miRNA-21 Inhibitor Research Articles

The Anti-Apoptotic Role of Berberine in Preimplantation Embryo In Vitro Development through Regulation of miRNA-21.

Traditional Chinese medicinal herbs containing berberine have been historically used to prevent miscarriage. Here, we investigated whether the anti-apoptotic effects of berberine on pre-implantation embryonic development are regulated by miRNA-21. Mouse pronuclear embryos were cultured in medium with or without berberine, and some were then microinjected with a miRNA-21 inhibitor. The in vitro developmental rates of 2- and 4-cell embryos and blastocysts, blastocyst cell numbers, apoptotic rates, and apoptotic cell numbers were measured in each group. Furthermore, we examined the transcription levels of miRNA-21 and its target genes (caspase-3, PTEN, and Bcl-2) and their translation levels. Comparisons were made with in vivo-developed and untreated embryos. We found that berberine significantly increased the developmental rates and cell numbers of mouse blastocysts and decreased apoptotic cell rates in vitro. Berberine also significantly increased miRNA-21 and Bcl-2 transcription levels and significantly decreased caspase-3 and PTEN transcription levels. In embryos treated with a miRNA-21 inhibitor, the results followed the opposite trend; PTEN and caspase-3 transcription levels increased significantly, while the transcription level of Bcl-2 decreased significantly. Additionally, berberine treatment significantly increased the Bcl-2 protein level and significantly decreased the caspase-3 and PTEN protein levels in blastocysts, but there were no significant differences observed in the levels of these proteins in 2- and 4-cell embryos. This study revealed that miRNA-21 is important for pre-implantation embryonic development, especially blastocyst development in vitro. Berberine elevates miRNA-21 expression, decreases PTEN and caspase-3 levels, increases Bcl-2 levels, and exerts anti-apoptotic and pro-growth effects.

Macrophage-secreted Exosomes Delivering miRNA-21 Inhibitor can Regulate BGC-823 Cell Proliferation.

Exosomes, membranous nanovesicles, naturally carry bio-macromolecules or miRNA and play impoetant roles in tumor pathogenesis. Here, we showed that macrophages cell-derived exosomes can function as vehicles to deliver exogenous miR-21 inhibitor into BGC-823 gastric cancer cells. Exosomes loaded with miR-21 inhibitor significantly increased miR-21 levels in BGC-823, but miR-21 inhibitor loaded in exosomes exerted an opposite effect. miRNA transfected with exosomes had less cellular toxicity to host cells compared to conventional transfection methods. The miR-21 inhibitor loaded exosomes promoted the migration ability and reduced apoptosis of BGC-823 gastric cancer cells. These observations indicate that miR-21 acts as a tumor promoter by targeting the PDCD4 gene and preventing apoptosis of gastric cancer cells through inhibition of PDCD4 expression. Furthermore, exosome -mediated miR-21 inhibitor delivery resulted in functionally more efficient inhibition and less cellular toxicity compared to conventional transfection methods. Similar approaches could be useful in modification of target biomolecules in vitro and in vivo. These findings contribute to our understanding of the functions of miR-21 and exosomes as a carrier for therapy of gastric cancer.

Colony stimulating factor-1 receptor signaling networks inhibit mouse macrophage inflammatory responses by induction of microRNA-21

AbstractMacrophage polarization between the M2 (repair, protumorigenic) and M1 (inflammatory) phenotypes is seen as a continuum of states. The detailed transcriptional events and signals downstream of colony-stimulating factor 1 receptor (CSF-1R) that contributes to amplification of the M2 phenotype and suppression of the M1 phenotype are largely unknown. Macrophage CSF-1R pTyr-721 signaling promotes cell motility and enhancement of tumor cell invasion in vitro. Combining analysis of cellular systems for CSF-1R gain of function and loss of function with bioinformatic analysis of the macrophage CSF-1R pTyr-721–regulated transcriptome, we uncovered microRNA-21 (miR-21) as a downstream molecular switch controlling macrophage activation and identified extracellular signal-regulated kinase1/2 and nuclear factor-κB as CSF-1R pTyr-721–regulated signaling nodes. We show that CSF-1R pTyr-721 signaling suppresses the inflammatory phenotype, predominantly by induction of miR-21. Profiling of the miR-21–regulated messenger RNAs revealed that 80% of the CSF-1–regulated canonical miR-21 targets are proinflammatory molecules. Additionally, miR-21 positively regulates M2 marker expression. Moreover, miR-21 feeds back to positively regulate its own expression and to limit CSF-1R–mediated activation of extracellular signal-regulated kinase1/2 and nuclear factor-κB. Consistent with an anti-inflammatory role of miRNA-21, intraperitoneal injection of mice with a miRNA-21 inhibitor increases the recruitment of inflammatory monocytes and enhances the peritoneal monocyte/macrophage response to lipopolysaccharide. These results identify the CSF-1R–regulated miR-21 network that modulates macrophage polarization.

MicroRNA-21 controls hTERT via PTEN in human colorectal cancer cell proliferation.

Colorectal cancer is a major health problem worldwide. The aim of this study was to determine a role of microRNA-21 (miRNA-21) in colorectal cancer (CRC) and to elucidate miRNA-21 regulation of hTERT by phosphatase and tensin homologue (PTEN). Protein and mRNA samples were extracted with 30 CRC samples and one CRC cell lines. The expression of miRNA-21, hTERT, PTEN in CRC tissues and CRC cell lines was measured by real-time fluorescent quantitative PCR, Western blotting. Cell viability was detected by MTT and cell cycle assay. In this study, we show that the expression of miRNA-21 was overexpressed in CRC tissue and CRC cell lines compared with the control group. The effects of miRNA-21 were then assessed in MTT assays through in vitro transfection with a miRNA-21 mimic or inhibitor. PTEN has been identified as a target gene of miRNA-21 in CRC cell lines. Moreover, Western blot and qRT-PCR analyses revealed that miRNA-21 increased the expression of human telomerase reverse transcriptase (hTERT) via the PTEN/ERK1/2. In addition, Western blot analyses confirmed that an inverse correlation between PTEN and hTERT levels of high miRNA-21 RNA-expressing CRC tissues and cell lines. Finally, these data indicate that miRNA-21 regulates hTERT expression via the PTEN/ERK1/2 signaling pathway, therefore controlling CRC cell line growth. MiRNA-21 may serve as a novel therapeutic target in CRC.

MiRNA-21 inhibition enhances RANTES and IP-10 release in MCF-7 via PIAS3 and STAT3 signalling and causes increased lymphocyte migration

MicroRNAs (miRNAs) are a class of small endogenous gene regulators that have been implicated in various developmental and pathological processes. However, the precise identities and functions of miRNAs involved in antitumor immunity are not yet well understood. miRNA-21 is an oncogenic miRNA that can be detected in various tumours. In this study, we report that a miRNA-21 inhibitor enhances the release of chemoattractants RANTES and IP-10 in the MCF-7 breast cancer cell line and results in increased lymphocyte migration. Thus, miRNA-21 is a potential therapeutic target for cancer immunotherapy. We further demonstrated that PIAS3, a protein inhibitor of activated STAT3, is a target of miRNA-21 in MCF-7. Thus, miRNA-21 is a novel miRNA regulating immune cell recruitment, which acts at least in part via its inhibition of PIAS3 expression and oncogenic STAT3 signalling in tumour cells.

MiR-210 expression in tumor tissue and in vitro effects of its silencing in renal cell carcinoma

Renal cell carcinoma (RCC) is the most common neoplasm of adult kidney accounting for about 3% of adult malignancies. MicroRNAs (miRNAs) are a class of naturally occurring, short non-coding RNAs that regulate gene expression at the post-transcriptional level. We determined global miRNA expression profiles of RCC and parallel renal parenchyma tissues by using quantitative reverse transcriptase-polymerase chain reaction-based TaqMan low-density arrays. Afterward, we validated the difference in miR-210 expression levels on the larger group of RCC patients (35 RCC versus 10 non-tumorous parenchyma samples). Functional in vitro experiments were performed on ACHN and CAKI-2 RCC cell lines transfected with miRNA-210 inhibitor. Cell viability, apoptosis, cell cycle, scratch wound migration assay, and invasion assay (xCELLigence) were performed. We have identified original ccRCC-specific miRNA signature in clinical samples (73 miRNAs were significantly downregulated and five miRNAs upregulated (P < 0.003)). Increased expression levels of miR-210 in RCC tumor tissue were independently validated. We observed decreased viability of ACHN and CAKI-2 cells and accumulation of CAKI-2 in G2 phase of cell cycle after silencing of miR-210 expression. Downregulation of miR-210 also reduced the migratory and invasive potential of ACHN metastatic RCC cells. Moreover, we showed downregulation of HIF1a protein in both cell lines after miR-210 silencing indicating participation of miR-210 in hypoxic processes of RCC not only through regulation of its target mRNAs but also by indirect regulation of HIF1a. To our knowledge, this is the first report to show miR-210 regulatory effects on cell migration, invasive potential, and HIF1a protein in RCC cells.

Endogenous microRNAs induced by heat-shock reduce myocardial infarction following ischemia–reperfusion in mice

We investigated the role of microRNAs (miRNA) in protection against ischemia/reperfusion (I/R) injury in heart. Mice subjected to cytoprotective heat-shock (HS) showed a significant increase of miRNA-1, miRNA-21 and miRNA-24 in the heart. miRNAs isolated from HS mice and injected into non-HS mice significantly reduced infarct size after I/R injury, which was associated with the inhibition of pro-apoptotic genes and increase in anti-apoptotic genes. Chemically synthesized miRNA-21 also reduced infarct size, whereas a miRNA-21 inhibitor abolished this effect. Overall, these studies for the first time provide evidence for the potential role of endogenously synthesized miRNA’s in cardioprotection following I/R injury.