miR-15a-5p and miR-21-5p contribute to chemoresistance in cytogenetically normal acute myeloid leukaemia by targeting PDCD4, ARL2 and BTG2

Cytarabine and daunorubicin are old drugs commonly used in the treatment of acute myeloid leukaemia (AML). Refractory or relapsed disease because of chemotherapy resistance is a major issue. microRNAs (miRNAs) were incriminated in resistance. This study aimed to identify miRNAs involved in chemoresistance in AML patients and to define their target genes. We focused on cytogenetically normal AML patients with wild-type NPM1 without FLT3-ITD as the treatment of this subset of patients with intermediate-risk cytogenetics is not well established. We analysed baseline AML samples by small RNA sequencing and compared the profile of chemoresistant to chemosensitive AML patients. Among the miRNAs significantly overexpressed in chemoresistant patients, we revealed miR-15a-5p and miR-21-5p as miRNAs with a major role in chemoresistance in AML. We showed that miR-15a-5p and miR-21-5p overexpression decreased apoptosis induced by cytarabine and/or daunorubicin. PDCD4, ARL2 and BTG2 genes were found to be targeted by miR-15a-5p, as well as PDCD4 and BTG2 by miR-21-5p. Inhibition experiments of the three target genes reproduced the functional effect of both miRNAs on chemosensitivity. Our study demonstrates that miR-15a-5p and miR-21-5p are overexpressed in a subgroup of chemoresistant AML patients. Both miRNAs induce chemoresistance by targeting three pro-apoptotic genes PDCD4, ARL2 and BTG2.     

Evaluation of exosomal miR-9 and miR-155 targeting PTEN and DUSP14 in highly metastatic breast cancer and their effect on low metastatic cells

Breast cancer is one of the most prevalent cancers in women. Triple-negative breast cancer consists 15% to 20% of breast cancer cases and has a poor prognosis. Cancerous transformation has several causes one of which is dysregulation of microRNAs (miRNAs) expression. Exosomes can transfer miRNAs to neighboring and distant cells. Thus, exosomal miRNAs can transfer cancerous phenotype to distant cells. We used gene expression omnibus (GEO) datasets and miRNA target prediction tools to find overexpressed miRNA in breast cancer cells and their target genes, respectively. Exosomes were extracted from MDA-MB-231 and MCF-7 cells and characterized. Overexpression of the miRNAs of MDA-MB-231 cells and their exosomes were analyzed using quantitative Real-time PCR. The target genes expression was also evaluated in the cell lines. Luciferase assay was performed to confirm the miRNAs: mRNAs interactions. Finally, MCF-7 cells were treated with MDA-MB-231 cells’ exosomes. The target genes expression was evaluated in the recipient cells. GSE60714 results indicated that miR-9 and miR-155 were among the overexpressed miRNAs in highly metastatic triple negative breast cancer cells and their exosomes. Bioinformatic studies showed that these two miRNAs target PTEN and DUSP14 tumor suppressor genes. Quantitative Real-time PCR confirmed the overexpression of the miRNAs and downregulation of their targets. Luciferase assay confirmed that the miRNAs target PTEN and DUSP14. Treatment of MCF-7 cells with MDA-MB-231 cells’ exosomes resulted in target genes downregulation in MCF-7 cells. We found that miR-9 and miR-155 were enriched in metastatic breast cancer exosomes. Therefore, exosomal miRNAs can transfer from cancer cells to other cells and can suppress their target genes in the recipient cells.     

Liquiritigenin suppresses the activation of hepatic stellate cells via targeting miR-181b/PTEN axis

BackgroundLiquiritigenin (LQ), an aglycone of liquiritin in licorice, has demonstrated antioxidant, anti-inflammatory and anti-tumor activities. Previously, LQ was found to inhibit liver fibrosis progression.PurposePhosphatase and tensin homolog (PTEN) has been reported to act as a negative regulator of hepatic stellate cell (HSC) activation. However, the roles of PTEN in the effects of LQ on liver fibrosis have not been identified to date.MethodsThe effects of LQ on liver fibrosis in carbon tetrachloride (CCl₄) mice as well as primary HSCs were examined. Moreover, the roles of PTEN and microRNA-181b (miR-181b) in the effects of LQ on liver fibrosis were examined.ResultsLQ markedly ameliorated CCl₄-induced liver fibrosis, with a reduction in collagen deposition as well as α-SMA level. Moreover, LQ induced an increase in PTEN and effectively inhibited HSC activation including cell proliferation, α-SMA and collagen expression, which was similar with curcumin (a positive control). Notably, loss of PTEN blocked down the effects of LQ on HSC activation. PTEN was confirmed as a target of miR-181b and miR-181b-mediated PTEN was involved in the effects of LQ on liver fibrosis. LQ led to a significant reduction in miR-181b expression. LQ-inhibited HSC activation could be restored by over-expression of miR-181b. Further studies demonstrated that LQ down-regulated miR-181b level via Sp1. Collectively, we demonstrate that LQ inhibits liver fibrosis, at least in part, via regulation of miR-181b and PTEN.ConclusionLQ down-regulates miR-181b level, leading to the restoration of PTEN expression, which contributes to the suppression of HSC activation. LQ may be a potential candidate drug against liver fibrosis.     

Differentially expressed miR-20, miR-21, miR-100, miR-125a and miR-146a as a potential biomarker for prostate cancer

Prostate cancer is the leading cause of death among men worldwide. Deregulation of microRNAs has been reported in many cancers. Expression of microRNAs miR-20a-5p, miR-21-5p, miR-100-5p, miR-125a-5p and miR-146a-5p in tissue blocks of histologically confirmed prostate cancer patients compared with BPH patients, to identify potential microRNA biomarker for prostate cancer. MicroRNA was isolated and expression was quantified by qRT-PCR using Taqman Advanced microRNA assay kits. The interactions between the microRNA:target mRNA were predicted by using bioinformatics tools such as miRwalk and miRTargetlink. The experimentally validated targets were analysed using gprofiler to identify their molecular function, biological process and related pathways. The expression analysis revealed that miR-21 and miR-100 were significantly down-regulated whereas miR-125a was up-regulated in prostate cancer patients. Comparative analysis of the expression levels with tumor grading reveal that miR-100 was significantly down-regulated (p?<?0.05) in high grade tumor, indicating that miR-100 associated with prostate cancer. ROC analysis revealed that combined analysis of down-regulated miRNAs (miR-21 and miR-100) shown AUC of 0.72 (95% CI 0.65–0.79). The combined analysis of all five miRNAs showed AUC of 0.87 (95% CI 0.81–0.92). The targets prediction analysis revealed several validated targets including BCL2, ROCK1, EGFR, PTEN, MTOR, NAIF1 and VEGFA. Our results provide evidence that combined analysis of all the five miRNAs as a panel can significantly improve the prediction level of the presence of prostate cancer and may be used as a potential diagnostic biomarker.

     

MicroRNA-21 (miR-21) Regulates Cellular Proliferation,Invasion, Migration,and Apoptosis by Targeting PTEN,RECK and Bcl-2 in Lung Squamous Carcinoma,Gejiu City,China

Background

In South China (Gejiu City, Yunnan Province), lung cancer incidence and associated mortality rate is the most prevalent and observed forms of cancer. Lung cancer in this area is called Gejiu squamous cell lung carcinoma (GSQCLC). Research has demonstrated that overexpression of miR-21 occurs in many cancers. However, the unique relationship between miR-21 and its target genes in GSQCLC has never been investigated. The molecular mechanism involved in GSQCLC must be compared to other non-small cell lung cancers in order to establish a relation and identify potential therapeutic targets.

Methodology/Principal Findings

In the current study, we initially found overexpression of miR-21 occurring in non-small cell lung cancer (NSCLC) cell lines when compared to the immortalized lung epithelial cell line BEAS-2B. We also demonstrated that high expression of miR-21 could increase tumor cell proliferation, invasion, viability, and migration in GSQCLC cell line (YTMLC-90) and NSCLC cell line (NCI-H157). Additionally, our results revealed that miR-21 could suppress YTMLC-90 and NCI-H157 cell apoptosis through arresting cell-cycle at G₂/M phase. Furthermore, we demonstrated that PTEN, RECK and Bcl-2 are common target genes of miR-21 in NSCLC. Finally, our studies showed that down-regulation of miR-21 could lead to a significant increase in PTEN and RECK and decrease in Bcl-2 at the mRNA and protein level in YTMLC-90 and NCI-H157 cell lines. However, we have not observed any remarkable difference in the levels of miR-21 and its targets in YTMLC-90 cells when compared with NCI-H157 cells.

Conclusions/Significance

miR-21 simultaneously regulates multiple programs that enhance cell proliferation, apoptosis and tumor invasiveness by targeting PTEN, RECK and Bcl-2 in GSQCLC. Our results demonstrated that miR-21 may play a vital role in tumorigenesis and progression of lung squamous cell carcinoma and suppression of miR-21 may be a novel approach for the treatment of lung squamous cell carcinoma.     

miR-181b promotes hepatic stellate cells proliferation by targeting p27 and is elevated in the serum of cirrhosis patients

MicroRNAs, as a kind of negative gene regulators, were demonstrated to be involved in many types of diseases. In this study, we found that transforming growth factor-beta 1 could induce the expression of miR-181a and miR-181b, and miR-181b increased in the much higher folds than miR-181a. Because of the important role of transforming growth factor-beta 1 in HSC activation and liver cirrhosis, we investigate the effect of miR-181a and miR-181b on HSC proliferation. The results showed that miR-181b could promote HSC-T6 cell proliferation by regulating cell cycle. Further study showed p27, the cell cycle regulator, was the direct target of miR-181b in HSC-T6 cell. But miR-181a had no effects on HSC-T6 cell proliferation and cell cycle, and did not target p27. Interestingly, miR-181b is elevated significantly in serum of liver cirrhosis cases comparing to that of normal persons, whereas miR-181a expression was in the similar level with that of normal persons. These results suggested that miR-181b could be induced by TGF-β1 and promote the growth of HSCs by directly targeting p27. The elevation of miR-181b in serum suggested that it may be potential diagnostic biomarkers for cirrhosis. As for miR-181a, it may work in TGF-β1 pathway by a currently unknown mechanism.     

miR-22 enhances the radiosensitivity of small-cell lung cancer by targeting the WRNIP1

Small-cell lung cancer (SCLC) is an aggressive malignancy characterized by high cellular proliferation and early distant metastasis. Our study aimed to explore the effect of miR-22-3p (miR-22, for short) on SCLC radiosensitivity and its molecular mechanisms. The expression level of miR-22 was evaluated in a human normal lung epithelial cell line and a human SCLC cell line, and cell apoptosis and migration were detected. The expression of the miR-22 direct target WRNIP1 mRNA and protein were explored. Five differentially expressed genes were detected. The miR-22 expression in NCI-H446 was significantly decreased, and miR-22 overexpression significantly promoted cell apoptosis. miR-22 overexpression could significantly inhibit the cell migration of SCLC cells, and miR-22 had a negative regulatory effect on WRNIP1 mRNA and protein levels. KLK8 was downregulated, and the messenger RNA (mRNA) of four other genes (PC, SCUBE1, STC1, and GPM6A) was upregulated mRNA in cells overexpressing miR-22, which was in accordance with the bioinformatics analysis. miR-22 could enhance the radiosensitivity of SCLC by targeting WRNIP1.     

miR-181b functions as an oncomiR in colorectal cancer by targeting PDCD4

Programmed cell death 4 (PDCD4) is a RNA-binding protein that acts as a tumor suppressor in many cancer types, including colorectal cancer (CRC). During CRC carcinogenesis, PDCD4 protein levels remarkably decrease, but the underlying molecular mechanism for decreased PDCD4 expression is not fully understood. In this study, we performed bioinformatics analysis to identify miRNAs that potentially target PDCD4. We demonstrated miR-181b as a direct regulator of PDCD4. We further showed that activation of IL6/STAT3 signaling pathway increased miR-181b expression and consequently resulted in downregulation of PDCD4 in CRC cells. In addition, we investigated the biological effects of PDCD4 inhibition by miR-181b both in vitro and in vivo and found that miR-181b could promote cell proliferation and migration and suppress apoptosis in CRC cells and accelerate tumor growth in xenograft mice, potentially through targeting PDCD4. Taken together, this study highlights an oncomiR role for miR-181b in regulating PDCD4 in CRC and suggests that miR-181b may be a novel molecular therapeutic target for CRC.     

MiRNA-181b suppresses IGF-1R and functions as a tumor suppressor gene in gliomas

MicroRNAs (miRNAs) are single-stranded, 18- to 23-nt RNA molecules that function as regulators of gene expression. Previous studies have shown that microRNAs play important roles in human cancers, including gliomas. Here, we found that expression levels of miR-181b were decreased in gliomas, and we identified IGF-1R as a novel direct target of miR-181b. MiR-181b overexpression inhibited cell proliferation, migration, invasion, and tumorigenesis by targeting IGF-1R and its downstream signaling pathways, PI3K/AKT and MAPK/ERK1/2. Overexpression of IGF-1R rescued the inhibitory effects of miR-181b. In clinical specimens, IGF-1R was overexpressed, and its protein levels were inversely correlated with miR-181b expression. Taken together, our results indicate that miR-181b functions in gliomas to suppress growth by targeting the IGF-1R oncogene and that miR-181b may serve as a novel therapeutic target for gliomas.     

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.     

miR-181a/b-5p regulates human umbilical vein endothelial cell angiogenesis by targeting PDGFRA

Type 2 diabetes mellitus (T2DM) is a growing burden in low-and middle-income countries. Changing lifestyles and lack of physical activity are some of the reasons contributing to this epidemic increase. Co-morbidities associated with T2DM are largely due to the complications which arise as a consequence of endothelial dysfunction. Platelet derived growth factor-alpha (PDGFRA) is a protein responsible for cell proliferation, angiogenesis, migration and invasion. Increased levels of PDGFRA have been reported in T2DM. This study assessed the epigenetic regulation of PDGFRA through microRNAs (miR-181a/b-5p).Using a bioinformatics-based approach, we assessed the binding of miR-181a/b-5p to PDGFRA. Experimentally, this binding was confirmed using a dual luciferase reporter assay. Further, we overexpressed miR-181a/b-5p in Human umbilical vein endothelial cells (HUVECs) and the influence of over-expression on cell proliferation, migration and angiogenesis was assessed using in-vitro approaches. The influence of miR-181a/b-5p over expression on cellular apoptosis was ascertained using a TUNEL assay with concomitant changes being observed in the levels of Bcl-2 and cleaved Caspase-3.In HUVECs, PDGFRA is a direct target for miR-181a/b-5p. Over expression of miR-181a/b-5p decreased cellular proliferation, migration, invasion, and tube formation—a surrogate marker for angiogenesis. miR-181a/b-5p may be used as a therapeutic intervention to restrict uncontrolled levels of PDGFRA and thereby rescue the phenotypes of increased cell proliferation, migration, invasion and tube formation. miR-181a/b negatively regulates PDGFRA levels. Significance of the study : T2DM and its associated complications emerge from endothelial dysfunction. The associated phenotypes are regulated by a number of proteins, one such member being, PDGFRA. PDGFRA is in turn regulated by miR-181a/b-5p. Complementation with miR-181a/b-5p resulted in reversion of phenotypes. Thus, miR-181a/b-5p-mediated suppression of PDGFRA may be used as a therapeutic intervention in the management of type 2 diabetes.     

miR-106b转基因小鼠的建立

目的:建立miR-106b转基因小鼠模型,探讨其在阿尔茨海默病(Alzheimer’s disease, AD)发病中的作用。方法:构建miR-106b表达载体,显微注射法建立miR-106b转基因小鼠。PCR鉴定转基因小鼠的基因型,real time RT-PCR检测miR-106b转基因小鼠脑组织中miR-106b的表达情况,Western blot检测miR-106b转基因小鼠脑组织中TGFBR2蛋白的表达。结果:构建了高表达miR-106b转基因小鼠;与对照相比,miR-106b转基因小鼠脑组织中TGFBR2蛋白的表达升高。结论:miR-106b转基因小鼠的建立为研究该microRNA在AD发病中的作用提供了工具。     

Roles and Mechanism of miR-199a and miR-125b in Tumor Angiogenesis

MicroRNAs (miRNAs) have been shown to be involved in different aspects of cancer biology including tumor angiogenesis. In this study, we identified that two miRNAs, miR-199a and miR-125b were downregulated in ovarian cancer tissues and cell lines. Overexpression of miR-199a and miR-125b inhibited tumor-induced angiogenesis associated with the decrease of HIF-1α and VEGF expression in ovarian cancer cells. Moreover, the levels of miR-199a and miR-125b were negatively correlated with VEGF mRNA levels in ovarian tissues. We further showed that direct targets of miR-199a and miR-125b HER2 and HER3 were functionally relevant. Forced expression of HER2 and HER3 rescued miR-199a- and miR-125b-inhibiting angiogenesis responses and Akt/p70S6K1/HIF-1α pathway. This study provides a rationale for new therapeutic approach to suppress tumor angiogenesis using miR-199a, miR-125b, or their mimics for ovarian cancer treatment in the future.