomarkers in various cancer types. However, the mechanism of LINC01018 and miR-499a-5p in AML requires further investigation. The mRNA expression of LINC01018, miR-499a-5p and PDCD4 in AML tissues and cells was detected using reverse transcription-quantitative polymerase chain reaction. Cell proliferation was measured using Cell Counting kit-8 and EdU assays. Cell apoptosis was monitored via a TUNEL staining assay. Protein expression of PDCD4, Bax and Bcl-2 was measured using western blot analysis. The interaction between PDCD4 and LINC01018 or miR-499a-5p was verified by RNA pull-down, RIP and dual-luciferase reporter assays. LINC01018 and PDCD4 were downregulated in AML, while miR-499a-5p was upregulated. LINC01018-overexpression suppressed AML cell proliferation and induced AML cell apoptosis, while miR-499a-5p transfection reversed these effects. LINC01018 acted as a sponge of miR-499a-5p, and PDCD4 was demonstrated to be targeted by miR-499a-5p. Knockdown of miR-499a-5p suppressed AML cell proliferation and promoted AML cell apoptosis, but silencing PDCD4 abolished this effect. LINC01018 inhibited AML cell growth by modulating PDCD4 through suppression of miR-499a-5p, providing a feasible theoretical basis for the treatment of AML.

MicroRNA-499-5p targets SIRT1 to aggravate lipopolysaccharide-induced acute lung injury.

Acute lung injury (ALI) is a life-threatening disease without effective and specific therapeutic strategies except the life-supporting treatments. Inflammation and oxidative stress are essential for the progression of ALI. MicroRNA-499-5p (miR-499-5p) has multiple pathophysiological actions; however, its function and mechanisms in ALI remain elusive. Mice were intravenously injected with miR-499-5p agomir, antagomir or the negative controls for 3 consecutive days and then received a single intratracheal injection of lipopolysaccharide (LPS, 5 mg/kg) to generate ALI model. Twenty four hours prior to LPS injection, EX-527 (1 mg/kg) was applied to inhibit SIRT1 activity. We identified a significant upregulation of miR-499-5p in LPS-treated lung tissues. miR-499-5p antagomir prevented, while miR-499-5p agomir promoted inflammation, oxidative stress and ALI in LPS-treated mice. Further studies indicated that miR-499-5p directly bound to the 3'-untranslated region of Sirtuin 1 (Sirt1) and decreased its protein level. SIRT1 inhibition blocked miR-499-5p antagomir-mediated pulmonary protection against LPS injury. miR-499-5p targets SIRT1 to aggravate LPS-induced ALI and it is a promising therapeutic target for the treatment of ALI.

Wharton's Jelly-Derived Mesenchymal Stem Cells Reduce Fibrosis in a Mouse Model of Duchenne Muscular Dystrophy by Upregulating microRNA 499.

The aim of this study was to evaluate the therapeutic effects and mechanisms of Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) in an animal model of Duchenne muscular dystrophy (DMD). Mdx mice (3-5 months old) were administered five different doses of WJ-MSCs through their tail veins. A week after injection, grip strength measurements, creatine kinase (CK) assays, immunohistochemistry, and western blots were performed for comparison between healthy mice, mdx control mice, and WJ-MSC-injected mdx mice. WJ-MSCs exerted dose-dependent multisystem therapeutic effects in mdx mice, by decreasing CK, recovering normal behavior, regenerating muscle, and reducing apoptosis and fibrosis in skeletal muscle. We also confirmed that miR-499-5p is significantly downregulated in mdx mice, and that intravenous injection of WJ-MSCs enhanced its expression, leading to anti-fibrotic effects via targeting TGFßR 1 and 3. Thus, WJ-MSCs may represent novel allogeneic "off-the-shelf" cellular products for the treatment of DMD and possibly other muscle disorders.

Ischemic postconditioning attenuates the inflammatory response in ischemia/reperfusion myocardium by upregulating miR­499 and inhibiting TLR2 activation.

Toll-like receptor 2 (TLR2)-mediated myocardial inflammation serves an important role in promoting myocardial ischemic/reperfusion (I/R) injury. Previous studies have shown that miR­499 is critical for cardioprotection after ischemic postconditioning (IPostC). Therefore, the present study evaluated the protective effect of IPostC on the myocardium by inhibiting TLR2, and also assessed the involvement of microRNA (miR)­499. Rat hearts were subjected to 30 min of ischemia and 2 h of reperfusion. The IPostC was 3 cycles of 30 sec of reperfusion and 30 sec of re­occlusion prior to reperfusion. In total, 90 rats were randomly divided into six groups (n=15 per group): Sham; I/R; IPostC; miR­499 negative control adeno­associated virus (AAV) vectors + IPostC; miR­499 inhibitor AAV vectors + IPostC; and miR­499 mimic AAV vectors + IPostC. It was identified that IPostC significantly decreased the I/R­induced cardiomyocyte apoptotic index (29.4±2.03% in IPostC vs. 42.64±2.27% in I/R; P<0.05) and myocardial infarct size (48.53±2.49% in IPostC vs. 66.52±3.1% in I/R; P<0.05). Moreover, these beneficial effects were accompanied by increased miR­499 expression levels (as demonstrated by reverse transcription­quantitative PCR) in the myocardial tissue and decreased TLR2, protein kinase C (PKC), interleukin (IL)­1ß and IL­6 expression levels (as demonstrated by western blotting and ELISA) in the myocardium and serum. The results indicated that IPostC + miR­499 mimics significantly inhibited inflammation and the PKC signaling pathway and enhanced the anti­inflammatory and anti­apoptotic effects of IPostC. However, IPostC + miR­499 inhibitors had the opposite effect. Therefore, it was speculated that IPostC may have a miR­499­dependent cardioprotective effect. The present results suggested that miR­499 may be involved in IPostC­mediated ischemic cardioprotection, which may occur via local and systemic TLR2 inhibition, subsequent inhibition of the PKC signaling pathway and a decrease in inflammatory cytokine release, including IL­1ß and IL­6. Moreover, these effects will ultimately lead to a decrease in the myocardial apoptotic index and myocardial infarct size via the induction of the anti­apoptotic protein Bcl­2, and inhibition of the pro­apoptotic protein Bax in myocardium.

microRNA-499-3p inhibits proliferation and promotes apoptosis of retinal cells in diabetic retinopathy through activation of the TLR4 signaling pathway by targeting IFNA2.

microRNAs (miRNAs) are involved in the physiological and pathophysiological processes of diabetes and its microvascular and macrovascular complications. Hence, the aim of the study was to investigate whether miR-499-3p played an important role in diabetic retinopathy. Diabetic retinopathy was developed in rats by intraperitoneal injection of streptozocin (STZ), followed by collection of retinal tissues and preparation of retinal cells. Immunohistochemical staining was used to detect expression of interferon alpha 2 (IFNA2). RT-qPCR was used to determine the expression of miR-499-3p. Bioinformatics website and dual luciferase reporter gene assay were used to validate the targeting relationship between miR-499-3p and IFNA2. Gain- and loss-of-function assays were performed to explore the functional roles of aberrantly expressed miR-499-3p and IFNA2 in retinal cell proliferation by MTT, and apoptosis by flow cytometry. In retinal tissues and cells of diabetic rats, IFNA2 expression was reduced, and miR-499-3p expression increased to activate the toll-like receptor 4 (TLR4) signaling pathway. IFNA2 was a target gene of miR-499-3p and negatively regulated by miR-499-3p. Further, downregulated miR-499-3p promoted retinal cell proliferation while suppressing apoptosis to alleviate diabetic retinopathy. All in all, miR-499-3p promoted retinopathy by enhancing activation of the TLR4 signaling pathway, which provides a new therapeutic target for diabetic retinopathy.