Inhibition Of miR-15b Research Articles

MiR-15b can target insulin receptor to regulate hepatic insulin signaling in mice

ABSTRACTNow diabetes is growing to be a health problems globally. However, its specific pathogenesis still needs further exploration. Here we showed that miR-15b was upregulated in the palmitate-induced HepG2 cells and livers of hyperglycemic mice. At the same time, we confirmed that the insulin receptor was a direct target of miR-15b. Then we found that the manipulation of miR-15b expression level could affect the insulin signaling pathway of HepG2 cells and the inhibition of miR-15b in liver of ob/ob mice can improve insulin sensitivity of mice. Furthermore, our study demonstrated that palmitate could upregulate the expression of miR-15b by activating PPARα. Our findings established PPARα-responsive miR-15b as a critical regulator of hepatic insulin signaling, thus serving as a new potential therapeutic target for diabetes.

MiR-15b and miR-322 inhibit SETD3 expression to repress muscle cell differentiation

SETD3 is a member of SET-domain containing methyltransferase family, which plays critical roles in various biological events. It has been shown that SETD3 could regulate the transcription of myogenic regulatory genes in C2C12 differentiation and promote myoblast determination. However, how SETD3 is regulated during myoblast differentiation is still unknown. Here, we report that two important microRNAs (miRNAs) could repress SETD3 and negatively contribute to myoblast differentiation. Using microRNA (miRNA) prediction engines, we identify and characterize miR-15b and miR-322 as the primary miRNAs that repress the expression of SETD3 through directly targeting the 3’-untranslated region of SETD3 gene. Functionally, overexpression of miR-15b or miR-322 leads to the repression of endogenous SETD3 expression and the inhibition of myoblast differentiation, whereas inhibition of miR-15b or miR-322 derepresses endogenous SETD3 expression and facilitates myoblast differentiation. In addition, knockdown SETD3 in miR-15b or miR-322 repressed myoblasts is able to rescue the facilitated differentiation phenotype. More interestingly, we revealed that transcription factor E2F1 or FAM3B positively or negatively regulates miR-15b or miR-322 expression, respectively, during muscle cell differentiation, which in turn affects SETD3 expression. Therefore, our results establish two parallel cascade regulatory pathways, in which transcription factors regulate microRNAs fates, thereby controlling SETD3 expression and eventually determining skeletal muscle differentiation.

A modest change in regulation of NOD1 expression has a major impact on inflammation and gastric cancer

Abstract Altered protein dosage by defects in single genes leads to haploinsufficiencies and monogenic disorders, but the impact of small changes in gene expression on multifactorial disease is unknown. Here we show that a persistent small (~1.5 fold) increase in expression of NOD1 (Nucleotide-binding oligomerization domain-containing protein 1), a key innate sensor of bacterial infection, precipitates a large physiological effect with dramatically altered cellular function associated with carcinogenesis. Inhibition of miR-15b and miR-16 microRNA function leads to a ~1.2–1.4 fold increase in NOD1 protein concentration, with even slightly greater increases leading to ligand-independent, switch-like NOD1 activation. miRNA regulation of NOD1 plays a crucial role in limiting the development of an inflammatory state is impaired in gastric cancer with a small increase in NOD1 being associated with greater early patient mortality. Overall, our data show that tight control of NOD1 expression by miRNA prevents this sensor from exceeding a physiological switching checkpoint that promotes persistent inflammation and lethal cancer progression, and reveal the impact of a single and modest cellular alteration on cancer. These findings also have broader implications for understanding how small expression changes caused by genetic variation impact development of complex diseases like autoimmunity and cancer.

Effects of the antagomiRs 15b and 200b on the altered healing pattern of diabetic mice.

Diabetic patients with non-healing ulcers have a reduced expression of VEGF. Genetically diabetic mice have an altered expression pattern of VEGF and its receptor, VEGF receptor 2 (VEGFR-2). In diabetic wounds, the microRNAs, miR15b and miR200b, which respectively inhibit VEGF and VEGF-R2 mRNAs, are up-regulated, further affecting the impaired angiogenesis. We investigated whether anti-miRs directed toward miR15b and miR200b could improve wound repair in genetically diabetic mice. Skin wounds were produced on the backs of female diabetic mice. The anti-miRs (antimiR15b, antimiR200b or antimiR15b/200b) at 10mg·kg-1 , or vehicle were applied to the wound edge. Mice were killed on days 7, 14 and at time of complete wound closure. Levels of mRNA and protein of angiogenic mediators and their receptors were measured with RT-qPCR and Western blotting. Wounds were examined by histological and immunochemical methods. mRNA expression of VEGF, VEGFR-2, angiopoietin-1 and its receptor TEK were evaluated after 7 and 14days. Protein levels of VEGF and transglutaminase II were measured at day 7, while VEGFR-2 and Angiopoietin-1 were measured at day 14. Histological features and the time to achieve a complete wound closure were also examined. Treatment with the anti-miRs improved the analysed parameters and the co-treatment resulted the most effective. The results suggest that the inhibition of miR15b and miR200b may have a potential application in diabetes-related wound disorders.

MicroRNA-15b promotes proliferation and invasion of non‑small cell lung carcinoma cells by directly targeting TIMP2.

MicroRNA-15b (miR-15b) plays an important role in tumor development and progression. miR-15b functions differently in various types of malignant tumors. However, the expression pattern and role of miR-15b in non-small cell lung cancer (NSCLC) have not been elucidated. In the present study, we investigated the effect of miR-15b on the occurrence and development of lung cancer and the underlying mechanism. Lung cancer cell lines A549 and LTEP-a-2 were transfected with miR-15b inhibitor or mimic, respectively. Real-time PCR revealed that the expression level of miR-15b was significantly higher in human NSCLC tissues and NSCLC cells, than that of normal tissues and cells, respectively (P<0.05). Moreover, the effect of miR-15b on A549 and LTEP-a-2 cell viability, cell cycle, migration and invasion was further evaluated. Experiments indicated that miR‑15b knockdown inhibited the viability, cell cycle, migration and invasion in A549 cells, while upregulation of miR-15b exhibited the opposite effect. Tissue inhibitor of metallopeptidases2 (TIMP2) protein and mRNA levels were downregulated after miR-15b overexpression in A549 and LTEP-a-2 cells, respectively. The dual-luciferase reporter gene assay implied that TIMP2 is a direct target gene of miR-15b. Our results indicate that high expression of miR-15b is associated with NSCLC and suggest that miR-15b expression may be a novel biomarker for predicting clinical outcomes in NSCLC patients. The inhibition of miR-15b may even provide helpful therapeutic strategies for the treatment of NSCLC.

Effects of microRNA-15b on proliferation of colorectal cancer cells and the preliminary mechanism

Objective To investigate the effect of the inhibition of microRNA (miR)-15b on proliferation of human colorectal carcinoma (CRC) cells and the preliminary mechanism. Methods Caco2, HCT116 and SW620 cells were examined for miR-15b and metastasis suppressor 1 (MTSS1) mRNA expression using reverse transcription-polymerase chain reaction (RT-PCR). We constructed the recombinant plasmids pMIR-Report-MTSS1 and pMIR-Report-MTSS1 mut. The targeting effect of miR-15b on MTSS1 gene was verified by the dual-luciferase reporter assay system. Then we transfected miR-15b inhibitor and miR-15b mimics into HCT116 cells by Lipofectamine 2000, and detected the cell proliferation, cell cycles, expression of MTSS1 mRNA and its protein by methyl thiazol tetrazolium (MTT), flow cytometric (FCM), RT-PCR and Western blotting, respectively. Results Caco2, HCT116 and SW620 cells showed significantly upregulated and downregulated expression of miR-15b and MTSS1 mRNA respectively. miR-15b could inhibit MTSS1 expression as a target. miR-15b could promote HCT116 cell proliferation, inhibition of miR-15b could inhibit cell proliferation of HCT116. Meanwhile, the MTSS1 mRNA and its protein could be upregulated by miR-15b inhibitor (P=0.004, 0.001). Conclusion MiR-15b could promote colorectal cancer cell proliferation by downregulating MTSS1 expression. Inhibition of miR-15b could inhibit CRC cell proliferation by up-regulating expression of MTSS1. MiR-15b can be used as a new pharmacological target gene for CRC therapy in further study. Key words: MicroRNA-15b; Metastasis suppressor 1; Colorectal carcinoma; Cell proliferation; Cell cycles

Maternal Low Protein Isocaloric Diet Suppresses Pancreatic β-Cell Proliferation in Mouse Offspring via miR-15b.

The mechanism underlying the increased susceptibility of type 2 diabetes in offspring of maternal malnutrition is poorly determined. Here we tested the hypothesis that functional microRNAs (miRNAs) mediated the maternal low-protein (LP) isocaloric diet induced pancreatic β-cell impairment. We performed miRNA profiling in the islets from offspring of LP and control diet mothers to explore the potential functional miRNAs responsible for β-cell dysfunction. We found that LP offspring exhibited impaired glucose tolerance due to decreased β-cell mass and insulin secretion. Reduction in the β-cell proliferation rate and cell size contributed to the decreased β-cell mass. MiR-15b was up-regulated in the islets of LP offspring. The up-regulated miR-15b inhibited pancreatic β-cell proliferation via targeting cyclin D1 and cyclin D2. Inhibition of miR-15b in LP islet cells restored β-cell proliferation and insulin secretion. Our findings demonstrate that miR-15b is critical for the regulation of pancreatic β-cells in offspring of maternal protein restriction, which may provide a further insight for β-cell exhaustion originated from intrauterine growth restriction.

MicroRNA-15b regulates mitochondrial ROS production and the senescence-associated secretory phenotype through sirtuin 4/SIRT4.

Mammalian sirtuins are involved in the control of metabolism and life-span regulation. Here, we link the mitochondrial sirtuin SIRT4 with cellular senescence, skin aging, and mitochondrial dysfunction. SIRT4 expression significantly increased in human dermal fibroblasts undergoing replicative or stress-induced senescence triggered by UVB or gamma-irradiation. In-vivo, SIRT4 mRNA levels were upregulated in photoaged vs. non-photoaged human skin. Interestingly, in all models of cellular senescence and in photoaged skin, upregulation of SIRT4 expression was associated with decreased levels of miR-15b. The latter was causally linked to increased SIRT4 expression because miR-15b targets a functional binding site in the SIRT4 gene and transfection of oligonucleotides mimicking miR-15b function prevented SIRT4 upregulation in senescent cells. Importantly, increased SIRT4 negatively impacted on mitochondrial functions and contributed to the development of a senescent phenotype. Accordingly, we observed that inhibition of miR-15b, in a SIRT4-dependent manner, increased generation of mitochondrial reactive oxygen species, decreased mitochondrial membrane potential, and modulated mRNA levels of nuclear encoded mitochondrial genes and components of the senescence-associated secretory phenotype (SASP). Thus, miR-15b is a negative regulator of stress-induced SIRT4 expression thereby counteracting senescence associated mitochondrial dysfunction and regulating the SASP and possibly organ aging, such as photoaging of human skin.

MicroRNA-15b target Sall4 and diminish in vitro UCB-derived HSCs expansion.

Hematopoietic Stem Cells (HSCs) are cells that have the ability to self-renewal and differentiate into all of hematopoietic lineages. The lack of donors and unavailable efficient protocols for ex vivo expansion of HSCs, are obstacles in successful cell therapies. MicroRNAs (also refer as miRNAs or miRs) have significant roles in hematopoiesis; they can effect on HSCs expansion, maintaining undifferentiated state, self-renewal and differentiation. Recently attentions have been given to these small regulatory molecules to utilize them in order to expand HSCs. Using bioinformatics analysis we identified Sall4 as putative target of miR-15b and miR-219-5p. Relative expression levels of miRNAs and Sall4 were evaluated by qRT-PCR. Here we show 247-fold and 4.2-fold increasing Sall4 expression level compared to control group in CD34+ cells nucleofected by anti-miR-15b and anti-miR-219-5p, respectively. These data showed that anti-miR-15b can promote clonogenic capacity of HSCs and also we found that miR-15b alone was able to increase the number of CD34+HSCs in vitro by more than 2 fold by targeting Sall4. Moreover, level of CD34 marker in HSCs nucleofected by anti-miR-15b increased more than 50 %. Our analysis showed no statistically difference in mRNA level of Sall4 after nucleofection of anti-miR-219-5p. Sall4 is a factor capable of enhancing HSC expansion significantly. We demonstrated that inhibition of miR-15b can enhance ex vivo expansion of UCB-derived HSCs and also expression of Sall4 allowed expansion and preserve self- renewal of CD34+ HSCs.

The microRNA-15 family inhibits the TGFβ-pathway in the heart

The overloaded heart remodels by cardiomyocyte hypertrophy and interstitial fibrosis, which contributes to the development of heart failure. Signalling via the TGFβ-pathway is crucial for this remodelling. Here we tested the hypothesis that microRNAs in the overloaded heart regulate this remodelling process via inhibition of the TGFβ-pathway. We show that the miRNA-15 family, which we found to be up-regulated in the overloaded heart in multiple species, inhibits the TGFβ-pathway by targeting of TGFBR1 and several other genes within this pathway directly or indirectly, including p38, SMAD3, SMAD7, and endoglin. Inhibition of miR-15b by subcutaneous injections of LNA-based antimiRs in C57BL/6 mice subjected to transverse aorta constriction aggravated fibrosis and to a lesser extent also hypertrophy. We identified the miR-15 family as a novel regulator of cardiac hypertrophy and fibrosis acting by inhibition of the TGFβ-pathway.

Abstract 28: In Vivo Knockdown of Mirna-15b Results in Increased Cardiac Hypertrophy and Fibrosis in Response to Pressure Overload of the Mouse Heart

MiRNAs play an important role in the control of diverse aspects of cardiac function. MiR-15b is highly expressed in the heart and is found consistently upregulated in hypertrophic and failing hearts. To investigate the function of miR-15b in the heart we set out two experiments. In the first experiment we generated two independent transgenic mouse lines that drive miR-15b expression under the αMHC-promotor and show a three and four fold overexpression of miR-15b. Strikingly, both lines show a decrease in heart weight/tibia length of 20% in adult and aged mice when compared to littermate controls. We investigated the response of these transgenic mice to thoracic aorta constriction (TAC) and found no differences in the hypertrophic response or in cardiac function measured by echocardiography between wild-type and transgenic mice. In a second experiment, we inhibited miR-15b using LNA-based antimiRs. In these mice, TAC resulted in an increased hypertrophic response and increased cardiac fibrosis when compared to a negative control antimiR. A wide range of predicted targets of miR-15 belong to the pathways of the TGFβ-superfamily and using a smad-dependent reporter we show that miR-15b inhibits TGFβ-induced Smad activity in HepG2 cells. One of the predicted targets in the TGFβ pathway is TGFβ receptor 1 (TGFβR1), of which the 3’UTR contains six predicted miR-15 binding sites. This suggests that the phenotype in the transgenic mice and after knockdown of miR-15b may be (partly) mediated by repression of TGFβR1. Indeed, in the adult miR-15b transgenic hearts we found a downregulation of TGFβR1 mRNA and protein and we confirmed binding of miR-15 to the TGFβR1 3’UTR by luciferase assays. In conclusion, miR-15b causes a cardiac hypotrophic phenotype at baseline in transgenic mice and inhibition of miR-15b leads to a stronger hypertrophic and fibrotic response after TAC. Furthermore miR-15b inhibits the TGFβ pathway by targeting the TGFβR1 and possibly other targets in this pathway. This research is funded by the Dutch Heart Foundation (NHF grant #2007B077).

MiR-15b and miR-16 regulate TNF mediated hepatocyte apoptosis via BCL2 in acute liver failure

Acute liver failure (ALF) still has an unacceptable high mortality rate, despite substantial improvements with multidisciplinary care. The precise underlying mechanism of ALF remains to be explored. It has been reported that microRNAs (miRNAs) are novel regulators in a number of liver diseases, but the role of miRNAs in the development of ALF is not fully understood. An ALF murine model was generated by ip injection of D: -GalN/LPS, which was confirmed with histopathology and biochemistry. The hepatic miRNA expression profile in ALF was determined by microarray and verified by qRT-PCR. The functions and signal pathways of the targeted genes of these deregulated miRNAs were predicted, using bioinformatics analysis. The possible underlying mechanism was investigated by exploring the relationship between miRNA modification and hepatocyte apoptosis. There were a total of 95 significantly changed miRNAs in ALF compared to mock-treated (P < 0.01). Among these 95 miRNAs, 20 were up-regulated and 26 were down-regulated at both 5 and 7 h time points. Bioinformatics analysis predicted that some of these 46 miRNAs were involved in apoptosis. Among the up-regulated miRNAs involved in apoptosis, miR-15b and miR-16 showed the highest enrichment and targeted the common anti-apoptotic gene, BCL2. Our in vitro data demonstrated that miR-15b and/or miR-16 regulated BCL2 at the protein level. Inhibition of miR-15b and/or miR-16 reduced hepatic apoptosis and TNF production. These data suggest that miR-15b and miR-16 regulate TNF mediated hepatic apoptosis via BCL2 during ALF, and may shed light on the development of a therapeutic strategy for treatment of ALF.