Osteosarcoma (OS) is the most common primary pediatric malignancy of the bone having poor prognosis and long-term survival rates of less than 30% in patients with metastasis. MicroRNA-509 was reported to be downregulated in OS. We and others previously published that miR-509-3p can strongly attenuate cellular migration/invasion and sensitize ovarian cancer to cisplatin. Here, we show that overexpression of miR-509-3p inhibited migration of primary OS cell lines U2OS, HOS, and SaOS2 as well as metastatic derivatives 143B and LM7. miR-509-3p overexpression also inhibited proliferation and invasion of HOS and 143B cells and sensitized cells to cisplatin. Luciferase reporter assays using 3′-UTRs of predicted miR-509-3p targets associated with metastatic phenotypes revealed ARHGAP1 could be one of the downstream effectors of miR-509-3p in HOS. To find the global impact of miR-509-3p overexpression and cisplatin treatment we performed Reverse Phase Protein Analysis (RPPA). AXL, which has been reported to play a critical role in cisplatin resistance and confirmed as direct target of miR-509-3p was downregulated upon miR-509-3p treatment and further down-regulated upon miR-509-3p + cisplatin treatment. We propose that the miR-509-3p/AXL and miR-509-3p/ARHGAP1 axes have the potential to uncover new druggable targets for the treatment of drug resistant metastatic osteosarcoma.
Overactivation of the renin–angiotensin system (RAS) during obesity disrupts adipocyte metabolic homeostasis and induces endoplasmic reticulum (ER) stress and inflammation; however, underlying mechanisms are not well known. We propose that overexpression of angiotensinogen (Agt), the precursor protein of RAS in adipose tissue or treatment of adipocytes with Angiotensin II (Ang II), RAS bioactive hormone, alters specific microRNAs (miRNA), that target ER stress and inflammation leading to adipocyte dysfunction. Epididymal white adipose tissue (WAT) from B6 wild type (Wt) and transgenic male mice overexpressing Agt (Agt-Tg) in adipose tissue and adipocytes treated with Ang II were used. Small RNA sequencing and microarray in WAT identified differentially expressed miRNAs and genes, out of which miR-690 and mitogen-activated protein kinase kinase 3 (MAP2K3) were validated as significantly up- and down-regulated, respectively, in Agt-Tg, and in Ang II-treated adipocytes compared to respective controls. Additionally, the direct regulatory role of miR-690 on MAP2K3 was confirmed using mimic, inhibitors and dual-luciferase reporter assay. Downstream protein targets of MAP2K3 which include p38, NF-κB, IL-6 and CHOP were all reduced. These results indicate a critical post-transcriptional role for miR-690 in inflammation and ER stress. In conclusion, miR-690 plays a protective function and could be a useful target to reduce obesity.
Brain epigenetic microRNAs (miRNA) can provide integrated and rapid brain regulation during recovery from suicidal ideation (SI), and because brain miRNA enter the circulation, plasma miRNA may be markers for SI recovery. Therefore, we used genome-wide miRNA expression profiling to measure plasma miRNA changes during resolution of SI and assessed mRNA-targets using functional annotation analyses. We correlated plasma miRNA with Columbia-Suicide Severity Rating Scores (C-SSRS), depression and anxiety in 42 SI and 26 non-SI inpatients at admission and 4–6 weeks later in recovery (C-SSRS = 0). The 42 SI patients showed down-regulation of four miRNAs (hsa-miR-424-5p, hsa-miR-378i, hsa-miR-6724-5p, and hsa-miR-10b-5p) after recovery from SI, while these four miRNAs showed no change for non-SI patients. We validated these differentially expressed (DE) miRNAs by qRT-PCR, and luciferase assays confirmed miRNA functional activity in Clusterin for miR-424-5p and in SDC1 for miR-10b-5p. Depression and anxiety improved and correlated with changes in miR-6724-5p and miR-378i, respectively. The serum protein Clusterin also increased in the plasma of the 42 recovered patients. mRNA target prediction for miR-378i, miR-10b-5p, and miR-424-5p yielded 37 hub genes. KEGG analyses showed enrichment in 5 to 15 hub genes within six neuronal pathways previously identified as critical in depression and suicidality: MAPK, ErbB, AMPK, Ras, p53, and PI3K-Akt. Thus, these four plasma miRNA changes and the associated modulation of six depression and suicidality pathways in brain may reflect brain-related epigenetic changes specifically involved in recovery from SI rather than reflecting non-specific changes in miRNA also found among non-SI inpatients.
Obesity is a complex disease of global epidemic proportions. Several endocrine systems contribute to obesity, including the renin angiotensin system (RAS), which is overexpressed in adipose tissue during obesity. Overactivation of RAS induces endoplasmic reticulum (ER) stress and inflammation, both hallmarks of obesity; however, mechanisms underlying these interactions are not well known. MicroRNAs (miRNA) plays a pivotal role in post transcriptionally regulating genes involved in obesity and other disorders. Here we propose that changes in RAS alters miRNAs, which thereby regulate ER stress and obesity associated inflammation. This hypothesis was tested using male wild type C57BL/6 mice (Wt) and transgenic mice (Agt‐Tg) overexpressing angiotensinogen (Agt) in the adipose tissue. Mice were fed a low fat (LF) diet and white adipose tissues (WAT) were used to perform microarray and small RNA Seq. High‐throughput computational analyses of differentially expressed genes and miRNAs led to top candidates, which were subsequently validated in WAT. Moreover, specific gene targets for top miRNA candidates were identified using bioinformatic analyses (KEGG pathways, TargetScan). To further validate these miRNA‐gene targets we treated 3T3‐L1 adipocytes with miRNA mimics and inhibitors and tested mRNA expression of target genes. We also mutated the complementary binding sequences on the 3’UTR for positive target genes and validated them in cell culture by dual‐luciferase reporter assay. Here we present data related to miRNA 690 which was significantly altered by Agt overexpression. 23 and 8 miRNAs were upregulated and down regulated respectively in adipose tissue from Agt‐Tg mice. miR690 was significantly upregulated by 3‐fold in Agt‐Tg mice compared to Wt mice and it was also recognized as a potential regulator of several MAPKs including mitogen‐activated protein kinase kinase 3 (MAP2K3). Interestingly, in contrast to other inflammatory genes such as interleukin 6 (IL6) and several MAPKs which were significantly (p<0.05) upregulated in adipose tissue from Agt‐Tg mice compared to Wt mice, MAP2K3 was significantly down regulated in both Agt‐Tg compared to Wt mice and also in 3T3‐L1 cells treated with angiotensin II (Ang II) vs non treated controls. MiRNA 690 mimic treated adipocytes showed significantly reduced Map2k3 expression compared to control. This was further confirmed by significantly reduced luciferase activity for mir690 mimic. Furthermore, mutation of complementary binding sequence of MAP2K3 was unable to reduce luciferase activity, confirming the direct regulatory role of miRNA 690 on MAP2K3. Lastly, a downstream target of MAP2K3, IL‐6 also showed similar results at protein level confirming the involvement of miRNA 690 in regulating inflammation when RAS is overexpressed. In conclusion, miRNA 690 play a protective function with RAS overactivation and miR690 could be used as a potential target to reduce RAS‐induced obesity and related metabolic diseases. Support or Funding Information American Heart Association (NM...
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