miRNA-1 regulates endothelin-1 in diabetes

Aims

MicroRNAs (miRNAs) play important roles in several biological processes. In this study, we investigated the role of miR-1, an endothelin-1 (ET-1) targeting miRNA, in endothelial cells (ECs) and tissues of diabetic animals. ET-1 is known to be of pathogenetic significance in several chronic diabetic complications.

Main methods

PCR array was used to identify alterations of miRNA expression in ECs exposed to glucose. miR-1 expression was validated by TaqMan real-time PCR assay. Human retinal ECs (HRECs) and human umbilical vein ECs (HUVECs) exposed to various glucose levels with or without miR-1 mimic transfection, and tissues from streptozotocin-induced diabetic animals after two months of follow-up, were examined for miR-1 expression, as well as ET-1 and fibronectin (FN) mRNA and protein levels.

Key findings

Array analyses showed glucose-induced alterations of 125 miRNAs (out of 381) in ECs exposed to 25 mM glucose compared to 5 mM glucose. Fifty-one miRNAs were upregulated and 74 were downregulated. 25 mM glucose decreased miR-1 expression and increased ET-1 mRNA and protein levels. miR-1 mimic transfection prevented HG-induced ET-1 upregulation. Furthermore, glucose induced upregulation of FN, which is mediated partly by ET-1, was also prevented by such transfection.Diabetic animals showed decreased miR-1 expression in the retina, heart and kidneys. In parallel, ET-1 mRNA expressions were increased in these tissues of diabetic animals, in association with upregulation of FN.

Significance

These results indicate a novel glucose-induced mechanism of tissue damage, in which miR-1 regulates ET-1 expressions in diabetes. Identifying such mechanisms may lead to RNA based treatment for diabetic complications.     

High expression of miR-210 predicts poor survival in patients with breast cancer: A meta-analysis

Background

Many microRNAs (miRNAs) exhibit altered expression levels in cancers, and they may be considered as valuable prognostic biomarkers for cancers. Here we aimed to summarize the recent advances in miR-210 involvement in human breast cancer and analyze the predicting role of miR-210 for survival.

Methods

A meta-analysis was performed by searching PubMed, Cochrane Library, and Science Direct databases. Data were extracted from studies comparing survival in patients with breast cancer having higher expression of miR-210 with those having lower expression. Pooled hazard ratios (HRs) and 95% confidence intervals (CI) were calculated.

Results

A total of 511 cases of breast cancer were involved for this global meta-analysis. For post-operational survival, the HR of higher miR-210 expression in breast cancer tissue was 3.39 (95% CI: 2.04–5.63, P < 0.05), which could significantly predict poorer survival.

Conclusions

High expression of miR-210 might predict poor survival in patients with breast cancer.     

Inorganic Phosphate Accelerates the Migration of Vascular Smooth Muscle Cells: Evidence for the Involvement of miR-223

Backgound

An elevated serum inorganic phosphate (Pi) level is a major risk factor for kidney disease and downstream vascular complications. We focused on the effect of Pi levels on human aortic vascular smooth muscle cells (VSMCs), with an emphasis on the role of microRNAs (miRNAs).

Methodology/Principal Findings

Exposure of human primary VSMCs in vitro to pathological levels of Pi increased calcification, migration rate and concomitantly reduced cell proliferation and the amount of the actin cytoskeleton. These changes were evidenced by significant downregulation of miRNA-143 (miR-143) and miR-145 and concomitant upregulation of their targets and key markers in synthetic VSMCs, such as Krüppel-like factors−4 and −5 and versican. Interestingly, we also found that miR-223 (a marker of muscle damage and a key factor in osteoclast differentiation) is expressed in VSMCs and is significantly upregulated in Pi-treated cells. Over-expressing miR-223 in VSMCs increased proliferation and markedly enhanced VSMC migration. Additionally, we found that the expression of two of the known miR-223 targets, Mef2c and RhoB, was highly reduced in Pi treated as well as miR-223 over-expressing VSMCs. To complement these in vitro findings, we also observed significant downregulation of miR-143 and miR-145 and upregulation of miR-223 in aorta samples collected from ApoE knock-out mice, which display vascular calcification.

Conclusions/Significance

Our results suggest that (i) high levels of Pi increase VSMC migration and calcification, (ii) altered expression levels of miR-223 could play a part in this process and (iii) miR-223 is a potential new biomarker of VSMC damage.     

The miR-182/SORT1 axis regulates vascular smooth muscle cell calcification in vitro and in vivo

Arterial calcification is a common feature of cardiovascular disease. Sortilin is involved in the development of atherosclerosis, but the specific mechanism is unclear. In this study, we established calcification models in vivo and in vitro by using vitamin D₃ and β-glycerophosphate, respectively. In vivo, the expression of SORT1 was up-regulated and the expression of miR-182 was down-regulated in calcified arterial tissues. Meanwhile there was a negative correlation between SORT1 expression and miR-182 levels. In vitro, downregulating SORT1 expression using shRNA inhibited β-glycerophosphoric induced vascular smooth muscle cells (VSMCs) calcification. Moreover, reduced sortilin levels followed transfection of miR-182 mimics, whereas there was a significant increase in sortilin levels after transfection of miR-182 inhibitors. A luciferase reporter assay confirmed that SORT1 is the direct target of miR-182. Our study suggests that SORT1 plays a vital role in the development of arterial calcification and is regulated by miR-182.     

MicroRNA-33a functions as a bone metastasis suppressor in lung cancer by targeting parathyroid hormone related protein

Background

Bone is a common site of metastasis for lung cancer, and is associated with significant morbidity and a dismal prognosis. MicroRNAs (miRNAs) are increasingly implicated in regulating the progression of malignancies.

Methods

The efficacy of miR-33a or anti-miR-33a plasmid was assessed by Real-time PCR. Luciferase assays were using One-Glo Luciferase Assay System. Measurement of secreted factors was determined by ELISA kit.

Results

We have found that miR-33a, which is downregulated in lung cancer cells, directly targets PTHrP (parathyroid hormone-related protein), a potent stimulator of osteoclastic bone resorption, leading to decreased osteolytic bone metastasis. We also found that miR-33a levels are inversely correlated with PTHrP expression between human normal bronchial cell line and lung cancer cell lines. The reintroduction of miR-33a reduces the stimulatory effect of A549 on the production of osteoclastogenesis activator RANKL (receptor activator of nuclear factor kappa-B ligand) and M-CSF (macrophage colony-stimulating factor) on osteoblasts, while the expression of PTHrP is decreased in A549 cells. miR-33a overexpression also reduces the inhibitory activity of A549 on the production of OPG (osteoprotegerin), an osteoclastogenesis inhibitor. In addition, miR-33a-mediated PTHrP downregulation results in decreased IL-8 secretion in A549, which contributes to decreased lung cancer-mediated osteoclast differentiation and bone resorption.

Conclusions

These findings have led us to conclude that miR-33a may be a potent tumor suppressor, which inhibits direct and indirect osteoclastogenesis through repression of PTHrP.

General significance

miR-33a may even predict a poor prognosis for lung cancer patients.     

Microarray analysis of microRNA expression in liver cancer tissues and normal control

Background

Recently, many studies have focused on microRNAs (miRNAs) expression profiling in liver cancer, due to the ability of these small RNAs to potently influence cellular behavior. In this study, to further investigate the relationship between them, the miRNA expression profiling of the cancer liver tissues and normal liver tissues were compared.

Methods

The datasets of miRNAs microarray in liver cancer and normal control were downloaded from Gene Expression Omnibus. Then the SOAP analysis was performed to identify the differentially expressed miRNAs.

Results

A total of 221 differentially expressed miRNAs were found. Five of them (including hsa-miR-15b, hsa-miR-1975, hsa-miR-199a-3p, hsa-miR-199b-3p and hsa-miR-421) were determined by t-test and may be involved in the pathogenesis of liver cancer.

Conclusion

There differentially expressed miRNAs may be potential molecular markers for liver cancer screening.     

MiR-517a-3p accelerates lung cancer cell proliferation and invasion through inhibiting FOXJ3 expression

Aims

Aberrant expression of microRNAs (miRNAs) results in alterations of various biological processes (e.g., cell cycle, cell differentiation, and apoptosis) and cell transformation. Altered miRNAs expression was associated with lung carcinogenesis and tumor progression. This study aimed to investigate the function and underlying molecular events of miR-517a-3p on regulation of lung cancer cell proliferation and invasion.

Main methods

Transfected miR-517a-3p mimics or inhibitors into 95D and 95C cells respectively, the effects of miR-517a-3p on lung cancer cell proliferation, migration, and invasion were detected. Bioinformatics software forecasted potential target genes of miR-517a-3p and dual luciferase reporter gene system and western blot verified whether miR-517a-3p regulates FOXJ3 expression directly.

Key findings

MiR-517a-3p was differentially expressed in lung cancer 95D and 95C cell lines that have different metastatic potential. Manipulation of miR-517a-3p expression changed lung cancer cell proliferation, migration and invasion capacity. MiR-517a-3p directly regulated FOXJ3 expression by binding to FOXJ3 promoter.

Significance

This study demonstrated that miR-517a-3p promoted lung cancer cell proliferation and invasion by targeting of FOXJ3 expression.     

miR-29b,miR-205 and miR-221 Enhance Chemosensitivity to Gemcitabine in HuH28 Human Cholangiocarcinoma Cells

Background and Aims

Cholangiocarcinoma (CCA) is highly resistant to chemotherapy, including gemcitabine (Gem) treatment. MicroRNAs (miRNAs) are endogenous, non-coding, short RNAs that can regulate multiple genes expression. Some miRNAs play important roles in the chemosensitivity of tumors. Here, we examined the relationship between miRNA expression and the sensitivity of CCA cells to Gem.

Methods

Microarray analysis was used to determine the miRNA expression profiles of two CCA cell lines, HuH28 and HuCCT1. To determine the effect of candidate miRNAs on Gem sensitivity, expression of each candidate miRNA was modified via either transfection of a miRNA mimic or transfection of an anti-oligonucleotide. Ontology-based programs were used to identify potential target genes of candidate miRNAs that were confirmed to affect the Gem sensitivity of CCA cells.

Results

HuCCT1 cells were more sensitive to Gem than were HuH28 cells, and 18 miRNAs were differentially expressed whose ratios over ± 2log2 between HuH28 and HuCCT1. Among these 18 miRNAs, ectopic overexpression of each of three downregulated miRNAs in HuH28 (miR-29b, miR-205, miR-221) restored Gem sensitivity to HuH28. Suppression of one upregulated miRNA in HuH28, miR-125a-5p, inhibited HuH28 cell proliferation independently to Gem treatment. Selective siRNA-mediated downregulation of either of two software-predicted targets, PIK3R1 (target of miR-29b and miR-221) or MMP-2 (target of miR-29b), also conferred Gem sensitivity to HuH28.

Conclusions

miRNA expression profiling was used to identify key miRNAs that regulate Gem sensitivity in CCA cells, and software that predicts miRNA targets was used to identify promising target genes for anti-tumor therapies.     

Diagnostic and prognostic value of circulating miR-21 for cancer: A systematic review and meta-analysis

Background

MicroRNAs (miRNAs) have been reported to be aberrantly expressed in patients with cancer. Many studies have shown that circulating miRNAs could play potential roles as diagnostic and prognostic biomarkers of cancers. The aim of this meta-analysis is to summarize the role of circulating miR-21 as a biomarker in patients with a variety of carcinomas.

Material and methods

Eligible studies were identified and assessed for quality through multiple search strategies. For diagnostic meta-analysis, the sensitivity, specificity, and other measures of miR-21 in the diagnosis of cancer were pooled using bivariate random-effects approach models. For prognostic meta-analysis, pooled hazard ratios (HRs) of circulating miR-21 for survival were calculated.

Results

A total of 36 studies dealing with various carcinomas were included for the systemic review. Among them, 23 studies were finally enrolled in the global meta-analysis (17 studies for diagnosis and 6 studies for prognosis). For diagnostic meta-analysis, the overall pooled results for sensitivity, specificity, positive likelihood ratio (LRP), negative likelihood ratios (LRN) and diagnostic odds ratio (DOR) were 75.7% (95% CI: 67.1%–82.6%), 79.3% (95% CI: 74.2%–83.5%), 3.65 (95% CI: 2.83–4.70), 0.31 (95% CI: 0.22–0.43), and 11.88 (95% CI: 6.99–20.19), respectively. For prognostic meta-analysis, the pooled HR of higher miR-21 expression in circulation was 2.37 (95% CI: 1.83–3.06, P < 0.001), which could significantly predict poorer survival in general carcinomas. Importantly, subgroup analysis suggested that higher expression of miR-21 correlated with worse overall survival (OS) significantly in carcinomas of digestion system (HR, 5.77 [95% CI: 2.65–12.52]).

Conclusions

Our findings suggest that circulating miR-21 may not suitable to be a diagnostic biomarker, but it has a prognostic value in patients with cancer.     

Circulating MiR-125b as a marker predicting chemoresistance in breast cancer

Background

Chemotherapy is an important component in the treatment paradigm for breast cancers. However, the resistance of cancer cells to chemotherapeutic agents frequently results in the subsequent recurrence and metastasis. Identification of molecular markers to predict treatment outcome is therefore warranted. The aim of the present study was to evaluate whether expression of circulating microRNAs (miRNAs) can predict clinical outcome in breast cancer patients treated with adjuvant chemotherapy.

Methodology/Principal Findings

Circulating miRNAs in blood serum prior to treatment were determined by quantitative Real-Time PCR in 56 breast cancer patients with invasive ductal carcinoma and pre-operative neoadjuvant chemotherapy. Proliferating cell nuclear antigen (PCNA) immunostaining and TUNEL were performed in surgical samples to determine the effects of chemotherapy on cancer cell proliferation and apoptosis, respectively. Among the miRNAs tested, only miR-125b was significantly associated with therapeutic response, exhibiting higher expression level in non-responsive patients (n = 26, 46%; p = 0.008). In addition, breast cancers with high miR-125b expression had higher percentage of proliferating cells and lower percentage of apoptotic cells in the corresponding surgical specimens obtained after neoadjuvant chemotherapy. Increased resistance to anticancer drug was observed in vitro in breast cancer cells with ectopic miR-125b expression; conversely, reducing miR-125b level sensitized breast cancer cells to chemotherapy. Moreover, we demonstrated that the E2F3 was a direct target of miR-125b in breast cancer cells.

Conclusions/Significance

These data suggest that circulating miR-125b expression is associated with chemotherapeutic resistance of breast cancer. This finding has important implications in the development of targeted therapeutics for overcoming chemotherapeutic resistance in novel anti-cancer strategies.     

miR-204/miR-211 downregulation contributes to Candidemia-induced kidney injuries via derepression of Hmx1 expression

Aims

This study was aimed to exploit the role of heme oxygenase Hmx1 and the potential miRNA mechanisms in the kidney injuries induced by urinary tract infection by Candida species/Candidemia.

Main methods

We employed a mouse model of systemic Candidiasis by injection of the Candida albicans strain SC5314 into C57BL/6 mice. Kidney injuries were assessed by measuring serum cystatin C (CysC), serum β₂-microglobulin (β₂-MG) and blood urea nitrogen (BUN). Validation of miRNA target gene was conducted by luciferase reporter gene assay, Western blot analysis and real-time RT-PCR.

Key findings

We showed here that Candidemia caused significant downregulation of microRNAs miR-204 and miR-211. In sharp contrast, Hmx1 expression was remarkably upregulated, particularly at the protein level. Computational analysis predicted Hmx1 as a target gene for both miR-204 and miR-211 that share the same seed site sequence. We then experimentally validated the targeting relationship between miR-204/miR-211 and Hmx1, which explains the reciprocal changes of expression of miR-204/miR-211 and Hmx1 in Candidemia. Administration of miR-204/miR-211 mimics substantially downregulated Hmx1 and mitigated the severity of the kidney injuries induced by Candidemia, as reflected by improved renal glomerular filtration rate (GFR) determined by serum cystatin C (CysC), serum β₂-microglobulin (β₂-MG) and blood urea nitrogen (BUN). Knockdown of miR-204/miR-211 worsened while forced expression of miR-204/miR-211 ameliorated kidney injuries in mice with systemic Candidiasis.

Significance

Our findings indicate that miR-204/miR-211 downregulation accounts at least partially for the Hmx1 upregulation and the miR-204/miR-211–Hmx1 signaling axis may contribute to immune-suppression in the host thereby the Candidemia-induced kidney dysfunction.     

Technical Factors Involved in the Measurement of Circulating MicroRNA Biomarkers for the Detection of Colorectal Neoplasia

Background

Circulating miRNAs are emerging as promising blood-based biomarkers for colorectal and other human cancers; however, technical factors that confound the development of these assays remain poorly understood and present a clinical challenge. The aim of this study was to systematically evaluate the effects of factors that may interfere with the accurate measurement of circulating miRNAs for clinical purposes.

Methods

Blood samples from 53 subjects, including routinely drawn serum samples, matched plasma from 30 subjects, and matched serum samples drawn before and after bowel preparation for colonoscopy from 29 subjects were collected. Additionally, 38 serum specimens stored in the clinical laboratory for seven days were used to test the stability of miRNAs. Hemolysis controls with serial dilutions of hemoglobin were prepared. RNA was extracted from serum, plasma or hemolyzed controls with spiked-in cel-miR-39, and levels of miR-21, miR-29a, miR-125b and miR-16 were examined by real-time RT-PCR. Hemolysis was measured by spectrophotometry.

Results

The expression levels of miR-16 and the degree of hemolysis were significantly higher in plasma than in serum (P<0.0001). Measured miR-21, miR-29a, miR-125b and miR-16 expression increased with hemoglobin levels in hemolyzed controls. The degree of hemolysis in serum samples correlated significantly with the levels of miR-21 (P<0.0001), miR-29a (P = 0.0002), miR-125b (P<0.0001) and miR-16 (P<0.0001). All four miRNAs showed significantly lower levels in sera that had been stored at 4°C for seven days (P<0.0001). Levels of miR-21 (P<0.0001), miR-29a (P<0.0001) and miR-16 (P = 0.0003), and the degree of hemolysis (P = 0.0002) were significantly higher in sera drawn after vs. before bowel preparation.

Conclusions

The measured levels of miRNAs in serum and plasma from same patients varied in the presence of hemolysis, and since hemolysis and other factors affected miRNA expression, it is important to consider these confounders while developing miRNA-based diagnostic assays.     

Site directed processing: Role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type O-glycan core 2

Background

The assembly of Ser/Thr-linked O-glycans of mucins with core 2 structures is initiated by polypeptide GalNAc-transferase (ppGalNAc-T), followed by the action of core 1 β3-Gal-transferase (C1GalT) and core 2 β6-GlcNAc-transferase (C2GnT). β4-Gal-transferase (β4GalT) extends core 2 and forms the backbone structure for biologically important epitopes. O-glycan structures are often abnormal in chronic diseases. The goal of this work is to determine if the activity and specificity of these enzymes are directed by the sequences and glycosylation of substrates.

Methods

We studied the specificities of four enzymes that synthesize extended O-glycan core 2 using as acceptor substrates synthetic mucin derived peptides and glycopeptides, substituted with GalNAc or O-glycan core structures 1, 2, 3, 4 and 6.

Results

Specific Thr residues were found to be preferred sites for the addition of GalNAc, and Pro in the + 3 position was found to especially enhance primary glycosylation. An inverse relationship was found between the size of adjacent glycans and the rate of GalNAc addition. All four enzymes could distinguish between substrates having different amino acid sequences and O-glycosylated sites. A short glycopeptide Galβ1–3GalNAcα-TAGV was identified as an efficient C2GnT substrate.

Conclusions

The activities of four enzymes assembling the extended core 2 structure are affected by the amino acid sequence and presence of carbohydrates on nearby residues in acceptor glycopeptides. In particular, the sequences and O-glycosylation patterns direct the addition of the first and second sugar residues by ppGalNAc-T and C1GalT which act in a site directed fashion.

General significance

Knowledge of site directed processing enhances our understanding of the control of O-glycosylation in normal cells and in disease.     

MiR-124 Promote Neurogenic Transdifferentiation of Adipose Derived Mesenchymal Stromal Cells Partly through RhoA/ROCK1, but Not ROCK2 Signaling Pathway

Objective

Some recent studies suggest that multiple miRNAs might regulate neurogenic transdifferentiation of mesenchymal stromal cells (MSCs). In the present study, we hypothesized that the miR-124 can repress the expression of RhoA upon the neurogenesis of adipose derived MSCs (ADMSCs).

Methods

MiRNA expression dynamics during neurogenic transdifferentiation of ADMSCs were measured. The expression of neuron-specific enolase (NSE), Tuj-1 (Neuron-specific class III beta-tubulin) and glial fibrillary acidic protein (GFAP), as well as electrophysiological properties, were detected after neurogenic transdifferentiation. The targeting of miR-124 over RhoA was verified by dual luciferase assay, qRT-PCR and western blot. The functions of miR-124 and the RhoA/ROCK signaling pathway were studied using gain and loss of function experiments in vitro.

Results

MiR-124 is significantly upregulated during neurogenic transdifferentiation of ADMSCs. Knockdown of endogenous miR-124 hampered neurogenic transdifferentiation and the acquired electrophysiological properties. MiR-124 could directly target RHOA mRNA and repress its expression, through which it increased the proportion of transdifferentiated (transdiff.) cells with positive NSE, Tuj-1 and GFAP. RhoA/ROCK1, but not ROCK2 is a downstream signaling pathway of miR-124 in the process of transdifferentiation.

Conclusion

MiR-124 is an important miRNA modulating neurogenic transdifferentiation of ADMSCs at least partly via the miR-124/RhoA/ROCK1 signaling pathway. These findings provided some fundamental information for future use of ADMSCs as an agent for regenerative medicine and cell therapy for neurological diseases.     

The appearance and modulation of osteocyte marker expression during calcification of vascular smooth muscle cells

Background

Vascular calcification is an indicator of elevated cardiovascular risk. Vascular smooth muscle cells (VSMCs), the predominant cell type involved in medial vascular calcification, can undergo phenotypic transition to both osteoblastic and chondrocytic cells within a calcifying environment.

Methodology/Principal Findings

In the present study, using in vitro VSMC calcification studies in conjunction with ex vivo analyses of a mouse model of medial calcification, we show that vascular calcification is also associated with the expression of osteocyte phenotype markers. As controls, the terminal differentiation of murine calvarial osteoblasts into osteocytes was induced in vitro in the presence of calcifying medium (containing ß-glycerophosphate and ascorbic acid), as determined by increased expression of the osteocyte markers DMP-1, E11 and sclerostin. Culture of murine aortic VSMCs under identical conditions confirmed that the calcification of these cells can also be induced in similar calcifying medium. Calcified VSMCs had increased alkaline phosphatase activity and PiT-1 expression, which are recognized markers of vascular calcification. Expression of DMP-1, E11 and sclerostin was up-regulated during VSMC calcification in vitro. Increased protein expression of E11, an early osteocyte marker, and sclerostin, expressed by more mature osteocytes was also observed in the calcified media of Enpp1^−/− mouse aortic tissue.

Conclusions/Significance

This study has demonstrated the up-regulation of key osteocytic molecules during the vascular calcification process. A fuller understanding of the functional role of osteocyte formation and specifically sclerostin and E11 expression in the vascular calcification process may identify novel potential therapeutic strategies for clinical intervention.     

MicroRNA-125b regulates the expression of aggrecanase-1 (ADAMTS-4) in human osteoarthritic chondrocytes

Introduction

Increased expression of aggrecanase-1 (ADAMTS-4) has emerged as an important factor in osteoarthritis (OA) and other joint diseases. This study aimed to determine whether the expression of ADAMTS-4 in human chondrocytes is regulated by miRNA.

Methods

MiRNA targets were identified using bioinformatics. Chondrocytes were isolated from knee cartilage and treated with interleukin-1 beta (IL-1β). Gene expression was quantified using TaqMan assays and protein production was determined by immunoblotting. Luciferase reporter assay was used to verify interaction between miRNA and target messenger RNA (mRNA).

Results

In silico analysis predicted putative target sequence of miR-125b on ADAMTS-4. MiR-125b was expressed in both normal and OA chondrocytes, with significantly lower expression in OA chondrocytes than in normal chondrocytes. Furthermore, IL-1β-induced upregulation of ADAMTS-4 was suppressed by overexpression of miR-125b in human OA chondrocytes. In the luciferase reporter assay, mutation of the putative miR-125b binding site in the ADAMTS-4 3''UTR abrogated the suppressive effect of miR125.

Conclusions

Our results indicate that miR-125b plays an important role in regulating the expression of ADAMTS-4 in human chondrocytes and this identifies miR-125b as a novel therapeutic target in OA.