Plasma miR-21 Expression Research Articles

The role of miR-21 as a predictive biomarker and a potential target to improve the effects of chemoradiotherapy against head and neck squamous cell carcinoma.

This study aimed to clarify whether circulating miR-21 represents a predictive biomarker in patients with head and neck squamous cell carcinoma (HNSCC) undergoing chemoradiotherapy, and to investigate the effect of miR-21 inhibitor for chemoradiation in human SCC cells. Plasma samples were obtained from 22 patients with HNSCC and 25 non-cancer volunteers. Plasma miR-21 expression was measured using real-time quantitative reverse transcription polymerase chain reaction. The effects of miR-21 inhibitor in human SCC cells were investigated by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry and western blot analysis. As a result, plasma miR-21 expression was higher in HNSCC patients than in control patients (P < 0.001). Seven patients with recurrence showed significantly higher plasma miR-21 than the 15 patients without recurrence. And high miR-21 expression group showed poor overall survival. Moreover, miR-21 inhibition significantly enhanced cisplatin- or radiation-induced apoptosis. Western blot analysis suggested the programmed cell death 4 protein as a potential target of miR-21 in relation to apoptosis. In conclusion, this study provides new insights into the role of miR-21 as a predictive biomarker for HNSCC treated with chemoradiotherapy and suggests a potential target to improve the effects of chemoradiotherapy against HNSCC.

The mir-21 Inhibition Enhanced HUVEC Cellular Viability during Hypoxia-Reoxygenation Injury by Regulating PDCD4.

The purpose of this study was to explore the clinical value of altered plasma mir-21 expression level as a biomarker for the severity of coronary artery disease (CAD) and its molecular impact on HUVEC cellular injuries. Angiographically validated 56 patients with single-vessel CAD disease, 92 patients with double-vessel CAD, 139 complex coronary artery stenosis patients, and 56 healthy individuals (n = 343) were enrolled in this study. The expressions of plasma mir-21 were evidently and progressively higher while PDCD4 levels were significantly and steadily lower in single-, dual-, and multivessel occluded CAD patients than in healthy participants (P < 0.001). The relative expressions of mir-21 in hypoxia-reoxygenation- (HR-) exposed HUVECs were markedly upregulated, but PDCD4 concentrations were obviously downregulated as compared with normal control cells (P < 0.001). Moreover, altered circulatory mir-21 expression levels were able to significantly differentiate single- (AUC 0.893), double- (AUC 0.914), and multivessel stenosis CAD (AUC 0.933) patients from healthy subjects. Besides, the plasma mir-21 expressions in elderly (66-85 years) groups were remarkably higher than those in younger aged (25-45 years) subjects. Caspase-3 and ROS expression levels were remarkably elevated, but cellular viability noticeably declined in HR-induced HUVECs than in normoxic cells (P < 0.001). In contrast, mir-21 inhibition markedly reduced caspase-3 activity and ROS concentrations while significantly ameliorating HUVEC cellular viability in HR conditions. PDCD4 expressions in HR-exposed HUVECs were prominently decreased whereas mir-21 inhibition significantly enhanced PDCD4 levels (P < 0.001). Upregulated plasma mir-21 can be a valuable clinical biomarker for the detection of the severity of coronary artery stenosis patients. Elevated circulatory mir-21 concentrations have a positive correlation with aging. Inhibitory mir-21 evidently increased HUVEC cellular viability through upregulation of targeting PDCD4 and recommended a newer possible therapeutic molecule for the management of CAD patients.

Correlation between the Expression Levels of miR-21 and miR-192 and Clinicopatological Features in Plasma of Patients with Gastric Cancer in Iran

Background: Gastric cancer (GC) is the most prevalent malignancy worldwide and a common cause of death in Iran. Studies have proved that a variety of dysregulated microRNAs is involved in the development and progression of gastric cancer. The present study aimed to evaluate the expression levels of plasma circulating oncogenic miR-21 and miR-192 and their association with clinical phenotypes of patients with gastric cancer in the north of Iran. Material and Methods: Clinico-pathological analysis was conducted using a standard protocol and pathological tests. The expression levels of miR-21 and miR-192 were measured using quantitative reverse transcription-polymerase chain reaction in the plasma of twenty pre/post-operative gastric cancer patients and twenty healthy subjects. The receiver operating characteristic (ROC) curve of these microRNAs was analyzed to investigate their diagnosis properties. Results: The study results indicated that plasma miR-21 expression was significantly associated with tumor stage and helicobacter pylori infection status (P=0.024, P=0.0004, respectively). However, no association was observed between clinic-pathological characteristics and miR-192 expression. The results showed that the plasma levels of miR-21 (P=0.0001) and miR-192 (P=0.0007) were significantly higher in GC patients compared to those in healthy individuals. Furthermore, the ROC analyses yielded the area under the curve (AUC) values of 0.9525±0.03 (P&lt;0.0001) and 0.5925±0.09 (P=0.316) for miR-21and miR-192, respectively. Pearson regression analysis showed that there was no significant correlation between the expression of miR-21 and miR-192 (P=0.1507). Conclusion: Based on the obtained results, the expression of the plasma level of miR-21 was significantly higher in gastric cancer patients compared to that in the healthy group. Furthermore, the higher levels of AUC in miR-21 indicated the potential role of miR-21 as a noninvasive biomarker for the prognosis of gastric cancer in the population of the north of Iran.

Diagnostic Role of Plasma MicroRNA-21 in Stable and Unstable Angina Patients and Association with Aging.

The present study explored the clinical value of plasma microRNA-21 as a novel biomarker for early prediction of stable and unstable angina patients and its relationship with aging. A total of 255 participants, 123 patients with chronic stable angina, 82 patients with unstable angina, and 50 healthy subjects, were included in our study. Stable coronary and unstable coronary patients were confirmed following AHA/ACC clinical protocols. Total RNA was extracted from plasma by using miRNA-based TRIzol reagent. Plasma miR-21 expression levels were determined by real-time polymerase chain reaction. To evaluate the diagnosis accuracy, the receiver operating characteristic (ROC) curves were used. Plasma microRNA-21 concentration levels were significantly elevated in stable and unstable angina patients as compared with control subjects (P < 0.001). The area under the ROC curves of circulating microRNA-21 was accurately distinguished in stable angina patients (AUC 0.921) and unstable angina patients (AUC 0.944) from healthy subjects. MicroRNA-21 expression gradually elevated with increasing aging in all the populations. Moreover, the current study also demonstrated that the expression of plasma miR-21 levels was significantly associated with different age groups within healthy subjects and stable and unstable angina patients (P < 0.001). This research finding suggested that plasma microRNA-21 may be considered as a suitable new biomarker for early detection of stable and unstable angina patients, and it has a strong correlation with aging.

A longitudinal study on circulating miR-21 as a therapeutic effect marker in head and neck squamous cell carcinoma.

The aim of the study is to investigate plasma miR-21 for a possible therapeutic effect determination marker in head and neck squamous cell carcinoma (HNSCC). Plasma samples are obtained from 86 HNSCC patients and 29 non-cancer volunteers who had been treated at Mie University Hospital between May 2015 and December 2016, and plasma miR-21 expression was measured using real-time quantitative reverse transcription polymerase chain reaction. In addition, plasma miR-21 level of advanced HNSCC patients including 22 non-recurrent cases and 11 recurrent cases before and after treatment was analyzed using a longitudinal design. Plasma miR-21 expression in 86 HNSCC patients was obviously higher than in 29 control patients (P < 0.0001). The area under the curve (AUC) for plasma miR-21 was 0.756 (95% confidence interval: 0.661-0.851). Furthermore, our longitudinal study of plasma miR-21 showed that the expression level of plasma miR-21 was significantly reduced at the time point of 2 months after treatment in case of no recurrence. On the other hand, plasma miR-21 was not decreased after treatment in case of 10 patients who had developed recurrences during the follow-up period. This study may provide new insights into the role of plasma miR-21 as a biomarker for HNSCC, and plasma miR-21 would be useful for early detection of tumor recurrence after operation or chemoradiotherapy.

Correlation of miR-21 and BNP with pregnancy-induced hypertension complicated with heart failure and the diagnostic value.

Correlation of miR-21 and B-type natriuretic peptide (BNP) with pregnancy-induced hypertension (PIH) complicated with heart failure and the diagnostic value was investigated. Sixty patients with PIH complicated with heart failure admitted to Affiliated Hospital of Chengde Medical University from July 2016 to July 2017 were enrolled as the experimental group, and 35 normal pregnant women as the control group. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method was used to determine the expression level of plasma miR-21 expression level. An automatic biochemical analyzer was used to determine plasma BNP expression level. Spearmans correlation analysis was used for the correlation analysis of miR-21 and BNP. ROC curve was used for evaluating the diagnostic values of miR-21 and BNP for PIH complicated with heart failure. miR-21 and BNP expression levels were higher in patients with PIH complicated with heart failure than those in the normal individuals, and were increased in line with the heart failure grade (P<0.001). The plasma miR-21 expression was positively correlated with BNP in patients with PIH complicated with heart failure (r=0.685, P<0.001). Both miR-21 and BNP had higher diagnostic values for PIH complicated with heart failure, in the diagnosis, the best cut-off value [odds ratio (OR)] of miR-21 was 1.113, with an area under curve (AUC) of 0.889 and a 95% confidence interval (CI) of 82.05–95.76%; the OR of BNP was 123, with an AUC of 0.747 and a 95% CI of 64.95–84.38%. Blood pressure, six-minute walk test (6MWT), left ventricular ejection fraction (LVEF) and left ventricular end diastolic diameter (LVEDD) were independent risk factors for the occurrence of PIH complicated with heart failure (P<0.05). In conclusion, miR-21 and BNP, highly expressed in patients with PIH complicated with heart failure, are expected to become important biomarkers for diagnosing PIH complicated with heart failure and judging the degree of heart failure in the patients, and worthy of clinical popularization and application.

Circulating miR-21 as a prognostic and predictive biomarker in oral squamous cell carcinoma.

Background & Objective:The high-throughput analysis of circulating microRNAs (miRNAs) is an active area of biomarker research. The oral cancer remains a common cancer among Pakistani males that continues to present at an advance stage, thus exhibiting poor survival. MiRNA 21 (miR-21) is the most consistently over-expressed miRNA in different types of tumor tissues. However, information regarding expression of miR-21 in plasma remains to be resolved. Therefore, present study was designed to investigate if miR-21 was expressed in plasma of patients with oral cancer, and further explore its diagnostic and prognostic potential.Methods:Present study was conducted at Ziauddin University and Karachi Institute of Radiotherapy and Nuclear Medicine (KIRAN). Histologically confirmed cases of oral squamous cell carcinoma were recruited from Oncology Department of Ziauddin Hospital between 2013 and 2017. Controls were carefully selected after considering age, gender and socioeconomic condition. MiRNA was extracted and immediately reverse transcribed to cDNA. MiR-21 expression was evaluated using probes specifically designed for Real time quantitative polymerase chain reaction.Results:A significant over expression of miRNA 21 was observed in histologically confirmed cases as compared to controls. The increased expression of miRNA 21 was also reported to be associated with tumor size, metastasis and local invasion (p<0.05).Conclusion:The expression of circulating miR-21 in plasma samples of oral cancer patients makes it a promising diagnostic and prognostic marker.

Identification of thyroid follicular carcinoma and papillary thyroid carcinoma by plasma microRNA-21

Objective To investigate the value of plasma microRNA (miRNAs, miR)-21 in differentiating thyroid follicular carcinoma from thyroid papillary carcinoma. Methods 45 patients with thyroid follicular carcinoma, 61 patients with thyroid papillary carcinoma and 50 normal subjects were selected as subjects. The expression of miR-21 in plasma was detected by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR). Receiver operating characteristic (ROC) curve was established to analyze the cut-off value, sensitivity and specificity of miR-21 in differentiating thyroid follicular carcinoma from papillary thyroid carcinoma. Results The relative expression levels of miR in plasma of normal persons, follicular thyroid carcinoma and papillary thyroid carcinoma were 0.254±0.123, 4.047±1.499 and 1.274±0.581, respectively. There was significant difference among the three groups (P=0.010). The relative expression level of miR-21 in plasma of follicular thyroid carcinoma was significantly higher than that of papillary thyroid carcinoma. The relative expression of miR-21 in plasma of patients with thyroid papillary carcinoma was significantly higher than that of normal subjects (P=0.010). The ROC curves were established with plasma levels of miR as test variables and thyroid follicular carcinoma and thyroid papillary carcinoma as state variables. The area under the curve (AUC) was 0.931 [95% confidence interval (CI): 0.890-0.991]. The optimum cut-off value (Cut-off value) for differentiating follicular thyroid carcinoma from papillary thyroid carcinoma was 2.325. The sensitivity and specificity were 85.11% and 96.72% respectively, the positive predictive value and negative predictive value were 95.24% and 89.39% respectively, and the Yorden index was 81.83%. Conclusion Detection of the expression of microRNA-21 in plasma is helpful to differentiate follicular thyroid carcinoma from papillary thyroid carcinoma and to differentiate follicular thyroid carcinoma from follicular thyroid carcinoma. Key words: MicroRNA-21; Thyroid follicular carcinoma; Papillary thyroid carcinoma; Differential diagnosis

Plasma miR-21 expression: an indicator for the severity of Type 2 diabetes with diabetic retinopathy.

To investigate the roles of plasma miR-21 in the pathogenic process of Type 2 diabetes (T2D) with diabetic retinopathy (DR). T2D patients included patients without DR (NDR) group, patients with non-proliferative/background DR (BDR) group and patients with proliferative DR (PDR) group. Healthy individuals served as control group. Fasting plasma glucose (FPG), glycosylated haemoglobin (HbA1c), triacylglycerol (TG), total cholesterol (TC), urine creatinine (Cr), fasting blood glucose (FBG), blood urea nitrogen (BUN), low-density lipoprotein cholesterol (LDL-C), fasting insulin (FINS) and plasma miR-21 expression were measured. Quantitative real-time PCR (qRT-PCR) was applied to detect miR-21 expression. Pearson analysis was used to conduct correlation analysis and receiver operating characteristic (ROC) curve was used to analyse the diagnostic value of miR-21 in T2D with DR. Compared with the control group, FBG and HbA1c increased in the NDR group; compared with the control and NDR groups, disease course, HbA1c, FPG levels and homoeostasis model assessment of insulin resistance (HOMA-IR) were increased in the BDR and PDR groups; and compared with the BDR group, disease course, HbA1c and FPG levels were higher in the PDR group. miR-21 expression was higher in the BDR group than the control group, and higher in the PDR group than the BDR group. miR-21 expression was positively related with disease course, HbA1C, FPG and HOMA-IR, and had diagnostic value for T2D with DR and PDR. The plasma miR-21 expression was increased in the development of T2D with DR and can be used as an indicator for the severity of T2D with DR.

Circulating miR-21 and eNOS in subclinical atherosclerosis in patients with hypertension

Objectives: The aim of this study is to evaluate the relationship of miR-21, nitric oxide (NOx) and endothelial nitric oxide synthase (eNOS) with subclinical atherosclerosis in carotid arteries by measuring carotid intima media thickness (CIMT) in patients with hypertension and healthy controls. Design and Methods: A total of 28 hypertensive and 28 healthy controls were enrolled. MiR-21 expression was analyzed by quantitative reverse transcription-PCR and NOx, and eNOS levels were measured by ELISA assay. CIMT was evaluated by ultrasonography and CIMT ≥ 0.8 mm was accepted as increased CIMT (iCIMT). Results: C-reactive protein (CRP) level, plasma miR-21 expression level and CIMT were found to be significantly higher in the hypertension group when compared to the control group (p = 0.009, p = 0.002 and p < 0.001, respectively). NOx and eNOS levels were significantly lower in the hypertension group compared to the control group (p < 0.001, both). MiR-21 level was positively correlated with the clinical systolic blood pressure, clinical diastolic blood pressure, CRP and CIMT. MiR-21 was also negatively correlated with NOx and eNOS. Eighteen patients with hypertension had iCIMT. MiR-21 and CRP levels were significantly higher (p < 0.001 and p = 0.001), whereas NOx and eNOS levels were significantly lower in patients with iCIMT (p < 0.001, both). Conclusion: The decreased levels of NOx and eNOS found in this study indicate the co-existence of endothelial dysfunction and hypertension once more. In the absence of microalbuminuria, the increased miR-21 expression in patients with iCIMT made us conclude that this miRNA might be involved in the early stages of atherosclerotic process in hypertensive patients.

Plasma Post-operative miR-21 Expression in the Prognosis of Gastric Cancers

Tumor-associated microRNAs have been detected in serum or plasma, but whether plasma microRNA-21 (miR-21) could be a potential circulating biomarker for gastric cancer (GC) prognosis in Chinese is still uncertain. Real-time quantitative reverse transcription PCR (qRT-PCR) was employed in this study to compare the relative expression of miR-21 between pre-operative and post-operative paired plasmas from 42 patients with primary GCs. The results showed that the expression levels of miR-21 in the post-operative plasmas were significantly reduced by an average of 18.2 times in all patients when compared to the pre-operative plasmas, and by 22.1 times in the subgroup of patients without family history, while only 1.76 times in the subgroup of patients with a family history. With respect of clinicopathological characteristics, the plasma miR-21 expression was highly associated with differentiation degree and lymph node metastasis rate. The results suggested plasma miR-21 could be a novel potential biomarker for GC prognosis and evaluation of surgery outcomes, especially in patients without a family history.

Abstract 18046: miR-21 is a Biomarker for Fibrosis and the Progression to Right Ventricular Systolic Failure in a Model of Pulmonary Volume and Pressure Overload

Introduction: Severe pulmonary insufficiency (PI) in combination with moderate pulmonary stenosis (PS) is a common long-term complication after repair of congenital heart diseases such as tetralogy of Fallot, causing progressive right ventricular (RV) dilation and failure. We characterize the physiologic and molecular characteristics of the first murine model of combined RV volume and pressure overload (PI/PS). Methods: Physiologic data was compared between sham controls and PI/PS at 1 and 3 mos. Histopathology and gene, microRNA (miR) and protein expression were assessed in the RV free wall. Plasma miR expression was assessed in children with severe PI/moderate PS vs controls (n=4). Results: Data are presented as sham vs. PI/PS at 1 and 3 mos (*p&lt;0.05). Mice developed diastolic heart failure at 1 mo progressing to systolic heart failure by 3 mos: restrictive RV filling (E/A ratio) (0.7±0.1 vs. 2.1±0.9 and 1.9±0.4*) and elevated RV end-diastolic pressure (2.7±0.3 vs. 7.8±2, and 8.5±2 mmHg*); followed by decreased RV outflow tract shortening (44±2 vs. 39±6 and 29±6%*) and exercise capacity (32±1.1 vs. 28±1.6 and 22±1.9 min*). RV fibrosis increased over time (0 vs. 4.0±2, 3.9±0.2%* sham vs 1 and 3 mos) along with pSMAD/total SMAD expression (0.2±0.1 vs. 0.4±0.1 and 0.6±0.1*), ANP expression (25±15 and 60±27* fold at 1 and 3 mos) and tissue miR-21 expression (2.7±0.3 and 2.9±0.6 fold). However, plasma miR-21 was upregulated with diastolic heart failure and switched to being downregulated with the development of systolic heart failure (2.6±1 and -1.8 ±0.2 fold). Finally, plasma miR-21 expression from children with severe PI/moderate PS at the time of pulmonary valve replacement was downregulated compared to controls (FC -3.6±1.2). Conclusion: We describe a novel murine model of chronic RV volume and pressure overload which recapitulates many aspects of the clinical disease. Gene and protein expression changes suggest enhanced miR-21 and TGF-β signaling as a mediator of RV fibrosis. miR-21 may serve as a novel biomarker of RV fibrosis, with a decline in expression heralding the need for valve replacement. This model provides investigators with a new tool for further evaluation of a form of RV failure unique to children with repaired congenital heart disease.

Ineffective delivery of diet-derived microRNAs to recipient animal organisms

Cross-kingdom delivery of specific microRNAs to recipient organisms via food ingestion has been reported recently. However, it is unclear if such delivery of microRNAs occurs frequently in animal organisms after typical dietary intake. We found substantial levels of specific microRNAs in diets commonly consumed orally by humans, mice, and honey bees. Yet, after ingestion of fruit replete with plant microRNAs (MIR156a, MIR159a, and MIR169a), a cohort of healthy athletes did not carry detectable plasma levels of those molecules. Similarly, despite consumption of a diet with animal fat replete in endogenous miR-21, negligible expression of miR-21 in plasma or organ tissue was observed in miR-21 −/− recipient mice. Correspondingly, when fed vegetarian diets containing the above plant microRNAs, wild-type recipient mice expressed insignificant levels of these microRNAs. Finally, despite oral uptake of pollen containing these plant microRNAs, negligible delivery of these molecules was observed in recipient honeybees. Therefore, we conclude that horizontal delivery of microRNAs via typical dietary ingestion is neither a robust nor a frequent mechanism to maintain steady-state microRNA levels in a variety of model animal organisms, thus defining the biological limits of these molecules in vivo.

Plasma MicroRNAs in Ovarian Cancer—Response

We are pleased that our research (1) has generated interest in the scientific community, and we are happy to provide our reply to the comments submitted by Dr. Haifeng Qiu.The major concern addressed by Dr. Qiu is that the upregulation of plasma miR-21 level in endometriosis-associated ovarian cancer (EAOC) cases is due to loss of estrogen possibly present in this group of patients. Dr. Qiu's argument is based on two lines of reasoning: (i) miR-21 expression is repressed by estrogen, and (ii) our patients with EAOC are significantly older than the healthy controls and those in the endometriosis group, and therefore they are likely postmenopausal women with low estrogen levels. In addition to miR-21, Dr. Qiu also has concerns about the increased expression of miR-16 and miR-195, which have also been previously reported as estrogen targets.Although the estrogen/progesterone imbalance is a well-documented pathogenic factor in ovarian cancer and endometriosis, the role of estrogen in regulating miR-16, 21, and 195 expressions remains inconclusive. Studies have shown miR-21 being either repressed (2, 3) or induced by estrogen (4). Furthermore, evidence from several studies on the effect of estrogen on miRNA expression (5) suggests that miR-21 was not a consistent estrogen-repressed miRNA, and miR-16 and 195 only were shown to be repressed by estrogen in one study (6) among 19 studies reviewed (5).Second, in our study the averaged fold change of miR-21 expression between EAOC and healthy controls is dramatic (fold change = 147.4) compared with the reported repressive effect of estrogen to miR-21 expression (ranging from 50%–70% repression), making it difficult to assume that estrogen has a big effect on our experimental results.Third, the average age of our healthy control group and endometriosis group is comparable (38.75 and 36.24 years, respectively). Although estrogen levels were not measured in our study, the patients with endometriosis are likely to have higher estrogen levels that were also suggested by Dr. Qiu. Thus, if miR-21 expression is repressed by estrogen, miR-21 levels in patients with endometriosis should be lower than that in the healthy controls. However, this was not what we found. Instead, patients with endometriosis displayed a much higher plasma miR-21 expression compared with healthy controls (fold change = 10.65).Fourth, differences in the above miRNAs were also present in comparisons with age-matched serous versus EAOC cases (all menopausal) and with age-matched female mice (tumor bearing vs. controls), further suggesting that factors other than estrogen may have impacted these changes.Finally, as we have shown in our study, plasma and tissue samples have distinct miRNA expression profiles. Currently, the effect of estrogen levels on plasma miRNA expression profile remains unclear.In summary, based on our study results and the above arguments, we do not believe that estrogen plays a significant role in regulating the plasma miRNA signature we have identified, which distinguishes EAOC patients with endometriosis from healthy controls.See the original Letter to the Editor, p. 3325No potential conflicts of interest were disclosed.