High Levels Of miR-21 Research Articles

A label-free colorimetric detection of microRNA via G-quadruplex-based signal quenching strategy

Signal amplification strategy have been the intensive direction for highly-sensitive microRNA (miRNA) detection, however, it is worth noting that signal quenching approach is rarely realized. Inspired by the unwinding of G-quadruplex with Klenow Fragment polymerase (KF), a simple and label-free signal quenching system for detection of miRNAs was developed. In the presence of target miRNA, miRNA was used as a primer to promote KF activity resulting in the disruption of G-quadruplex structure of probe and the biological catalysis inactivation of DNAzyme formed by G-quadruplex structure and hemin. As a result, the probe would be transformed from G-quadruplex to duplex and the obvious differential signal could be observed in most cases even with naked eyes. With the significant signal decreasing, the target miRNA can be rapidly analyzed by UV–vis spectra with a considerably low detection limit (4.5 nM). This method exhibits excellent selectivity and anti-interference ability by discriminating base-mismatched and other miRNA families from target miRNA. Meanwhile, a good recovery (90.7%∼102.4%) in 5% human serum was obtained and this strategy verified a high expression level of miR21 in total RNA of MCF-7 compared with 293T and HeLa. It implies that this assay is of great potential to be applied in biochemical research and clinical diagnosis.

Abstract 780: High expression of miR-155 promotes prostate cancer aggressiveness in African-Americans compared to Caucasians

Abstract Background: African-Americans have higher risk for developing prostate cancer and when diagnosed, the cancer is more aggressive and African-Americans are more than twice as likely to die of the disease compared to Caucasians. To clarify the mechanisms involved in this disparity, we analyzed the role of miR-155 in African-American and Caucasian prostate cancer tissues and cell lines. Materials and Methods: Human tissue samples from African-Americans and Caucasians clinical FFPE (Formaldehyde Fixed Paraffin Embedded) were used to analyzed miR-155 expression by Real Time PCR. We selected prostate cancer cell lines from Caucasians (DU-145, LNCaP, PC3), African-American (E006AA-hT) and normal epithelial cell line (PWR-1E) which express different levels of miR-155, to mimic the tissue samples and help identify the mechanisms related to racial disparity. We knocked down miR-155 in cells lines and performed cell viability assays using a CellTiter-Glo luminescent assay. For migration analysis, miR-155 knockdown cells were seeded in chambers and we measured the capacity of cell motility towards a chemo-attractant. To identify miR-155 mediated downstream regulators, three target prediction algorithms (miRDB, Targetscan and miRecords) were used. Results: We found miR-155 expression to be higher in African-American prostate cancer tissues compared to Caucasian patients with localized disease. Also, expression of miR-155 in African-American cell line, E006AA-hT, was significantly higher compared to Caucasian cell lines, DU-145, LNCaP and PC3. To determine the role of miR-155 in the differences between African-Americans and Caucasians, miR-155 was knocked down in E006AA-hT and DU-145 cells. miR-155 knockdown in E006AA-hT cells caused a decrease in cell viability compared with negative control, while there was no difference in DU-145 cells. We observed inhibition of migration E006AA-hT miR-155 knockdown cells but not on DU-145 miR-155 knockdown cells, suggesting that miR-155 knockdown have the more potent effect on African-American cell line. We analyzed three target prediction algorithms and found that four genes overlapped, SPI1, JARID2, ETS1 and CEBPB that may play a pivotal role in the treatment of aggressive African-American prostate cancers. Conclusion: We conclude that high levels of miR-155 is associated with prostate cancer aggressiveness in African-Americans. Citation Format: Marisa Shiina, Yutaka Hashimoto, Priyanka Kulkarni, Pritha Dasgupta, Varahram Shahryari, Guoren Deng, Divya Bhagirath, Laura Tabatabai, Sharanjot Saini, Shahana Majid, Soichiro Yamamura, Yuichiro Tanaka, Rajvir Dahiya. High expression of miR-155 promotes prostate cancer aggressiveness in African-Americans compared to Caucasians [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 780.

Dysregulated Expression of microRNA-21 and Disease-Related Genes in Human Patients and in a Mouse Model of Alport Syndrome

Alport syndrome is a genetic disease caused by mutations in type IV collagen and is characterized by progressive kidney disease. The Col4α3-/- mouse model recapitulates the main features of human Alport syndrome. Previously, it was reported that kidney microRNA-21 (miR-21) expression is significantly increased in Col4α3-/- mice, and administration of anti-miR-21 oligonucleotides (anti-miR-21) attenuates kidney disease progression in Col4α3-/- mice, indicating that miR-21 is a viable therapeutic target for Alport syndrome. However, the expression pattern of miR-21 in the kidneys of patients with human Alport syndrome has not been evaluated. Paraffin-embedded kidney specimens were obtained from 27 patients with Alport syndrome and from 10 normal controls. They were evaluated for miR-21 expression and for in situ hybridization and mRNA expression by quantitative polymerase chain reaction. In addition, anti-miR-21 was administrated to Col4α3-/- mice at different stages of disease, and changes in proteinuria, kidney function, and survival were monitored. Transcriptomic analysis of mouse kidney was conducted using RNA sequencing. miR-21 expression was significantly elevated in kidney specimens from patients with Alport syndrome compared to normal controls. Elevated renal miR-21 expression positively correlated with 24 h urine protein, serum blood urea nitrogen, serum creatinine, and severity of kidney pathology. On histological evaluation, high levels of miR-21 were localized to damaged tubular epithelial cells and glomeruli. Kidney specimens from both humans and mice with Alport syndrome exhibited abnormal expression of genes involved in kidney injury, fibrosis, inflammation, mitochondrial function, and lipid metabolism. Administration of anti-miR-21 to Alport mice resulted in slowing of kidney function decline, partial reversal of abnormal gene expression associated with disease pathology, and improved survival. Increased levels of miR-21 in human Alport kidney samples showed a correlation with kidney disease severity measured by proteinuria, biomarkers of kidney function, and kidney histopathology scores. These human data, combined with the finding that a reduction of miR-21 in Col4α3-/- mice improves kidney phenotype and survival, support miR-21 as a viable therapeutic target for the treatment of Alport syndrome.

MiR-155 controls follicular Treg cell-mediated humoral autoimmune intestinal injury by inhibiting CTLA-4 expression

High expression levels of miR-155 are involved in the pathogenesis of inflammatory bowel disease (IBD). We observed an increase in miR-155 in peripheral regulatory T (Treg) cells from IBD patients. Mice that specifically overexpress miR-155 in Foxp3+ Treg cells exhibit spontaneous autoimmunity and more severe dextran sulfate sodium (DSS)-induced intestinal injury. MiR-155 overexpression can lead to a lack of follicular Treg (Tfr) cells and central Treg (cTreg), whereas DSS treatment further depletes the Tfr cells. Furthermore, miR-155 can target the expression of CTLA-4 in cTreg and Tfr, directly inhibiting Tfr cell production and promoting enhanced germinal center (GC) B cell activation and autoantibody overproduction. This outcome may be the cause of severe intestinal injury in patients with autoimmune IBD.

Association between miR-21/146a/155 level changes and acute genitourinary radiotoxicity in prostate cancer patients: A pilot study

IntroductionNearly sixty percent of patients with prostate cancer (PCa) undergo radiation therapy (RT). During the course of treatment patients may experience normal tissue reactions. It is a well established fact that genetic and epigenetic mechanisms, such as microRNA (miRNA) level changes might be associated with radiotoxicity, as a response to irradiation. Materials and methodsThis is the first study that has investigated levels of radiosensory miRNAs in association with acute genitourinary radiotoxicity extracted from peripheral blood mononuclear cells (PBCs), in three points; before RT (BRT), after RT (ART) and on the first control examination (FCONT). We measured levels of miR-21/146a/155 expression by quantitative real-time PCR (qRT-PCR), comparative ΔΔCt method, in fifteen patients with localized prostate cancer, treated with three-dimensional conformal radiotherapy (3DCRT). Nine subjects have experienced acute genitourinary (GU) radiotoxicity whereas six where without GU radiotoxicity. ResultsFirstly, we detected the highest levels of miR-21 in ART group (p = 0.043) in the patients with acute GU radiotoxicity. Secondly, we found trend towards higher miR-21 levels and significantly higher levels of miR-146a/155 within the patients with acute GU toxicity than in patients without (p = 0.068, p = 0.016, and p = 0.010, respectively). Thirdly, we detected significant change in miR-146a/155 levels within the patients without acute GU radiotoxicity during RT p = 0.042, and p = 0.041, respectively). ConclusionmiR-21/146a/155 might be useful potential factors of radiosensitivity and acute genitourinary radiotoxicity in prostate cancer patients. miRNA might have great potential as predictors of various pathological conditions extracted from PBMCs.

Expressions of VEGF and miR-21 in tumor tissues of cervical cancer patients with HPV infection and their relationships with prognosis.

To investigate the expressions of vascular endothelial growth factor (VEGF) and micro-ribonucleic acid-21 (miR-21) in cervical cancer patients with human papillomavirus (HPV) infection and determine the potential relationships with prognosis. Expressions of VEGF in cervical cancer tissues and cancer-adjacent tissues were detected by immunohistochemistry, and the expressions of miR-21 and VEGF in both tissues were quantitatively analyzed using reverse transcription polymerase chain reaction (RT-PCR). Patients with cervical cancer were followed up after operation, and the survival rates of patients with different expression levels of miR-21 and VEGF were compared. VEGF was expressed in both cervical cancer tissues and cancer-adjacent tissues. The positive expression rate of VEGF in cervical cancer tissues (75.69%) was significantly higher than that in cancer-adjacent tissues (10.45%). RT-PCR results showed that the expression levels of miR-21 and VEGF in cervical cancer tissues were significantly higher than those in cancer-adjacent tissues (p<0.05). Correlation analyses revealed that miR-21 expression was significantly positively correlated with VEGF expression in cervical cancer tissues (r2=0.4174, p<0.0001). Prognostic analyses showed that the 5-year survival rate of patients was relatively high when miR-21 and VEGF were lowly expressed. VEGF and miR-21 are highly expressed in tumor tissues of cervical cancer patients with HPV infection. VEGF expression is significantly positively correlated with miR-21 expression, and the high levels of VEGF and miR-21 predict unfavorable prognosis of cervical cancer. Data provide a theoretical support for clinical treatment of cervical cancer patients with HPV infection.

PO-499 Comparison of expression of miR21’s guide strand (5P) and passenger strand (3P) in breast tumour progression

IntroductionmiR-21 is an oncomir that is overexpressed in many types of solid tumours. According to some studies miR-21 expression is higher in breast tumours than normal breast tissue and it is significantly correlated with shorter survival of patients. Many studies have focused on miR-21–5 p and its correlation with clinic pathological features and survival of breast tumours. In some of the studies miR-21–5 p expression has been shown to be associated with ER status, PR status, HER2 status, clinical stage, lymph node, tumour grade and size. The purpose of this study was to compare the expression of miR-21–3 p and miR-21–5 p in progression of breast tumours and their association with corresponding clinic pathological data from the breast cancer patients.Material and methodsSix non-neoplastic breast tissues and 144 tumours, from breast cancer patients diagnosed 1987–2003, were obtained from the Department of Pathology at Landspitali University Hospital. The expression levels of miR-21–3 p and miR-21–5 p were measured with StepOnePlus Real-Time PCR System by applying EXIQON primer sets and SYBR Green master mix using miR-16–5 p as endogenous control.Results and discussions miR-21–5 p was expressed at significantly higher levels than miR-21–3 p in both normal (p=0.0003) and tumour tissues (p=6.378e-12). A significant difference was not observed between miR-21–5 p expression in normal breast tissue and tumours. High miR-21–5 p expression correlated significantly with HER2 expression (p=0.001) but it did not correlate with other clinical and pathological factors or survival. miR-21–3 p was expressed at significantly higher levels in tumour tissues than normal breast tissue (p=0.003). Its expression was significantly higher in tumours from patients with metastasis than without metastasis (p=0.02). Furthermore, higher levels of miR-21–3 p correlated with shorter disease free survival (p=0.03, HR=1.72, 95%CI=1.03–2.9).ConclusionAlthough miR-21 is one of the most studied and upregulated miRNAs in human solid tumours the comparison of miR-21–3 p expression with miR-21–5 p expression in breast tumours has not been well studied. Our results suggest that miR-21–3 p could be a better indicator of breast cancer progression than miR-21–5 p. More studies in larger breast cohorts and functional cell based assays will be needed to shed light on the potential role of miR-21–3 p in breast tumours progression.

Human cancer cells suppress behaviors of endothelial progenitor cells through miR-21 targeting IL6R

Deep vein thrombosis (DVT) is a severe clinical process and has a high rate of fatality. Cancer patients have a high incidence rate of venous thrombosis complication and increase the mortality of cancer patients for 2–8 times. The mechanisms involved in human cancers and venous thrombosis remains unclear. In this study, we determined miR-21 expressed higher in human breast cancer, colon cancer and hepatocellular cancer tissues compared with normal tissues and expressed higher in exosomes of breast cancer and hepatocellular cancer cell lines compared with normal cells. MiR-21 dramatically suppressed proliferation, migration and invasion of endothelial progenitor cells (EPCs), which performed promoting role in thrombus repairment and resolution. High levels of miR-21 in exosomes of human cancers dramatically inhibited behaviors of EPCs, and depletion of miR-21 abrogated the decreased proliferation, migration and invasion of EPCs induced by human cancer cells. Moreover, IL6R (interleukin 6 receptor) was identified to be a direct target of miR-21 and promoted cell proliferation, migration and invasion of EPCs. Therefore, the miR-21-IL6R pathway contributed to behaviors of EPCs and consequently mediated the vein thrombosis in patients with cancer. MiR-21-IL6R pathway based therapeutic methods would be beneficial to decrease the complicated venous thrombosis in cancer patients and promote thrombus resolution.

Lung adenocarcinoma cell-derived exosomal miR-21 facilitates osteoclastogenesis

Osteoclastogenesis is the rate-limiting step in tumor osteolytic metastasis. MicroRNAs play crucial roles in tumor progression and osteoclastogenesis regulation. Recent studies have demonstrated that exosomes are able to function as messengers that deliver microRNAs between cells. However, the effects of lung adenocarcinoma cell-derived exosomal miRNAs in osteoclastogenesis remain poorly understood. In this study, we found that exosomes derived from A549 cells facilitate osteoclastogenesis. As miR-21 was involved in tumorigenesis and osteoclastogenesis, we further proved the existence of miR-21 in A549 cell-derived exosomes and investigated its function. MiR-21 overexpression in A549 cells led to increased levels of miR-21 in exosomes and facilitated osteoclastogenesis. Conversely, miR-21 depletion in A549 cells down-regulated miR-21 in exosomes and alleviated osteoclastogenesis. Mechanical experiments demonstrated that exosomal miR-21 exerted its promoting effects on osteoclastogenesis via targeting Pdcd4, which is a known target of miR-21 and a regulator of osteoclastogenesis. Finally, clinical data showed that higher miR-21 levels were associated with a worse overall survival in lung adenocarcinoma patients. In conclusion, we found that lung adenocarcinoma derived exosomal miR-21 may facilitate osteoclastogenesis, which suggests that it is a potential therapeutic target of bone metastasis.

MicroRNA miR-155 promotes medullary thymic epithelial cell maturity and regulatory T cell development

Abstract MicroRNA-155 (miR-155) is a small non-coding RNA highly upregulated in regulatory T (Treg) cells, an immune cell population specialized in maintaining immunological tolerance. While the importance of miR-155 in conferring Treg cell competitive fitness has been well established, how miR-155 regulates Treg cell development remains unknown. By taking cell-type specific gene targeting approaches, we demonstrate that miR-155 controls thymic Treg (tTreg) cell differentiation in both T cell-intrinsic and -extrinsic manners. Specifically, we show that mature CD80hiMHCIIhi medullary thymic epithelial cells (mTECs) crucial for the generation of tTreg cells express high levels of miR-155, where TEC-specific deletion of miR-155 diminishes this population and impairs tTreg cell development. Previously, the size of the mature mTEC compartment has been shown to be regulated by TGF-β signaling. Here, our results identify SMAD3, a key molecule downstream of TGF-β signaling, is targeted by miR-155 in mTECs. Thus, we propose a miR-155-SMAD3-TGF-β axis in the thymic medulla to determine mTEC maturity and, consequently, the quantity of tTreg cells.

Bone marrow mesenchymal stem cell-derived exosomal miR-21 protects C-kit+ cardiac stem cells from oxidative injury through the PTEN/PI3K/Akt axis.

Stem cell (SC) therapy for ischemic cardiomyopathy is hampered by poor survival of the implanted cells. Recently, SC-derived exosomes have been shown to facilitate cell proliferation and survival by transporting various proteins and non-coding RNAs (such as microRNAs and lncRNAs). In this study, miR-21 was highly enriched in exosomes derived from bone marrow mesenchymal stem cells (MSCs). Interestingly, exosomes collected from hydrogen peroxide (H2O2)-treated MSCs (H-Exo) contained higher levels of miR-21 than exosomes released from MSCs under normal conditions (N-Exo). The pre-treatment of C-kit+ cardiac stem cells (CSCs) with H-Exos resulted in significantly increased levels of miR-21 and phosphor-Akt (pAkt) and decreased levels of PTEN, which is a known target of miR-21. AnnexinV-FITC/PI analysis further demonstrated that the degree of oxidative stress-induced apoptosis was markedly lower in H-Exo-treated C-kit+ CSCs than that in N-Exo-treated cells. These protective effects could be blocked by both a miR-21 inhibitor and the PI3K/Akt inhibitor LY294002. Therefore, exosomal miR-21 derived from H2O2-treated MSCs could be transported to C-kit+ cardiac stem cells to functionally inhibit PTEN expression, thereby activating PI3K/AKT signaling and leading to protection against oxidative stress-triggered cell death. Thus, exosomes derived from MSCs could be used as a new therapeutic vehicle to facilitate C-kit+ CSC therapies in the ischemic myocardium.

Circulating miR-21, miR-210 and miR-146a as potential biomarkers to differentiate acute tubular necrosis from hepatorenal syndrome in patients with liver cirrhosis: a pilot study.

Acute kidney injury (AKI) in cirrhotic patients may be functional (hepatorenal syndrome [HRS]) or structural (acute tubular necrosis [ATN]). The differentiation between these two conditions remains challenging; no definite biomarker with a clear cutoff value had been declared. miRNAs seem to be attractive innovative biomarkers to identify the nature of kidney injury in cirrhotic patients. This study aimed to investigate the possibility of using miR-21, miR-210 and miR-146a as differentiating markers between HRS and ATN. This pilot case control study included 50 patients with liver cirrhosis; 25 with HRS and another 25 with ATN beside 30 healthy controls. Real-time qPCR was used to measure the circulating miRNA tested. Higher levels of miR-21 were observed in both ATN and HRS vs. controls with statistically significant difference between ATN and HRS. The means were 9.466±3.21 in ATN, 2.670±1.387 in HRS and 1.090±0.586 in controls. miR-146a and miR-210 were both significantly lower in ATN and HRS compared to controls with statistically significant differences between ATN and HRS. The means of miR-210 were 1.020±0.643, 1.640±0.605 and 3.0±0.532 in ATN, HRS and controls, respectively. The means of miR-146a were 2.543±1.929, 4.98±1.353 and 6.553±0.426 in ATN, HRS and controls, respectively. ROC analyses proved that the three studied mi-RNAs can be used as differentiating biomarkers between ATN and HRS with the best performance observed with mi-21 achieving specificity and sensitivity equal 96%. miR-21, miR-210 and miR-146a may be candidate differentiating markers between HRS and ATN in cirrhotic patients.

Analysis of Plasma MicroRNAs as Predictors and Biomarkers of Aging and Frailty in Humans

Although circulating microRNAs (miRNAs) can modulate gene expression and affect immune system response, little is known about their participation in age-associated frailty syndrome and sarcopenia. The aim of this study was to determine miRNAs as possible biomarkers of age and frailty and their correlation with oxidative and inflammatory state in human blood. Three inflammation-related miRNAs (miR-21, miR-146a, and miR-223) and one miRNA related with the control of melatonin synthesis (miR-483) were analyzed. Twenty-two healthy adults, 34 aged robust, and 40 aged fragile patients were selected for this study. The expression of plasma miRNAs was assessed by RT-qPCR; plasma cytokines (IL-6, IL-8, IL-10, and TNFα) were analyzed by commercial kits, and plasma advanced oxidation protein products (AOPP) and lipid oxidation (LPO) were spectrophotometrically measured. Fragile subjects had higher miR-21 levels than control subjects, whereas miR-223 and miR-483 levels increased at a similar extend in both aged groups. All cytokines measured increased in aged groups compared with controls, without differences between robust and fragile subjects. The fragile group had a TNFα/IL-10 ratio significantly higher than robust and control groups. Aged groups also had higher AOPP and LPO levels than controls. Women presented higher AOPP and LPO levels and increased expression of miR-483 compared with men. Positive correlations between miR-21 and AOPP and between miR-483 and IL-8 were detected. The expression of miR-21 and the TNFα/IL-10 ratio were correlated positively with the presence of frailty, which suggests that these markers can be considered as possible biomarkers for age-related frailty.

Micro RNA-155 plays a critical role in the initiation of food allergen-related inflammation in the intestine.

The pathogenesis of food allergy (FA) is to be further investigated. Regulatory B cells (B10 cell) play a critical in the maintenance of the homeostasis in the intestine. Deregulation of B10 cell is associated with immune inflammation. Micro RNA (miR) 155 is involved in affecting immune cell function. This study tests a hypothesis that miR-155 affects the B10 cell function to facilitate the initiation of FA. In this study, BALB/c mice were sensitized to ovalbumin (OVA) to induce FA-like inflammation in the intestine. B cells were isolated from the intestine by magnetic cell sorting. The expression of miR-155 and IL-10 in B cells was assessed by real time RT-PCR. The results showed that mice sensitized to OVA showed FA-like inflammation and lower frequency of B10 cell in the intestine. B cells isolated from the intestine of FA mice showed higher levels of miR-155 and lower levels of IL-10. Although all the three T helper (Th)2 cytokines, including interleukin (IL)-4, IL-5 and IL-13, were higher in the serum, only IL-13 was positively correlated with the levels of miR-155 in the intestinal B cells. Exposure to IL-13 in the culture markedly increased the expression of miR-155 and suppressed the expression of IL-10 in B cells. Blocking miR-155 abolished the IL-13-induced IL-10 suppression in B cells and inhibited FA response in mice. In conclusion, miR-155 plays a critical role in the initiation of FA in mice. Blocking miR-155 has therapeutic potential in the treatment of FA.

MicroRNA-155, induced by FOXP3 through transcriptional repression of BRCA1, is associated with tumor initiation in human breast cancer.

MicroRNA (miR)-155 is upregulated in breast cancer cells and in sera of patients with breast cancer, but its clinical relevance remains uncertain. The objective of the present effort was to address the transcriptional regulation of miR-155. A bioinformatics analysis of public datasets validated upregulation of miR-155 in tumor cells of patients with breast cancer, particularly those who were at early stages and had triple-negative cancers. The expression profiling and clinical relevance of miR-155 in tumor cells and blood cells were characterized by TaqMan miR assays and, in plasma and exosomes, by nest-quantitative PCR analysis. There was a positive correlation between expression of FOXP3 and miR-155 in breast cancer cell lines and primary breast cancers. In breast cancer cells, FOXP3 induced miR-155 through transcriptional repression of BRCA1. Furthermore, in an Alabama cohort, blood and plasma samples were collected from 259 participants, including patients with breast cancer or benign breast tumors, members of breast cancer families, and matched healthy female controls. For patients with early stage or localized breast cancer, there were high levels of miR-155 in both plasma and blood cells. In cultured breast cancer cells, expression of miR-155 was induced by FOXP3 but was not significantly changed in culture medium or exosomes, suggesting that circulating miR-155 originated from blood cells. These findings reveal a transcriptional axis of FOXP3-BRCA1-miR-155 in breast cancer cells and show that plasma miR-155 may serve as a non-invasive biomarker for detection of early stage breast cancer.

MicroRNA-155 and Anti-Müllerian Hormone: New Potential Markers of Subfertility in Men with Chronic Kidney Disease

Background/Aims: Men with terminal renal failure are often infertile. Anti-müllerian hormone (AMH), a marker of Sertoli cell function, is decreased among men with chronic kidney disease (CKD). Recently, a microRNA, miR-155, has been shown to be a potential marker for subfertility. We studied miR-155 and semen parameters in patients with CKD who were not yet on dialysis. We also aimed to study possible associations between AMH, miR-155, and semen parameters to evaluate them as markers of fertility. Methods: Thirty male patients with CKD 1–4 as well as 18 healthy controls were enrolled. Results: Serum levels of miR-155 were significantly higher among men with CKD stages 1–2 (4.51 ± 3.81 [p = 0.01]) and stages 3–4 (2.75 ± 1.77 [p = 0.006]) than in controls (1.09 ± 0.44). Sperm concentration was significantly lower among men with CKD 3–4 (42 ± 29) ×10⁶/mL compared to controls (88 ± 42) ×10⁶/mL (p = 0.011). High levels of miR-155 were associated with a relatively low sperm concentration (p = 0.02) and with a low total sperm number (p = 0.005). Low AMH levels were associated with a decreased percentage of motile sperm cells (p = 0.02). Conclusions: We conclude that men with stage 3–4 CKD had lower sperm concentrations than healthy fertile men and that increased serum miR-155 in men with stage 1–4 CKD was associated with semen parameters that indicate subfertility. Low AMH levels were associated with a low percentage of the total number of motile sperm cells. miR-155 and AMH may be potential markers of subfertility in men with CKD.

Glioma stem cells-derived exosomes promote the angiogenic ability of endothelial cells through miR-21/VEGF signal.

Glioma stem cells (GSCs) play an important role in glioblastoma prognosis. Exosomes (EXs) mediate cell communication by delivering microRNAs (miRs). Glioblastoma has a high level of miR-21 which could upregulate vascular endothelial growth factor (VEGF) expression. We hypothesized GSC-EXs can promote the angiogenic ability of endothelial cells (ECs) through miR-21/VEGF signal. GSCs were isolated from U-251 cells with stem cell marker CD133. GSCs transfected without or with scramble or miR-21 mimics were used to produce GSC-EXscon, GSC-EXssc and GSC-EXsmiR-21. Human brain ECs were co-cultured with vehicle, GSC-EXscon, GSC-EXssc or GSC-EXsmiR-21 plus VEGF siRNAs (siRNAVEGF). After 24 hours, the angiogenic abilities of ECs were evaluated. The levels of miR-21, VEGF and p-Flk1/VEGFR2 were determined. Results showed: 1) Over 90% of purified GSCs expressed CD133; 2) The levels of miR-21 and VEGF in GSCs and GSC-EXs were up-regulated by miR-21 mimic transfection; 3) Compared to GSC-EXscon or GSC-EXssc, GSC-EXsmiR-21 were more effective in elevating the levels of miR-21 and VEGF, and the ratio of p-Flk1/VEGFR2 in ECs; 4) GSC-EXsmiR-21 were more effective in promoting the angiogenic ability of ECs than GSC-EXscon or GSC-EXssc, which were remarkably reduced by siRNAVEGF pretreatment. In conclusion, GSC-EXs can promote the angiogenic ability of ECs by stimulating miR-21/VEGF/VEGFR2 signal pathway.

Urinary sediment miRNAs reflect tubulointerstitial damage and therapeutic response in IgA nephropathy

BackgroundImmunoglobulin A nephropathy (IgAN) is the most common glomerulonephritis worldwide. The clinical spectrum of IgAN varies from minor urinary abnormalities to rapidly progressive renal failure. Evaluation of the disease by repeated renal biopsy is not practical due to its invasive procedure. Urinary sediment miRNAs promise to serve as non-invasive biomarkers to assess kidney injury of IgAN.MethodsFifty two biopsy-proven IgAN patients and twenty five healthy controls were enrolled in the study. Urinary sediment miRNAs were extracted. Expressions of miR-34a, miR-205, miR-21, miR-146a and miR-155 were quantified by real-time quantitative polymerase chain reaction (RT-QPCR). The receiver operating characteristic (ROC) curve was used to investigate the value of the miRNAs for predicting diagnosis of IgAN and evaluating histopathological injury. The patients were treated according to the Kidney Disease: Improving Global Outcomes (KDIGO) guidelines and followed up. The roles of miRNAs in reflecting therapeutic efficacy and disease progression were analyzed.Results1. The IgAN group had significantly lower urinary miR-34a, miR-205, and miR-155, but higher miR-21 levels than controls. The ROC revealed that urinary miR-34a ≤ 0.047, miR-205 ≤ 0.209, miR-21 ≥ 0.461 and miR-155 ≤ 0.002 could distinguish patients with IgAN from healthy ones. In addition, miR-205 ≤ 0.125 and miR-21 ≥ 0.891 can distinguish IgAN patients with severe tubular atrophy/interstitial fibrosis from those with mild tubular atrophy/interstitial fibrosis. 2. After a mean 15.19 months follow-up, the reduction of proteinuria (g/24 h/year) was positively correlated with baseline urinary miR-21 and inversely correlated with miR-205. The levels of baseline eGFR and miR-205 in the complete remission group were significantly higher than non-complete remission group (p < 0.001; p = 0.018), while proteinuria, miR-21 and miR-146a were lower than non-complete remission group (p = 0.002; p = 0.021; p = 0.009). But multivariate analysis revealed that only baseline eGFR correlated with the remission of IgAN (p = 0.001, OR = 1.042).ConclusionsThe levels of some urinary sediment miRNAs, especially baseline miR-21 and miR-205, may be used as potential prognostic markers for evaluating the tubulointerstitial damage of IgAN. Furthermore, baseline levels of urinary miRNAs may be predictors of therapeutic efficacy and disease progression.

Over-Expression of miR-21 and Lower PTEN Levels in Wilms' Tumor with Aggressive Behavior.

Wilms' tumor (WT) is the most common pediatric kidney tumor. MiR-21 is one of the most frequently overexpressed microRNAs in solid tumors, while phosphatase and tensin homolog deleted from chromosome 10 (PTEN) is the most highly mutated tumor suppressor gene. The aim of this study was to investigate the relationship between miR-21 and PTEN in WT. The expression levels of miR-21 and the PTEN protein were determined by qRT-PCR and Western blot analyses in WT specimens, respectively. In WT tissues, the miR-21 expression levels were significantly higher and the PTEN protein levels were significantly lower, compared to the adjacent non-tumorous renal tissues. The higher levels of miR-21 and lower levels of PTEN were correlated with age (> 24 months), late clinical stage, unfavorable histopathological type and lymphatic metastasis. A univariate linear regression analysis indicated a significant correlation between higher miR-21 levels and lower PTEN levels. Using the SK-NEP-1 WT cell line, we showed that the decreased expression levels of miR-21 promoted cell proliferation and invasion, but inhibited apoptosis. Importantly, lowered expression levels of miR-21 increased the expression levels of PTEN protein and decreased the expression levels of phosphoinositide 3-kinase (PI3K) and phosphorylated protein kinase B (p-AKT), each of which functions in the downstream signaling pathway. Dual luciferase-reporter assays indicated that PTEN mRNA was a direct target of miR-21. In conclusion, higher miR-21 levels and lower PTEN protein levels are predictive biomarkers for poor prognosis of WT patients. Over-expression of miR-21 promotes aggressive behavior of WT by targeting PTEN.

Multiple Myeloma-Derived Exosomes Regulate the Functions of Mesenchymal Stem Cells Partially via Modulating miR-21 and miR-146a.

Exosomes derived from cancer cells can affect various functions of mesenchymal stem cells (MSCs) via conveying microRNAs (miRs). miR-21 and miR-146a have been demonstrated to regulate MSC proliferation and transformation. Interleukin-6 (IL-6) secreted from transformed MSCs in turn favors the survival of multiple myeloma (MM) cells. However, the effects of MM exosomes on MSC functions remain largely unclear. In this study, we investigated the effects of OPM2 (a MM cell line) exosomes (OPM2-exo) on regulating the proliferation, cancer-associated fibroblast (CAF) transformation, and IL-6 secretion of MSCs and determined the role of miR-21 and miR-146a in these effects. We found that OPM2-exo harbored high levels of miR-21 and miR-146a and that OPM2-exo coculture significantly increased MSC proliferation with upregulation of miR-21 and miR-146a. Moreover, OPM2-exo induced CAF transformation of MSCs, which was evidenced by increased fibroblast-activated protein (FAP), α-smooth muscle actin (α-SMA), and stromal-derived factor 1 (SDF-1) expressions and IL-6 secretion. Inhibition of miR-21 or miR-146a reduced these effects of OPM2-exo on MSCs. In conclusion, MM could promote the proliferation, CAF transformation, and IL-6 secretion of MSCs partially through regulating miR21 and miR146a.