Elevated Muscle-Specific miRNAs in Serum of Myotonic Dystrophy Patients Relate to Muscle Disease Progress

The discovery of reliable and sensitive blood biomarkers is useful for the diagnosis, monitoring and potential future therapy of diseases. Recently, microRNAs (miRNAs) have been identified in blood circulation and might have the potential to be used as biomarkers for several diseases and clinical conditions. Myotonic Dystrophy type 1 (DM1) is the most common form of adult-onset muscular dystrophy primarily characterized by muscle myotonia, weakness and atrophy. Previous studies have shown an association between miRNAs and DM1 in muscle tissue and, recently, in plasma. The aim of this study was to detect and assess muscle-specific miRNAs as potential biomarkers of DM1 muscle wasting, an important parameter in the disease’s natural history. Disease stable or progressive DM1 patients with muscle weakness and wasting were recruited and enrolled in the study. RNA isolated from participants’ serum was used to assess miRNA levels. Results suggest that the levels of muscle-specific miRNAs are correlated with the progression of muscle wasting and weakness observed in the DM1 patients. Specifically, miR-1, miR-133a, miR133b and miR-206 serum levels were found elevated in DM1 patients with progressive muscle wasting compared to disease stable DM1 patients. Based on these results, we propose that muscle-specific miRNAs might be useful molecular biomarkers for monitoring the progress of muscle atrophy in DM1 patients.     

Expression and regulation of miR-1, -133a, -206a, and MRFs by thyroid hormone during larval development in Paralichthys olivaceus

MiR-1, miR-133a, and miR-206a have been identified as muscle-specific miRNAs. They play multiple crucial roles in the regulation of muscle development. Here, we show that these miRNAs were differentially expressed during the larval development of flounder, and specifically expressed in skeletal muscle and heart in adult tissues/organs. The expression levels of these miRNAs were significantly changed by thyroid hormone (TH) or thiourea (TU) treatment during metamorphosis from 17 dph (days post hatching) to 42 dph. In addition, the expression levels of MyoD and Myf5 mRNAs markedly increased at 14 dph (pre-metamorphosis) compared to metamorphic stages, and their expression levels are far above the myogenin during larval development. Moreover, these MRFs (myogenic regulatory factors) expression were directly or indirectly regulated by thyroid hormone or thiourea during metamorphosis. All the results suggest that miRNAs and MRFs might be involved in signaling pathway of TH or TU-mediated flounder metamorphosis.     

Circulating Exosomal microRNAs as Biomarkers of Colon Cancer

Purpose

Exosomal microRNAs (miRNAs) have been attracting major interest as potential diagnostic biomarkers of cancer. The aim of this study was to characterize the miRNA profiles of serum exosomes and to identify those that are altered in colorectal cancer (CRC). To evaluate their use as diagnostic biomarkers, the relationship between specific exosomal miRNA levels and pathological changes of patients, including disease stage and tumor resection, was examined.

Experimental Design

Microarray analyses of miRNAs in exosome-enriched fractions of serum samples from 88 primary CRC patients and 11 healthy controls were performed. The expression levels of miRNAs in the culture medium of five colon cancer cell lines were also compared with those in the culture medium of a normal colon-derived cell line. The expression profiles of miRNAs that were differentially expressed between CRC and control sample sets were verified using 29 paired samples from post-tumor resection patients. The sensitivities of selected miRNAs as biomarkers of CRC were evaluated and compared with those of known tumor markers (CA19-9 and CEA) using a receiver operating characteristic analysis. The expression levels of selected miRNAs were also validated by quantitative real-time RT-PCR analyses of an independent set of 13 CRC patients.

Results

The serum exosomal levels of seven miRNAs (let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a) were significantly higher in primary CRC patients, even those with early stage disease, than in healthy controls, and were significantly down-regulated after surgical resection of tumors. These miRNAs were also secreted at significantly higher levels by colon cancer cell lines than by a normal colon-derived cell line. The high sensitivities of the seven selected exosomal miRNAs were confirmed by a receiver operating characteristic analysis.

Conclusion

Exosomal miRNA signatures appear to mirror pathological changes of CRC patients and several miRNAs are promising biomarkers for non-invasive diagnosis of the disease.     

Serum MicroRNAs Related with Chemoradiotherapy Resistance in Advanced-Stage Cervical Squamous Cell Carcinoma

OBJECTIVE: To investigate the serum microRNAs as biomarkers in predicting chemoradiotherapy resistance in advanced-stage cervical squamous cell carcinoma (ACSCC) patients. METHODS: Serum samples were collected from International Federation of Gynecology and Obstetrics (FIGO) stage IIB to IIIB cervical squamous cell carcinoma patients treated with platinum based Concomitant Chemoradiotherapy (CCRT) in our hospital during September 2013 to November 2015. Twenty well-matched samples (10 resistant and 10 sensitive) were chosen to screen the miRNA expression profile using serum samples pooled with microarrays. miRNAs expressed significantly different between two groups were further verified in 131 patients (29 resistant and 102 sensitive) serum samples with TaqMan Real-time PCR. The AUC was used to evaluate the accuracy of the biomarkers for prediction. RESULTS: MiR-136-5, miR-152-3p and miR-206 were expressed significantly different between sensitive and resistant groups. Results of 131 patients verification showed that the levels of miR-206 in sensitive samples and resistant samples were 2.715 ± 0.2115 and 14.64 ± 1.184, respectively, which was significantly different (P < .0001), while miR-136-5p and miR-152-3p could not be tested without pre-amplification reactions. Univariate analysis revealed that miR-206 expression was significantly associated with patients' DFS. Multivariate analysis demonstrated that miR-206 expression, tumor differentiation and pelvic lymph nodes metastasis were the independent prognostic factors associated with DFS in this cohort (P = .008, 0.000, 0.000, respectively). The probability of the prognostic accuracy of miR-206 expression in predicting chemoradiotherapy sensitivity of ACSCC patients was 91.3% (79.3% sensitivity and 92.2% specificity). CONCLUSION: Serum miR-206 is a powerful tool in predicting chemoradiotherapy sensitivity in ACSCC patients.     

MicroRNAs: new players in heart failure

MicroRNAs (miRNAs) are a class of non-coding small RNAs representing one of the most exciting areas of modern medical science. miRNAs modulate a large and complex regulatory network of gene expression of the majority of the protein-coding genes. Currently, evidences suggest that miRNAs play a crucial role in the pathogenesis of heart failure. Some miRNAs as miR-1, miR-133 and miR-208a are highly expressed in the heart and strongly associated with the development of cardiac hypertrophy. Recent data indicate that these miRNAs as well as miR-206 change their expression quickly in response to physical activity. The differential regulation of miRNAs in response to exercise suggests a potential value of circulating miRNAs (c-miRNAs) as biomarkers of physiological mediators of the cardiovascular adaptation induced by exercise. Likewise, serum levels of c-miRNAs such as miR-423-5p have been evaluated as potential biomarkers in the diagnosis and prognosis of heart failure. On the other hand, the manipulation of miRNAs levels using techniques such as ‘miR mimics’ and ‘antagomiRs’ is becoming evident the enormous potential of miRNAs as promising therapeutic strategies in heart failure.     

Serum exosomal miR-210 as a potential biomarker for clear cell renal cell carcinoma

Exosomal microRNAs (miRNAs) are suggested to reflect molecular changes occurring in their cells of origin and are potential indicators in the early detection of cancers. This study aimed to determine whether certain exosomal miRNAs from tumor tissue can be used as noninvasive biomarkers for clear cell renal cell carcinoma (ccRCC). Based on ccRCC miRNA expression profiles and the literature, we selected six miRNAs (miR-210, miR-224, miR-452, miR-155, miR-21, and miR-34a) and analyzed their expression in tissues, sera, and serum exosomes through quantitative real-time polymerase chain reaction in hypoxia-induced (with CoCl₂) renal cell lines. miR-210, miR-224, miR-452, miR-155, and miR-21 were upregulated in tumor tissues compared with normal tissues. Serum miR-210 and miR-155 levels were higher in patients with ccRCC than in healthy controls (HCs). Furthermore, only exosomal miR-210 was significantly upregulated in patients with ccRCC than in HCs. Moreover, receiver operating characteristic (ROC) curve analysis revealed an area under the ROC curve of 0.8779 (95% confidence interval, 0.7987-0.9571) and a sensitivity and specificity of 82.5% and 80.0%, respectively. Moreover, exosomal miR-210 was upregulated at an advanced stage, and Fuhrman grade and metastasis decreased significantly one month after surgery. Acute hypoxia exposure activates miR-210 and release of exosomes with upregulated miR-210 in both normal and tumor RCC cell lines and interferes with vacuole membrane protein 1 mRNA expression, especially in the metastatic ccRCC cell line. In conclusion, Serum exosomal miR-210 originating from tumor tissue has potential as a novel noninvasive biomarker for the detection and prognosis of ccRCC.     

高通量miRNA活性谱检测发现BHK21细胞中miR-206高活性

用我们建立的活细胞miRNA活性谱检测技术测定BHK21、HEK293和Vero三种肾来源细胞系中58种miRNA活性谱,发现miR-206在BHK21细胞中具有特征性高活性.鉴于miR-206是典型的横纹肌特征性miRNA,进一步以小鼠成肌细胞C2Cl2及人胚肾细胞HEK293为对照,检测了BHK21细胞中miR-206的活性及表达水平.之后,用马血清诱导培养BHK21细胞,检测诱导前后BHK21细胞中骨骼肌肌球蛋白重链(Slow skeletal myosin heavy chain,MHC)表达情况,miR-206活性和表达水平以及受miR-206负调控的Connexin43(Cx43)的表达水平.结果发现,miR-206在BHK21细胞中活性和表达水平都明显高于C2C12细胞;马血清诱导后,BHK21细胞中MHC表达水平升高,miR-206活性和表达水平都升高,而Cx43表达水平下降.结果提示BHK21细胞具有成肌细胞特性.本研究首次发现BHK21细胞中miR-206的高活性,从miRNA角度证实了BHK21细胞来源于肾间质细胞,而不是肾实质细胞.研究结果还提示BHK21细胞有可能作为一种体外模型用于miR-206的功能研究.     

MiR-133b Is Down-Regulated in Human Osteosarcoma and Inhibits Osteosarcoma Cells Proliferation,Migration and Invasion,and Promotes Apoptosis

MicroRNAs (miRNAs) decrease the expression of specific target oncogenes or tumor suppressor genes and thereby play crucial roles in tumorigenesis and tumor growth. To date, the potential miRNAs regulating osteosarcoma growth and progression are not fully identified yet. In this study, the miRNA microarray assay and hierarchical clustering analysis were performed in human osteosarcoma samples. In comparison with normal human skeletal muscle, 43 miRNAs were significantly differentially expressed in human osteosarcomas (fold change ≥2 and p≤0.05). Among these miRNAs, miR-133a and miR-133b expression was decreased by 135 folds and 47 folds respectively and the decreased expression was confirmed in both frozen and paraffin-embedded osteosarcoma samples. The miR-133b precursor expression vector was then transfected into osteosarcoma cell lines U2-OS and MG-63, and the stable transfectants were selected by puromycin. We found that stable over-expression of miR-133b in osteosarcoma cell lines U2-OS and MG-63 inhibited cell proliferation, invasion and migration, and induced apoptosis. Further, over-expression of miR-133b decreased the expression of predicted target genes BCL2L2, MCL-1, IGF1R and MET, as well as the expression of phospho-Akt and FAK. This study provides a new insight into miRNAs dysregulation in osteosarcoma, and indicates that miR-133b may play as a tumor suppressor gene in osteosarcoma.     

小鼠外周血心肌特异性microRNAs 的变化与病毒性心肌炎 相关关系初探

目的:通过识别病毒性心肌炎小鼠和正常小鼠血浆中miR-1,miR-133,miR-206表达量的差异,分析外周血中心肌特异性microRNAs的变化与病毒性心肌炎相关关系,为探索miRNAs作为病毒性心肌炎诊断的生物标志物提供可行性的研究资料。方法:在小鼠病毒性心肌炎模型的基础上,采用荧光定量PCR方法,检测病毒性心肌炎急性期小鼠组和正常小鼠组血浆中相关miRNAs含量,并进行统计学分析。再于病毒注射后1d,3d,5d,7d,9d,11d,分别处死病毒小鼠,观察血浆miRNAs的动态变化规律。同时用Elisa检测心肌肌钙蛋白的变化,对目的 miRNAs与心肌肌钙蛋白进行相关性分析。结果:急性期时三种miRNAs血浆含量显著上调。病毒注射后3d开始上升明显,并持续保持在较高水平到7d,于9d时开始下降。发病期间,血浆miRNAs含量与心肌肌钙蛋白呈现良好的正相关性。结论:小鼠外周血中心肌特异性microRNAs在病毒性心肌炎发病过程中的呈现明显上调并与病程和相应指标存在相关关系,为miRNAs作为病毒性心肌炎诊断的生物标志物提供了重要线索。     

Dystromirs as Serum Biomarkers for Monitoring the Disease Severity in Duchenne Muscular Dystrophy

Duchenne muscular Dystrophy (DMD) is an inherited disease caused by mutations in the dystrophin gene that disrupt the open reading frame, while in frame mutations result in Becker muscular dystrophy (BMD). Ullrich congenital muscular dystrophy (UCMD) is due to mutations affecting collagen VI genes. Specific muscle miRNAs (dystromirs) are potential non-invasive biomarkers for monitoring the outcome of therapeutic interventions and disease progression. We quantified miR-1, miR-133a,b, miR-206 and miR-31 in serum from patients with DMD, BMD, UCMD and healthy controls. MiR-1, miR-133a,b and miR-206 were upregulated in DMD, but unchanged in UCMD compared to controls. Milder DMD patients had higher levels of dystromirs than more severely affected patients. Patients with low forced vital capacity (FVC) values, indicating respiratory muscle weakness, had low levels of serum miR-1 and miR-133b. There was no significant difference in the level of the dystromirs in BMD compared to controls.We also assessed the effect of dystrophin restoration on the expression of the five dystromirs in serum of DMD patients treated systemically for 12 weeks with antisense oligomer eteplirsen that induces skipping of exon 51 in the dystrophin gene. The dystromirs were also analysed in muscle biopsies of DMD patients included in a single dose intramuscular eteplirsen clinical trial. Our analysis detected a trend towards normalization of these miRNA between the pre- and post-treatment samples of the systemic trial, which however failed to reach statistical significance. This could possibly be due to the small number of patients and the short duration of these clinical trials.Although longer term studies are needed to clarify the relationship between dystrophin restoration following therapeutic intervention and the level of circulating miRNAs, our results indicate that miR-1 and miR-133 can be considered as exploratory biomarkers for monitoring the progression of muscle weakness and indirectly the remaining muscle mass in DMD.     

MicroRNAs Involved in Skeletal Muscle Differentiation

MicroRNAs (miRNAs) negatively regulate gene expression by promoting degradation of target mRNAs or inhibiting their translation. Previous studies have expanded our understanding that miRNAs play an important role in myogenesis and have a big impact on muscle mass, muscle fiber type and muscle-related diseases. The muscle-specific miRNAs, miR-206, miR-1 and miR-133, are among the most studied and best characterized miRNAs in skeletal muscle differentiation. They have a profound influence on multiple muscle differ-entiation processes, such as alternative splicing, DNA synthesis, and cell apoptosis. Many non-muscle-specific miRNAs are also required for the differentiation of muscle through interaction with myogenic factors. Studying the regulatory mechanisms of these miRNAs in muscle differentiation will extend our knowledge of miRNAs in muscle biology and will improve our understanding of the myogenesis regulation.     

The diagnostic efficacy of circulating miRNAs in monitoring the early development of colitis-induced colorectal cancer

Early detection of colorectal cancer and monitoring the progress in colon carcinogenesis stages is essential to reduce mortality. Therefore, there is continuous search for noninvasive biomarkers with high stability and good sensitivity and specificity. miRNAs have attracted attention as promising biomarkers as they are stably expressed in circulation. The aim of our study is to evaluate the aberrant expression of circulating miRNAs during the stepwise progress of colitis-associated colon cancer. This was accomplished through assessing the expression levels of five miRNAs (miR-141, miR-15b, miR-17-3p, miR-21, and miR-29a) in serum and their corresponding tissue samples through the different cycles of colorectal carcinogenesis cascade using the azoxymethane/dextran sulfate sodium murine model. We also compared the diagnostic performance of these selected miRNAs with the conventional tumor biomarkers CEA and CA 19-9. The results of our study revealed that the expression levels of those miRNAs were dynamically changing in accordance with the tumor development state. Moreover, their aberrant expression in serum was statistically correlated with that in tissue. Our data also revealed that serum miR-15b, miR-21, and miR-29a showed the best performance in terms of diagnostic power. Our findings highlight the efficiency of these circulating miRNAs not only for early diagnostics purposes, but also for monitoring progress in the colorectal carcinogenesis process, and therefore encouraging integrating these noninvasive biomarkers into the clinical diagnostic settings beside the traditional diagnostic markers for accurate screening of the early progress of colon carcinogenesis.