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• Research Paper Volume 14, Issue 8 pp 3569-3596

  Effective biomarkers and therapeutic targets of nerve-immunity interaction in the treatment of depression: an integrated investigation of the miRNA-mRNA regulatory networks

  Relevance score: 4.8987412

  Zixuan Wu, Zhixiang Cai, Hongshuo Shi, Xuyan Huang, Minjie Cai, Kai Yuan, Peidong Huang, Guoqi Shi, Tao Yan, Zhichao Li

  Keywords: major depressive disorder (MMD), microRNAs (miRNAs), DE-miRNAs, regulatory networks, bioinformatics

  Published in Aging on April 25, 2022

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  Background: Major depressive disorder (MDD) is an emotional condition that interferes with sufferers’ work and daily life. Numerous studies have found that miRNAs play a significant role in the development of MDD and can be utilized as a biomarker for its diagnosis and therapy. However, there have been few studies on nerve-immunity interaction treatment for the brains of MMD patients. Methods: The work is performed on microarray data. We analyzed the differences of miRNAs (GSE58105, GSE81152, GSE152267, and GSE182194) and mRNA (GSE19738, GSE32280, GSE44593, GSE53987, and GSE98793) in MDD and healthy samples from GEO datasets. FunRich was used to predict the transcription factors and target genes of the miRNAs, and TF and GO enrichment analyses were performed. Then, by comparing the differential expression of the anticipated target genes and five mRNAs, intersecting mRNAs were discovered. The intersecting genes were submitted to GO and KEGG analyses to determine their functions. These intersecting potential genes and pathways that linked to MDD in neurological and immunological aspects have been identified for future investigation. Results: We discovered five hub genes: KCND2, MYT1L, GJA1, CHL1, and SNAP25, which were all up-regulated genes. However, in MMD, the equivalent miRNAs, hsa-miR-206 and hsa-miR-338-3p, were both down-regulated. These miRNAs can activate or inhibit the T cell receptor signal pathway, JAK-STAT and other signal pathways, govern immune-inflammatory response, neuronal remodeling, and mediate the onset and development of MMD Conclusions: The results of a thorough bioinformatics investigation of miRNAs and mRNAs in MDD showed that miR-338-3P and miR-206 might be effective biomarkers and possible therapeutic targets for the treatment of MDD via nerve-immunity interaction.

• Research Paper Volume 13, Issue 18 pp 21991-22029

  Identification of colorectal cancer associated biomarkers: an integrated analysis of miRNA expression

  Relevance score: 6.7795024

  André Fonseca, Sara Ventura Ramalhete, André Mestre, Ricardo Pires das Neves, Ana Marreiros, Pedro Castelo-Branco, Vânia Palma Roberto

  Keywords: microRNAs, biomarker, colorectal cancer, diagnosis, prognosis

  Published in Aging on September 21, 2021

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  Colorectal cancer is one of the leading causes of cancer-related deaths worldwide. This complex disease still holds severe problems concerning diagnosis due to the high invasiveness nature of colonoscopy and the low accuracy of the alternative diagnostic methods. Additionally, patient heterogeneity even within the same stage is not properly reflected in the current stratification system. This scenario highlights the need for new biomarkers to improve non-invasive screenings and clinical management of patients.

  MicroRNAs (miRNAs) have emerged as good candidate biomarkers in cancer as they are stable molecules, easily measurable and detected in body fluids thus allowing for non-invasive diagnosis and/or prognosis.

  In this study, we performed an integrated analysis first using 4 different datasets (discovery cohorts) to identify miRNAs associated with colorectal cancer development, unveil their role in this disease by identifying putative targets and regulatory networks and investigate their ability to serve as biomarkers. We have identified 26 differentially expressed miRNAs which interact with frequently deregulated genes known to participate in commonly altered pathways in colorectal cancer. Most of these miRNAs have high diagnostic power, and their prognostic potential is evidenced by panels of 5 miRNAs able to predict the outcome of stage II and III colorectal cancer patients. Notably, 8 miRNAs were validated in three additional independent cohorts (validation cohorts) including a plasma cohort thus reinforcing the value of miRNAs as non-invasive biomarkers.

• Research Paper Volume 13, Issue 15 pp 19282-19292

  Evaluation of the cargo contents and potential role of extracellular vesicles in osteoporosis

  Relevance score: 7.186321

  Jifeng Xu, Yu Chen, Dongsheng Yu, Li Zhang, Xiaofan Dou, Gang Wu, Yaping Wang, Shuijun Zhang

  Keywords: osteoporosis, extracellular vesicles, microRNAs, Wnt

  Published in Aging on August 10, 2021

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  Osteoporosis is a common aging-related disease diagnosed primarily using bone mineral density (BMD). Extracellular vesicles (EVs) remain unexplored in the context of osteoporosis. Towards this, EVs were isolated from plasma of a discovery cohort with 8 non-osteoporotic and 8 osteoporotic individuals, and nanoparticle tracking analysis (NTA) revealed a significantly higher EV concentration in osteoporotic individuals (P = 0.003). Moreover, EVs concentration showed a linear correlation with bone mineral density (BMD) values (linear correlation coefficient r = 0.9542, deviation from zero, p < 0.001). Results using a mouse model of osteoporosis confirmed that the number of EVs in mice from hindlimb unloading group was significantly higher than that from the age-matched control group (p = 0.015). TaqMan Real-Time PCR demonstrated that miR-335-5p, -320a, -483-5p, and miR-21-5p, were significantly higher expressed in osteoporotic patients compared with non-osteoporotic individuals. Quantitative real-time PCR shown that Wnt1, Wnt5a, Wnt7a, and Wnt9a mRNAs were lower expressed in osteoporosis derived EVs. In vitro functional assay indicated that osteoporosis derived EVs resulted in reduced mineralization in SaOS-2 cells. In conclusion, these results suggest that osteoporosis increased the secretion of EVs which carry higher expression of miRNAs and decreased expression of Wnt signals, further decreased the mineralization capacity in human osteoblasts.

• Research Paper Volume 13, Issue 15 pp 19805-19821

  MiR-195-5p and miR-205-5p in extracellular vesicles isolated from diabetic foot ulcer wound fluid decrease angiogenesis by inhibiting VEGFA expression

  Relevance score: 6.1791496

  Jing Liu, Jiahuan Wang, Wan Fu, Xiaoyi Wang, Hongxing Chen, Xiaoying Wu, Guojuan Lao, Yuxi Wu, Mengdie Hu, Chuan Yang, Li Yan, Meng Ren

  Keywords: extracellular vesicles, diabetic foot ulcers, microRNAs, angiogenesis, wound fluid

  Published in Aging on August 9, 2021

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  Diabetic foot ulcers are recalcitrant to healing, and poor angiogenesis is considered as the main contributing factor. We aimed to explore the effect of extracellular vesicles (EVs) derived from wound fluids on new vessel formation in diabetic foot ulcers. EVs were isolated from wound fluids of diabetic foot ulcers (DF-EVs). The inhibitory effect of DF-EVs on human umbilical vein endothelial cells (HUVECs) and wound healing was tested. To elucidate the potential mechanism of these effects, we screened the differentially expressed microRNAs (miRNAs) in DF-EVs via microarray analysis and verified the upregulation of miR-195-5p and miR-205-5p in DF-EVs via quantitative real-time polymerase chain reaction (qRT-PCR). Further dual-luciferase reporter assays and overexpression experiments proved these two miRNAs inhibited the expression of vascular endothelial growth factor A (VEGFA) directly to the 3′ untranslated region (UTR) of VEGFA and, in turn, promoted an inhibitory effect of DF-EVs on angiogenesis and wound healing in patients with diabetic foot ulcers. Our study shows EVs in the wound fluids of diabetic foot ulcer lesions carrying antiangiogenic miR-195-5p and miR-205-5p negatively regulated angiogenesis and wound healing in patients with diabetic foot.

• Research Paper Volume 13, Issue 15 pp 19657-19677

  Diagnostic performance of microRNAs in testicular germ cell tumors: a systematic review and meta-analysis

  Relevance score: 5.8846555

  Xi-Yi Zhao, Yu-Lu Gao, Dan-Feng Li, Hong-Chao Liu, Rui-Fang Zhu, Chang-Tai Zhu

  Keywords: microRNAs, testicular germ cell tumors, diagnosis, biomarker, meta-analysis

  Published in Aging on August 3, 2021

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  The sensitivity (Sen) of classic biomarkers for the diagnosis of testicular germ cell tumors (TGCTs) is currently low. Previous studies have shown the diagnostic potential of microRNAs (miRNAs) for TGCTs; however, the results of these studies are inconsistent. Therefore, we conducted a systematic review and meta-analysis to evaluate their diagnostic value. PubMed, EMBASE, Cochrane Library, and Web of Science databases were systematically searched until September 30, 2020 and 18 trials from 11 studies involving 2,068 participants were included in this meta-analysis. Using a bivariate mixed-effects meta-analysis model, the pooled Sen, specificity (Spe), positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and area under the curve (AUC) with 95% confidence interval values of total miRNAs were 0.83 (0.73–0.90), 0.95 (0.89–0.98), 15.79 (7.41–33.66), 0.18 (0.11–0.29), 87.13 (41.99–180.82), and 0.95 (0.93–0.97), respectively; however, the observed values of single miR-371a-3p were  0.84 (0.76–0.90), 0.95 (0.91–0.98), 18.41 (9.69–34.97), 0.17 (0.11–0.26), 111.56 (47.72–260.80), and 0.97 (0.95–0.98), respectively. Subgroup analysis revealed that miRNAs that included miR-371a-3p showed higher predictive performance than those that did not (P < 0.05). This research identified that miR-371a-3p has a high diagnostic value for TGCTs, except teratoma.

• Research Paper Volume 13, Issue 11 pp 15384-15399

  CXCR5 induces perineural invasion of salivary adenoid cystic carcinoma by inhibiting microRNA-187

  Relevance score: 4.9822216

  Mei Zhang, Jia-Shun Wu, Hong-Chun Xian, Bing-Jun Chen, Hao-Fan Wang, Xiang-Hua Yu, Xin Pang, Li Dai, Jian Jiang, Xin-Hua Liang, Ya-Ling Tang

  Keywords: salivary adenoid cystic carcinoma (SACC), CXCR5, perineural invasion (PNI), microRNAs (miRNAs), Schwann cells

  Published in Aging on June 10, 2021

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  CXCR5 played critical roles in tumorigenesis and metastasis. Nevertheless, little was known about the involvement of CXCR5 in perineural invasion (PNI) of salivary adenoid cystic carcinoma (SACC). Here, we confirmed upregulation of CXCR5 in SACC specimens and cells and identified that CXCR5 exhibited a significant positive correlation with PNI. Functionally, knockdown of CXCR5 suppressed SACC cells migration, invasion and PNI ability, whereas CXCR5 overexpression displayed the opposite effects. Moreover, CXCR5 downregulated microRNA (miR)-187, which could competitively sponge S100A4. The PNI-inhibitory effect of CXCR5 knockdown or miR-187 overexpression could be reversed by elevated expression of S100A4. Conjointly, our data revealed that CXCR5 facilitated PNI through downregulating miR-187 to disinhibit S100A4 expression in SACC.

• Research Paper Volume 13, Issue 6 pp 8177-8203

  Genetic predisposition and bioinformatics analysis of ATP-sensitive potassium channels polymorphisms with the risks of elevated apolipoprotein B serum levels and its related arteriosclerosis cardiovascular disease

  Relevance score: 4.5208597

  Cheng Liu, Tianwang Guan, Yanxian Lai, Junfang Zhan, Yan Shen

  Keywords: polymorphism, ATP-sensitive potassium channels, apolipoprotein B, arteriosclerosis cardiovascular disease, exosome-derived microRNAs

  Published in Aging on March 3, 2021

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  Serum concentration of apolipoprotein B (Apo B) is causally associated with arteriosclerosis cardiovascular disease (ASCVD) risk. Whether ATP-sensitive potassium channels (KATP) variants predict the risk of increased Apo B concentration (≥ 80 mg/dL) and related ASCVD remain less clear. We recruited 522 subjects with elevated Apo B concentration (≥ 80 mg/dL) and 522 counterpart subjects (< 80 mg/dL) from South China to assess the associations of KATP variants (rs11046182, rs78148713, rs145456027 and rs147265929) with the risks of increased Apo B serum concentration (≥ 80 mg/dL), carotid artery stenosis (CAS) ≥ 50% and new-onset ischemic stroke (IS). Our results showed that only KATP SNP rs11046182 (GG genotype) was associated with increased risk of Apo B ≥ 80 mg/dL (adjusted OR=2.17, P<0.001) and CAS ≥ 50% (adjusted OR=2.63, P=0.011). After median 50.6-months follow-up, subjects carrying GG genotype of rs11046182 were associated with higher risk of new-onset IS (adjusted HR=2.24, P=0.024). Further, the exosome-derived microRNAs (exo-miRs) expression profile was identified by next-generation sequencing. 41 exo-miRs were significantly differentially expressed under cross-talk status between high Apo B level (≥ 80 mg/dL) and KATP rs11046182. Our study demonstrated that KATP variant rs11046182 was associated with higher risks of elevated serum Apo B levels and its related ASCVD, and the possible mechanism was related to specific exo-miRs expression profile of KATP rs11046182.

• Research Paper Volume 13, Issue 5 pp 7120-7132

  Hypoxic pancreatic stellate cell-derived exosomal mirnas promote proliferation and invasion of pancreatic cancer through the PTEN/AKT pathway

  Relevance score: 5.6283875

  Wenpeng Cao, Zhirui Zeng, Zhiwei He, Shan Lei

  Keywords: pancreatic cancer, exosomal microRNAs, PTEN/AKT pathway, metastasis

  Published in Aging on February 26, 2021

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  Pancreatic stellate cells (PSCs) are important components of the tumor microenvironment in pancreatic cancer (PC) and contribute to its development and metastasis through mechanisms that remain incompletely characterized. Tumor hypoxia affects the function and behavior of PC and stromal cells, and can alter exosomal content to modify cell-cell communication. The present study explored the effects of exosomal miRNAs produced by hypoxia-preconditioned PSCs on the growth and metastatic potential of PC cells. Subcutaneous xenografts and liver metastasis mouse models revealed increased tumorigenic potential upon co-implantation of PC cells and PSCs as compared to PC cells alone. Screening miRNA profiles of mouse plasma exosomes and cultured PSCs, followed by miRNA overexpression and inhibition assays, enabled us to identify miR-4465 and miR-616-3p as prominent hypoxia-induced, PSC-derived, exosomal miRNAs promoting PC cell proliferation, migration, and invasion. Proteomics analysis of PC cells incubated with exosomes derived from hypoxic PSCs showed significant downregulation of PTEN. Dual-luciferase reporter assays and western blotting showed that both miR-4465 and miR-616-3p target PTEN and activate AKT signaling in PC cells. We conclude that hypoxia upregulates miR-4465 and miR-616-3p expression in PSC-derived exosomes. Following exosome uptake, these miRNAs promote PC progression and metastasis by suppressing the PTEN/AKT pathway.

• Research Paper Volume 13, Issue 4 pp 5946-5966

  Multi-omics analysis identifies potential mechanisms of AURKB in mediating poor outcome of lung adenocarcinoma

  Relevance score: 6.192831

  Jie Huang, Qianyun Zhang, Juan Shen, Xueqin Chen, Shenglin Ma

  Keywords: AURKB, lung adenocarcinoma, prognosis, methylation, microRNAs

  Published in Aging on February 17, 2021

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  Aurora kinases B (AURKB), which plays a critical role in chromosomal segmentation and mitosis, greatly promotes cell cycle progression and aggressive proliferation of cancers. So far, its role and underlying mechanisms in mediating poor outcome of lung adenocarcinoma (LUAD) remained largely unclear. Analyses on multiple omics data of lung adenocarcinoma cohort in The Cancer Genome Atlas (TCGA) were performed based on AURKB expression, and demonstrated its association with clinical characteristics and the potential of using AURKB as a biomarker in predicting patients’ survival. This study found aberrant alterations of genomics and epigenetics, including up-regulation and down-regulation of oncogenic genes and tumor suppressors, pathways involved in the cell cycle, DNA repair, spliceosome, and proteasome, hypermethylation enrichments around transcriptional start sites, which are all related to AURKB expression. We further discovered the possible role of tumor suppressors DLC1 and HLF in AURKB-mediated adverse outcome of LUAD. To conclude, this study proved AURKB as a potential prognostic factor and therapeutic target for lung adenocarcinoma treatment and provide a future research direction.

• Research Paper Volume 12, Issue 17 pp 17062-17078

  Evidence that dysplasia related microRNAs in Barrett’s esophagus target PD-L1 expression and contribute to the development of esophageal adenocarcinoma

  Relevance score: 5.3455567

  Juanjuan Xu, Zhongyuan Yin, Lin Yang, Feng Wu, Jinshuo Fan, Qi Huang, Yang Jin, Guanghai Yang

  Keywords: microRNAs, PD-L1, Barrett’s esophagus, esophageal adenocarcinoma

  Published in Aging on September 9, 2020

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  Esophageal adenocarcinoma (EAC) is the cancer arising from the esophagus, which frequently develop from Barrett’s esophagus (BE). Extracellular vesicles (EVs), particularly exosomes, are nanosized vesicles of endosomal origin released from various types of cells that have been implicated in cancers. However, the significance of circulating exosomes during the progression of BE to EAC remains unknown. Sera exosmal microRNAs were profiled from 13 EAC and 12BE patients compared to 12 healthy controls. We found a substantial dysregulation of exosomal miRNA levels in BE compared to healthy control, and identified a unique signature of 24 up regulated and 14 down regulated miRNAs. Further validation showed exosomal miR-196a, -26b, -21, and -143 expression was significantly higher in BE and continued to have higher levels in EAC compared to healthy controls; while sera exosomal miR-378, -210, -205, and -200c-3p were significantly lower expressed in BE patients compared to compared to controls. Further, miR-378, -210, -205, and -200c-3p continue to have even lower levels in EAC patients compared to BE. Interestingly, sera expression levels of exosomal miR-15a, -16, and -193a-3p were significantly down regulated in BE PD-L1(+) patients; Sera exosomal miR-15a, -15b, -16, and -193a-3p expression levels in EAC PD-L1(+) patients were significantly lower (all p < 0.01) when compared to EAC PD-L1(-) patients. More importantly, the BE-EAC group had longitudinally decreased exosomal expression levels of miR-15a, -15b, -16, and -193a-3p from BE status to their EAC progression. In conclusion, distinct microRNA expression patterns were demonstrated in circulating exosomes from Barrett’s esophagus and esophageal adenocarcinoma; Furthermore exosomal microRNAs potentially targeting PD-L1 mRNA were down regulated in PD-L1 (+) BE and EAC patients.

• Research Paper Volume 12, Issue 13 pp 12841-12849

  miR-27-3p inhibition restore fibroblasts viability in diabetic wound by targeting NOVA1

  Relevance score: 6.554378

  Peng Zhang, Xiaomei Song, Qirong Dong, Long Zhou, Lei Wang

  Keywords: microRNAs, fibroblast, NOVA1, proliferation, migration

  Published in Aging on June 26, 2020

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  Diabetic wounds increase morbidity and decrease quality of life in patients with type 2 diabetes. Serum miR-27-3p levels are reportedly elevated in type 2 diabetic patients. In the present study, we explored the role of miR-27-3p during wound healing. We found that miR-27-3p is overexpressed in cutaneous fibroblasts of diabetic patients and mice. miR-27-3p knockdown enhanced the proliferation and migration of fibroblasts, while suppressing the incidence of fibroblast apoptosis. Overexpressing miR-27-3p in fibroblasts had the opposite effects. We also identified neuro-oncological ventral antigen 1 (NOVA1) as a target of miR-27-3p in fibroblasts. Knocking down NOVA1 using targeted siRNA mimicked the effects of miR-27-3p overexpression in fibroblasts. Administration of miR-27-3p to the area around wounds inflicted in mice delayed healing of those wounds. This suggests that miR-27-3p suppresses fibroblast function by targeting NOVA1, which results in the slowing of wound healing. These findings may offer a new approach to the treatment of diabetic wound healing.

  

  miR-27-3p is upregulated in fibroblasts from diabetic wounds. (A) miR-27-3p levels in fibroblasts from wounds in diabetic and otherwise healthy patients. (B) miR-27-3p level in fibroblasts from normal and wounds in diabetic and healthy mice.

  
  
  

  

  miR-27-3p impairs fibroblast function in vitro. (A) qRT-PCR was used to detected levels of miR-27-3p expression. (B) CCK8 assays were used to assess the viability of fibroblasts. (C) Fibroblast migration was evaluated using transwell assays. (D) Flow cytometry was used to evaluate apoptosis (Q2+Q3) among fibroblasts: Q1, dead cells; Q2, later apoptosis; Q3, early apoptosis; Q4, living cells. (E, F) pro-apoptotic and anti-apoptotic proteins were assessed with Western blotting and qRT-PCR. (G, H) The ECM-related proteins collagen III, MMP1 and MMP3 were evaluated with Western blotting and qRT-PCR.

  
  
  

  

  NOVA1 is a novel target of miR-27-3p. (A) The predicted miR-27-3p binding site within the NOVA1 3’-UTR was determined by Targetscan (Upper). miR-27-3p suppresses NOVA1 3’-UTR reporter activity (Lower). (B) qRT-PCR analysis showing that NOVA1 expression is suppressed in fibroblasts transfected with agomiR-27-3p. (C) CCK8 assays used to assess fibroblast proliferation. (D) Transwell assays were used to assess fibroblast migration capacity. (E, F) Expression of pro-apoptotic and anti-apoptotic proteins was detected with qRT-PCR and Western blotting. (G) Flow cytometry evaluating the cell cycle in fibroblasts. (H, I) The ECM-related proteins collagen III, MMP1 and MMP3 were evaluated with Western blotting and qRT-PCR.

  
  
  

  

  Downregulation of miR-27-3p promotes wound healing in vivo. (A) Digital photo of wounds treated with PBS, agomiR-27-3p, or antagomiR-27-3p. (B) Rate of wound-closure of the three groups. (C) Masson’s trichrome staining of wound sections treated with PBS, agomiR-27-3p, or antagomiR-27-3p. (D) Quantitative analysis of the mean intensity of Masson-stained areas in the three groups. (E) qRT-PCR evaluating the level of collagen 3, MMP1 and MMP3 expression. n=5. *p<0.05, **p<0.01, ***p<0.001.

  
  
  

• Research Paper Volume 12, Issue 12 pp 12324-12341

  Ovarian aging increases small extracellular vesicle CD81⁺ release in human follicular fluid and influences miRNA profiles

  Relevance score: 6.442945

  Rosalia Battaglia, Paolo Musumeci, Marco Ragusa, Davide Barbagallo, Marina Scalia, Massimo Zimbone, Josè Maria Lo Faro, Placido Borzì, Paolo Scollo, Michele Purrello, Elena Maria Vento, Cinzia Di Pietro

  Keywords: reproductive aging, extracellular vesicles, microRNAs, follicular fluid

  Published in Aging on June 17, 2020

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  Ovarian aging affects female reproductive potential and is characterized by alterations in proteins, mRNAs and non-coding RNAs inside the ovarian follicle. Ovarian somatic cells and the oocyte communicate with each other secreting different molecules into the follicular fluid, by extracellular vesicles. The cargo of follicular fluid vesicles may influence female reproductive ability; accordingly, analysis of extracellular vesicle content could provide information about the quality of the female germ cell.

  In order to identify the most significant deregulated microRNAs in reproductive aging, we quantified the small extracellular vesicles in human follicular fluid from older and younger women and analyzed the expression of microRNAs enclosed inside the vesicles. We found twice as many small extracellular vesicles in the follicular fluid from older women and several differentially expressed microRNAs. Correlating microRNA expression profiles with vesicle number, we selected 46 deregulated microRNAs associated with aging. Bioinformatic analyses allowed us to identify six miRNAs involved in TP53 signaling pathways. Specifically, miR-16-5p, miR214-3p and miR-449a were downregulated and miR-125b, miR-155-5p and miR-372 were upregulated, influencing vesicle release, oocyte maturation and stress response. We believe that this approach allowed us to identify a battery of microRNAs strictly related to female reproductive aging.

  

  Morphological and Molecular Characterization of Extracellular Vesicles (EVs) from Follicular Fluid (FF) of older and younger women. (A, B) Scanning Electron Micrographs of EVs isolated from the FF of older (A) and younger women (B) showing the presence of vesicles of spherical shape with a higher abundance in FFs from older women. (C) Diameter distribution of EVs from FFs of older and younger women. Gauss fit of the diameters measured on SEM microscopies shows an EV average diameter of 149 ± 50 nm for FFs from older women (red curve) and of 142 ± 20 nm for those of FFs from younger women (blue trend). (D) The TEM analysis shows that a small Gold (Au) nanoparticle functionalized with an antibody specific for the CD81 protein marker binds to the membrane of small EVs from the FF of younger women.

  
  
  

  

  DLS and NTA analysis. (A) EV size characterization by DLS and NTA. No variation is observed for the EV size in older and younger women. A small difference in the average diameters of EVs is generally found between DLS (where the hydrodynamic radius is measured) and SEM and NTA (that measure the actual radius). (B) Dynamic Light Scattering (DLS) measurements and Nanoparticle tracking analysis (NTA) of small EVs from FFs of older and younger women. Scattered light Intensity, expressed as Kilocounts-per-second (Kcps), and nanoparticle concentration for FFs are shown as red and blue bars respectively. (C) Significant positive linear relationship between DLS and NTA measurements on FF samples. Pearson’s coefficient of correlation (r), R2, and P-value are reported. (D) DLS and (E) NTA show a significantly higher concentration of exosomes in FFs from older women. Kcps and concentration differences related to small EVs of younger and older women are reported as Box plots with Whiskers. The ratio of small EV concentrations ® between younger and older women for DLS and NTA is shown. ** P-values<0.01; * P-value <0.05.

  
  
  

  

  Comparison of DE miRNA sets obtained by two normalization methods for RT-qPCR. Differential expression analysis of miRNAs from FF EVs, comparing older women to younger women and using two normalization methods revealed 64 DE miRNAs (gFC), without considering the number of EVs, and 80 DE miRNAs (vFC) taking into account the number of vesicles. Comparison between gFC and vFC datasets identified 4 classes of DE miRNAs (A-D) for which we show a possible distribution (represented by vertical bars) within the individual EVs from the FF of the two groups of women according to an EV ratio of 2:1 (older vs younger).

  
  
  

  

  GO enrichment analysis for 46 DE common miRNAs identified by two normalization methods. Bar chart representing the most highly statistically enriched Gene Ontologies, in terms of Biological Processes, for DE miRNA targets in female reproductive aging. The x-axis represents the -log₁₀(P-value). The number of target genes in each GO category is shown.

  
  
  

  

  Signaling Pathway enrichment analysis for 46 DE common miRNAs with KEGG. The x-axis represents the -log₁₀(P-value). The number of target genes in each molecular pathway is shown.

  
  
  

  

  DE miRNAs with female aging control target mRNAs within complex regulatory networks of (A) vesicle secretion, (B) oocyte maturation and (C) stress response. DE common miRNAs are highlighted.

  
  
  

  

  Hypothetical model of miRNAs mediating the regulation of TP53 in the ovarian follicle in female reproductive aging. Potential mechanisms by which specific ovarian follicle cell subpopulations can send and receive stress signaling, via EVs, in ovarian aging. MiR-155 expression is induced by stress in granulosa cells. TP53 activation, mediated by the downregulation of miR-16 and miR-214, can initiate apoptosis or lead to cellular senescence and the activation of miR-372 or cause the increase of EV secretion. The upregulation of miR-125 and the downregulation of miR-449 could represent a defense mechanism implemented by follicular cells and oocyte to repress TP53 expression and stress-induced apoptosis.

  
  
  

• Research Paper Volume 11, Issue 24 pp 12278-12294

  Insulin-like growth factor-1 enhances neuroprotective effects of neural stem cell exosomes after spinal cord injury via an miR-219a-2-3p/YY1 mechanism

  Relevance score: 6.472707

  Ke Ma, Huiyou Xu, Jian Zhang, Fei Zhao, Haiqian Liang, Hongtao Sun, Ping Li, Sai Zhang, Renjie Wang, Xuyi Chen

  Keywords: spinal cord injury (SCI), microRNAs (miRNAs), exosomes, apoptosis

  Published in Aging on December 17, 2019

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  Spinal cord injury (SCI) remains the most common cause of paralysis, and there are no effective therapies for SCI patients. Neural stem cell (NSC)-derived exosomes can attenuate apoptosis and neuroinflammation after traumatic spinal cord injury, but the mechanisms underlying these effects remain unclear. Here, we examined the efficacy of miRNAs isolated from exosomes as treatments for SCI and characterized their mechanisms of action. Furthermore, we evaluated the effects of exosomes formed in the presence of insulin growth factor-1 (IFG-1, IGF-Exo), which promotes neural proliferation and regeneration, as well as normal exosomes (Nor-Exo) and compared control and H₂O₂-treated groups both in vitro and in vivo. Using microRNA sequencing and qRT-PCR, we identified miR-219a-2-3p, levels of which were higher in the IGF-Exo than Nor-Exo group and played crucial anti-inflammatory and anti-apoptosis roles. Additional experiments revealed that IGF-Exo inhibits YY1 expression through up-regulation of miR-219a-2-3p. This in turn inhibits the NF-κB pathway, partly inhibiting neuroinflammation and promoting the neuroprotective effects after SCI.

  

  Characteristics of neural stem cells (NSCs) and exosomes derived from NSCs. (A) Morphology of neurospheres with typical shape examined by light microscopy. (B) Nestin immunofluorescence (red), a marker of NSCs, in neurospheres. (C) Exosome morphology examined by transmission electron microscopy. (D) Western blot analysis of exosome surface markers. (E) Particle size distribution of Nor-Exo and IGF-Exo by Nano Sight.

  
  
  

  

  IGF-Exo inhibited H₂O₂-induced neural apoptosis in PC12 cells invitro. (A) Morphology for each experimental group examined by light microscopy (control group: PC12 cells without treatment; H₂O₂ group: PC12 cells treated with H₂O₂ for 24h; Nor-Exo group: PC12 cells pretreated with Nor-Exo for 24h followed by H₂O₂ for 24h; IGF-Exo group: PC12 cells pretreated with IGF-Exo for 24h followed by H₂O₂ for 24h). (B) Cell viability in each experimental group; (C) TUNEL staining (red) indicating cell apoptosis in each experimental group; nuclear DAPI stain in blue. (D) TUNEL-positive cell numbers per field for each experimental group. (E) Western Blot analysis of apoptotic and anti-apoptotic proteins. (F–I) Relative expression of BAX, Bcl-2, Beclin-1 and Caspase-3. Data are expressed as means ± SD (analysis of variance followed by Student-Newman-Keuls post hoc test). **P < 0.01 vs. control group; ##P < 0.01 vs. H₂O₂ group.

  
  
  

  

  IGF-Exo inhibited neural apoptosis and neuroinflammation after SCI invivo. (A) Schematic of tail intravenous injections of Nor-Exo and IGF-Exo in SCI model rats. (B) DTIs constructed for the SCI, Nor-Exo, and IGF-Exo groups at 1 day and 28 days after surgery. (C) Neuroelectrophysiological examination results for each experimental group (control, SCI, Nor-Exo, and IGF-Exo) at 3 days and 28 days after surgery. (D) MEP amplitudes for each experimental group at 3 days and 28 days after surgery. (E) Hematoxylin-Eosin staining of sections containing SCI lesions in each experimental group at 3 days and 28 days after surgery. (F) Cavity space percentages for each experiment group at 3 days and 28 days after surgery. (G) BrdU and NeuN immunofluorescence indicative of neuron regeneration in each experimental group at 28 days after surgery. (H) TUNEL staining (green) indicative of apoptosis after SCI in each experimental group; DAPI in blue. (J) Numbers of TUNEL-positive cells per field for each experimental group. (K–O) Western Blot analysis of pro- and anti-apoptotic proteins (BAX, Bcl-2, Beclin-1, and Caspase-3). Data are expressed as means ± SD (analysis of variance followed by Student-Newman-Keuls posthoc test). **P < 0.01, vs. control group; ##P < 0.01, vs. SCI group.

  
  
  

  

  Screening and identification of upregulated miRNAs in Nor-Exo and IGF-Exo. (A) Heat map showing the 6 miRNAs for which expression was upregulated ≥ 2-fold in IGF-Exo compared to Nor-Exo. (B) IGF-1-induced miRNA regulation network. (C) Among the 6 upregulated miRNAs, miR-219a-2-3p expression increased the most. (D) qRT-PCR comparison of rno-miR-219a-2-3p, 138-5p, 92b-3p, 92b, 25-5p, and 674-3p expression between Nor-Exo and IGF-Exo from 3 independent experiments. *P < 0.05, **P < 0.01 for IGF-Exo vs. Nor-Exo.

  
  
  

  

  Inhibition of miR-219a-2-3p in exosomes reduced the protective effects of IGF-Exo. (A) Luciferase activity was detected in PC12 cells co-transfected with pGL3 vector, pGL3-YY1, miR-219a-2-3p mimics, or miR-219a-2-3p mutant. (B) Relative YY1 expression after upregulation of miR-219a-2-3p measured by qRT-PCR. B) PC12 cells were transfected with miR-219a-2-3p mimics or miR-NC; relative YY1 expression was analyzed by qRT-PCR. (C) PC12 cells were transfected with Anti-miR-219a-2-3p (miR-219a-2-3p inhibitor) or Anti-NC (control); relative YY1 expression was analyzed by qRT-PCR. (D) Morphology in each experimental group examined by light microscopy (control group: PC12 cells without treatment; H₂O₂ group: PC12 cells treated with H₂O₂ for 24h; IGF-Exo group: PC12 cells pretreated with IGF-Exo for 24h followed by H₂O₂ for 24h); Anti-miR-219a-2-3p-IGF-Exo group: PC12 cells pretreated with miR-219a-2-3p inhibitor-transfected IGF-Exo for 24h followed by H₂O₂ for 24h). (E) Cell viability in each experimental group when miR-219a-2-3p was downregulated in IGF-Exo; (F) TUNEL staining (red) indicative of apoptosis in each experimental group; DAPI in blue. (G) Numbers of TUNEL-positive cells per field for each experiment group. (H–J) Western Blot analysis of YY1 and NF-kB-p65 (si-NC: siRNA blank vector; miR-NC: miRNA blank vector; si-YY1: YY1 siRNA; miR-219a-2-3p: miR-219a-2-3p mimics). Data are expressed as means ± SD (analysis of variance followed by Student-Newman-Keuls post hoc test). **P < 0.01, vs. control group; ##P < 0.01, vs. SCI group.

  
  
  

• Research Paper Volume 11, Issue 22 pp 10374-10384

  Hypoxia-induced microRNA-10b-3p promotes esophageal squamous cell carcinoma growth and metastasis by targeting TSGA10

  Relevance score: 6.442945

  Qiang Zhang, Jingjing Zhang, Zhanzhao Fu, Lixin Dong, Yong Tang, Chunlei Xu, Haifeng Wang, Tao Zhang, Yue Wu, Chao Dong, Shasha Shao, Guangxia Wang

  Keywords: microRNAs, hypoxia, esophageal squamous cell carcinoma, metastasis

  Published in Aging on November 26, 2019

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  Evidence has shown that hypoxia promotes esophageal squamous cell carcinoma (ESCC) growth and metastasis, but the molecular mechanisms underlying that response remain poorly understood. MicroRNAs (miRNAs) are post-transcriptional regulators that participate in various cancer-related processes. Here, we demonstrated that hypoxia along with hypoxia-inducible factor 1α significantly increased expression of miR-10b-3p. Inhibition of miR-10b-3p weakened the effects of hypoxia on ESCC cell proliferation, migration and invasion, while miR-10b-3p overexpression had the opposite effects. Mechanistically, miR-10b-3p acted as cancer-promoting gene by targeting testis specific 10. Using a xenograft model, we observed that administration of miR-10b-3p agomir to tumors enhanced their growth and metastasis in vivo. These findings verified the potent regulatory role played by hypoxia-induced miR-10b-3p expression in ESCC progression. These results suggest that miR-10b-3p may be a useful therapeutic target for treating ESCC.

  

  Hypoxia induced miR-10b-3p expression and its target validation. ECA109 cells transfected with sh-HIF-1α or pcDNA- HIF-1α were incubated for 48 hours under normoxic or hypoxic conditions. (A) Western blotting showed HIF-1α expression. (B) Quantification of miR-10b-3p expression using qRT-PCR. (C) ECA109 and KYSE410 cells were transfected with miR-10b-3p mimics or mimics NC, after which TSGA10 expression was assessed by western blotting. (D) Predicted miR-10b-3p wild-type binding sites (WT) or mutant binding sites (MUT) were cloned into a luciferase reporter. Cells co-transfected with miR-10b-3p mimics or controls and WT or MUT luciferase constructs were subjected to luciferase assay. (E) Measurement of luciferase activity. *P < 0.05.

  
  
  

  

  Hypoxia-induced miR-10b-3p enhances ESCC cell proliferation. (A) ECA109 and KYSE410 cells transfected with miR-10b-3p mimics or pc-DNA TSGA10 were cultured for 48 hours under normoxic conditions, after which cell viability was assessed. (B) ECA109 and KYSE410 cells transfected with miR-10b-3p inhibitor or sh-TSGA10 were cultured under hypoxic conditions, after which cell viability was assessed. (C) ECA109 and KYSE410 cells transfected with miR-10b-3p mimics or pc-DNA TSGA10 were cultured for 10 days under normoxic conditions, after which colonies were fixed, photographed and counted. (D) ECA109 and KYSE410 cells transfected with miR-10b-3p inhibitor or sh-TSGA10 were cultured for 10 days under hypoxic conditions, after which colonies were fixed, photographed, and counted. *P < 0.05.

  
  
  

  

  Hypoxia-induced miR-10b-3p enhances ESCC cell migration and invasion. (A and B) ECA109 and KYSE410 cells transfected with miR-10b-3p mimics or pc-DNA TSGA10 were seeded in serum-medium into Matrigel-free (A) or Matrigel-coated (B) upper chambers, while the lower chamber contained complete medium. After incubation for 20 hours under normoxic conditions, invading cells were fixed, photographed, and counted. (C and D) For hypoxia assays, ECA109 and KYSE410 cells transfected with miR-10b-3p inhibitor or sh-TSGA10 were seeded in serum-free medium into Matrigel-free (C) or Matrigel-coated (D) upper chambers, while the lower contained complete medium. After incubation for 20 hours under hypoxic condition, invading cells were fixed, photographed, and counted. *P < 0.05.

  
  
  

  

  MiR-10b-3p contributes to ESCC tumor growth and metastasis in vivo. (A) An ESCC xenograft model in BALB/c nude mice was constructed using ECA109 cells. MiR-10b-3p agomir or agomir NC was injected into the tumor mass as indicated. After 40 days, tumors that developed were excised and photographed. (B) Tumor volumes were measured every 5 days and plotted. (C and D) To assess cell proliferation, tumor sections were immunostained for Ki-67 and photographed (C), after which Ki-67-positive cells were quantified (D). (E) Representative images of hematoxylin- and eosin-stained metastases in lungs. (F) Quantification of metastatic lung nodules. *P < 0.05.

  
  
  

  

  Schematic diagram of hypoxia-induced miR-10b-3p promoting tumor growth and metastasis by targeting TSGA10 in ESCC.

  
  
  

• Research Paper Volume 11, Issue 8 pp 2420-2429

  Diagnostic performance of new and classic CSF biomarkers in age-related dementias

  Relevance score: 6.7955465

  Francesca Marchegiani, Giulia Matacchione, Deborah Ramini, Fiorella Marcheselli, Rina Recchioni, Tiziana Casoli, Elisa Mercuri, Marco Lazzarini, Belinda Giorgetti, Valentina Cameriere, Susy Paolini, Lucia Paciaroni, Tommaso Rossi, Roberta Galeazzi, Rosamaria Lisa, Anna Rita Bonfigli, Antonio Domenico Procopio, Maria De Luca, Giuseppe Pelliccioni, Fabiola Olivieri

  Keywords: neurofilament-light, microRNAs, age-related dementias, CSF

  Published in Aging on April 27, 2019

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  The identification of diagnostic-prognostic biomarkers of dementia has become a global priority due to the prevalence of neurodegenerative diseases in aging populations. The objective of this study was to assess the diagnostic performance of cerebrospinal fluid (CSF) biomarkers across patients affected by either Alzheimer’s disease (AD), tauopathies other than AD (TP), or vascular dementia (VD), and cognitively normal subjects (CNS). One hundred fifty-three patients were recruited and tested for classical AD CSF biomarkers- Amyloid-ß42 and tau proteins - and novel candidate biomarkers - neurofilament (NF-) light and microRNA (miR) -21, -125b, -146a, and -222.

  All dementia patients had significantly higher concentrations of NF-light compared to CNS, with the TP group displaying the highest NF-light values. A significant inverse correlation was also observed between NF-light and cognitive impairment. Of the four miRNAs analyzed, miR-222 levels were significantly increased in VD patients compared to both CNS and AD. In addition, while NF-light showed a better diagnostic performance than miR-222 and classical AD biomarkers in differentiating TP and VD from CNS, classical AD biomarkers revealed higher performance in discriminating AD from non-AD disorders.

  Overall, our results suggest that CSF NF-light and miR-222 are promising biomarkers that may help to diagnose non-AD disorders.

  

  CSF NF-light concentration levels. (A) in CNS, AD, VD and TP and (B) in CNS, AD, VD and TP grouped by gender. Data are presented as median (Interquartile Range). *p<0.05; **p<0.01.

  
  
  

  

  CSF miR-222 expression levels in CNS, AD, VD and TP. Data are presented as median [interquartile range]; **p<0.01

  
  
  

  

  ROC curve analysis of NF-Light and miR-222. (A) CNS vs. VD. (B) AD vs. VD. AUC= Area Under the Curve.

  
  
  

  

  ROC curve analysis of NF-Light and classical AD biomarkers. (A) CNS vs. AD. (B) CNS vs. TP. (C) CNS vs. VD. (D) AD vs. VD and (E) AD vs. TP. AUC= Area Under the Curve.

  
  
  

• Editorial Volume 11, Issue 5 pp 1329-1330

  microRNA-based biomarker for dementia

  Relevance score: 5.3752522

  Kensuke Toyama, Masaki Mogi, Philip S. Tsao

  Keywords: microRNAs, exosome, dementia, blood-brain barriers, miR-501-3p

  Published in Aging on March 12, 2019

  

  Concept of microRNA-based diagnostic assessment in cognitive decline and/or in predicting blood-brain barrier dysfunction status.

  
  
  

• Research Paper Volume 10, Issue 12 pp 3881-3896

  Accelerated aging induced by deficiency of Zmpste24 protects old mice to develop bleomycin-induced pulmonary fibrosis

  Relevance score: 6.9245253

  Jazmín Calyeca, Yalbi I. Balderas-Martínez, Raúl Olmos, Rogelio Jasso, Vilma Maldonado, Quetzali Rivera, Moisés Selman, Annie Pardo

  Keywords: pulmonary fibrosis, aging, microRNAs

  Published in Aging on December 10, 2018

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  Idiopathic pulmonary fibrosis is a devastating aging-associated disease of unknown etiology. Despite that aging is a major risk factor, the mechanisms linking aging with this disease are uncertain, and experimental models to explore them in lung fibrosis are scanty. We examined the fibrotic response to bleomycin-induced lung injury in Zmpste24-deficient mice, which exhibit nuclear lamina defects developing accelerated aging. We found that young WT and Zmpste24(-/-) mice developed a similar fibrotic response to bleomycin. Unexpectedly, while old WT mice developed severe lung fibrosis, accelerated aged Zmpste24-/- mice were protected showing scant lung damage. To investigate possible mechanisms associated with this resistance to fibrosis, we compared the transcriptome signature of the lungs and found that Zmpste24(-/-) mice showed downregulation of several core and associated matrisome genes compared with WT mice. Interestingly, some microRNAs that target extracellular matrix molecules such as miR23a, miR27a, miR29a, miR29b-1, miR145a, and miR491 were dysregulated resulting in downregulation of profibrotic pathways such as TGF-β/SMAD3/NF-κB and Wnt3a/β-catenin signaling axis. These results indicate that the absence of Zmpste24 in aging mice results in impaired lung fibrotic response after injury, which is likely associated to the dysregulation of fibrosis-related miRNAs.

  

  Differentially expressed genes and pathways in lungs from old compared to young C57/BL6 wild-type mice. (A) Volcano plot of the global gene expression profiling in lungs from old wild type versus young WT mice. Each point represents the difference in expression (log fold-change) between the two groups of mice plotted against the level of statistical significance. Right blue dots represent overexpressed genes, while the left blue dots represent downregulated genes at a significant level of *p< 0.05 and ** p< 0.01. Some Core or associated genes to the Matrisome are plotted in red. (B) The expression of these genes was evaluated by quantitative RT-PCR. Bars represent fold-change of old mice over young ones (dotted line). (C and D) Gene ontology (C) and KEGG (D) functional analysis of dysregulated genes in old WT mice compared to young littermates. The most significant 20 terms are shown in this figure. Threshold criteria considered for the analysis are log Fold change > 1 or <-1 and p-value < 0.05. KEGG: Kyoto Encyclopedia of Genes and Genomes.

  
  
  

  

  Differentially expressed genes and pathways in lungs from old compared to young Zmpste24 deficient mice. (A) Volcano plot of the global gene expression profiling in lungs from old Zmpste24 deficient mice vs young littermates. Each point represents the difference in expression (log fold-change) between the two groups of mice plotted against the level of statistical significance. Right blue dots represent overexpressed genes; left blue dots represent downregulated genes at a significant level of p< 0.05. Some Core Matrisome genes are plotted in red. (B) The expression of these genes was evaluated by quantitative RT-PCR. Data are shown as fold-change values as compared with young -/- (dotted lines). (C and D) Gene ontology (C) and KEGG (D) functional analysis of dysregulated genes in old Zmpste24 deficient mice compared to young littermates. The most significant 20 terms are shown in this figure. Threshold criteria considered for the analysis are log Fold change > 1 or <-1 and p-value < 0.05.

  
  
  

  

  Aging increases lung collagen content in WT and Zmpste24^-/- mice. (A) OH-Proline content. Data represent mean ± SD (n=4); *p< 0.05. (B) Representative morphological images of two WT and two Zmpste24 deficient mice stained with Masson’s trichrome. Scale bar, 100 μm.

  
  
  

  

  Young WT and Zmpste24 deficient mice develop similar fibrotic response after bleomycin injury. (A) Representative Masson Trichrome staining of lung sections from young saline control WT (panel a), and 21 days after bleomycin (panels b, c), and Zmpste24 deficient mice saline control (panel d) and at 21 d after bleomycin (panels e, f). Scale bar, 100 μm. (B) Fibrosis score for grading lung histopathological changes. Graphs represent means ± SD. (C) OH-Proline content in lungs after saline or 21 days of bleomycin injury. *p< 0.05; (n=6).

  
  
  

  

  Aging protects Zmpste24 deficient mice from bleomycin-induced pulmonary fibrosis. (A) Representative Masson Trichrome staining of lung sections from old saline control WT (panel a), and 21 days after bleomycin (panels b, c), and Zmpste24 deficient mice saline control (panel d) and at 21 d after bleomycin (panels e, f). Scale bar, 100 μm. (B) Fibrosis score for grading lung histopathological changes. Graphs represent means ± SD. *p< 0.05. (C) OH-Proline content in lungs after saline or 21 days of bleomycin injury. *p< 0.05; Old WT (n=3); Zmpste24 deficient mice (n=6).

  
  
  

  

  Microarray analysis of bleomycin-injured lungs of old Zmpste24 deficient mice (n=3) compared with old wildtype littermates (n=3). Lungs were obtained 21 days after bleomycin injury. (A) Volcano plot of the global gene expression profiling in lungs from old injured Zmpste24 deficient mice vs old WT mice. Each point represents the difference in expression (log fold-change) between the two groups of mice plotted against the level of statistical significance. Right blue dots represent overexpressed genes; left blue dots represent relatively downregulated genes at a significant level of p< 0.05. (B and C) Gene ontology (B) and KEGG (C) functional analysis. The most significant 20 terms are shown. Threshold criteria considered for the analysis are log fold-change > 1 or <-1 and p-value < 0.05 for genes, and >0.5 or z-0.5 for miRnas.

  
  
  

  

  Dysregulated miRNA expression and gene targets of bleomycin-treated lungs of old Zmpste24 deficient mice (n=3) compared to old WT (n=3) analyzed by quantitative RT-PCR. (A) Pie graph showing significantly different mRNA (up= 627, down= 538) and miRNAs (up= 22, down= 20) in lungs of Zmpste24 deficient mice compared with their WT counterparts after bleomycin damage. (B) Expression levels of selected miRNAs. Gene expression of (C) miR29 targets, (D) miR27a targets, (E) mir145a targets. White bars represent mean expression in WT lungs, and black bars represent mean expression in Zmpste24 deficient mice ± S.E.M. *p < 0.05 and ** p< 0.01.

  
  
  

• Editorial Volume 10, Issue 2 pp 156-157

  Biological age of transplanted livers

  Relevance score: 6.163769

  Miriam Capri, Claudio Franceschi, Matteo Cescon

  Keywords: biological age, microRNAs, liver, transplant, donor-recipient interaction

  Published in Aging on February 1, 2018

  

  Donor-recipient biological age mismatch. The main scientific questions and possible answers in terms of biomarkers are focused in the context of orthotopic liver transplantation. GM = Gut microbiome; DAMPs = Danger-Associated Molecular Patterns; PRRs = Pattern Recognition Receptors.

  
  
  

• Research Paper Volume 9, Issue 12 pp 2559-2586

  Central role of the p53 pathway in the noncoding-RNA response to oxidative stress

  Relevance score: 5.6283875

  Paola Fuschi, Matteo Carrara, Christine Voellenkle, Jose Manuel Garcia-Manteiga, Paolo Righini, Biagina Maimone, Elena Sangalli, Francesco Villa, Claudia Specchia, Mario Picozza, Giovanni Nano, Carlo Gaetano, Gaia Spinetti, Annibale A. Puca, Alessandra Magenta, Fabio Martelli

  Keywords: oxidative stress, long noncoding RNAs, microRNAs, p53, endothelium

  Published in Aging on December 12, 2017

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  Oxidative stress plays a fundamental role in many conditions. Specifically, redox imbalance inhibits endothelial cell (EC) growth, inducing cell death and senescence. We used global transcriptome profiling to investigate the involvement of noncoding-RNAs in these phenotypes. By RNA-sequencing, transcriptome changes were analyzed in human ECs exposed to H₂O₂, highlighting a pivotal role of p53-signaling. Bioinformatic analysis and validation in p53-silenced ECs, identified several p53-targets among both mRNAs and long noncoding-RNAs (lncRNAs), including MALAT1 and NEAT1. Among microRNAs (miRNAs), miR-192-5p was the most induced by H₂O₂ treatment, in a p53-dependent manner. Down-modulated mRNA-targets of miR-192-5p were involved in cell cycle, DNA repair and stress response. Accordingly, miR-192-5p overexpression significantly decreased EC proliferation, inducing cell death. A central role of the p53-pathway was also confirmed by the analysis of differential exon usage: Upon H₂O₂ treatment, the expression of p53-dependent 5’-isoforms of MDM2 and PVT1 increased selectively. The transcriptomic alterations identified in H₂O₂-treated ECs were also observed in other physiological and pathological conditions where redox control plays a fundamental role, such as ECs undergoing replicative senescence, skeletal muscles of critical limb-ischemia patients and the peripheral-blood mononuclear cells of long-living individuals. Collectively, these findings indicate a prominent role of noncoding-RNAs in oxidative stress response.

  

  mRNAs differentially expressed upon HUVEC exposure to H₂O₂. Gene Ontology enrichment analysis of the transcriptomic changes induced by HUVEC treatment with H₂O₂ as assessed by rRNA-depleted RNA-sequencing. Circles represent specific ontology terms or KEGG pathways that were significantly enriched in the list of differentially expressed genes after 16 hrs (A) or 36 hrs (B) of H₂O₂ treatment (n= 3). Edges represent term connections within the ontology tree and colors highlight terms correlated in meaning. Terms are captioned if they are the most significant of the group or if they show a biological meaning connected to the system under analysis.

  
  
  

  

  Differential MDM2 exon usage. (A) Genomic structure of the human MDM2 promoter region. The locus has two independent promoters before exons 1a (P1) and 1b (P2), yielding long- and short-5’ UTR isoforms, respectively. Translation start site (ATG) in exon 2 is indicated. Red triangles indicate p53 protein binding sites involved in P2 activation. (B) In H₂O₂ treated HUVEC, short-5’ UTR isoforms of MDM2 were induced, as assessed by qPCR using primers spanning from exon 1b to exon 2. Modulation of long-5’ UTR isoforms of MDM2 was not statistically significant, as assessed by qPCR using primers spanning from exon 1a to exon 2. The bar graph represents average values ±SEM (n= 3; *p<0.05, ***p<0.001).

  
  
  

  

  LncRNAs differentially expressed upon HUVEC exposure to H₂O₂. HUVEC were exposed to H₂O₂ for 16 hrs (A) and 36 hrs (B) and lncRNA expression was measured by rRNA-depleted RNA-sequencing (n= 3). Volcano plots show adjusted p values in a negative Log₁₀ scale and fold changes in a log₂ scale. LncRNAs significantly (adjusted p values <0.05) modulated by H₂O₂ treatment are indicated in red. The names of top 10 modulated lncRNAs and of qPCR validated lncRNAs are indicated. (C) In independent HUVEC cultures treated as in A and B (n= 3), the modulation of the indicated lncRNAs was assayed by qPCR. The heat map shows modulated lncRNAs as log₂ values. Green= down-modulation; red= up-regulation.

  
  
  

  

  miRNAs differentially expressed upon HUVEC exposure to H₂O₂. (A) HUVEC were exposed to H₂O₂ for 16 hrs and miRNA expression was measured by small RNA-sequencing (n= 3). Validation was performed by qPCR in independent HUVEC cultures treated with H₂O₂ for 16 hrs and 36 hrs (n= 3). The heat map shows modulated lncRNAs as log₂ values. Green= down-modulation; red= up-regulation. (B) Interactions between qPCR-validated miRNAs and their potential targets, as reported by miRTarBase, that showed a significant down-regulation at both 16 hrs and 36 hrs time points in rRNA-depleted RNA-sequencing data. Results were represented using Cytoscape: For each gene, the inner color represents the log₂ fold change at 16 hrs and the border color represents the log₂ fold change at 36h. (C) miR-192-5p targets enrichment analysis. Enrichment analysis performed with ClueGO on miR-192-5p targets that showed a significant down regulation at both time points in rRNA-depleted RNA-sequencing data. Circles represent specific ontology terms or KEGG pathways that are significantly enriched. Edges represent terms connections within the ontology tree and colors highlight terms correlated in meaning. Terms are captioned if they are the most significant of the group or if they show a biological meaning connected to the system under analysis.

  
  
  

  

  miR-192-5p overexpression inhibits EC growth. HUVEC were transfected with miR-192-5p mimic or control oligonucleotides. The next day, cells were plated at 200 000 cells/p3 plate density (T0) and harvested 24 and 48 hrs later. (A) The expression of the indicated genes was measured by qPCR at 24 hrs from T0. The bar graph represents average values ±SEM (n= 3; *p<0.05 **p<0.01). (B) Cells were counted at 24 hrs and 48 hrs from plating (T0) and plotted as % of the T0 cell number. miR192-5p expression inhibited HUVEC growth significantly. The graph represents average values ±SEM (n=8 independent transfections; **** p<0.0001).

  
  
  

  

  p53 silencing inhibits the induction of target ncRNAs by H₂O₂. HUVEC were transfected with p53 or control (ctr) siRNAs and, 24 hrs later, treated with either 200 μM H₂O₂ or solvent alone. After 16 hrs, total RNA was extracted and the indicated RNAs analyzed by qPCR. (A) coding RNAs; (B) ncRNAs. The bar graphs show average fold changes ±SEM of the indicated RNAs referred to untreated cells transfected with control siRNA (n= 3; *p<0.05,**p<0.01, ***p<0.001).

  
  
  

  

  ncRNA alterations identified in H₂O₂-treated ECs are also present in senescent HUVEC. Total RNA was extracted from early and late passage HUVEC and the indicated RNAs were tested by qPCR. The bar graphs represent average values ±SEM (early passage n= 3, late passage n= 7; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). (A) ncRNAs. (B) Differential exon usage. Exon 1b isoforms of MDM2 and PVT1 are p53-regulated.

  
  
  

  

  Increased miR-192-5p levels in ischemic muscles. Skeletal muscle samples were harvested from the ischemic limb of patients undergoing above the knee amputations for CLI. Tibialis anterior and sartorious muscle samples were harvested and total RNA was extracted. The bar graph shows average ±SEM miR-192-5p levels measured by qPCR. Values of individual patients are indicated as black squares. For each individual, ischemic sartorius muscle values are referred to non-ischemic sartorius values (n= 7; **p< 0.01).

  
  
  

  

  Association between H₂O₂-responsive RNA levels and life-span. Box plots of the indicated RNAs in LLIs (n= 53) versus controls (n= 65); (*p≤0.05, **p≤0.01, ***p<0.001, ****p<0.0001 after adjustment for sex distribution). (A) Coding RNAs. (B) LncRNAs. (C) miRNAs.

  
  
  

  

  Association of MALAT1 and NEAT1 levels to healthy life-span. Box plots of the indicated RNAs in LLIs subdivided based on the presence/absence of age-associated diseases (healthy n= 25; non-healthy n= 28; *p<0.05 after adjustment for sex distribution).

  
  
  

• Research Paper Volume 3, Issue 12 pp 1178-1191

  Molecular links between cellular senescence, longevity and age-related diseases – a systems biology perspective

  Relevance score: 5.3455567

  Robi Tacutu, Arie Budovsky, Hagai Yanai, Vadim E. Fraifeld

  Keywords: cellular senescence, age-related diseases, genes, microRNAs, pathways, networks

  Published in Aging on December 18, 2011

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  The role of cellular senescence (CS) in age-related diseases (ARDs) is a quickly emerging topic in aging research. Our comprehensive data mining revealed over 250 genes tightly associated with CS. Using systems biology tools, we found that CS is closely interconnected with aging, longevity and ARDs, either by sharing common genes and regulators or by protein-protein interactions and eventually by common signaling pathways. The most enriched pathways across CS, ARDs and aging-associated conditions (oxidative stress and chronic inflammation) are growth-promoting pathways and the pathways responsible for cell-extracellular matrix interactions and stress response. Of note, the patterns of evolutionary conservation of CS and cancer genes showed a high degree of similarity, suggesting the co-evolution of these two phenomena. Moreover, cancer genes and microRNAs seem to stand at the crossroad between CS and ARDs. Our analysis also provides the basis for new predictions: the genes common to both cancer and other ARD(s) are highly likely candidates to be involved in CS and vice versa. Altogether, this study shows that there are multiple links between CS, aging, longevity and ARDs, suggesting a common molecular basis for all these conditions. Modulating CS may represent a potential pro-longevity and anti-ARDs therapeutic strategy.

  

  Fraction of CS, longevity and ARD proteins forming a continuous PPI network. Values obtained from simulations with sets of randomly selected proteins are presented as dots. For all the sets of interest, the fraction of interconnected proteins was significantly higher than expected by chance (p < E-25). Insert: average connectivity (number of first-order protein partners) of the sets analyzed in this study. For more details, see Materials and Methods.

  
  
  

  

  MicroRNA-regulated cellular senescence PPI network. Genes are depicted as red circles and miRNAs as green squares.

  
  
  

  

  Fraction of genes which are essential to growth and development in each of the gene sets under analysis. The difference between each set and all genes (control) was highly significant (p < E-25). Insert: The correlation between essentiality and average connectivity (R – Pearson's coefficient of correlation; p = 0.004).

  
  
  

  

  Evolutionary conservation of human CS genes. The difference between CS genes and all genes in InParanoid was significant for D. rerio (p = 0.0001), X. tropicalis (p = 0.003), G. gallus (p = 0.002), R. norvegicus (p = 0.006) and M. musculus (p = 0.02).

  
  
  

  

  Enrichment of genes involved in ARDs and aging-associated conditions among CS genes. The fold-increase was computed as the ratio between the number of observed genes vs. the expected value. In all cases, the fold-increase was highly significant (p < E-25).

  
  
  

  

  Common pathways enriched across CS and ARDs. Pathways directly involved in specific pathologies were excluded in order to remove bias. See also Suppl. Table 3.