Population density regulates Drosophila synaptic morphology in a Fasciclin‐II‐dependent manner

Genetic analysis of the Drosophila larval neuromuscular junction has identified some of the key molecules that regulate synaptic plasticity. Among these molecules, the expression level of Fasciclin II (FasII), a homophilic cell adhesion molecule, is critically important for determining the final form of the neuromuscular junction. Genetic reduction of FasII expression by 50% yields more elaborate nerve terminals, while a greater reduction in expression, to 10% of wild‐type, yields a substantial reduction in the nerve terminal morphology. Importantly, regulation of FasII expression seems to be the final output for several genetic manipulations that transform NMJ morphology. In an effort to understand the importance of this regulatory pathway in the normal animal, we have undertaken studies to identify environmental cues that might be important for initiating FasII‐dependent changes in synaptic plasticity. Here we report on the relationship between larval population density and synaptic morphology, synaptic strength, and FasII levels. We raised Drosophila larvae under conditions of increasing population density and found an inverse exponential relationship between population density and the number of synaptic boutons, the number of branches, and the length of branches. We also observed population‐dependent alteration in FasII levels, with lower densities having less FasII at the synapse. The correlation between density and morphological change was abrogated in larvae constitutively expressing FasII, and in wild‐type larvae grown on soft culture medium. Together these data show that environmental cues can induce regulation of FasII. Interestingly, however, the quantal content of synaptic transmission was not different among the different population densities, suggesting that other factors contribute to maintaining synaptic strength at a defined level. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2004     

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‐21 promotes cardiac fibroblast‐to‐myofibroblast transformation and myocardial fibrosis by targeting Jagged1

Myocardial fibrosis after myocardial infarction (MI) is a leading cause of heart diseases. MI activates cardiac fibroblasts (CFs) and promotes CF to myofibroblast transformation (CMT). This study aimed to investigate the role of miR‐21 in the regulation of CMT and myocardial fibrosis. Primary rat CFs were isolated from young SD rats and treated with TGF‐β1, miR‐21 sponge or Jagged1 siRNA. Cell proliferation, invasion and adhesion were detected. MI model was established in male SD rats using LAD ligation method and infected with recombinant adenovirus. The heart function and morphology was evaluated by ultrasonic and histological analysis. We found that TGF‐β1 induced the up‐regulation of miR‐21 and down‐regulation of Jagged1 in rat CFs. Luciferase assay showed that miR‐21 targeted 3′‐UTR of Jagged1 in rat CFs. miR‐21 sponge inhibited the transformation of rat CFs into myofibroblasts, and abolished the inhibition of Jagged1 mRNA and protein expression by TGF‐β1. Furthermore, these effects of miR‐21 sponge on rat CFS were reversed by siRNA mediated knockdown of Jagged1. In vivo, heart dysfunction and myocardial fibrosis in MI model rats were partly improved by miR‐21 sponge but were aggravated by Jagged1 knockdown. Taken together, these results suggest that miR‐21 promotes cardiac fibroblast‐to‐myofibroblast transformation and myocardial fibrosis by targeting Jagged1. miR‐21 and Jagged1 are potential therapeutic targets for myocardial fibrosis.     

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.     

The relationship between myocardial fibrosis and myocardial microRNAs in dilated cardiomyopathy: A link between mir‐133a and cardiovascular events

It is unknown whether fibrosis‐associated microRNAs: miR‐21, miR‐26, miR‐29, miR‐30 and miR‐133a are linked to cardiovascular (CV) outcome. The study evaluated the levels of extracellular matrix (ECM) fibrosis and the prevalence of particular microRNAs in patients with dilated cardiomyopathy (DCM) to investigate any correlation with CV events. Methods: Seventy DCM patients (48 ± 12 years, EF 24.4 ± 7.4%) underwent right ventricular biopsy. The control group was comprised of 7 patients with CAD who underwent CABG and intraoperative biopsy. MicroRNAs were measured in blood and myocardial tissue via qPCR. The end‐point was a combination of CV death and urgent HF hospitalization at the end of 12 months. There were differential levels of circulating and myocardial miR‐26 and miR‐29 as well as myocardial miR‐133a when the DCM and CABG groups were compared. Corresponding circulating and myocardial microRNAs did not correlate with one another. There was no correlation between microRNA and ECM fibrosis. By the end of the 12‐month period of the study, CV death had occurred in 6 patients, and a further 19 patients required urgent HF hospitalization. None of the circulating microRNAs was a predictor of the combined end‐point; however, myocardial miR‐133a was an independent predictor in unadjusted models (HR 1.53; 95% CI 1.14‐2.05; P < .004) and adjusted models (HR 1.57; 95% CI 1.14‐2.17; P < .005). The best cut‐off value for the miR‐133a level for the prediction of the combined end‐point was 0.74 ΔCq, with an AUC of 0.67. The absence of a correlation between the corresponding circulating and myocardial microRNAs calls into question their cellular source. This study sheds new light on the role of microRNAs in ECM fibrosis in DCM, which warrants further exploration.     

Crosstalk between AMPK activation and angiotensin II‐induced hypertrophy in cardiomyocytes: the role of mitochondria

AMP‐kinase (AMPK) activation reduces cardiac hypertrophy, although underlying molecular mechanisms remain unclear. In this study, we elucidated the anti‐hypertrophic action of metformin, specifically, the role of the AMPK/eNOS/p53 pathway. H9c2 rat cardiomyocytes were treated with angiotensin II (AngII) for 24 hrs in the presence or absence of metformin (AMPK agonist), losartan [AngII type 1 receptor (AT1R) blocker], Nω‐nitro‐L‐arginine methyl ester (L‐NAME, pan‐NOS inhibitor), splitomicin (SIRT1 inhibitor) or pifithrin‐α (p53 inhibitor). Results showed that treatment with metformin significantly attenuated AngII‐induced cell hypertrophy and death. Metformin attenuated AngII‐induced activation (cleavage) of caspase 3, Bcl‐2 down‐regulation and p53 up‐regulation. It also reduced AngII‐induced AT1R up‐regulation by 30% (P < 0.05) and enhanced AMPK phosphorylation by 99% (P < 0.01) and P‐eNOS levels by 3.3‐fold (P < 0.01). Likewise, losartan reduced AT1R up‐regulation and enhanced AMPK phosphorylation by 54% (P < 0.05). The AMPK inhibitor, compound C, prevented AT1R down‐regulation, indicating that metformin mediated its effects via AMPK activation. Beneficial effects of metformin and losartan converged on mitochondria that demonstrated high membrane potential (Δψ_m) and low permeability transition pore opening. Thus, this study demonstrates that the anti‐hypertrophic effects of metformin are associated with AMPK‐induced AT1R down‐regulation and prevention of mitochondrial dysfunction through the SIRT1/eNOS/p53 pathway.     

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.     

MiR‐133a‐3p Inhibits Oral Squamous Cell Carcinoma (OSCC) Proliferation and Invasion by Suppressing COL1A1

The aim of our study was to investigate the effects of miR‐133a‐3p on human oral squamous cell carcinoma (OSCC) cells by regulating gene COL1A1. OSCC tissues, adjacent tongue epithelial tissues, the immortalized oral epithelial cell line HIOEC, and OSCC cell lines (CAL‐27, TCA‐8113, SCC‐4, SCC‐9, and SCC‐15) were used in this research. Quantitative real‐time PCR (RT‐qPCR) was employed to determine the expression of miR‐133a‐3p and COL1A1. Dual luciferase reporter gene assay and Western blot were applied to verify the binding relationship between miR‐133a‐3p and COL1A1. Functional assays were also conducted in this study, including CCK‐8 assay, colony formation assay, flow cytometry analysis as well as Transwell assay. MiR‐133a‐3p was found low‐expressed both in OSCC tissues and cells lines compared with normal tissues and cell line, respectively, whereas COL1A1 was just the opposite. The over‐expression of miR‐133a‐3p or the down‐regulation of COL1A1 suppressed the proliferation, invasion, and mitosis of OSCC cells, whereas simultaneous down‐regulation of miR‐133a‐3p and up‐regulation of COL1A1 led to no significant alteration of cell activities. MiR‐133a‐3p could inhibit the proliferation and migration of OSCC cells through directly targeting COL1A1 and reducing its expression. J. Cell. Biochem. 119: 338–346, 2018. © 2017 Wiley Periodicals, Inc.     

MiR‐451 is decreased in hypertrophic cardiomyopathy and regulates autophagy by targeting TSC1

The molecular mechanisms that drive the development of cardiac hypertrophy in hypertrophic cardiomyopathy (HCM) remain elusive. Accumulated evidence suggests that microRNAs are essential regulators of cardiac remodelling. We have been suggested that microRNAs could play a role in the process of HCM. To uncover which microRNAs were changed in their expression, microRNA microarrays were performed on heart tissue from HCM patients (n = 7) and from healthy donors (n = 5). Among the 13 microRNAs that were differentially expressed in HCM, miR‐451 was the most down‐regulated. Ectopic overexpression of miR‐451 in neonatal rat cardiomyocytes (NRCM) decreased the cell size, whereas knockdown of endogenous miR‐451 increased the cell surface area. Luciferase reporter assay analyses demonstrated that tuberous sclerosis complex 1 (TSC1) was a direct target of miR‐451. Overexpression of miR‐451 in both HeLa cells and NRCM suppressed the expression of TSC1. Furthermore, TSC1 was significantly up‐regulated in HCM myocardia, which correlated with the decreased levels of miR‐451. As TSC1 is a known positive regulator of autophagy, we examined the role of miR‐451 in the regulation of autophagy. Overexpression of miR‐451 in vitro inhibited the formation of the autophagosome. Conversely, miR‐451 knockdown accelerated autophagosome formation. Consistently, an increased number of autophagosomes was observed in HCM myocardia, accompanied by up‐regulated autophagy markers, and the lipidated form of LC3 and Beclin‐1. Taken together, our findings indicate that miR‐451 regulates cardiac hypertrophy and cardiac autophagy by targeting TSC1. The down‐regulation of miR‐451 may contribute to the development of HCM and may be a potential therapeutic target for this disease.     

Novel mechanism of cardiac protection by valsartan: synergetic roles of TGF‐β1 and HIF‐1α in Ang II‐mediated fibrosis after myocardial infarction

Transforming growth factor (TGF)‐β1 is a known factor in angiotensin II (Ang II)‐mediated cardiac fibrosis after myocardial infarction (MI). Hypoxia inducible factor‐1 (Hif‐1α) was recently demonstrated to involve in the tissue fibrosis and influenced by Ang II. However, whether Hif‐1α contributed to the Ang II‐mediated cardiac fibrosis after MI, and whether interaction or synergetic roles between Hif‐1α and TGF‐β pathways existed in the process was unclear. In vitro, cardiac cells were incubated under hypoxia or Ang II to mimic ischaemia. In vivo, valsartan was intravenously injected into Sprague–Dawley rats with MI daily for 1 week; saline and hydralazine (another anti‐hypertensive agent like valsartan) was used as control. The fibrosis‐related proteins were detected by Western blotting. Cardiac structure and function were assessed with multimodality methods. We demonstrated in vitro that hypoxia would induce the up‐regulation of Ang II, TGF‐β/Smad and Hif‐1α, which further induced collagen accumulation. By blocking with valsartan, a blocker of Ang II type I (AT1) receptor, we confirmed that the up‐regulation of TGF‐β/Smad and Hif‐1α was through the Ang II‐mediated pathway. By administering TGF‐β or dimethyloxalylglycine, we determined that both TGF‐β/Smad and Hif‐1α contributed to Ang II‐mediated collagen accumulation and a synergetic effect between them was observed. Consistent with in vitro results, valsartan significantly attenuated the expression of TGF‐β/Smad, Hif‐1α and fibrosis‐related protein in rats after MI. Heart function, infarcted size, wall thickness as well as myocardial vascularization of ischaemic hearts were also significantly improved by valsartan compared with saline and hydralazine. Our study may provide novel insights into the mechanisms of Ang II‐induced cardiac fibrosis as well as into the cardiac protection of valsartan.     

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.