MiR‐139‐5p,miR‐940 and miR‐193a‐5p inhibit the growth of hepatocellular carcinoma by targeting SPOCK1

The study was aimed to screen out miRNAs with differential expression in hepatocellular carcinoma (HCC), and to explore the influence of the expressions of these miRNAs and their target gene on HCC cell proliferation, invasion and apoptosis. MiRNAs with differential expression in HCC were screened out by microarray analysis. The common target gene of these miRNAs (miR‐139‐5p, miR‐940 and miR‐193a‐5p) was screened out by analysing the target genes profile (acquired from Targetscan) of the three miRNAs. Expression levels of miRNAs and SPOCK1 were determined by quantitative real time polymerase chain reaction (qRT‐PCR). The target relationships were verified by dual luciferase reporter gene assay and RNA pull‐down assay. Through 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide,thiazolyl blue tetrazolium bromide (MTT) and transwell assays and flow cytometry, HCC cell viability, invasion and apoptosis were determined. In vivo experiment was conducted in nude mice to investigate the influence of three miRNAs on tumour growth. Down‐regulation of miR‐139‐5p, miR‐940 and miR‐193a‐5p was found in HCC. Overexpression of these miRNAs suppressed HCC cell viability and invasion, promoted apoptosis and inhibited tumour growth. SPOCK1, the common target gene of miR‐139‐5p, miR‐940 and miR‐193a‐5p, was overexpressed in HCC. SPOCK1 overexpression promoted proliferation and invasion, and restrained apoptosis of HCC cells. MiR‐139‐5p, miR‐940 and miR‐193a‐5p inhibited HCC development through targeting SPOCK1.     

Developmental wave of Brn3b expression leading to RGC fate specification is synergistically maintained by miR‐23a and miR‐374

Differential regulation of Brn3b is essential for the Retinal Ganglion Cell (RGC) development in the two phases of retinal histogenesis. This biphasic Brn3b regulation is required first, during early retinal histogenesis for RGC fate specification and secondly, during late histogenesis, where Brn3b is needed for RGC axon guidance and survival. Here, we have looked into how the regulation of Brn3b at these two stages happens. We identified two miRNAs, miR‐23a and miR‐374, as regulators of Brn3b expression, during the early stage of RGC development. Temporal expression pattern of miR‐23a during E10–19, PN1–7, and adult retina revealed an inverse relation with Brn3b expression. Though miR‐374 did not show such a pattern, its co‐expression with miR‐23a evidently inhibited Brn3b. We further substantiated these findings by ex vivo overexpression of these miRNAs in E14 mice retina and found that miR‐23a and miR‐374 together brings about a change in Brn3b expression pattern in ganglion cell layer (GCL) of the developing retina. From our results, it appears that the combined expression of these miRNAs could be regulating the timing of the wave of Brn3b expression required for early ganglion cell fate specification and later for its survival and maturation into RGCs. Taken together, here we provide convincing evidences for the existence of a co‐ordinated mechanism by miRNAs to down regulate Brn3b that will ultimately regulate the development of RGCs from their precursors. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1155–1171, 2014     

Long non‐coding RNA deleted in lymphocytic leukaemia 1 promotes hepatocellular carcinoma progression by sponging miR‐133a to regulate IGF‐1R expression

Long non‐coding RNA (lncRNA) deleted in lymphocytic leukaemia 1 (DLEU1) was reported to be involved in the occurrence and development of multiple cancers. However, the exact expression, biological function and underlying mechanism of DLEU1 in hepatocellular carcinoma (HCC) remain unclear. In this study, real‐time quantitative polymerase chain reaction (qRT‐PCR) in HCC tissues and cell lines revealed that DLEU1 expression was up‐regulated, and the increased DLEU1 was closely associated with advanced tumour‐node‐metastasis stage, vascular metastasis and poor overall survival. Function experiments showed that knockdown of DLEU1 significantly inhibited HCC cell proliferation, colony formation, migration and invasion, and suppressed epithelial to mesenchymal transition (EMT) process via increasing the expression of E‐cadherin and decreasing the expression of N‐cadherin and Vimentin. Luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay demonstrated that DLEU1 could sponge miR‐133a. Moreover, miR‐133a inhibition significantly reversed the suppression effects of DLEU1 knockdown on HCC cells. Besides, we found that silenced DLEU1 significantly decreased insulin‐like growth factor 1 receptor (IGF‐1R) expression (a target of miR‐133a) and its downstream signal PI3K/AKT pathway in HCC cells, while miR‐133a inhibitor partially reversed this trend. Furthermore, DLEU1 knockdown impaired tumour growth in vivo by regulating miR‐133a/IGF‐1R axis. Collectively, these findings indicate that DLEU1 promoted HCC progression by sponging miR‐133a to regulate IGF‐1R expression. Deleted in lymphocytic leukaemia 1/miR‐133a/IGF‐1R axis may be a novel target for treatment of HCC.     

Loss of PPM1F expression predicts tumour recurrence and is negatively regulated by miR‐590‐3p in gastric cancer

Objectives

MicroRNAs (miRNAs) as small non‐coding RNA molecules act by negatively regulating their target genes. Recent studies have shown that protein phosphatase Mg2+/Mn2+‐dependent 1F (PPM1F) plays a critical role in cancer metastasis. But, the regulation mechanisms of PPM1F by miRNAs in gastric cancer (GC) remain undefined.

Methods

The correlation of PPM1F or miR‐590‐3p (miR‐590) expression with clinicopathological features and prognosis of the patients with GC was analysed by TCGA RNA‐sequencing data. The miRNAs that target PPM1F gene were identified by bioinformatics and Spearman correlation analysis, and the binding site between miR‐590 and PPM1F 3′UTR was confirmed by dual luciferase assay. MTT and Transwell assays were conducted to evaluate the effects of miR‐590 or (and) PPM1F on cell proliferation and invasion.

Results

We found that PPM1F expression was downregulated in GC tissues and cell lines and was correlated with tumour recurrence in patients with GC. The decreased expression of PPM1F was attributed to the dysregulation of miR‐590 expression rather than its genetic or epigenetic alterations. Overexpression of miR‐590 promoted cell proliferation and invasion capability of GC cells, while knockdown of miR‐590 reversed these effects. Moreover, PPM1F was validated as a direct target of miR‐590 and counteracted the tumour‐promoting effects caused by miR‐590. The expression of miR‐590 presented the negative correlation with PPM1F expression and acted as an independent prognostic factor for tumour recurrence in patients with GC.

Conclusion

PPM1F may function as a suppressive factor and is negatively regulated by miR‐590 in GC.

     

MicroRNAs mir‐184 and let‐7 alter Drosophila metabolism and longevity

MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression associated with many complex biological processes. By comparing miRNA expression between long‐lived cohorts of Drosophila melanogaster that were fed a low‐nutrient diet with normal‐lived control animals fed a high‐nutrient diet, we identified miR‐184, let‐7, miR‐125, and miR‐100 as candidate miRNAs involved in modulating aging. We found that ubiquitous, adult‐specific overexpression of these individual miRNAs led to significant changes in fat metabolism and/or lifespan. Most impressively, adult‐specific overexpression of let‐7 in female nervous tissue increased median fly lifespan by ~22%. We provide evidence that this lifespan extension is not due to alterations in nutrient intake or to decreased insulin signaling.     

IGF2‐derived miR‐483‐3p associated with Hirschsprung's disease by targeting FHL1

HSCR (Hirschsprung's disease) is a serious congenital defect, and the aetiology of it remains unclear. Many studies have highlighted the significant roles of intronic miRNAs and their host genes in various disease, few was mentioned in HSCR although. In this study, miR‐483‐3p along with its host gene IGF2 (Insulin‐like growth factor 2) was found down‐regulated in 60 HSCR aganglionic colon tissues compared with 60 normal controls. FHL1 (Four and a half LIM domains 1) was determined as a target gene of miR‐483‐3p via dual‐luciferase reporter assay, and its expression was at a higher level in HSCR tissues. Here, we study cell migration and proliferation in human 293T and SH‐SY5Y cell lines by performing Transwell and CCK8 assays. In conclusion, the knockdown of miR‐483‐3p and IGF2 both suppressed cell migration and proliferation, while the loss of FHL1 leads to opposite outcome. Furthermore, miR‐483‐3p mimics could rescue the negative effects on cell proliferation and migration caused by silencing IGF2, while the FHL1 siRNA may inverse the function of miR‐483‐3p inhibitor. This study revealed that miR‐483‐3p derived from IGF2 was associated with Hirschsprung's disease by targeting FHL1 and may provide a new pathway to understand the aetiology of HSCR.     

Serum miRNAs are potential biomarkers for the detection of disc degeneration,among which miR‐26a‐5p suppresses Smad1 to regulate disc homeostasis

Disc degeneration is a common clinical condition in which damaged discs cause chronic pain; however, a laboratory diagnosis method for its detection is not available. As circulating miRNAs have potential as biomarkers, their application in disc degeneration has not been explored. Here, we prepared serum miRNAs from a mouse disc degeneration model and performed miRNA‐Seq and quantitative PCR to characterize disc degeneration–associated miRNAs. We identified three miRNAs, including miR‐26a‐5p, miR‐122‐5p and miR‐215‐5p, undergoing perturbation during the pathogenesis of disc degeneration. Specifically, the levels of miR‐26a‐5p in the serum demonstrated steady increases in the model of disc degeneration, compared with those in the pre‐injury samples of younger age or compared with normal controls of the same age but without disc degeneration, whereas the miRNAs miR‐122‐5p and miR‐215‐5p exhibited lower expression in post‐injury samples than in their counterparts without the surgery. Moreover, we found that miR‐26a‐5p targets Smad1 expression, and Smad1 negatively regulates Vegfa expression in disc cells, and thus, miR‐26a‐5p promotes disc degeneration. In summary, we established a method that consistently profiles circulating miRNAs and identified multiple miRNAs as promising biomarkers for disc degeneration, among which miR‐26a‐5p enhances VEGF expression during disc degeneration through targeting Smad1 signalling.     

Aryl hydrocarbon‐estrogen alpha receptor‐dependent expression of miR‐206, miR‐27b,and miR‐133a suppress cell proliferation and migration in MCF‐7 cells

The underlying functions of miR‐206, miR‐133a, miR‐27b, and miR‐21, and their link to the estrogen receptor alpha (ERα) and aryl hydrocarbon receptor (AhR) signaling pathways remain largely unexplored. In this study, we detect the expression of miR‐206, miR‐133a, miR‐27b, and miR‐21 in MCF‐7 through quantificational real‐time polymerase chain reaction assay along with the activation/inhibition of ERα and AhR receptors. Aside from this, cell proliferation and migration as well as AhR‐dependent CYP1A1 enzyme activity were measured. Here, we found that the forced increased expression of miR‐206, miR‐133a, and miR‐27b were closely associated with the suppression of MCF‐7 cell proliferation and migration. The anti‐proliferative‐metastatic effect of miR‐206, miR‐133a, and miR‐27b was probably mediated by targeting the ERα and AhR signaling pathways. Considered together, our study indicated that the overexpression of miR‐206, miR‐133a, and miR‐27b might be potential biomarkers for prognosis and therapeutic strategies in breast cancer.     

miR‐1‐3p and miR‐206 sensitizes HGF‐induced gefitinib‐resistant human lung cancer cells through inhibition of c‐Met signalling and EMT

Hepatocyte growth factor (HGF) overexpression is an important mechanism in acquired epidermal growth factor receptor (EGFR) kinase inhibitor gefitinib resistance in lung cancers with EGFR activating mutations. MiR‐1‐3p and miR‐206 act as suppressors in lung cancer proliferation and metastasis. However, whether miR‐1‐3p and miR‐206 can overcome HGF‐induced gefitinib resistance in EGFR mutant lung cancer is not clear. In this study, we showed that miR‐1‐3p and miR‐206 restored the sensitivities of lung cancer cells PC‐9 and HCC‐827 to gefitinib in present of HGF. For the mechanisms, we demonstrated that both miR‐1‐3p and miR‐206 directly target HGF receptor c‐Met in lung cancer. Knockdown of c‐Met mimicked the effects of miR‐1‐3p and miR‐206 transfections Meanwhile, c‐Met overexpression attenuated the effects of miR‐1‐3p and miR‐206 in HGF‐induced gefitinib resistance of lung cancers. Furthermore, we showed that miR‐1‐3p and miR‐206 inhibited c‐Met downstream Akt and Erk pathway and blocked HGF‐induced epithelial‐mesenchymal transition (EMT). Finally, we demonstrated that miR‐1‐3p and miR‐206 can increase gefitinib sensitivity in xenograft mouse models in vivo. Our study for the first time indicated the new function of miR‐1‐3p and miR‐206 in overcoming HGF‐induced gefitinib resistance in EGFR mutant lung cancer cell.     

miR‐129‐2‐3p directly targets SYK gene and associates with the risk of ischaemic stroke in a Chinese population

Spleen tyrosine kinase (SYK) gene has been identified as novel susceptibility locus for ischaemic stroke (IS) previously. However, regulation of SYK gene remains unknown in IS. In this study, we aimed to identify miRNAs that might be involved in the development of IS by targeting SYK gene. miRNAs were firstly screened by bioinformatics predicting tool. The expression levels of SYK gene were detected by qRT‐PCR and western blotting, respectively, after miRNA transfection. Luciferase reporter assay was applied to investigate the direct binding between miRNAs and target gene. miRNA levels were detected by miRNA TaqMan assays in the blood cells of 270 IS patients and 270 control volunteers. Results suggest that SYK gene might be a direct target of miR‐129‐2‐3p. The blood level of miR‐129‐2‐3p was significantly lower in IS patients (P < 0.05), and negatively associated with the risk of IS (adjusted OR: 0.88; 95% CI: 0.80‐0.98; P = 0.021) by multivariable logistic regression analysis. The blood levels of SYK gene were significantly higher in IS patients, and miR‐129‐2‐3p expression was negatively correlated with mean platelet volume. In summary, our study suggests that miR‐129‐2‐3p might be involved in the pathogenesis of IS through interrupting SYK expression and the platelet function, and further investigation is needed to explore the underlying mechanism.     

Modified level of miR‐376a is associated with Parkinson's disease

Parkinson's disease (PD) is a frequent progressive neurodegenerative disorder. Impaired mitochondrial function is a major feature of sporadic PD. Some susceptibility or causative genes detected in PD are strongly associated with mitochondrial dysfunction including PGC1α, TFAM and GSK3β. microRNAs (miRNAs) are non‐coding RNAs whose altered levels are proven in disparate PD models and human brains. Therefore, the aim of this study was to detect modulations of miRs upstream of PGC1α, TFAM and GSK3β in association with PD onset and progress. In this study, a total of 33 PD subjects and 25 healthy volunteers were recruited. Candidate miRNA (miR‐376a) was selected through target prediction tools and literature survey. Chronic and acute in vitro PD models were created by MPP⁺‐intoxicated SHSY5Y cells. The levels of miR‐376a and aforementioned genes were assessed by RT‐qPCR. The expression of target genes was decreased in chronic model while there were dramatically up‐regulated levels of those genes in acute model of PD. miR‐376a was strongly altered in both acute and chronic PD models as well as PBMCs of PD patients. Our results also showed overexpression of PGC1α, and TFAM in PBMCs is inversely correlated with down‐regulation of miR‐376a, suggesting that miR‐376a possibly has an impact on PD pathogenesis through regulation of these genes which are involved in mitochondrial function. miR‐376a expression in PD‐derived PBMCs was also correlated with disease severity and may serve as a potential biomarker for PD diagnosis. This is the first study showing altered levels of miR‐376a in PD models and PBMCs, suggesting the probable role of this miRNA in PD pathogenesis. The present study also proposed TFAM and PGC1α as target genes of miR‐376a for the first time, through which it possibly can exert its impact on PD pathogenesis.