Decrease of alpha crystallin A by miR-325-3p in retinal cells under blue light exposure.

Exposure to blue light can lead to retinal degeneration, causing adverse effects on eye health. Although the loss of retinal cells due to blue light exposure has been observed, the precise molecular mechanisms underlying this process remain poorly understood. In this study, we investigate the role of alpha crystallin A (CRYAA) in neuro-retinal degeneration and their regulation by blue light. We observed significant apoptotic cell death in both the retina of rats and the cultured neuro-retinal cells. The expressions of Cryaa mRNA and protein were significantly down-regulated in the retina exposed to blue light. We identified that miR-325-3p reduces Cryaa mRNA and protein by binding to its 3'-untranslated region (UTR). Up-regulation of miR-325-3p destabilized Cryaa mRNA and suppresses CRYAA, whereas down-regulation of miR-325-3p increased both expressions. Blue light-induced neuro-retinal cell death was alleviated by CRYAA overexpression. These results highlight the critical role of Cryaa mRNA and miR-325-3p molecular axis in blue light-induced retinal degeneration. Consequently, targeting CRYAA and miR-325-3p presents a potential strategy for protecting against blue light-induced retinal degeneration.

MicroRNA-21a-5p inhibition alleviates systemic sclerosis by targeting STAT3 signaling.

BACKGROUND: MicroRNA (miRNA)-21-5p participates in various biological processes, including cancer and autoimmune diseases. However, its role in the development of fibrosis in the in vivo model of systemic sclerosis (SSc) has not been reported. This study investigated the effects of miRNA-21a-5p overexpression and inhibition on SSc fibrosis using a bleomycin-induced SSc mouse model. METHODS: A murine SSc model was induced by subcutaneously injecting 100 µg bleomycin dissolved in 0.9% NaCl into C57BL/6 mice daily for 5 weeks. On days 14, 21, and 28 from the start of bleomycin injection, 100 µg pre-miRNA-21a-5p or anti-miRNA-21a-5p in 1 mL saline was hydrodynamically injected into the mice. Fibrosis analysis was conducted in lung and skin tissues of SSc mice using hematoxylin and eosin as well as Masson's trichrome staining. Immunohistochemistry was used to examine the expression of inflammatory cytokines, phosphorylated signal transducer and activator of transcription-3 (STAT3) at Y705 or S727, and phosphatase and tensin homologue deleted on chromosome-10 (PTEN) in skin tissues of SSc mice. RESULTS: MiRNA-21a-5p overexpression promoted lung fibrosis in bleomycin-induced SSc mice, inducing infiltration of cells expressing TNF-α, IL-1ß, IL-6, or IL-17, along with STAT3 phosphorylated cells in the lesional skin. Conversely, anti-miRNA-21a-5p injection improved fibrosis in the lung and skin tissues of SSc mice, reducing the infiltration of cells secreting inflammatory cytokines in the skin tissue. In particular, it decreased STAT3-phosphorylated cell infiltration at Y705 and increased the infiltration of PTEN-expressing cells in the skin tissue of SSc mice. CONCLUSION: MiRNA-21a-5p promotes fibrosis in an in vivo murine SSc model, suggesting that its inhibition may be a therapeutic strategy for improving fibrosis in SSc.

The microRNA-195-5p/hnRNP A1 axis contributes to the progression of hepatocellular carcinoma by regulating the migration of cancer cells.

Dysregulation of gene expression is critical for the progression of cancer. The augmented expression of hnRNP A1 in patients with hepatocellular carcinoma (HCC) has been related to its oncogenic functions. However, the underlying mechanisms responsible for upregulation of hnRNP A1 have not been fully elucidated. In the present study, we identified microRNA-195-5p (miR-195-5p), a miRNA downregulated in HCC, as a novel regulator governing hnRNP A1 expression. Notably, our investigations showed an inverse correlation between hnRNP A1 level, which was increased in HCC, and miR-195-5p level, which was decreased. Our findings demonstrated that hnRNP A1 significantly enhanced the migration and invasion of PLC/PRF/5 cells through its association with mRNAs regulating metastasis. MiR-195-5p also interfered with the hnRNP A1-mediated cell migration by targeting hnRNP A1. Our results underscore the significance of the miR-195-5p/hnRNP A1 axis in regulating the migratory potential of cancer cells and its role in promoting HCC by orchestrating cell migration processes.

RNA binding protein HuD mediates the crosstalk between ß cells and islet endothelial cells by the regulation of Endostatin and Serpin E1 expression.

RNA binding protein HuD plays essential roles in gene expression by regulating RNA metabolism, and its dysregulation is involved in the pathogenesis of several diseases, including tumors, neurodegenerative diseases, and diabetes. Here, we explored HuD-mediated differential expression of secretory proteins in mouse insulinoma ßTC6 cells using a cytokine array. Endostatin and Serpin E1 that play anti-angiogenic roles were identified as differentially expressed proteins by HuD. HuD knockdown increased the expression of α chain of collagen XVIII (Col18a1), a precursor form of endostatin, and Serpin E1 by associating with the 3'-untranslated regions (UTRs) of Col18a1 and Serpin E1 mRNAs. Reporter analysis revealed that HuD knockdown increased the translation of EGFP reporters containing 3'UTRs of Col18a1 and Serpin E1 mRNAs, which suggests the role of HuD as a translational repressor. Co-cultures of ßTC6 cells and pancreatic islet endothelial MS1 cells were used to assess the crosstalk between ß cells and islet endothelial cells, and the results showed that HuD downregulation in ßTC6 cells inhibited the growth and migration of MS1 cells. Ectopic expression of HuD decreased Col18a1 and Serpin E1 expression, while increasing the markers of islet vascular cells in the pancreas of db/db mice. Taken together, these results suggest that HuD has the potential to regulate the crosstalk between ß cells and islet endothelial cells by regulating Endostatin and Serpin E1 expression, thereby contributing to the maintenance of homeostasis in the islet microenvironment.

Loss of RNA binding protein HuD facilitates the production of the senescence-associated secretory phenotype.

HuD, an RNA binding protein, plays a role in the regulation of gene expression in certain types of cells, including neuronal cells and pancreatic ß-cells, via RNA metabolism. Its aberrant expression is associated with the pathogenesis of several human diseases. To explore HuD-mediated gene regulation, stable cells expressing short hairpin RNA against HuD were established using mouse neuroblastoma Neuro2a (N2a) cells, which displayed enhanced phenotypic characteristics of cellular senescence. Two approaches, RNA immunoprecipitation (RNA IP)-NanoString profiling and cytokine array, were used to subsequently identify a subset of putative HuD targets that act as senescence-associated secretory phenotype (SASP), including C-C motif ligand 2 (CCL2), CCL20, C-X-C motif chemokine ligand 2 (CXCL2), and interleukin-6 (IL-6). Here, we further demonstrated that HuD regulates the expression of CCL2, a SASP candidate upregulated in cells following HuD knockdown, by binding to the 3'-untranslated region (UTR) of Ccl2 mRNA. Downregulation of HuD increased the level of CCL2 in N2a cells and the brain tissues of HuD knockout (KO) mice. Exposure to γ-irradiation induced cellular senescence in N2a cells and HuD knockdown facilitated stress-induced cellular senescence. Our results reveal that HuD acts as a novel regulator of CCL2 expression, and its aberrant expression may contribute to cellular senescence by regulating SASP production.

Superoxide dismutase 3 prevents early stage diabetic retinopathy in streptozotocin-induced diabetic rat model.

PURPOSE: To identify the effects of superoxide dismutase (SOD)3 on diabetes mellitus (DM)-induced retinal changes in a diabetic rat model. METHODS: Diabetic models were established by a single intraperitoneal injection of streptozotocin (STZ) in Sprague-Dawley rats. After purification of the recombinant SOD3, intravitreal injection of SOD3 was performed at the time of STZ injection, and 1 and 2 weeks following STZ injection. Scotopic and photopic electroretinography (ERG) were recorded. Immunofluorescence staining with ɑ-smooth muscle actin (SMA), glial fibrillary acidic protein (GFAP), pigment epithelium-derived factor (PEDF), Flt1, recoverin, parvalbumin, extracellular superoxide dismutase (SOD3), 8-Hydroxy-2'deoxyguanosine (8-OHdG) and tumor necrosis factor-ɑ (TNF-ɑ) were evaluated. RESULTS: In the scotopic ERG, the diabetic group showed reduced a- and b-wave amplitudes compared with the control group. In the photopic ERG, b-wave amplitude showed significant (p < 0.0005) reduction at 8 weeks following DM induction. However, the trend of a- and b-wave reduction was not evident in the SOD3 treated group. GFAP, Flt1, 8-OHdG and TNF-ɑ immunoreactivity were increased, and ɑ-SMA, PEDF and SOD3 immunoreactivity were decreased in the diabetic retina. The immunoreactivity of these markers was partially recovered in the SOD3 treated group. Parvalbumin expression was not decreased in the SOD3 treated group. In the diabetic retinas, the immunoreactivity of recoverin was weakly detected in both of the inner nuclear layer and inner plexiform layer compared to the control group but not in the SOD3 treated group. CONCLUSIONS: SOD3 treatment attenuated the loss of a/b-wave amplitudes in the diabetic rats, which was consistent with the immunohistochemical evaluation. We also suggest that in rod-dominant rodents, the use of blue on green photopic negative response (PhNR) is effective in measuring the inner retinal function in animal models of diabetic retinopathy. SOD3 treatment ameliorated the retinal Müller cell activation in diabetic rats and pericyte dysfunction. These results suggested that SOD3 exerted protective effects on the development of diabetic retinopathy.

Long Non-Coding RNA GAS5 Promotes BAX Expression by Competing with microRNA-128-3p in Response to 5-Fluorouracil.

The acquisition of drug resistance is a major hurdle for effective cancer treatment. Although several efforts have been made to overcome drug resistance, the underlying mechanisms have not been fully elucidated. This study investigated the role of long non-coding RNA (lncRNA) growth arrest-specific 5 (GAS5) in drug resistance. GAS5 was found to be downregulated in colon cancer cell lines that are resistant to 5-fluorouracil (5-FU). Downregulation of GAS5 decreased the viability of HCT116 cells and the level of the pro-apoptotic BAX protein, while GAS5 overexpression promoted cell death in response to 5-FU. The interaction between GAS5 and BAX mRNA was investigated using MS2-tagged RNA affinity purification (MS2-trap) followed by RT-qPCR, and the results showed that GAS5 bound to the 3'-untranslated region of BAX mRNA and enhanced its expression by interfering with the inhibitory effect of microRNA-128-3p, a negative regulator of BAX. In addition, ectopic expression of GAS5 increased the sensitivity of resistant cells in response to anti-cancer drugs. These results suggest that GAS5 promoted cell death by interfering with miR-128-3p-mediated BAX downregulation. Therefore, GAS5 overexpression in chemo-resistant cancer cells may be a potential strategy to improve the anti-cancer efficacy of drugs.

Early Microglial Changes Associated with Diabetic Retinopathy in Rats with Streptozotocin-Induced Diabetes.

Although morphological changes in microglia have been reported to be associated with diabetic retinopathy, little is known about the early changes in the microglia and macrophages during the progression of this condition. The present study was aimed at characterizing retinal microglial activation in the early stages of experimental diabetic retinopathy. Toward this end, a model of diabetic retinopathy was generated by intraperitoneally injecting male Sprague-Dawley rats with streptozotocin. No apparent histological changes were observed during the early stages of experimental diabetic retinopathy. However, at 4 to 16 weeks after the onset of diabetes, the retinas from diabetic rats exhibited higher density of microglia than those from age-matched normal controls, with microglial density peaking at 12 weeks. In particular, the proportion of the activated microglia increased significantly in the diabetic rats, specifically in the nerve fiber and ganglion cell layers, whereas it decreased in the inner plexiform layer within 12 weeks. Furthermore, the resident retinal microglial cells were activated immediately after diabetes induction, peaked at 12 weeks, and remained for up to 16 weeks after disease onset. Thus, experimental diabetic retinopathy causes gradual hypoxia and neuroinflammation, followed by the activation of microglia and the migration of macrophages. The distribution and density of retinal microglial activation changed typically with the progression of the disease in early-stage diabetic rats.

Role of long non-coding RNAs in metabolic control.

Long non-coding RNAs (lncRNAs) have emerged as pivotal regulators of gene expression by influencing various biological processes including proliferation, apoptosis, differentiation, and senescence. Accumulating evidence implicates lncRNAs in the maintenance of metabolic homeostasis; dysregulation of certain lncRNAs promotes the progression of metabolic disorders such as diabetes, obesity, and cardiovascular diseases. In this review, we discuss our understanding of lncRNAs implicated in metabolic control, focusing on in particular diseases arising from chronic inflammation, insulin resistance, and lipid homeostasis. We have analyzed lncRNAs and their molecular targets involved in the pathogenesis of chronic liver disease, diabetes, and obesity, and have discussed the rising interest in lncRNAs as diagnostic and therapeutic targets improving metabolic homeostasis. This article is part of a Special Issue entitled: ncRNA in control of gene expression edited by Kotb Abdelmohsen.

RNA binding protein HuD and microRNA-203a cooperatively regulate insulinoma-associated 1 mRNA.

RNA binding protein HuD regulates translation and turnover of target mRNAs, thereby affecting gene expression at the posttranscriptional level in mainly neuronal as well as pancreatic ß-cells. Here, we identified insulinoma-associated 1 (INSM1), an essential factor governing differentiation and proliferation of neuroendocrine cells, as a novel target of HuD and demonstrated the regulatory mechanism of INSM1 expression by HuD. HuD bound to 3'untranslated region (3'UTR) of Insm1 mRNA and negatively regulated its expression; knockdown of HuD increased INSM1 expression, while HuD overexpression repressed it by destabilizing its mRNA. In addition, we further demonstrated that HuD enhanced reduction of INSM1 by miR-203a, a novel miRNA targeting Insm1 mRNA 3'UTR. These results suggest that HuD and miR-203a cooperatively regulate INSM1 expression and it provides a novel regulatory mechanism of INSM1 expression by HuD and miR-203a.

RNA binding protein HuD contributes to ß-cell dysfunction by impairing mitochondria dynamics.

Imbalanced mitochondrial dynamics in pancreatic ß-cells contributes to ß-cell dysfunction in diabetes; however, the molecular mechanisms underlying mitochondrial dynamics in the pathology of diabetes are not fully elucidated. We previously reported the reduction of RNA binding protein HuD in pancreatic ß-cells of diabetes. Herein, we demonstrate that HuD plays a novel role in the regulation of mitochondrial dynamics by promoting mitochondrial fusion. We show enhanced mitochondrial fragmentation in the pancreas of db/db mice and HuD KO mice. Downregulation of HuD increases the number of cells with fragmented mitochondria and reduces the mitochondrial activity determined by mitochondrial membrane potential and ATP production in mouse insulinoma ßTC6 cells. HuD binds to 3'-untraslated region of mitofusin 2 (Mfn2) mRNA and positively regulates its expression. Ectopic expression of Mfn2 in ßTC6 cells stably expressing short hairpin RNA against HuD (shHuD) restores HuD-mediated mitochondrial dysfunction. Taken together, our results suggest that HuD regulates mitochondrial dynamics by regulating Mfn2 level and its reduced expression leads to mitochondrial dysfunction in pancreatic ß-cells.

The MicroRNA-551a/MEF2C Axis Regulates the Survival and Sphere Formation of Cancer Cells in Response to 5-Fluorouracil.

microRNAs regulate a diverse spectrum of cancer biology, including tumorigenesis, metastasis, stemness, and drug resistance. To investigate miRNA-mediated regulation of drug resistance, we characterized the resistant cell lines to 5-fluorouracil by inducing stable expression of miRNAs using lenti-miRNA library. Here, we demonstrate miR-551a as a novel factor regulating cell survival after 5-FU treatment. miR-551a-expressing cells (Hep3B-lenti-miR-551a) were resistant to 5-FU-induced cell death, and after 5-FU treatment, and showed significant increases in cell viability, cell survival, and sphere formation. It was further shown that myocyte-specific factor 2C is the direct target of miR-551a. Our results suggest that miR-551a plays a novel function in regulating 5-FU-induced cell death, and targeting miR-551a might be helpful to sensitize cells to anti-cancer drugs.

RNA Binding Protein HuR Promotes Autophagosome Formation by Regulating Expression of Autophagy-Related Proteins 5, 12, and 16 in Human Hepatocellular Carcinoma Cells.

Autophagy is a process of lysosomal self-degradation of cellular components by forming autophagosomes. Autophagosome formation is an essential process in autophagy and is fine-tuned by various autophagy-related gene (ATG) products, including ATG5, ATG12, and ATG16. Although several reports have shown that numerous factors affect multiple levels of gene regulation to orchestrate cellular autophagy, the detailed mechanism of autophagosome formation still needs further investigation. In this study, we demonstrate that the RNA binding protein HuR (human antigen R) performs an essential function in autophagosome formation. We observe that HuR silencing leads to inhibition of autophagosome formation and autophagic flux in liver cells. Ribonucleoprotein immunoprecipitation (RIP) assay allows the identification of ATG5, ATG12, and ATG16 mRNAs as the direct targets of HuR. We further show that HuR mediates the translation of ATG5, ATG12, and ATG16 mRNAs by binding to their 3' untranslated regions (UTRs). In addition, we show that HuR expression positively correlates with the levels of ATG5 and ATG12 in hepatocellular carcinoma (HCC) cells. Collectively, our results suggest that HuR functions as a pivotal regulator of autophagosome formation by enhancing the translation of ATG5, ATG12, and ATG16 mRNAs and that augmented expression of HuR and ATGs may participate in the malfunction of autophagy in HCC cells.

Reduced expression of the RNA-binding protein HuD in pancreatic neuroendocrine tumors correlates with low p27Kip1 levels and poor prognosis.

For the majority of patients diagnosed with pancreatic neuroendocrine tumors (NETs), there is significant malignant potential with a poor prognosis; however, the molecular abnormalities and pathogenesis of pancreatic NETs have not been firmly established. Here, we report that loss of expression of the RNA-binding protein HuD correlates with low p27Kip1 (p27) levels and poor prognosis in pancreatic NETs. HuD expression was frequently lost in many human pancreatic NETs, and these pancreatic NETs showed aggressive clinicopathological phenotypes with low p27 levels, increased tumor size, higher World Health Organization grade and pT stage of the tumor, and the presence of angioinvasion. Furthermore, loss of HuD was an independent, progression-free prognostic factor in multivariate survival analysis. However, the level of HuR, a member of the same Hu protein family as HuD, was not significantly correlated with pancreatic NET size and progression. Mechanistically, HuD enhanced p27 mRNA translation by interacting with both the 5'-untranslated region (UTR) and the 3'-UTR of p27 mRNA, and consequently suppressed cell cycle progression and tumor growth. In addition, HuD competed with miR-30a-3p for binding to the 3'-UTR of p27 mRNA, suggesting an interplay between HuD and miR-30a-3p in controlling p27 translation. Our results identify HuD as a pivotal suppressor of pancreatic NET growth, and suggest that HuD has potential value as a prognostic factor of pancreatic NETs. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

MicroRNA-195 desensitizes HCT116 human colon cancer cells to 5-fluorouracil.

Multidrug resistance is one major barrier to successful chemotherapy. Although several studies have attempted to overcome resistance of cancer cells to anti-cancer drugs, key determinants of resistance remain largely unknown. The objective of this study was to investigate whether microRNAs might play a role in the acquisition of resistance. Human colorectal cancer HCT-116 cell lines were transduced with a lentivirus library containing 578 precursor microRNAs (miRNAs) to establish cell lines resistant to 5-fluorouracil (5-FU). Specific miRNAs were identified from four different resistant clones and a miR-195-expressing resistant clone (HCT-116_lenti-miR-195) was further investigated. The HCT-116_lenti-miR-195 cells showed resistant phenotype. These cells grew faster after 5-FU treatment compared to control cells (HCT-116_lenti-control). Check point kinase 1 (CHK1) and G2 check point kinase WEE1 were found to be direct targets of miR-195. Downregulation of miR-195 sensitized HCT-116 cells after 5-FU treatment. Our results demonstrate that miR-195 can promote acquisition of drug resistance to 5-FU.

Long Noncoding RNAs and RNA-Binding Proteins in Oxidative Stress, Cellular Senescence, and Age-Related Diseases.

Cellular senescence is a complex biological process that leads to irreversible cell-cycle arrest. Various extrinsic and intrinsic insults are associated with the onset of cellular senescence and frequently accompany genomic or epigenomic alterations. Cellular senescence is believed to contribute to tumor suppression, immune response, and tissue repair as well as aging and age-related diseases. Long noncoding RNAs (lncRNAs) are >200 nucleotides long, poorly conserved, and transcribed in a manner similar to that of mRNAs. They are tightly regulated during various cellular and physiological processes. Although many lncRNAs and their functional roles are still undescribed, the importance of lncRNAs in a variety of biological processes is widely recognized. RNA-binding proteins (RBPs) have a pivotal role in posttranscriptional regulation as well as in mRNA transport, storage, turnover, and translation. RBPs interact with mRNAs, other RBPs, and noncoding RNAs (ncRNAs) including lncRNAs, and they are involved in the regulation of a broad spectrum of cellular processes. Like other cell fate regulators, lncRNAs and RBPs, separately or cooperatively, are implicated in initiation and maintenance of cellular senescence, aging, and age-related diseases. Here, we review the current understanding of both lncRNAs and RBPs and their association with oxidative stress, senescence, and age-related diseases.