MicroRNA‐379 suppresses osteosarcoma progression by targeting PDK1

Osteosarcoma is the most common primary bone tumour. Increasing evidence has demonstrated the pathogenic role of microRNA (miRNAs) dysregulation in tumour development. miR‐379 was previously reported to function as an oncogenic or tumour‐suppressing miRNA in a tissue‐dependent manner. However, its function in osteosarcoma remains unknown. In this study, we found that the expression of miR‐379 was downregulated in osteosarcoma tissues and cell lines. Further functional characterization revealed that miR‐379 suppressed osteosarcoma cell proliferation and invasion in vitro and retarded the growth of osteosarcoma xenografts in vivo. Mechanistically, PDK1 was identified as the direct target of miR‐379 in osteosarcoma, in which PDK1 expression was up‐regulated and showed inverse correlation with miR‐379. Enforced expression of PDK1 promoted osteosarcoma cell proliferation and rescued the anti‐proliferative effect of miR‐379. These data suggest that miR‐379 could function as a tumour‐suppressing miRNA via targeting PDK1 in osteosarcoma.     

Evaluation of polymorphisms in microRNA‐binding sites and pancreatic cancer risk in Chinese population

As promising biomarkers and therapy targets, microRNAs (miRNAs) are involved in various physiological and tumorigenic processes. Genetic variants in miRNA‐binding sites can lead to dysfunction of miRNAs and contribute to disease. However, systematic investigation of the miRNA‐related single nucleotide polymorphisms (SNPs) for pancreatic cancer (PC) risk remains elusive. We performed integrative bioinformatics analyses to select 31 SNPs located in miRNA‐target binding sites using the miRNASNP v2.0, a solid database providing miRNA‐related SNPs for genetic research, and investigated their associations with risk of PC in two large case‐control studies totally including 1847 cases and 5713 controls. We observed that the SNP rs3802266 is significantly associated with increased risk of PC (odds ratio (OR) = 1.21, 95% confidence intervals (CI) = 1.11‐1.31, P = 1.29E‐05). Following luciferase reporter gene assays show that rs3802266‐G creates a stronger binding site for miR‐181a‐2‐3p in 3′ untranslated region (3′UTR) of the gene ZHX2. Expression quantitative trait loci (eQTL) analysis suggests that ZHX2 expression is lower in individuals carrying rs3802266‐G with increased PC risk. In conclusion, our findings highlight the involvement of miRNA‐binding SNPs in PC susceptibility and provide new clues for PC carcinogenesis.     

Proteomic screening and identification of microRNA‐128 targets in glioma cells

Brain‐enriched miR‐128 is repressed in glioma cells, and could inhibit the proliferation of gliomas by targeting genes such as E2F3a and BMI1. To identify more targets of miR‐128 in glioblastoma multiforme, the pulse stable isotope labeling with amino acids in cell culture (pSILAC) technique was used to test its impact on whole protein synthesis in T98G glioma cells. We successfully identified 1897 proteins, of which 1459 proteins were quantified. Among them, 133 proteins were downregulated after the overexpression of miR‐128. Through predictions using various bioinformatics tools, 13 candidate target genes were chosen. A luciferase assay validated that 11 of 13 selected genes were potential targets of miR‐128, and a mutagenesis experiment confirmed CBFB, CORO1C, GLTP, HnRNPF, and TROVE2 as the target genes. Moreover, we observed that the expression of CORO1C, TROVE2, and HnRNPF were higher in glioma cell lines compared to normal brain tissues and presented a tendency toward downregulation after overexpression of miR‐128 in T98G cells. Furthermore, we have validated that CORO1C, TROVE2, and HnRNPF could inhibit glioma cell proliferation. In sum, our data showed that the integration of pSILAC and bioinformatics analysis was an efficient method for seeking the targets of miRNAs, and plentiful targets of miR‐128 were screened and laid the foundation for research into the miR‐128 regulation network.     

Quantitative proteomics reveals that miR‐222 inhibits erythroid differentiation by targeting BLVRA and CRKL

miR‐222 plays an important role in erythroid differentiation, but the potential targets of miR‐222 in the regulation of erythroid differentiation remain to be determined. The target genes of miR‐222 were identified by proteomics combined with bioinformatics analysis in this study. Thirteen proteins were upregulated, and 13 were downregulated in K562 cells following transfection with miR‐222 inhibitor for 24 and 48 hours. Among these proteins, BLVRA and CRKL were upregulated after transfection of miR‐222 inhibitor in K562 cells and human CD34+ HPCs. Moreover, miR‐222 mimics reduced and miR‐222 inhibitor enhanced the mRNA and protein levels of both BLVRA and CRKL. Luciferase assay showed that miR‐222 directly targeted 3′‐UTR of BLVRA and CRKL. In addition, overexpression of either BLVRA or CRKL or both increased the erythroid differentiation of K562 cells, while silencing of either BLVRA or CRKL or both by siRNA significantly attenuated hemin‐induced erythroid differentiation of K562 cells. Our results indicated that BLVRA and CRKL are targets of miR‐222.     

MicroRNA‐30a ameliorates hepatic fibrosis by inhibiting Beclin1‐mediated autophagy

We explored the role of microRNA‐30a (miR‐30a) and the mechanism involved in hepatic fibrosis. MiR‐30a overexpression was achieved by miR‐30a mimics transfection in hepatic stellate cells (HSCs) (HSC‐T6, LX‐2), and miR‐30a agomir (ago‐miR‐30a) treatment in mice. MiR‐30a levels were measured using TaqMan miRNA assay system, and the localization of miR‐30a was detected by fluorescence in situ hybridization (FISH). The interaction of miR‐30a and Beclin1 was confirmed by dual‐luciferase reporter assay. Autophagic flux was analysed using tandem mRFP‐GFP‐LC3 fluorescence microscopy, electron microscopy and Western blot of LC3‐II/I ratio. MiR‐30a was notably down‐regulated in activated HSCs and LX‐2‐exosomes induced by TGF‐β1; overexpression of miR‐30a down‐regulated extracellular matrix (ECM), such as α‐SMA, TIMP‐1, and Collagen I expression, and suppressed cell viability in HSCs. MiR‐30a was significantly down‐regulated in hepatic fibrosis mice and overexpression of miR‐30a prevented BDL‐induced fibrogenesis, concomitant with the down‐regulation of ECM. MiR‐30a inhibited HSCs autophagy and increased lipid accumulation in HSCs and in mice fibrotic hepatic tissues. MiR‐30a inhibited its downstream effector of Beclin1 by direct targeting its 3′‐UTR region. Moreover, Knock‐down of Beclin1 by small interfering RNA (siRNA) inhibited HSC autophagy and activation in LX‐2 cells. In conclusion, miR‐30a is down‐regulated in hepatic fibrosis models and its overexpression prevents liver fibrogenesis by directly suppressing Beclin1‐mediated autophagy; therefore, miR‐30a may be a new potential therapeutic target for controlling hepatic fibrosis.     

Sucrose induction of <Emphasis Type="Italic">Arabidopsis</Emphasis> miR398 represses two Cu/Zn superoxide dismutases

MicroRNAs (miRNAs) are approximately 21-nt RNAs that reduce target accumulation through mRNA cleavage or translational repression. Arabidopsis miR398 regulates mRNAs encoding two copper superoxide dismutase (CSD) enzymes and a cytochrome c oxidase subunit. miR398 itself is down-regulated in response to copper and stress. Here we show that miR398 is positively regulated by sucrose, resulting in decreased CSD1 and CSD2 mRNA and protein accumulation. This sucrose regulation is maintained both in the presence and absence of physiologically relevant levels of supplemental copper. Additionally, we show that plants expressing CSD1 and CSD2 mRNAs with altered miR398 complementarity sites display increased mRNA accumulation, whereas CSD1 and CSD2 protein accumulation remain sensitive to miR398 levels, suggesting that miR398 can act as a translational repressor when target site complementarity is reduced. These results reveal a novel miR398 regulatory mechanism and demonstrate that plant miRNA targets can resist miRNA regulation at the mRNA level while maintaining sensitivity at the level of protein accumulation. Our results suggest that even in plants, where miRNAs are thought to act primarily through target mRNA cleavage, monitoring target protein levels along with target mRNA levels is necessary to fully assess the consequences of disrupted miRNA-mRNA pairing. Moreover, the limited complementarity required to maintain robust miR398-directed repression of target protein accumulation suggests that similarly regulated endogenous plant miRNA targets may have eluded detection.     

miR‐214 activates TP53 but suppresses the expression of RELA,CTNNB1, and STAT3 in human cervical and colorectal cancer cells

High Mobility Group AT‐hook 1 (HMGA1) was identified as a target of miR‐214 in human cervical and colorectal cancers (CaCx and CRC) in a previous study. While the expression of miR‐214 remains suppressed, HMGA1 behaves as a potent oncogene and plays crucial roles in several aberrant signalling pathways by interacting with intermediates like RELA, CTNNB1, STAT3, and TP53 in CaCx and CRC. Hypothetically, miR‐214 should be able to regulate the stabilization of some of these intermediates through the regulation of HMGA1. This was assessed by ectopically expressing miR‐214 or complementarily, by inhibiting the expression of HMGA1. In promoter luciferase assays, miR‐214 inhibited NF‐κB and Wnt activities but elevated TP53 activity in cancer cells. Further, miR‐214 suppressed the expression of HMGA1, RELA, CTNNB1, and STAT3 while elevating TP53 levels, similar to when small interfering RNA (siRNA) against HMGA1 was used, as revealed by Western blotting. It is suggested that poor expression of miR‐214, commonly reported in CaCx and CRC tissues, may not only result in the sustained expression of HMGA1 but also that of RELA, CTNNB1, and STAT3, and a congruent suppression of TP53 during cancer initiation/progression. These several states are, however, reversed when miR‐214 is reintroduced and could explain the tumour suppressive functions observed in earlier studies. Further studies are, however, required to reveal how microRNA‐mediated regulation of HMGA1 expression may affect individual signalling pathways in CaCx and CRC. Current results reveal that miR‐214 is not only able to regulate the expression of its direct target, HMGA1, but also that of a few signalling intermediates like TP53, RELA, CTNNB1, and STAT3, with which HMGA1 interacts. These intermediates play crucial roles in signalling pathways commonly deregulated in human CaCx and CRC. Hence, it is proposed that miR‐214 might act as a tumour suppressor by regulating several aberrant signalling pathways through HMGA1. This knowledge has the potential to help design novel therapeutic strategies in CaCx and CRC.     

miR‐181c‐5p mediates simulated microgravity‐induced impaired osteoblast proliferation by promoting cell cycle arrested in the G2 phase

Impaired osteoblast proliferation plays fundamental roles in microgravity‐induced bone loss, and cell cycle imbalance may result in abnormal osteoblast proliferation. However, whether microgravity exerts an influence on the cell cycle in osteoblasts or what mechanisms may underlie such an effect remains to be fully elucidated. Herein, we confirmed that simulated microgravity inhibits osteoblast proliferation. Then, we investigated the effect of mechanical unloading on the osteoblast cell cycle and found that simulated microgravity arrested the osteoblast cell cycle in the G₂ phase. In addition, our data showed that cell cycle arrest in osteoblasts from simulated microgravity was mainly because of decreased cyclin B1 expression. Furthermore, miR‐181c‐5p directly inhibited cyclin B1 protein translation by binding to a target site in the 3′UTR. Lastly, we demonstrated that inhibition of miR‐181c‐5p partially counteracted cell cycle arrest and decreased the osteoblast proliferation induced by simulated microgravity. In conclusion, our study demonstrates that simulated microgravity inhibits cell proliferation and induces cell cycle arrest in the G₂ phase in primary mouse osteoblasts partially through the miR‐181c‐5p/cyclin B1 pathway. This work may provide a novel mechanism of microgravity‐induced detrimental effects on osteoblasts and offer a new avenue to further investigate bone loss induced by mechanical unloading.     

Long non‐coding RNA NR2F1‐AS1 promoted proliferation and migration yet suppressed apoptosis of thyroid cancer cells through regulating miRNA‐338‐3p/CCND1 axis

Thyroid cancer (TC) is a prevalent endocrine malignant cancer whose pathogenic mechanism remains unclear. The aim of the study was to investigate the roles of long non‐coding RNA (lncRNA) NR2F1‐AS1/miRNA‐338‐3P/CCND1 axis in TC progression. Differentially expressed lncRNAs and mRNAs in TC tissues were screened out and visualized by R program. Relative expression of NR2F1‐AS1, miRNA‐338‐3p and cyclin D1 (CCND1) was determined by quantitative real time polymerase chain reaction. In addition, Western blot analysis was adopted for evaluation of protein expression of CCND1. Targeted relationships between NR2F1‐AS1 and miRNA‐338‐3p, as well as miRNA‐338‐3p and CCND1 were predicted using bioinformatics analysis and validated by dual‐luciferase reporter gene assay. Besides, tumour xenograft assay was adopted for verification of the role of NR2F1‐AS1 in TC in vivo. NR2F1‐AS1 and CCND1 were overexpressed, whereas miRNA‐338‐3p was down‐regulated in TC tissues and cell lines. Down‐regulation of NR2F1‐AS1 and CCND1 suppressed proliferation and migration of TC cells yet greatly enhanced cell apoptotic rate. Silence of NR2F1‐AS1 significantly suppressed TC tumorigenesis in vivo. NR2F1‐AS1 sponged miRNA‐338‐3p to up‐regulate CCND1 expression to promote TC progression. Our study demonstrated that up‐regulation of NR2F1‐AS1 accelerated TC progression through regulating miRNA‐338‐3P/CCND1 axis.     

MiR‐590‐3p regulates proliferation,migration and collagen synthesis of cardiac fibroblast by targeting ZEB1

Previous studies have implicated the attractive and promising role of miR‐590‐3p to restore the cardiac function following myocardial infarction (MI). However, the molecular mechanisms for how miR‐590‐3p involves in cardiac fibrosis remain largely unexplored. Using human cardiac fibroblasts (HCFs) as the cellular model, luciferase report assay, mutation, EdU assay and transwell migration assay were applied to investigate the biological effects of miR‐590‐3p on the proliferation, differentiation, migration and collagen synthesis of cardiac fibroblasts. We found that miR‐590‐3p significantly suppressed cell proliferation and migration of HCFs. The mRNA and protein expression levels of α‐SMA, Col1A1 and Col3A were significantly decreased by miR‐590‐3p. Moreover, miR‐590‐3p directly targeted at the 3’UTR of ZEB1 to repress the translation of ZEB1. Interfering with the expression of ZEB1 significantly decreased the cell proliferation, migration activity, mRNA and protein expressions of α‐SMA, Col1A1 and Col3A. Furthermore, the expressions of miR‐590‐3p and ZEB1 were identified in infarct area of MI model in pigs. Collectively, miR‐590‐3p suppresses the cell proliferation, differentiation, migration and collagen synthesis of cardiac fibroblasts by targeting ZEB1. These works will provide useful biological information for future studies on potential roles of miR‐590‐3p as the therapeutic target to recover cardiac function following MI.     

Hormone replacement therapy enhances IGF‐1 signaling in skeletal muscle by diminishing miR‐182 and miR‐223 expressions: a study on postmenopausal monozygotic twin pairs

MiRNAs are fine‐tuning modifiers of skeletal muscle regulation, but knowledge of their hormonal control is lacking. We used a co‐twin case–control study design, that is, monozygotic postmenopausal twin pairs discordant for estrogen‐based hormone replacement therapy (HRT) to explore estrogen‐dependent skeletal muscle regulation via miRNAs. MiRNA profiles were determined from vastus lateralis muscle of nine healthy 54–62‐years‐old monozygotic female twin pairs discordant for HRT (median 7 years). MCF‐7 cells, human myoblast cultures and mouse muscle experiments were used to confirm estrogen's causal role on the expression of specific miRNAs, their target mRNAs and proteins and finally the activation of related signaling pathway. Of the 230 miRNAs expressed at detectable levels in muscle samples, qPCR confirmed significantly lower miR‐182, miR‐223 and miR‐142‐3p expressions in HRT using than in their nonusing co‐twins. Insulin/IGF‐1 signaling emerged one common pathway targeted by these miRNAs. IGF‐1R and FOXO3A mRNA and protein were more abundantly expressed in muscle samples of HRT users than nonusers. In vitro assays confirmed effective targeting of miR‐182 and miR‐223 on IGF‐1R and FOXO3A mRNA as well as a dose‐dependent miR‐182 and miR‐223 down‐regulations concomitantly with up‐regulation of FOXO3A and IGF‐1R expression. Novel finding is the postmenopausal HRT‐reduced miRs‐182, miR‐223 and miR‐142‐3p expression in female skeletal muscle. The observed miRNA‐mediated enhancement of the target genes' IGF‐1R and FOXO3A expression as well as the activation of insulin/IGF‐1 pathway signaling via phosphorylation of AKT and mTOR is an important mechanism for positive estrogen impact on skeletal muscle of postmenopausal women.