Expression Levels Of miR-125b Research Articles

Quantification of microRNA-181b in Egyptian Acute Myeloid Leukemia Patients and its Relation to Prognosis

Authors Iman M Omar, Dina A Fouad, Deena M M Habashy, Doaa G Eissa, Mariam F Abdel-Maksoud Clinical Pathology Department, Hematology Unit, Faculty of Medicine, Ain Shams University, Cairo-Egypt Correspondence Doaa Gamal Eissa MD, Clinical Pathology, Department, Hematology Unit, Faculty of Medicine, Ain Shams University, Cairo, Egypt Address: 20 Nagatysarag Street, Nasr city, Cairo Email: doaaeissa3@yahoo.com Tel: + 02 27772933 Abstract Unique micro RNA (miRNA) expression signatures are aberrantly expressed in acute myeloid leukemia (AML). Certain miRNA signatures have been associated with prognosis of AML, one of these is miR-181b. We aimed to quantify miR-181b in AML Egyptian adult patients and evaluate its prognostic significance in those patients. Eighty newly diagnosed AML patients with 40 age and sex matching healthy volunteers as a control group were studied for the expression of miR-181b by stem-loop quantitative reverse-transcription polymerase chain reaction (qRTPCR).miR-181b expression level was higher in patients’ group than controls (p=0.013). Patients with miR-181b  6.0 were associated with favorable molecular abnormalities and most of them achieved complete remission (CR), whereas most of patients with miR-181b< 6.0 were associated with unfavorable ones and failed to achieve CR. Patients with miR181b values ≥ 6 showed significantly higher mean survival than patients with values <6 (35±0.7, 14.6±2 month respectively, p<0.001).Low value of miRNA181b was identified as an independent predictor of bad prognosis. High expression level of miR-181b is detected in Egyptian AML adult patients identifying a distinct group with favorable prognosis

Overexpression of tissue microRNA10b may help predict glioma prognosis

We investigated the relationship between microRNA-10b (miR-10b) expression and prognosis in human glioma patients. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to characterize the expression patterns of miR-10b in 128 glioma and 20 normal brain tissues. Clinical information – age, sex, Karnofsky Performance Status (KPS) and World Health Organization (WHO) grade – were also collected. The associations between miR-10b expression and the clinicopathological factors and outcome of glioma patients were statistically analyzed. Expression levels of miR-10b in glioma tissue were significantly higher than in normal brain tissue (P<0.001). High-grade glioma (WHO grade III and IV) had much higher miR-10b expression levels than low-grade tumors (WHO grade I and II). Additionally, the increased miR-10b expression in the glioma tissues was significantly associated with a low KPS (P=0.03). Kaplan–Meier survival curves and Cox regression analyses showed that overexpression of miR-10b (P=0.01) and high grade (P=0.02) were independent factors predicting poor outcome for glioma patients. Furthermore, subgroup analyses showed that the miR-10b expression level was significantly associated with poor overall survival in glioma patients with high grades (P<0.001). Up-regulation of miR-10b may have value in predicting clinical outcome in glioma patients, particularly for those with high pathological grades.

Dynamic changes in circulating miRNA levels in response to antitumor therapy of lung cancer

ABSTRACTPurpose: Expression levels of cancer-associated microRNAs were reported to be altered in serum/plasma samples from lung cancer patients compared with healthy subjects. The purpose of this study was to estimate the value of five selected miRNAs plasma levels as markers of response to antitumor therapy in lung cancer patients. Materials and Methods: Expression levels of miR-19b, miR-126, miR-25, miR-205, and miR-125b have been evaluated by quantitative reverse transcription PCR versus control miR-16 in blood plasma samples from 23 lung cancer (LC) patients. Plasma samples were obtained from LC patients before treatment (untreated-UT), within 30 days after completing two courses of chemotherapy (postchemotherapy-PC) and 15 days after surgery (postoperative-PO). Results: Repeated Measures ANOVA demonstrated that miR-19b expression levels were decreased in PC and increased in PO samples. These changes were characterized by a significant quadratic trend (p = 0.03). Expression levels of miR-125b increased both after chemotherapy and again after surgery and demonstrated a significant linear trend (p = 0.03). The miR-125b/miR-19b ratio changed during the course of the antitumor treatment with a significant linear trend (p = 0.04). Individual analysis in the groups of patients with partial response to chemotherapy and patients with stable or progressive disease showed different trends for miR-19b, miR-125b, and miR-125b/miR-19b ratio between the groups. The Kaplan–Meier survival curves demonstrated an association of miR-125b/miR-19b ratio value with the survival time without the tumor relapse (p < 0.1). Conclusions: Dynamic change of trends for miR-19b and miR-125b expression levels and miR-125b/miR-19b ratio in the blood plasma have shown a potentiality to discriminate types of response to antitumor therapy in lung cancer patients. Further in-depth investigation is needed to establish a direct link the miRNAs expression levels in blood plasma with therapy response and patient's survival.

MicroRNA-26b suppresses the metastasis of non-small cell lung cancer by targeting MIEN1 via NF-κB/MMP-9/VEGF pathways

MicroRNAs (miRNAs) are involved in tumor initiation and progression. MiR-26b was reported to be significantly downregulated in non-small cell lung cancer (NSCLC). However, the underlying mechanisms of miR-26b involvement in the development and progression of NSCLC remains poorly understood. In the present study, we report that miR-26b suppresses cell metastasis in NSCLC through targeting migration and invasion enhancer 1 (MIEN1). We found that miR-26b was significantly downregulated and MIEN1 was significantly upregulated in both NSCLC tissues and cells lines. The expression levels of miR-26b were negatively related to those of MIEN1 mRNA in clinical NSCLC tissues. Furthermore, MIEN1 was confirmed to be a direct target of miR-26b by dual-luciferase reporter assay and MIEN1 expression was downregulated by miR-26b in NSCLC cells. In terms of function, transwell and wound healing assays demonstrated that the miR-26b remarkably inhibited invasion and migration of NSCLC cells, which was simulated by siRNA knockdown of MIEN1 and reversed by pcDNA/MIEN1 overexpression of MIEN1. Finally, we found that miR-26b could regulate NF-κB/MMP-9/VEGF pathway in NSCLC cells. In conclusion, this study revealed that miR-26b suppresses NSCLC metastasis by targeting MIEN1 via NF-κB/MMP-9/VEGF pathways, implicating a potential prognostic biomarker and therapeutic target for NSCLC treatment.

Expression and Function Analysis of MicroRNA-29b in Xuanwei Lung Cancer.

In lung cancer, miR-29b can act as a tumor suppressor, influencing epigenetic regulation, cell proliferation, differentiation, apoptosis, metastasis, and chemosensitivity. However, its expression and biological function in Xuanwei lung cancer (XWLC) remains unknown. Fifty pairs of XWLC and matched normal tissues were collected and analyzed for the expression of miR29b by quantitative RT-PCR. MiR-29b precursor and inhibitor were transfected into XWLC-05 cells to investigate its role on regulating cell proliferation by MTT assay and cell apoptosis by caspase 3/7/9 assay in vitro. The target genes of miR-29b were predicted and determined by bioinformatics analysis and luciferase reporter assay. qRT-PCR results showed that the expression level of miR-29b in XWLC tissues was significantly lower than that in the matched normal tissues, with a proportion of 86%. The low expression of miR-29b was significantly associated with lymphatic metastasis. MiR-29b could suppress cell proliferation and promote apoptosis in XWLC-05 cells. According to the bioinformatics analysis and luciferase reporter assay results, PUMA was a target gene of miR-29b in XWLC. Our studies provided evidence for the involvement of miR-29b in XWLC lymphatic metastasis and suggested that miR-29b may serve as a new biomarker for diagnosis of XWLC or therapeutic targets.

MicroRNA-26b Enhances the Radiosensitivity of Hepatocellular Carcinoma Cells by Targeting EphA2.

Sensitizing hepatocellular carcinoma (HCC) cells to irradiation is important to achieve satisfactory therapeutic effect with low-dose radiotherapy. Erythropoietin-producing hepatocellular carcinoma A2 (EphA2) is a member of the Eph receptor family that constitutes the largest family of tyrosine kinase receptors. EphA2 overexpression is one of the poor prognostic factors in many progressive cancers. Importantly, EphA2 is a potential target of microRNA-26b (miR-26b), and miR-26b expression is down-regulated in several types of cancer. In this study, we measured the expression levels of miR-26b and EphA2 protein in seven human HCC cell lines by quantitative PCR and western blot analysis, respectively. Overall, lower miR-26b expression levels tended to be associated with higher EphA2 levels in HCC cell lines. Among the cell lines examined, 97H HCC cells expressed the lowest level of miR-26b and highest level of EphA2 protein. Thus, using 97H HCC cells, EphA2 mRNA was verified as the target of miR-26b by the luciferase reporter assay. Accordingly, a synthetic miR-26b, miR-26b mimics, was used to mimic the function of endogenous miR-26b. In 97H HCC cells transfected with miR-26b mimics or short-hairpin RNA targeting EphA2 mRNA, expression of EphA2 protein was reduced, which was associated with significantly lower proliferation rate and invasion ability and with higher apoptosis rate in response to low-dose irradiation, compared to control cells. In contrast, 97H HCC cells over-expressing EphA2 showed higher proliferation rate and invasion ability and lower apoptosis rate upon irradiation. These data suggest that miR-26b enhances the radiosensitivity of 97H HCC cells by targeting EphA2 protein.

Down-Regulation of Mir-26b and Its Inhibition on the Growth of T Cell Acute Lymphoblastic Leukemia

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy accounting for 15% of pediatric and 25% of adult acute lymphoblastic leukemia (ALL) cases. With current chemotherapies and transplantation therapy, there are still 25-50% T-ALL patients that suffer from relapse and have a poor outcome. MicroRNAs (miRNAs or miRs) are endogenous small non-coding RNAs (containing about 22 nucleotides in length). miRs function at posttranscriptional level as negative regulators of gene expression and exert their regulatory function through binding to target mRNAs and silencing gene expression. To better understand the pathogenesis and develop the new therapeutic targets of T-ALL, we have developed a Pten tumor suppressor knockout T-ALL mouse model and profiled miRs from the mouse Pten deficient T-ALL. miR-26b was one of the miRs that were found down-regulated in the mouse Pten deficient T-ALL. Recent studies showed that the aberrant expression of miR-26b is implicated in several types of cancer. The expression level of miR-26b and its role of in T-ALL, however, are unknown. We investigated if the expression level of miR-26b is aberrant in T-ALL and the effect of potentially altered expression on the growth of human T-ALL cells.Methods: We conducted miR array profiling to identify differentially expressed miRs in the mouse Pten deficient T-ALLs compared with preneoplastic thymocyte controls. We validated expression levels of several miRs, including miR-26b, that are differentially expressed in mouse and human T-ALL cells using quantitative RT-PCR. We also overexpressed miR-26b using a lentivirus based vector in human T-ALL cell lines to assess its effect on cell growth and apoptosis.Results: Employing miR array profiling, we identified a subset of miRs that exhibited marked altered expression in the mouse Pten deficient T-ALL cells. Quantitative RT-PCR validated that the expression level of miR-26b in the mouse Pten deficient T-ALL cells was markedly lower in comparison to that of preneoplastic thymocytes. To determine if miR-26b expression level is also altered in human T-ALL, we performed quantitative RT-PCR on a panel of human T-ALL cell lines. Indeed, the expression level of miR-26b is significantly lower in the human T-ALL cell lines when compared with that of normal thymocytes. To functionally assess if miR-26b plays a role in the cell growth of human T-ALL cells, we expressed exogenous miR-26b in a panel of human T-ALL cell lines. We demonstrated that the expression of exogenous miR-26b significantly reduced the proliferation and promoted apoptosis of several human T-ALL cell lines.Conclusions: Our results demonstrated that miR-26b is down-regulated in T-ALL and the expression of exogenous miR-26b elicits deceased cell proliferation and increased apoptosis of human T-ALL. These results suggest that miR-26b may function as a tumor suppressor in the development of T-ALL and further characterization of the target and regulation of miR-26b may have therapeutic implications. DisclosuresNo relevant conflicts of interest to declare.

MiR-181b regulates cisplatin chemosensitivity and metastasis by targeting TGFβR1/Smad signaling pathway in NSCLC.

MicroRNAs (miRNAs) have been identified as important post-transcriptional regulators involved in various biological and pathological processes of cells, but their underlying mechanisms in chemosensitivity and metastasis have not been fully elucidated. The objective of this study was to identify miR-181b and its mechanism in the chemosensitivity and metastasis of NSCLC. We found that miR-181b expression levels were lower in A549/DDP cells compared with A549 cells. Functional assays showed that the overexpression of miR-181b inhibited proliferation, enhanced chemosensitivity to DDP, attenuated migration and metastatic ability in NSCLC cell lines in vitro and in vivo. TGFβR1 was subsequently identified as a novel functional target of miR-181b. TGFβR1 knockdown revealed similar effects as that of ectopic miR-181b expression, whereas overexpression of TGFβR1 rescued the function of miR-181b-mediated growth, chemosensitivity and metastasis in NSCLC cells. In addition, miR-181b could inactivate the TGFβR1/Smad signaling pathway. We also observed that decreased miR-181b expression and increased TGFβR1 expression were significantly associated with chemosensitivity to DDP and tumor metastasis in NSCLC patients. Consequently, miR-181b functions as a tumor suppressor and has an important role in proliferation, chemosensitivity to DDP and metastasis of NSCLC by targeting TGFβR1/Smad signaling pathway.

P90: Dynamic changes of circulating microRNA expression in response to the lung cancer combined therapy

Background The analysis of circulating tumor nucleic acids (DNAs and RNAs) in the blood seems to be a promising approach for the development of the low-invasive methods of tumor detection, valuable for clinical practice. Detection of oncogenic and tumor suppressor miRNAs in the blood plasma/serum is evidence of their participation in pathogenesis and suggest the possibility of their use as tumor markers and targets for therapy. Thus, the determination of expression level of tumor-associated miRNAs in plasma can lead to significant progress in understanding the tumor development and treatment. Aim Estimation the changes in expression level of miRNAs (miR-19b, miR-25, miR-125b, miR-126, miR-205) in blood plasma from lung cancer patients during combined therapy and to estimate their value as disease monitoring markers. Materials and methods Blood samples were taken from patients (n = 23) with non-small cell lung cancer treated at the Tomsk Cancer Research Institute. These samples were stabilized and fractionated into plasma and blood cells. MicroRNA was isolated from blood plasma using single-phase phenol-free extraction protocol and purified on silica-based spin columns (BioSilica Ltd, Novosibirsk, Russia). Concentration of five above mentioned miRNAs was measured by quantitative RT-PCR and normalized to miR-16 using dCt method. Results In this study we analyzed the dynamic expression changes of circulating DNA in blood plasma from lung cancer patients during the combined therapy. Circulating miRNAs were isolated from plasma samples of non-small cell lung cancer patients before treatment, within 30 days after completing chemotherapy and 15 days after surgery, by using developed methodological approach. In case of miR-19b and miR-125b analysis was found that the miRNA expression level correlates with clinical response to chemotherapy and surgery. Increasing level of miR-19b and decreasing level of miR-125b were associated with therapeutic response. Using Repeated measures ANOVA analysis we demonstrated that the miR-19b and miR-125b expression levels changes throughout three check-up points during the combined treatment are characterized by the significant cubic trend (P = 0.00284 and P = 0.029, respectively). Changes of miRNA-126 expression level during post-treatment follow-up were not characterized by the definite trend and not correlated with changes of other miRNAs expression level. It was shown the significant correlation between miRNA-25 and miRNA-205 expression levels, but was not found the trend of these miRNAs level changes. Conclusion The clinical utility of the circulating miR-19b and miR-125b expression analysis from this study remains to be validated in large cohorts of patients with different histological types of tumors, at the different stage of disease and outcomes. One of the criteria for inclusion in the group must be susceptibility and/or resistance to therapy. The research has been carried out with support of the grant from the Russian Science Support Foundation 14-04-01881 , Post-doctorate program in TPU.

Early intervention with Interleukin-1 Receptor Antagonist Protein modulates catabolic microRNA and mRNA expression in cartilage after impact injury

The purpose of this controlled laboratory study was to determine the efficacy of Interleukin-1 Receptor Antagonist Protein (IRAP) treatment as an early intervention strategy by examining the changes in microRNA and mRNA expression in cartilage in an ex-vivo porcine knee joint impact model. Custom impact device was used to create replicable injury ex-vivo to intact porcine knee joint. Injury was caused by dropping a 10kg weight one time from 1m directly above the knee in extension. One hour after impact 20μg/ml IRAP solution was intra-articularly injected. At 8h post-injury, cartilage samples were harvested for cell viability and genetic expression analysis. Genetic expression of miR-27b, miR-140, miR-125b, ADAMTS-4, ADAMTS-5, MMP-3, IL-1β, and TNF-α were analyzed by RT-PCR. Cell viability image analysis was performed using ImageJ software. Groups were compared by analysis of variance (ANOVA) followed by Tukey's post-hoc test. A P-value <0.05 was considered significant. At 8h after IRAP treatment, expressions of ADAMTS-4, ADAMTS-5, MMP-3, IL-1β, and TNF-α in cartilage were significantly down-regulated from injury group (all P<0.001). MiR-140, miR-125b, and miR-27b expressions were significantly up-regulated after treatment as compared to control and injury groups (all P<0.001). This study demonstrates that IRAP treatment administered during acute phase of cartilage impact injury increases expression levels of miR-140, miR-125b, and miR-27b in cartilage, indicating increased inhibition of their respective matrix-degrading enzymes. Clinically, these findings support the potential of IRAP treatment as an early intervention strategy for the prevention of cartilage degeneration after impact injury.

Tissue expression levels of miR-29b and miR-422a in children, adolescents, and young adults' age groups and their association with prediction of poor prognosis in human osteosarcoma.

Osteosarcoma is the most common type of bone cancer in children and adolescents. MicroRNAs (miRNAs) play important roles in the development, differentiation, and function of different cell types and in the pathogenesis of various human diseases. miRNAs are differentially expressed in normal and cancer cells. The investigation of miRNA expression between healthy subjects and patients with osteosarcoma is crucial for future clinical trials. In this study, the expression levels of miRNAs were detected by qRT-PCR. Correlation between expression levels of tow miRNAs and different clinicopathological characteristics were analyzed using the χ (2) test. Survival rate was detected using the log-rank test and Kaplan-Meier method. qRT-PCR was shown that expression levels of miR-29b and miR-422a were strongly decreased in osteosarcoma bone tissue compared with noncancerous bone tissues. Our result indicated that the low expression levels of miR-29b and miR-422a showed strong correlation with large tumor size (P = 0.20; 0.029), advanced TNM stage (P = 0.001; 0.012), distant metastasis (P = 0.008; 0.019), and grade of tumor (P = 0.009; 0.016). Kaplan-Meier survival analysis showed that the low expressions of miR-29b/miR-422a were correlated with shorter time overall survival (log-rank test, P = 0.009; P = 0.013). Moreover, multivariate Cox proportional hazards model indicated that miR-29b and miR-422a (P = 0.024; P = 0.016) were independent prognostic markers of overall survival of patients. Our result indicated that downregulation of miR-29b and miR-422a may be linked to the prediction of poor prognosis, indicating that miR-29b and miR-422a may be a valuable prognostic marker for osteosarcoma patients.

Respiratory syncytial virus infection inhibits TLR4 signaling via up-regulation of miR-26b.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illnesses in infants worldwide. TLR4 signal pathway plays a critical role in regulating immune response against RSV infection. However, the activation of TLR4 in RSV infection is still unclear. In present study, the expression levels of miR-26b and TLR4 mRNA were detected in peripheral blood mononuclear cells (PBMCs) of children with or without RSV infected bronchiolitis. The expression levels of TLR4 and its downstream genes IFNβ and CCL5 were also quantified in PBMCs infected with RSVΔG or RSV A2 in vitro. The results showed that children with RSV infection had higher miR-26b level and lower TLR4 mRNA level in PBMCs. miR-26b was predicted to target TLR4. In vitro, miR-26b mimic markedly down-regulated TLR4 mRNA/protein expression and IFNβ/CCL5 concentrations while miR-26b inhibitor up-regulated these levels. This study reveals that RSV infection inhibits TLR4 signaling via up-regulation of miR-26b, which provides a potential therapeutic target for preventing and treating RSV infection.

Investigation of the Application of miR10b and miR135b in the Identification of Semen Stains.

To evaluate the identification method using the microRNA markers miR10b and miR135b to distinguish semen stains from menstrual blood, peripheral blood, vaginal fluid and so on body fluid stains. The expression levels of miR10b and miR35b in semen stains and menstrual blood and so on were detected utilizing a real-time quantitative PCR technique with a specific fluorescence-labeled TaqMan probe. RNU6b was used as the internal reference gene; the difference in their expression was analyzed, and the specificity, sensitivity, and detection capability of the techniques were evaluated. The expression of miR10b and miR135b in semen stains was significantly higher than that of other body fluid stains, with a mean value of ΔCт from-6 to-7. However, it ranged from-2 to-4 for other body fluid stains. The initial criteria for judging which semen stains can be identified were determined by analyzing the research results. When the threshold value was set to 0.04, the CT value could be detected in the target genes miR10b, miR135b and in the internal reference gene RNU6b, and CT values are<40, ΔCT[10b-U6]<-5.5, and ΔCT[135b-U6]<-6, respectively, and the semen stain could be identified. The expression levels of miR10b and miR135b are higher in semen with strong tissue specificity; thus, they can be used to differentiate semen stains from other body fluid stains in forensic science.

MicroRNA-133b inhibits cell migration and invasion by targeting matrix metalloproteinase 14 in glioblastoma.

Increasing evidence has suggested that microRNA-133b (miR-133b) is important in regulating the genesis of different types of cancer. However, the effects and the underlying mechanisms of miR-133b in the development of glioblastoma (GBM) remain largely unknown. The aim of the present study was to investigate the role of miR-133b in GBM and to determine the molecular mechanisms underlying its action. Reverse transcription-quantitative polymerase chain reaction was used to measure the expression levels of miR-133b in 21 human GBM samples and 9 normal brain tissue samples. A wound healing assay, and Transwell migration and invasion assays were used to evaluate the effects of miR-133b on cell migration and invasion. Western blotting and a luciferase reporter assay were used to identify the target genes of miR-133b. It was found that miR-133b suppressed GBM cell migration and invasion, and matrix metalloproteinase 14 (MMP14) was identified as a direct target gene. In conclusion, miR-133b may suppress GBM migration and invasion through directly targeting MMP14, highlighting its potential as a novel agent for the treatment of GBM invasion.

RETRACTED ARTICLE: Upregulation of miR-300 and downregulation of miR-125b act as potential predictor biomarkers in progression, metastasis, and poor prognosis of osteosarcoma.

Abstract MiRNAs play crucial roles in development of cancer. However, the underlying mechanisms of miRNAs in osteosarcoma (OS) are poorly understood. This study is aimed to investigate the clinical importance of miR-125b and miR-300 expression in OS tissue samples using real-time PCR.Total RNA was purified from samples and noncancerous bone tissues and then real-time PCR was used to evaluate the expression rate of microRNAs. Furthermore, the association of miR-125b and MiR-300 level with pathological factors and prognosis were evaluated. Our results suggested that miR-125b expression was significantly downregulated in osteosarcoma bone tissue compared with noncancerous bone tissues. On the other hand, miR-300 expression was significantly upregulated in OS bone tissue than noncancerous bone tissues.Our findings suggested that a lower expression level of miR-125b was linked to advanced clinical stage (P = 0.005), metastasis (P = 0.004), and a large size of tumor (P = 0.023). In addition, a higher expression level of miR-300 was related to advanced tumor, node, and metastasis (TNM) stage (P = 0.002), metastasis (P = 0.001), and a large size of tumor (P = 0.038). The lower expression and higher expression of mentioned microRNAs was significantly linked to shorter overall survival according to the Kaplan-Meier survival and log-rank analysis (log-rank test P = 0.026; P = 0.017).Moreover, lower expression of miR-125b (P = 0.001), a decreased expression level of miR-300 (P = 0.004), TNM stage (P = 0. 0.004; P = 0.003), metastasis (P = 0.027; P = 0.001), and a large tumor size (P = 0.006; P = 0.025) were independent prognostic markers of overall survival of patients according to the multivariate Cox proportional hazards model. In conclusion, our results indicated that downregulation of miR-125b and miR-300 was associated with progression of OS and both of them may act as tumor suppressor in OS.

Enhancing HOTAIR/MiR-10b Drives Normal Liver Stem Cells Toward a Tendency to Malignant Transformation Through Inducing Epithelial- to-Mesenchymal Transition.

Previously, other groups and our team consistently have demonstrated that the possible origination of liver cancer stem cells (LCSCs) is the malignant transformation from liver normal stem cells (LNSCs). However, this complex and multi-step process is far from clear due to the accumulation of various gene dysregulations. Because non-coding RNAs (ncRNAs) could regulate multiple genes, a family of genes, and even whole chromosomes, this study further investigated the effect of dysregulated short ncRNA microRNA-10b and long ncRNA HOX transcript antisense RNA (HOTAIR) between LNSCs and LCSCs on phenotype reversion. To clarify the role of ncRNA in malignant transformation of LNSCs, we used lentivirus transduction to enhance the miR-10b and HOTAIR expression levels in our previously isolated rat LNSCs. The malignant abilities of proliferation, invasiveness, and tumorigenesis were observed and compared in cells before and after ncRNAs enhancement. After microRNA-10b and HOTAIR were enhanced separately, several cancer stem cell (CSC)-like traits appeared in these LNSCs, including in vitro-enhanced proliferative capacity, expression of putative LCSC markers, progressive invasive ability, and even in vivo aggravation into and taking the place of normal liver tissue. Furthermore, strengthened expression of these ncRNAs partially degraded E-cadherin in LNSCs, which is one of the classic markers in epithelial-to-mesenchymal transition (EMT). HOTAIR or miR-10b enhanced in LNSCs may drive the LNSCs to a tendency toward malignant transformation. This study partially uncovers the mechanism by which miR-10b or HOTAIR promotes malignant transformation of LNSCs through down-regulating E-cadherin and inducing EMT.

Association between miR-181b and PKG 1 in myocardial hypertrophy and its clinical implications.

The aim of this study was to explore the microRNA (miR)-181b expression in myocardial hypertrophy and to investigate its association with cGMP-dependent protein kinase type I (PKG 1) in an in vitro model. The miR-181b level in the peripheral blood was determined in patients with myocardial hypertrophy, and an in vitro model was established via phenylephrine (PE) treatment. Reverse transcription-quantitative polymerase chain reaction analysis and western blotting were performed to detect the expression levels of miR-181b, PKG 1 and hypertrophy-related genes. The results revealed that the expression of miR-181b was elevated in the peripheral blood of patients with myocardial hypertrophy, and this may have contributed to the pathology and progression of the disease. When the primary myocardial cells were treated with PE, microscopic observation and flow cytometry revealed significant hypertrophy. Furthermore, upregulation of myocardial hypertrophy-related genes, including β-myosin heavy chain, α-sarcomeric actinin and atrial natriuretic peptide, was observed. The miR-181b expression level in the PE-treated cells was elevated, while the mRNA and protein expression levels of PKG 1 were decreased, indicating a negative correlation between miR-181b and PKG 1 in myocardial hypertrophy. In addition, when the PE-treated primary myocardial cells were transfected with miR-181b inhibitor, the reduced PKG 1 expression was restored and the myocardial hypertrophy alleviated, as indicated by the reduced cellular sizes and decreased expression levels of the myocardial hypertrophy-related genes. In conclusion, miR-181b expression has been shown to be upregulated in myocardial hypertrophy, and this may play a role in the pathogenesis of the disease by regulating the expression of PKG 1. The present findings suggest that miR-181b is a promising molecular indicator for the clinical diagnosis and treatment of cardiac hypertrophy.

MiR-125b inhibits stromal cell proliferation in giant cell tumor of bone by targeting parathyroid hormone 1 receptor.

miR-125b has been identified as a tumor suppressor in many tumors, but its role in giant cell tumor (GCT) of bone remains poorly understood. The current study aimed to investigate the potential role and mechanism of miR-125b in GCT. Expression levels of miR-125b in GCT tissues were determined using RT-PCR. The cell proliferation was surveyed by direct cell counting, MTS and CCK-8, and the apoptotic cells were evaluated by Annexin V-FITC and propidium iodine staining assay. The target gene expression was determined using RT-PCR and western blot. Parathyroid hormone 1 receptor (PTH1R) 3'-UTR was cloned into luciferase reporter plasmid to confirm direct targeting. We found that miR-125b was significantly down-regulated in GCT tissues. Using both gain- and loss-of-function analyses, we further revealed that miR-125b suppressed GCT stromal cell proliferation and induced cell apoptosis. Furthermore, we revealed that PTH/PTHrP type 1 receptor is a direct and functional target of miR-125b. Our results suggest that miR-125b acts as a tumor suppressor through suppression of the PTH1R/RANKL signaling pathway. These findings contribute to our understanding of the functions of miR-125b in GCT.

MiR-9 and miR-21 as prognostic biomarkers for recurrence in papillary thyroid cancer.

Despite low mortality rates, nodal recurrence in papillary thyroid carcinoma occurs in up to 20 % of patients. Emerging evidences indicate that dysregulated microRNAs are implicated in the process of metastasis. In the present study, we investigated whether miR-9, miR-10b, miR-21 and miR-146b levels are predictive of papillary thyroid carcinoma recurrence. Using macro-dissection followed by quantitative real-time PCR, we measured miR-9, miR-10b, miR-21 and miR-146b expression levels in formalin-fixed, paraffin-embedded samples of 66 patients with papillary thyroid carcinoma categorized into two groups: the recurrent group (n = 19) and the non-recurrent group (n = 47). All patients underwent total thyroidectomy and were followed for at least 120 months after surgery to be considered recurrence-free. Univariate and multivariate analysis were performed using the Cox proportional hazard model in order to identify associations between multiple clinical variables and microRNA expression levels and papillary thyroid carcinoma recurrence. MiR-9 and miR-21 expression levels were found to be significant prognostic factors for recurrence in patients with papillary thyroid carcinoma (HR = 1.48; 95 % CI 1.24-1.77, p < 0.001; and HR = 1.52; 95 % CI 1.18-1.94, p = 0.001; respectively). Multivariate analysis involving the expression level of miR-9 and miR-21 and various clinical parameters identified the expression of these microRNAs as independent prognostic factors for papillary thyroid cancer patients. In conclusion, our results support the potential clinical value of miR-9 and miR-21 as prognostic biomarkers for recurrence in papillary thyroid carcinoma.

MiR-26b modulates insulin sensitivity in adipocytes by interrupting the PTEN/PI3K/AKT pathway.

MicroRNAs (miRNAs) have emerged as epigenetic regulators of metabolism and energy homeostasis. There is a growing body of evidence pointing to miRNAs that have important regulatory roles in insulin sensitivity. The aim of this work was to explore the expression and mechanism of action of miR-26b in obesity-related insulin resistance (IR) in adipocytes. Quantitative real-time PCR was performed to determine miR-26b expression in obese rodent models, human obesity subjects and insulin-resistant adipocytes. We analysed the roles of miR-26b overexpression and inhibition on glucose uptake in adipocytes. Western blotting was used to detect the levels of protein molecules involved in the phosphoinositide-3-kinase (PI3K) pathway. Bioinformatics and the Dual Luciferase Assay were used to identify the target gene of miR-26b. We assessed the regulatory roles of miR-26b on the phosphatase and tensin homologue (PTEN)/PI3K/AKT pathway and the relationship between miR-26b and the metabolism of human obese subjects. Levels of miR-26b are reduced in visceral adipose tissue (VAT) in obese rodent models, human obesity and insulin-resistant adipocytes. MiR-26b promotes insulin-stimulated glucose uptake and increases insulin-stimulated glucose transporter type 4 translocation to the plasma membrane in human mature adipocytes. MiR-26b modulates insulin-stimulated AKT activation via inhibition of its target gene, PTEN, and significantly increases insulin sensitivity via the PTEN/PI3K/AKT pathway. The expression level of miR-26b negatively correlates with increasing body mass index and homeostasis model assessment for IR in human obese subjects. Decreased miR-26b expression in VAT may be involved in obesity-related IR by interrupting the PTEN/PI3K/AKT pathway.