Diagnostic and prognostic value of circulating miRNA-499 and miRNA-22 in acute myocardial infarction.

BACKGROUND: Currently, acute myocardial infarction (AMI) represents a serious cardiovascular disease with high morbidity and mortality. Therefore, this study aimed to systematically evaluate the roles of miRNA-499 and miRNA-22 as potential biomarkers for AMI. METHODS: According to the inclusion and exclusion criteria, we measured circulating levels of miRNAs in 50 AMI patients and 50 non-MI populations. The expression levels of plasma miRNA-499 and miRNA-22 were analyzed by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). A statistical analysis of clinical data of AMI patients was conducted by 90-day follow-up. RESULTS: Real-time PCR analysis showed that the relative expression level of miRNA-499 increased gradually among the three groups (P < .05). However, the expression of miRNA-22 showed a downward trend (P < .05). According to logistic analysis, the relative levels of miRNA-499 and miRNA-22 were important predictors of AMI. When the miRNA-499 and miRNA-22 levels were 0.377 and 0.946 separately, the diagnostic value of miRNA-499 and miRNA-22 for AMI was 86.00% and 86.00% for sensitivity, and 98.00% and 94.00% for specificity, respectively. In addition, compared to the baseline GRACE scoring system, the combination of miRNA-499, miRNA-22, and GRACE scores had a stronger discriminating power for MACE occurrence, with a sensitivity of 100.00% and a specificity of 79.40%. CONCLUSIONS: The results showed that plasma miRNA-499 and miRNA-22 were more sensitive and specific for the diagnosis of AMI, suggesting that they can be used as potential biomarkers for clinical diagnosis of AMI.

Association of MiRNA-146a, MiRNA-499, IRAK1 and PADI4 Polymorphisms with Rheumatoid Arthritis in Egyptian Population.

BACKGROUND/AIMS: Rheumatoid arthritis (RA) is a systemic autoimmune disease affecting up to 1% of the population worldwide. The aim of the present study was to investigate whether miRNA-146a rs2910164, miRNA-499 rs3746444, IRAK1 rs3027898 and PADI4 rs1748033 polymorphisms are associated with susceptibility to RA in Egyptians and whether they influence disease severity and activity. METHODS: The study was performed on 104 unrelated RA patients and 112 healthy subjects. RA patients were further subdivided into active and inactive RA groups. Polymorphisms were genotyped by using real-time polymerase chain reaction with TaqMan allelic discrimination assay. RESULTS: Significant differences in the frequency of miRNA-146a rs2910164, miRNA-499 rs3746444, IRAK1 rs3027898 and PADI4 rs1748033 alleles and genotypes were observed between RA patients and controls. Only CA and AA genotypes of IRAK1 rs3027898 shows a significant difference between active and inactive subgroups. MiRNA-146a rs2910164 and IRAK1 rs3027898 polymorphisms were a risk factor for predisposition to RA in codominant and dominant tested inheritance models, while, the miRNA-499 rs3746444 and PADI4 rs1748033 polymorphisms were a risk factor in codominant and recessive one. CG and GG genotypes of miRNA-146a rs2910164 were associated with positive erosions. CA genotype of IRAK1 rs3027898 was associated with low disease activity and negative erosions, while, the AA genotype was associated with high disease activity. CC genotype of PADI4 rs1748033 was associated with negative rheumatoid factor. CONCLUSION: The 4 studied SNPs were likely to play an important role in the susceptibility to RA and can influence disease severity and activity in Egyptian population.

Possible use of miRNAs-146a and -499 expression and their polymorphisms as diagnostic markers for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder affecting the peripheral joints. Different microRNAs had been investigated in RA including miRNA-146a meanwhile, miRNA-499 there were no studies to prove its expression in RA serum samples. This study was performed to investigate expression of both miRNAs-146a and -499 and their polymorphisms in Egyptian patients with RA and to evaluate their relationship with clinico-pathological data. The present study includes 108 subjects classified into two main groups: 52 RA patients and 56 unrelated healthy controls. RA patients were subclassified according to DAS28 score into inactive (23 patients) and active (29 patients). Quantitative expression of serum miRNA-146a, miRNA-499 as well as their Genotyping rs2910164 (C/G) and rs3746444 (T/C), respectively, were done to all subjects using real-time PCR. Serum miRNA-146a and -499 were significantly over expressed in RA patients, but they were not correlated to disease activity. Serum miRNA-146a was negatively correlated with anti-nuclear antibodies (ANA). miRNA-146a (rs2910164) genotyping revealed that the GG genotype and the frequency of the G allele were significantly higher in RA patients compared to the controls. miRNA-499 (rs3746444), genotyping revealed that the CC genotype and the frequency of the C allele were significantly higher. It can be concluded that both miRNAs-146a and -499 can be used as diagnostic markers for RA patients. Both miRNA-146a (rs2710164) and miRNA-499 (rs3746444) were significantly associated with RA susceptibility. The C allele of miRNA-146a (rs2710164) can be considered to be protective. On the other hand, the C allele of miRNA-499 (rs3746444) was significantly associated with RA susceptibility.

Serum miRNA-499 and miRNA-210: A potential role in early diagnosis of acute coronary syndrome.

In clinical practice, there is still a need for novel biomarkers, which can reliably rule in or rule out acute coronary syndrome (ACS) immediately on admission. This is of particular interest in patients with unstable angina (UA) and non-ST-segment elevation myocardial infarction (NSTEMI) in whom diagnostic uncertainty is high. The aim of the present study is to evaluate the potential role of miRNA-499 and miRNA-210 as novel molecular biomarkers for early diagnosis of UA and NSTEMI suspected patients presented at the emergency unit. A total of 110 patients presenting to the intensive care unit (ICU) within 24 h of onset of chest pain suggestive of ACS were enrolled in the study. They included 37 UA, 48 NSTEMI and 25 noncardiac chest pain (NCCP) patients. Immediately at enrollment, blood samples were taken for estimation of serum miRNA-499 and miRNA-210 expression levels by real time PCR. miRNA-499 and miRNA-210 expression levels were significantly increased in UA and NSTEMI patients compared with NCCP patients (P < 0.001). Receiver operating characteristic (ROC) curve analysis revealed that the area under curve (AUC) of miR-499 for the diagnosis of UA and NSTEMI was 0.98 and 0.97, respectively; while the AUC of miRNA-210 was 0.84 and 0.90, respectively. The important finding of our study was that the AUC of miRNA-499 for the diagnosis of ACS patients with symptoms onset <3 h was 0.89, while the AUC of miRNA-210 was 0.86. Interestingly, combining miRNA-499 and miRNA-210 significantly improved the diagnostic value by increasing the AUC to 0.96, P < 0.001. In conclusion, serum miRNA-499 and miRNA-210 are associated with UA and NSTEMI and with those presenting within 3 h of symptom onset. Both miRNAs might be potentially novel biomarkers for accelerating the diagnosis of ACS patients in emergency unit. © 2016 IUBMB Life, 68(8):673-682, 2016.

Association analysis of miRNA-146a and miRNA-499 polymorphisms with rheumatoid arthritis: a case-control and trio-family study.

Single nucleotide polymorphism is known to alter the expression and processing of miRNAs leading to a variety of diseases including rheumatoid arthritis (RA). However, disagreement is present up to date regarding the association of miRNA-146a and miRNA-499 polymorphisms with RA. The goal of this study was to assess the association of polymorphisms at miRNA-146a and miRNA-499 with the pathogenesis of RA in patients originating from Pakistan. Initially, eleven hundred subjects (1100) comprises of 550 RA patients and 550 healthy controls were investigated in the case-control analysis. Spectrophotometric measurement of lipids and C-reactive protein was used, whereas interleukin-1 receptor associated kinase-1 and TNF-receptor associated factor-6 values were quantified by an enzyme-linked immunosorbent assay. Secondly, heritability of susceptible alleles was tested from 70 trio-families. The miRNA-146a rs2910164 and miRNA-499 rs3746444 polymorphisms were genotyped using the polymerase chain reaction followed by restriction digestion. A Significant association of miRNA-146a and miRNA-499 genotypes was observed with RA patients (P < 0.05, respectively). The miRNA-146a rs2910164 G (OR = 1.4, P < 0.05) and miRNA-499 rs3746444 C (OR = 1.6, P < 0.0001) allele was significantly associated with RA in comparison with controls, respectively. Besides, the transmission analysis revealed a significant (P < 0.05) inheritance of rs2910164 G and rs3746444 C allele from parents to affected offspring. The current research concludes that miRNA-146a (rs2910164; C > G) and miRNA-499 (rs3746444; T > C) polymorphisms are linked to RA in the population studied. Furthermore, it was demonstrated for the first time in our high-risk cohort that the rs2910164 G and rs3746444 C allele was strongly related to familial RA.

Copeptin, miRNA-208, and miRNA-499 as New Biomarkers for Early Detection of Acute Coronary Syndrome.

cTn and CK-MB are gold standard biomarkers for acute coronary syndrome (ACS) but are less sensitive in the first 3 h after onset of symptoms. A need thus exists for novel biomarkers for early detection of ACS. We evaluated circulating copeptin, miRNA-208, and miRNA-499 as possible biomarkers for early detection of unstable angina (UA) and non-ST-segment elevation myocardial infarction (NSTEMI). Sixty-five patients with probable ACS that presented within 4 h of the onset of chest pain (23 UA and 42 NSTEMI) and 25 apparently healthy individuals were studied. Two sets of blood samples collected in the first 3 h and at 6 h after onset were analyzed for copeptin levels via ELISA and miRNA-208 and miRNA-499 expression via real-time PCR. Copeptin, miRNA-208, and miRNA-499 expression levels were significantly increased in UA and NSTEMI patients compared with controls (p < 0.001) and in NSTEMT compared with UA patients (p < 0.001). Levels were also significantly elevated in UA and NSTEMI patients with negative cardiac troponin in the first 3 h (p < 0.001). ROC curves displayed AUC for prediction of ACS of 0.96 for copeptin, 0.97 for miRNA-208, and 0.97 for miRNA-499. Their combination improved AUC to 0.98. Copeptin and miRNA-208 and miRNA-499 expression are promising biomarkers for UA and NSTEMI that present in the first 3 h of pain onset. A combination of these markers with cTn may increase the accuracy of diagnosis by avoiding the gray zone of cTn as a biomarker.

Associations of the miRNA-146a rs2910164 and the miRNA-499a rs3746444 Polymorphisms With Plasma Lipid Levels: A Meta-Analysis.

Background: The studies of miRNAs are vibrant and remain at the forefront in the cardiovascular system. Emerging studies indicate that the genetic polymorphisms of the miRNA gene may affect lipid metabolism; this study aims to clarify the specific correlations between the rs2910164 and rs3746444 polymorphisms and lipid levels. Methods and Results: A comprehensive search of literature was performed from December 31, 2020, to May 31, 2021, by searching of the PubMed and the Cochrane databases. The standardized mean difference (SMD) and 95% confidence interval (CI) were used to evaluate the differences in lipid levels between the genotypes. rs2910164, a functional polymorphism in the miRNA-146a gene, was associated with increased triglycerides (TG) (SMD = 0.35, 95% CI = 0.15-0.54, p < 0.001), total cholesterol (TC) (SMD = 0.43, 95% CI = 0.16-0.70, p < 0.001), and low-density lipoprotein cholesterol (LDL-C) (SMD = 0.37, 95% CI = 0.11-0.63, p = 0.01) as well as decreased high-density lipoprotein cholesterol (HDL-C) (SMD = -0.27, 95% CI = -0.47-0.07, p = 0.01) levels. rs3746444, a functional polymorphism in the miRNA-499a gene, was only correlated with decreased TG (SMD = -0.09, 95% CI = -0.17-0.01, P = 0.03) levels. Conclusions: The miRNA-146a rs2910164 polymorphism is significantly associated with atherogenic dyslipidemia.

Inhibition of breast cancer stem-like cells by a triterpenoid, ursolic acid, via activation of Wnt antagonist, sFRP4 and suppression of miRNA-499a-5p.

Breast cancer, one of the leading causes of death in the world, has been largely considered to be drug resistant because of a small population of drug refractory cells, the cancer stem cells (CSCs). The CSCs are tightly regulated by self-renewal pathways such as the Wnt pathway, which is further regulated by a gamut of microRNAs. In this study, we investigated the effect of ursolic acid (UA), a natural triterpene, on breast CSCs enriched from breast cancer cell lines, MCF7, MDA-MB-231 and T47D and analysed the interplay of the Wnt inhibitor, sFRP4 and an miRNA, miR-499a-5p, in mediating the effect of UA. By using caspase 3/7, ROS, migration, TCF/LEF and CAM assays, overexpressing and inhibiting miR-499a-5p and NanoString PanCancer analysis, we observed that UA had significant anti-CSC ability. There was a link between UA and Wnt/ß-catenin pathway wherein, Wnt was suppressed by upregulation of the antagonist, sFRP4. Furthermore, expression of the oncogenic miR-499a-5p was substantially diminished in CSCs after UA treatment. Notably, the axis by which miR-499a-5p acts is via the TCF/LEF machinery of the Wnt/ß-catenin pathway. Our findings indicate for the first time that UA can target breast CSCs via Wnt by suppressing miR-499a-5p and upregulating the Wnt antagonist, sFRP4.

MiR-499a-5p promotes 5-FU resistance and the cell proliferation and migration through activating PI3K/Akt signaling by targeting PTEN in pancreatic cancer.

BACKGROUND: Pancreatic cancer (PC) can be considered a representative cancer type of the human body. As demonstrated by some studies, microRNA (miR)-499 is dysregulated in various cancer types including PC, for which chemotherapy involving 5-fluorouracil (5-FU) has long been considered the first-line therapy. However, there are complex and comprehensive mechanisms related to 5-FU, which have not been fully elucidated. This study thus aimed to examine the molecular mechanisms of 5-FU resistance through miR-499a-5p in PC. METHODS: The expression of miR-499a-5p in PC was measured using quantitative polymerase chain reaction (PCR). MiR-499a-5p was examined in-vivo for its effects on the malignant phenotypes of PC cells. RESULTS: The results of the present study demonstrated miR-499a-5p to be upregulated in PC and 5-FU resistant PC tissues. According to in vitro assays in PC cells (PANC1/FR), miR-499a-5p was found to affect adenosine triphosphate (ATP) binding cassette subfamily B member 1 (P-gp), ATP binding cassette subfamily C member 1 (MRP1), and ATP binding cassette subfamily G member 2 (BCRP), thereby facilitating 5-FU resistance in PC cells. Functions assays indicated that suppressed miR-499a-5p expression inhibited the proliferation and migration of cells but facilitated apoptosis in the PC cell line; by contrast, miR-499a-5p overexpression triggered the inverse phenotypic changes of cells. Concerning the mechanisms involved, miR-499a-5p increased PI3K/Akt signaling by targeting phosphatase and tensin homolog (PTEN). CONCLUSIONS: Taken together, these findings demonstrate that miR-499a-5p can be potentially applied to PC therapy.

The relationships between maternal and placental polymorphisms of miR-196a2 and miRNA-499 genes and preeclampsia.

Background: miRNAs are small non-coding RNAs with potential roles in the complications of pregnancy. We hypothesised links between polymorphisms in miRNA-196a2 and miRNA-499 in maternal blood and the placentas of patients with preeclampsia. Methods: The blood of 315 women with preeclampsia and 317 controls and the placentas of 103 PE and 133 healthy women were collected. The genotyping of both polymorphisms was performed by PCR-RFLP. Results: The maternal blood rs11614913 was unrelated to preeclampsia in genotype and allele models, but in placental tissue, the CT (odds ratio [95% CI] 0.5 [0.3-0.9, p = 0.018) and TT (0.4 [0.2-0.9] p = 0.033) genotypes alone and together (CT+TT v CC 0.5 [0.3-0.8] p = 0.009), and the T allele (0.6 [0.4-0.9], p = 0.019) were associated with lower risk of preeclampsia. The maternal blood rs3746444 CC genotype was more frequent in preeclampsia (2.2 [1.2-3.8] p = 0.008) and the recessive model (CC v TC+TT) was also significant (1.9 [1.1-3.3], p = 0.018), as was the C allele (1.4 [1.1-1.7] p = 0.014). In placental tissue, the increase in the frequency of the CC genotype was marginally significant (2.4 [1.0-5.8] p = 0.046). The maternal or placental miRNA-196a2 rs11614913 and miRNA-499 rs3746444 polymorphisms were unrelated to the severity of preeclampsia. Conclusion: The placental but not maternal miRNA-196a2 rs11614913 variant could be a protective factor for preeclampsia predisposition in all models except the recessive model. The maternal/placental rs3746444 CC genotype was in association with higher preeclampsia risk.

Investigation of the Association of miRNA-499, miRNA-146a, miRNA-196a2 Loci with Hepatocellular Carcinoma Risk: A Case-Control Study Involving 1507 Subjects.

microRNAs' (miRNAs) loci may influence hepatocellular carcinoma (HCC) development. Many recent studies have assessed the relationship between miRNA-499, miRNA-146a, and miRNA-196a2 loci and HCC risk. However, the observed results are conflicting. A total of 584 HCC patients and 923 age- and sex-matched controls were recruited. The correlation of miRNA-499 rs3746444, miRNA-146a rs2910164, and miRNA-196a2 rs11614913 with HCC development was assessed. In the <53-year-old subgroup, a correlation of the rs2910164 locus with HCC risk was found (GG/CG vs. CC: adjusted p = 0.011, GG vs. CC: adjusted p = 0.021 and CG vs. CC: adjusted p = 0.027). The association between miRNA-146a rs2910164 and the risk of HCC was also found in the never smoking (GG/CG vs. CC: adjusted p = 0.011 and CG vs. CC: adjusted p = 0.018). Using false-positive report probability method and power value, we identified that miRNA-146a rs2910164 conferred a risk to HCC in the <53-year-old and never-smoking subgroups. In conclusion, this study indicates rs2910164 may be a risk factor for HCC, especially in the <53-year-old and never-smoking subgroups.

Association of miRNA-499 rs3746444 A>G variants with adenocarcinoma of esophagogastric junction (AEG) risk and lymph node status.

BACKGROUND: MicroRNAs (miRNAs) miRNA-499 rs3746444 A>G polymorphism may be complicated in the susceptibility to cancer. However, the correlation of this polymorphism with adenocarcinoma of esophagogastric junction (AEG) was unknown. PATIENTS AND METHODS: A total of 1063 AEG patients and 1677 controls were included in this study to assess the association of miR-499 rs3746444 A>G with AEG risk. SNPscanTM genotyping assay was harnessed to obtain the genotypes of miRNA-499 rs3746444 A>G polymorphism. RESULTS: We identified that SNP miR-499 rs3746444 A>G increased the susceptibility of AEG (AG vs AA: adjusted OR=1.25, 95% CI=1.05-1.49, P=0.012 and AG/GG vs AA: adjusted OR=1.30, 95% CI=1.10-1.54, P=0.002). In a stratified analysis, we found that miR-499 rs3746444 A>G polymorphism had an increased susceptibility of AEG in several subgroups (male subgroup: AG vs AA: adjusted P=0.004 and AG/GG vs AA: adjusted P=0.002; female subgroup: GG vs AG/AA: adjusted P=0.046; <64 years subgroup: AG vs AA: adjusted P=0.006 and AG/GG vs AA: adjusted P=0.003; never smoking subgroup: AG vs AA: adjusted P=0.003 and AG/GG vs AA: adjusted P=0.001; and never drinking subgroup: AG vs AA: adjusted P=0.008 and AG/GG vs AA: adjusted P=0.002). The results of power calculation indicated that miR-499 rs3746444 A>G polymorphism increased the risk of AEG in overall comparison, male, <64 years, never smoking, and never drinking subgroups. Among the AEG cases, 625 patients accompanied by positive lymph node. However, the distribution of miRNA-499 rs3746444 A>G variants was no significant difference between different lymph node status. CONCLUSION: Our findings indicate that miR-499 rs3746444 A>G polymorphism is significantly associated with AEG susceptibility. In the future, further exploration of this genetic factor in relation to AEG susceptibility with an adequate methodological quality is needed.

Identification of key microRNAs affecting drip loss in porcine longissimus dorsi by RNA-Seq.

MicroRNAs (miRNAs) are a class of ~22-nt non-coding small RNA that play an important role in various metabolic processes, mainly through suppressing the expression of protein coding genes at post-transcriptional level. Drip loss (DL) is one of the most important meat quality traits affecting the end product yield and quality of pork. To date, the underlying regulatory factors involved in DL trait are still incompletely understood. In the present study, we constructed two small RNA libraries with longissimus dorsi muscles from the higher DL (WJJ-H group) and the lower DL (WJJ-L group) individuals, and applied RNA-Seq technology to identify the differentially expressed miRNAs between the two extreme phenotypes of DL groups. A total of 184 and 176 porcine known miRNAs were detected from WJJ-H and WJJ-L groups, respectively. Moreover, 73 differentially expressed miRNAs were identified between two groups, of which 40 were up-regulated and 33 were down-regulated. In addition, 133 and 140 novel potential miRNAs were predicted from WJJ-H and WJJ-L groups, respectively. Notably, we preliminary confirmed that both miRNA-499 and miRNA-22 were potential candidates influencing DL trait by their expression pattern analysis. Overall, our data enhance the knowledge of porcine skeletal muscle miRNAs, and provide foundation for clarifying the miRNA regulatory mechanisms involved in DL trait.

Circulating miR-499 as a potential biomarker for acute myocardial infarction.

Acute myocardial infarction (AMI), a common heart disease that may lead to chronic heart failure, is the leading cause of morbidity and mortality worldwide. MicroRNAs (miRNAs) are small non-coding RNAs that mediate the expression of target genes. Recently, a number of miRNAs are emerging as potential biomarkers of AMI. MiRNA-499 is a newly discovered member of miRNAs, and is mainly expressed in myocardium, the circulating levels of miRNA-499 was increased in AMI patients. This review summarizes the latest advances in the miRNA-499 study and discusses the potential of miRNA-499 to be a biomarker of AMI.

miRNA-15a, miRNA-15b, and miRNA-499 are Reduced in Erythrocytes of Pre-Diabetic African-American Adults.

AIMS: The use of circulatory miRNAs as biomarkers and therapeutic targets for T2DM is an explosive area of study. However, no study has investigated circulatory miRNA expression exclusively in African-American adults. The aim of this study was to identify the expression of nine selected miRNAs in erythrocytes of pre-diabetic and type 2 diabetic African-American adults. MAIN METHODS: Patients were recruited from the Howard University Hospital Diabetes Treatment Center following an 8 to 10 hour overnight fast. Expression of the nine selected miRNAs (miRNA-499, miRNA-146, miRNA-126, miRNA-223, miRNA-15a, miRNA-15b, miRNA-224, miRNA-326, and miRNA-375) was evaluated using quantitative real time PCR. KEY FINDINGS: miRNA-15a, miRNA-15b, and miRNA-499 were significantly reduced in erythrocytes of pre-diabetic African-American adults. In the T2DM group, we found significant correlations between miRNA-15a and BMI (r=0.59, p=0.04), miRNA-15a and weight (r=0.52, p=0.01), and miRNA-15b and diastolic blood pressure (r=-0.52, p=0.02). In the pre-diabetic group, we found significant correlations between miRNA-15b and weight (r=0.90, p=0.02) and miRNA-499 and HbA1c (r=-0.89, p=0.01). SIGNIFICANCE: To our knowledge, this is the first study investigating miRNA expression in erythrocytes of non-diabetic high-risk obese--pre-diabetic and type 2 diabetic African-American adults. The findings of this study are consistent with previous reports of reduced expression of miRNA-15a, miRNA-15b, and miRNA-499 in human plasma or serum and in animal models. The current findings support the use of circulating miRNA-15a, miRNA-15b, and miRNA-499 as potential biomarkers for T2DM in African-American adults.

Lack of Association between miRNA-146a rs2910164 and miRNA-499 rs3746444 Gene Polymorphisms and Susceptibility to Pulmonary Tuberculosis.

Single-nucleotide polymorphisms (SNPs) in miRNAsmay alter its expression levels or processing and contribute to susceptibility to a wide range of diseases. Our study aimed to evaluate the possible association between miRNA-146a rs2910164 and miRNA-499 rs3746444 polymorphisms and susceptibility to pulmonary tuberculosis (PTB) in a sample of Iranian population. This case- control study was performed on 202 PTB patients and 204 healthy individuals. Genotyping was performed using tetra amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR). The results indicated that neither miRNA-499 rs3746444 nor miRNA-146a rs2910164 are associated with the risk of PTB in a sample of Iranian population. Larger studies with different ethnicities are required to validate our findings.