Mir-204-5p alleviates mitochondrial dysfunction by targeting IGFBP5 in diabetic cataract.

BACKGROUND: Cataract contributes to visual impairment worldwide, and diabetes mellitus accelerates the formation and progression of cataract. Here we found that the expression level of miR-204-5p was diminished in the lens epithelium with anterior lens capsule of cataract patients compared to normal donors, and decreased more obviously in those of diabetic cataract (DC) patients. However, the contribution and mechanism of miR-204-5p during DC development remain elusive. METHODS AND RESULT: The mitochondrial membrane potential (MMP) was reduced in the lens epithelium with anterior lens capsule of DC patients and the H2O2-induced human lens epithelial cell (HLEC) cataract model, suggesting impaired mitochondrial functional capacity. Consistently, miR-204-5p knockdown by the specific inhibitor also attenuated the MMP in HLECs. Using bioinformatics and a luciferase assay, further by immunofluorescence staining and Western blot, we identified IGFBP5, an insulin-like growth factor binding protein, as a direct target of miR-204-5p in HLECs. IGFBP5 expression was upregulated in the lens epithelium with anterior lens capsule of DC patients and in the HLEC cataract model, and IGFBP5 knockdown could reverse the mitochondrial dysfunction in the HLEC cataract model. CONCLUSIONS: Our results demonstrate that miR-204-5p maintains mitochondrial functional integrity through repressing IGFBP5, and reveal IGFBP5 may be a new therapeutic target and prognostic factor for DC.

LncRNA GABPB1-IT1 Is Upregulated in Ischemia-Induced Acute Kidney Injury and Downregulates miR-204-5p to Promote Hypoxia-Induced Human Renal Proximal Tubular Epithelial Cell Apoptosis.

INTRODUCTION: The present study investigated the role of long non-coding RNA (lncRNA) GABPB1-IT1 in ischemia-induced acute kidney injury (AKI). METHODS: The expression of GABPB1-IT1 in the plasma of patients with ischemia-induced AKI and healthy controls was detected by RT-qPCR. GABPB1-IT1 and miR-204-5p were overexpressed in human renal proximal tubular epithelial cells (HRPTEpCs), followed by RT-qPCR to assess the overexpression effect and the regulatory relationship between GABPB1-IT1 and miR-204-5p. Methylation-specific PCR was performed to assess the promoter methylation status of miR-204-5p. Additionally, a cell apoptosis assay was carried out to evaluate the correlation between miR-204-5p and GABPB1-IT1 in the context of hypoxia-induced apoptosis of HRPTEpCs. RESULTS: GABPB1-IT1 was upregulated in the plasma of patients with ischemia-induced AKI. In HRPTEpCs, hypoxia upregulated the expression of GABPB1-IT1. MiR-204-5p was downregulated in ischemia-induced AKI, and the expression of miR-204-5p was inversely correlated with GABPB1-IT1. In HRPTEpCs, overexpression of GABPB1-IT1 decreased the expression levels of miR-204-5p and increased miR-204-5p gene methylation. In addition, overexpression of GABPB1-IT1 reduced the inhibitory effects of miR-204-5p on the apoptosis of HRPTEpC induced by hypoxia. Furthermore, overexpression of GABPB1-IT1 promoted kidney injury, renal tissue injury scores, and the level of serum creatinine. However, miR-204-5p had the opposite effect. CONCLUSION: GABPB1-IT1 was upregulated in ischemia-induced AKI and may induce hypoxia-induced apoptosis of HRPTEpC by methylation of miR-204-5p.

MicroRNA-204-5p Attenuates Oxidative Stress, Apoptosis and Inflammation by Targeting TXNIP in Diabetic Cataract.

Diabetic cataract (DC) is a major cause of blindness in diabetic patients and it is characterized by early onset and rapid progression. MiR-204-5p was previously identified as one of the top five down-regulated miRNAs in human DC lens tissues. We aimed to determine the expression of miR-204-5p in human lens epithelial cells (HLECs) and explore its effects and mechanisms in regulating the progression of DC. The expression of miR-204-5p in the anterior capsules of DC patients and HLECs was examined by RT-qPCR. Bioinformatics tools were then used to identify the potential target of miR-204-5p. The relationship between miR-204-5p and the target gene was confirmed through a dual luciferase reporter assay. Additionally, the regulatory mechanism of oxidative stress, apoptosis, and inflammation in DC was investigated by overexpressing miR-204-5p using miR-204-5p agomir. The expression of miR-204-5p was downregulated in the anterior capsules of DC patients and HLECs. Overexpression of miR-204-5p reduced ROS levels, pro-apoptosis genes (Bid, Bax, caspase-3), and IL-1ß production in HG-treated HLECs. TXNIP was the direct target of miR-204-5p by dual luciferase reporter assay. Therefore, this study demonstrated that miR-204-5p effectively reduced oxidative damage, apoptosis, and inflammation in HLECs under HG conditions by targeting TXNIP. Targeting miR-204-5p could be a promising therapeutic strategy for the potential treatment of DC.

Exosomal circRNA RHOT1 promotes breast cancer progression by targeting miR-204-5p/ PRMT5 axis.

BACKGROUND: Circular RNA RHOT1 (circRHOT1) plays crucial roles in tumorigenesis by competing with microRNAs. It is largely abundant in tumor cell-derived exosomes. Meanwhile, cancer-derived exosomes participate in diverse biological processes. However, the expression patterns and functions of exosomal circRHOT1 in breast cancer remain unknown. This study is aimed to investigate and elucidate the exosomal circRHOT1/miR-204-5p/PRMT5 axis in breast cancer. METHODS: The exosomes derived from serum samples of breast cancer patients and breast cancer cell lines were characterized using transmission electron microscopy and Western blot. MTT, colony formation, wound healing, and transwell assays were utilized to analyze cell proliferation, migration, and invasion of breast cancer cells. Flow cytometry was used for apoptosis analysis. The bioinformatics method was employed to screen differentially expressed novel circRNAs and predict the microRNA targets of circRHOT1. Dual-luciferase reporter gene assays were performed to verify their direct interaction. Finally, Xenograft experiments were used to investigate the effect of exosomal circRHOT1 on tumor growth in vivo. RESULTS: CircRHOT1 exhibited significantly high expression in exosomes derived from the serum of breast cancer patients and breast cancer cell lines, which suggested its potential diagnostic value. Breast cancer-derived exosomes promoted the cell proliferation, migration, invasion, and epithelial-mesenchymal transition of breast cancer cells while inhibiting apoptosis. However, exosomes with downregulated circRHOT1 inhibited the growth of co-cultured cells. Mechanistically, circRHOT1 acted as a sponge of miR-204-5p and promoted protein arginine methyltransferase 5 (PRMT5) expression. Moreover, miR-204-5p inhibitor and pcPRMT5 could reverse the tumor suppressive effects mediated by circRHOT1-knockdown. Furthermore, treatment with exosomes derived from breast cancer cells with circRHOT1 knockdown attenuated tumor growth in tumor-bearing nude mice, which was accompanied by a reduction in PRMT5 expression and an enhancement of miR-204-5p expression. CONCLUSION: The exosomal circRHOT1 may promote breast cancer progression by regulating the miR-204-5p/PRMT5 axis. The current study strengthens the role of circRHOT1, miR-204-5p, and PRMT5 in breast cancer development and provides a potential treatment strategy for breast cancer.

Up-regulated miR-204-5p promoted the migration, invasion, and angiogenesis of endothelial progenitor cells to enhance the thrombolysis of rats with deep venous thrombosis by targeting SPRED1.

Deep venous thrombosis (DVT) endangers human health. Endothelial progenitor cells (EPCs) were proven to promote thrombolysis and miR-204-5p was discovered to be low-expressed in DVT patients. This study concentrated on exploring whether miR-204-5p had a regulatory effect on EPCs and DVT. Concretely, the expression of miR-204-5p in DVT patients' blood was detected by qRT-PCR. The target of miR-204-5p was predicted by bioinformatics and verified by dual-luciferase reporter assay. After rat EPCs were isolated, identified, and transfected with miR-204-5p agomiR, antagomiR, or SPRED1 plasmids, the viability, migration, invasion, and tube formation of EPCs were detected by MTT, wound healing, Transwell, and tube formation assays, respectively. MiR-204-5p, SPRED1, p-PI3K, PI3K, p-AKT, AKT, VEGFA, and Ang1 expressions in EPCs were measured by qRT-PCR or Western blot. EPCs transfected with miR-204-5p overexpression lentivirus plasmid were injected into the DVT rat model. The histopathology of the thrombus and the homing of EPCs to thrombus in the DVT rats were observed by hematoxylin-eosin staining and confocal microscopy, respectively. We found that miR-204-5p was low-expressed in DVT patients and SPRED1 was a target gene of miR-204-5p. MiR-204-5p agomiR promoted the viability, migration, invasion, and tube formation of EPCs, the levels of VEGFA and Ang1 and the activation of PI3K/AKT pathway in EPCs, while miR-204-5p antagomiR and SPRED1 worked oppositely. SPRED1 reversed the effect of miR-204-5p agomiR on EPCs. Up-regulated miR-204-5p inhibited thrombosis and promoted EPCs homing to thrombus in DVT rats. Collectively, up-regulated miR-204-5p enhanced the angiogenesis of EPCs and thrombolysis in DVT rats by targeting SPRED1.

Exosomal lncRNA LINC02191 Promotes Laryngeal Squamous cell Carcinoma Progression by Targeting miR-204-5p/RAB22A Axis and Regulating PI3K/Akt/mTOR Pathway.

Recent research has explored the potential use of serum-derived biomarkers in cancer screening, and mounting evidence has illustrated the pivotal roles of long noncoding RNAs (lncRNAs) in regulating laryngeal squamous cell carcinoma (LSCC) progression. LINC02191 is a newly identified lncRNA and no studies have investigated its role in malignant tumors. This study aims to explore the functions and mechanisms of lncRNA LINC02191 in LSCC. LINC02191 was knocked down in LSCC cells using shRNAs for loss-of-function experiments. RT-qPCR revealed that LINC02191 was upregulated in LSCC patients' serum exosomes, tissues and cells. Western blotting and RT-qPCR were implemented for detecting molecular protein and RNA levels. Colony formation, CCK-8, wound healing and Transwell assays were employed for examining LSCC cell malignant behaviors in vitro. A tumor-bearing mouse model (n = 4/group) was established for examining LINC02191 role in vivo. The results showed that LINC02191 silencing hindered LSCC cell proliferation, invasiveness, migration as well as EMT in vitro and impeded tumorigenesis in xenograft mouse model. Luciferase reporter assay was utilized for verifying the interaction between LINC02191, miR-204-5p and RAB22A. Pearson correlation analysis was employed to evaluate their expression correlation in LSCC tissue specimens (N = 30). Mechanistically, LINC02191 upregulated RAB22A by binding to miR-204-5p, and knocking down LINC02191 inhibited PI3K/Akt/mTOR signaling transduction in LSCC cells and tumor-bearing mice. Moreover, RAB22A overexpression reversed LINC02191 depletion-triggered suppression of LSCC cell aggressiveness and inactivation of PI3K/Akt/mTOR signaling. In conclusion, LINC02191 aggravates LSCC by targeting miR-204-5p/RAB22A/PI3K/Akt/mTOR signaling pathway, which indicates that LINC02191 may serve as a promising target for LSCC treatment.

Circ_LRP6 facilitates osteosarcoma progression via the miR-122-5p/miR-204-5p/HMGB1 axis.

Circ_LRP6 is participated in the occurrence and development of numerous tumors. Nevertheless, its roles and mechanism in osteosarcoma (OS) is unknown. This study aims to illustrate this point. With the use of qRT-PCR, the level of circ_LRP6, miR-122-5p, miR-204-5p and HMGB1 was identified. To observe cell proliferation, migration and invasion, we adopted CCK-8 and Transwell assays in the present study. Besides, to prove the existing interaction, bioinformatics analysis and dual luciferase reporting assays were employed. The influence of circ_LRP6 on osteosarcoma in vivo was evaluated by subcutaneous tumor formation model in nude mice. In osteosarcoma tissues, circ_LRP6 and HMGB1 are strongly denoted, whereas miR-122-5p and miR-204-5p are under-expressed. Circ_LRP6 knockdown could significantly hinder the proliferation, migration and invasion of osteosarcoma cells. Circ_LRP6 hindered the proliferation of osteosarcoma in vivo. Bioinformatics predicted that miR-122-5p and miR-204-5p functioned as direct targets of circ_LRP6, and HMGB1 were possible target genes of miR-122-5p and miR-204-5p. The findings indicated that the low level of miR-122-5p and miR-204-5p and the overexpression of HMGB1 could partially restore and reduce the inhibitory impact of circ_LRP6 on the proliferation, migration and invasion of osteosarcoma cells. Circ_LRP6 affects osteosarcoma progression via the miR-122-5p/miR-204-5p/HMGB1 axis, and is shown to be a molecular biomarker.

Unraveling the Roles of miR-204-5p and HMGA2 in Papillary Thyroid Cancer Tumorigenesis.

Thyroid cancer is the most common endocrine malignant tumor with an increasing incidence rate. Although differentiated types of thyroid cancer generally present good clinical outcomes, some dedifferentiate into aggressive and lethal forms. However, the molecular mechanisms governing aggressiveness and dedifferentiation are still poorly understood. Aberrant expression of miRNAs is often correlated to tumor development, and miR-204-5p has previously been identified in papillary thyroid carcinoma as downregulated and associated with aggressiveness. This study aimed to explore its role in thyroid tumorigenesis. To address this, gain-of-function experiments were performed by transiently transfecting miR-204-5p in thyroid cancer cell lines. Then, the clinical relevance of our data was evaluated in vivo. We prove that this miRNA inhibits cell invasion by regulating several targets associated with an epithelial-mesenchymal transition, such as SNAI2, TGFBR2, SOX4 and HMGA2. HMGA2 expression is regulated by the MAPK pathway but not by the PI3K, IGF1R or TGFß pathways, and the inhibition of cell invasion by miR-204-5p involves direct binding and repression of HMGA2. Finally, we confirmed in vivo the relationship between miR-204-5p and HMGA2 in human PTC and a corresponding mouse model. Our data suggest that HMGA2 inhibition offers promising perspectives for thyroid cancer treatment.

[Effects of lncRNA-UCA1 targeting miR-204-5p on the proliferation, migration, apoptosis and immune escape of endometrial carcinoma cells].

Objective: To investigate the effect of long non-coding RNA urothelial carcinoma-associated 1 (UCA1) gene on the proliferation, migration, apoptosis and immune escape of endometrial cancer cells and its molecular mechanism. Methods: Endometrial cancer tissues and adjacent normal tissues of patients with endometrioid adenocarcinoma who underwent total or partial hysterectomy in Henan Provincial People's Hospital from 2017 to 2019 were collected. The expressions of UCA1 and miR-204-5p were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), and the cell proliferation, migration and apoptosis were detected by cell counting kit 8 (CCK8) method, Transwell method, flow cytometry, and dual-luciferase reporter assay was used to explore the target relationship between UCA1 and miR-204-5p. HEC-1A-sh-NC or HEC-1A-sh-UCA1 cells were co-cultured with peripheral blood mononuclear cells (PBMC) or cytokine-induced killer cells in vitro to explore the role of UCA1 in immune escape. Results: The expression level of UCA1 in endometrial cancer tissue (17.08±0.84) was higher than that in adjacent normal endometrial tissue (3.00±0.37), and the expression level of miR-204-5p (0.98±0.16) was lower than that in adjacent normal endometrial tissue (2.00±0.20, P<0.05). Pearson correlation analysis showed that the expression of miR-204-5p was negatively correlated with the expression of UCA1 (r=-0.330, P=0.030). The expressions of UCA1 and miR-204-5p were associated with the International Federation of Gynecology and Obstetrics stage of endometrial cancer, lymph node metastasis and vascular invasion (P<0.05). The relative ratio of absorbance (0.58±0.11) and the number of cell migration [(199.68±18.44)] in the sh-UCA1 group were lower than those in the sh-NC group (1.24±0.17 and 374.76±24.83), respectively. The apoptosis rate of sh-UCA1 group [(28.64±7.80)%] was higher than that of sh-NC group [(14.27±4.38)%, P<0.05]. After different ratios of effector cells and target cells were cultured, the cell survival rate of HEC-1A-sh-UCA1 group was lower than that of HEC-1A-sh-NC group, and the difference was statistically significant (P<0.05). UCA1 had a binding site for miR-204-5p. The relative ratio of absorbance (1.74±0.08) and the number of cell migration (426.00±18.00) cells in the UCA1+ anti-miR-204-5p group were higher than those in the control group [1.00±0.03 and (284.00±8.00) cells, respectively]. The apoptosis rate of UCA1+ anti-miR-204-5p group [(5.42±0.93)%] was lower than that of control group [(14.82±1.48)%, P<0.05]. HEC-1A-sh-UCA1 cells could induce higher interferon gamma (IFN-γ) expression when co-cultured with PBMC, and the levels of IFN-γ expression in PHA group and PHA+ pre-miR-204-5p group cells were 2.42±0.49 and 1.88±0.26, which were higher than that in the PHA+ pre-NC group (0.85±0.10, P<0.05). When co-cultured with cytokine-induced killer cells (different ratios) in vitro, the HEC-1A-sh-UCA1 group and the HEC-1A-pre-miR-204-5p group had lower survival rates than that in the HEC-1A-pre-miR-204-5p group. In the HEC-1A-pre-NC group, the differences were statistically significant (P<0.05). Conclusion: UCA1/miR-204-5p may play an important role in human endometrial cancer.

Down-regulation of miR-204-5p ameliorates sevoflurane-induced brain injury in neonatal rats through targeting VCAM1.

To confirm the regulation of miR-204-5p on VCAM1 and its effect on sevoflurane-induced brain injury in neonatal rats. We adopted the sevoflurane-induced brain injury model, and the double luciferase reporter gene assay was applied to explore the targeting relationship between vascular adhesion factor 1 (VCAM1) and miR-204-5p. RT-qPCR was applied to assess the levels of miR-204-5. VCAM1, LC3, P62 and cleaved-caspase 3 levels in the hippocampus were estimated by western blot. The number of autophagosomes in the cerebral cortex was assessed via Transmission electron microscopy (TEM), and histopathological changes within the hippocampus by HE staining. miR-204-5p levels were remarkably increased, but VCAM1 expression was decreased after neonatal rat brain injury. Furthermore, miR-204-5p directly targeted VCAM1. The escape latency, swimming distance, autophagosome number, neuronal apoptosis ratio, LC3 II and Cleaved-caspase 3 expression were reduced after miR-204-5p inhibition interference, whereas crossing times, and P62 expression increased in the sevoflurane-induced brain injury model. Furthermore, down-regulation of VCAM1 reversed the trend of these indices. These results suggest that down-regulation of miR-204-5p ameliorates sevoflurane-induced cognitive impairment and hippocampal pathology and inhibits neuronal autophagy and apoptosis by targeting VCAM1.

Circular RNA circMET contributes to tamoxifen resistance of breast cancer cells by targeting miR-204/AHR signaling.

Breast cancer is a prevalent female malignancy and tamoxifen remains the first-line treatment for breast cancer, but tamoxifen resistance is a frequent clinical problem. Circular RNAs (circRNAs) are a bunch of noncoding RNAs with circular structures and play crucial roles in cancer development. Here, we aimed to explore the unreported function of circMET in the modulation of tamoxifen resistance of breast cancer cells. The expression of circMET was upregulated in tamoxifen-resistant breast cancer cells. The depletion of circMET significantly reduced the cell viability and proliferation in tamoxifen-resistant breast cancer cells and the co-treatment of tamoxifen promoted the effect. Mechanically, the luciferase activity of circMET and was repressed by miR-204-5p and AHR 3'UTR in the cells. The expression of miR-204-5p was elevated by circMET knockdown. The expression of AHR was downregulated by miR-204-5p or circMET depletion, while the miR-204-5p inhibitor reversed the effect of circMET depletion in cells. The overexpression of circMET enhanced the cell viability and proliferation of MCF7-Re and T47D-Re cells but miR-204-5p or AHR depletion blocked the phenotype. Clinically, the expression of circMET and AHR has enhanced in tamoxifen-resistant samples compared with tamoxifen sensitive samples, but miR-204-5p presented a revered expression in the samples. Consequently, we concluded that circular RNA circMET contributed to tamoxifen resistance of breast cancer cells by targeting miR-204-5p/AHR signaling.

LncRNA UCA1 regulates silicosis-related lung epithelial cell-to-mesenchymal transition through competitive adsorption of miR-204-5p.

The main clinical manifestations are pulmonary fibrosis, silicosis, is one of the most common types of pneumoconiosis, and its pathogenesis is still unclear. The proliferation and transdifferentiation of fibroblasts are considered to be the key link leading to pulmonary fibrosis. Type II alveolar epithelial cells can be transformed into lung fibroblasts through epithelial-mesenchymal transition (EMT) to promote lung fibrosis. Involved in the EMT process of a variety of cancers, lncRNA UCA1 (UCA1) has been shown to competitively adsorb miR-204-5p, and play an effect on the downstream target gene E-box binding zinc finger protein 1 (ZEB1), thereby promoting EMT to facilitate the invasion and migration of cancer cells. This is an important potential intervention target that affects the process of EMT, but it has not been reported in the study of EMT related to silicosis. Therefore, this study established a SiO2 dust-treated mouse silicosis model and an in vitro EMT model of A549 cells to observe the changes and effects of UCA1 and miR-204-5p, and intervene on the two respectively. The results showed that the EMT process existed in the aforementioned models, while UCA1 was upregulated in the in vitro model. Double luciferase reporter assay demonstrated the targeted binding of UCA1 and miR-204-5p. Silencing UCA1 can up-regulate the expression of miR-204-5p and reduce the level of ZEB1, thus inhibiting EMT process, while intervention of miR-204-5p can change the level of ZEB1 and regulate EMT. Therefore, UCA1 may release its target gene ZEB1 through competitive adsorption of miR-204-5p to regulate EMT process.

miR-204-5p inhibits cell proliferation and induces cell apoptosis in esophageal squamous cell carcinoma by regulating Nestin.

Esophageal cancer (EC) is a highly malignant gastrointestinal tumor, and esophageal squamous cell carcinoma (ESCC) is one of the most common histological types of EC. MicroRNAs (miRNAs) are small noncoding RNAs closely related to tumorigenesis and tumor progression. In addition, Nestin is an intermediate filament protein (class VI) and contributes to the progression of numerous tumors. However, the correlation between miRNAs and Nestin in ESCC remains unclear. This study aimed to investigate the relationship between miR-204-5p and Nestin in ESCC. First, Nestin-related miRNAs in ESCC were explored using RNA sequencing. In ESCC tissues and cell lines, the expression of miR-204-5p was decreased detected by quantitative real-time polymerase chain reaction (qPCR), whereas Nestin protein level was upregulated identified by Western blotting (WB). Besides, Nestin was the direct target of miR-204-5p in ESCC determined via the luciferase reported assay. Moreover, miR-204-5p regulated Nestin to inhibit ESCC cell proliferation detected by the colony formation assay and promote ESCC cell apoptosis identified using the flow cytometry and TUNEL assay. Furthermore, miR-204-5p suppressed tumorigenesis in vivo evaluated by the murine xenograft tumor model. In conclusion, these results indicated that miR-204-5p inhibited cell proliferation and induced cell apoptosis in ESCC through targeting Nestin, which might provide novel therapeutic targets for ESCC therapy.

Circ_0057583 facilitates brain microvascular endothelial cell injury through modulating miR-204-5p/NR4A1 axis.

Lipopolysaccharide (LPS) can induce vascular endothelial injury. Circular RNAs (circRNAs) have been verified to regulate different cellular processes in various diseases. This study intended to explore the potential role and mechanism of circ_0057583 in brain microvascular endothelial cell injury. Human brain microvascular endothelial cells (hBMECs) were exposed to different doses of LPS to induce cell damage. The levels of circ_0057583, microRNA-204-5p (miR-204-5p) and nuclear receptor 4A1 (NR4A1) were detected by quantitative real-time PCR or Western blot assays. Cell viability, apoptosis, inflammation and angiogenesis were assessed by Counting Kit-8 (CCK-8), flow cytometry, enzyme linked immunosorbent assay (ELISA) and tube formation assays. The targeting relationship between miR-204-5p and circ_0057583 or NR4A1 was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. LPS treatment elevated the expression of circ_0057583 and NR4A1, but decreased the expression of miR-204-5p in LPS-induced hBMECs. Downregulation of circ_0057583 abated LPS-induced hBMEC injury by inducing cell proliferation and angiogenesis, as well as inhibiting cell apoptosis, autophagy and inflammation. Circ_0057583 aggravated LPS-evoked hBMEC injury by regulating miR-204-5p. Also, miR-204-5p suppressed LPS-evoked hBMEC damage via targeting NR4A1. Moreover, circ_0057583 sponged miR-204-5p to up-regulate NR4A1 level. Depletion of circ_0057583 alleviated LPS-triggered brain microvascular endothelial endothelial cell injury through modulating miR-204-5p/NR4A1 axis.

Remote ischemic preconditioning protects against cerebral ischemia injury in rats by upregulating miR-204-5p and activating the PINK1/Parkin signaling pathway.

Remote ischemic preconditioning (RiPC) is the process where preconditioning ischemia protects the organs against the subsequent index ischemia. RiPC is a protective method for brain damage. This study is to explore the effect and mechanism of RiPC in cerebral ischemia injury in rats through regulation of miR-204-5p/BRD4 expression. Middle cerebral artery occlusion (MCAO) rat model and glucose deprivation (OGD) neuron model were established. The effect of RiPC on neurological deficits, cerebral infarct size, autophagy marker, inflammatory cytokines and apoptosis was evaluated. miR-204-5p expression was analyzed using RT-qPCR, and then downregulated using miR-204-5p antagomir to estimate its effect on MCAO rats. The downstream mechanism of miR-204-5p was explored. RiPC promoted autophagy, reduced cerebral infarct volume and neurological deficit score, and alleviated apoptosis and cerebral ischemia injury in rats, with no significant effects on healthy rat brains. RiPC up-regulated miR-204-5p expression in MCAO rats. miR-204-5p knockdown partially reversed the effect of RiPC. RiPC promoted autophagy in OGD cells, and attenuated inflammation and apoptosis. miR-204-5p targeted BRD4, which partially reversed the effect of miR-204-5p on OGD cells. RiPC activated the PINK1/Parkin pathway via the miR-204-5p/BRD4 axis. In conclusion, RiPC activated the PINK1/Parkin pathway and prevented cerebral ischemia injury by up-regulating miR-204-5p and inhibiting BRD4.

LINC00922 acts as a novel oncogene in gastric cancer.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been discovered to participate in various cancer developments. However, the biological function of lncRNAs associated with gastric cancer (GC) has not been fully elucidated. METHODS: Quantitative RT-PCR (qRT-PCR) assay was performed to measure lncRNAs, microRNAs (miRNAs) and message RNA (mRNA) expression. Cell Counter Kit-8 (CCK-8), clone formation, wound healing, and transwell assays were performed to investigate cell proliferation, migration, invasion, and apoptosis. Fluorescence in situ hybridization (FISH) assay was used to analyze LINC00922 in either the cytoplasm or nucleus. The potential binding among lncRNA, miRNA, and mRNA was evidenced by bioinformatics, luciferase reporter assay. Mouse-xenograft experiments were used to explore the tumorigenesis in vivo. RESULTS: LINC00922 was upregulated in GC, and high LINC00922 expression was associated with poor prognosis. Inhibition of LINC00922 suppressed GC cell proliferation, migration, invasion, and activated cell apoptosis in vitro and inhibited tumorigenesis in vivo. Besides, LINC00922 was markedly located in the cytoplasm. The mechanistic analysis demonstrated that LINC00922 acted as a sponge of miR-204-5p, thereby inhibiting the expression of the target gene-High Mobility Group AT-hook 2 (HMGA2). CONCLUSION: LINC00922 accelerated the progression of GC by miR-204-5p/HMGA2 axis. These findings support LINC00922 may be a promising option for the diagnosis and therapy of GC.

CircTLK1 Downregulation Attenuates High Glucose-Induced Human Mesangial Cell Injury by Blocking the AKT/NF-κB Pathway Through Sponging miR-126-5p/miR-204-5p.

Diabetic nephropathy (DN) is the main cause of end-stage renal disease. Circular RNA hsa_circ_0004442 (circTLK1) accelerates the progression of renal cell carcinoma. However, the role of circTLK1 in DN pathogenesis is indistinct. The expression of circTLK1, microRNA-126-5p (miR-126-5p), and microRNA-204-5p (miR-204-5p) was tested by quantitative real-time polymerase chain reaction. The levels of interleukin-6 and interleukin-1ß were measured by enzyme-linked immunosorbent assay. The levels of reactive oxygen species and malondialdehyde and the activity of superoxide dismutase were determined with corresponding kits. Several protein levels were evaluated with western blotting. The relationship between circTLK1 and miR-126-5p/miR-204-5p was verified by dual-luciferase reporter assay. CircTLK1 was highly expressed in DN patient's serum and high-glucose (HG)-treated human mesangial cells. Functionally, circTLK1 inhibition reduced HG-induced inflammation, oxidative stress, and ECM accumulation in human mesangial cells. CircTLK1 was verified as a sponge for miR-126-5p and miR-204-5p, which were downregulated in DN patient's serum and HG-treated human mesangial cells. Both miR-126-5p and miR-204-5p upregulation decreased inflammation, oxidative stress, and ECM accumulation in HG-treated human mesangial cells and circTLK1 silencing-mediated influence on HG-induced human mesangial cell injury was overturned by miR-126-5p or miR-204-5p inhibition. Moreover, circTLK1 knockdown blocked the AKT/NF-κB pathway by sponging miR-126-5p/miR-204-5p. CircTLK1 downregulation alleviated HG-induced inflammation, oxidative stress, and ECM accumulation through blocking the AKT/NF-κB pathway via sponging miR-126-5p/miR-204-5p, providing a new mechanism to comprehend the pathogenesis of DN.

Fibrogenic signals persist in DAA-treated HCV patients after sustained virological response.

BACKGROUND & AIMS: Patients with HCV who achieve a sustained virological response (SVR) on direct-acting antiviral (DAA) therapy still need to be monitored for signs of liver disease progression. To this end, the identification of both disease biomarkers and therapeutic targets is necessary. METHODS: Extracellular vesicles (EVs) purified from plasma of 15 healthy donors (HDs), and 16 HCV-infected patients before (T0) and after (T6) DAA treatment were utilized for functional and miRNA cargo analysis. EVs purified from plasma of 17 HDs and 23 HCV-infected patients (T0 and T6) were employed for proteomic and western blot analyses. Functional analysis in LX2 cells measured fibrotic markers (mRNAs and proteins) in response to EVs. Structural analysis was performed by qPCR, label-free liquid chromatography-mass spectrometry and western blot. RESULTS: On the basis of observations indicating functional differences (i.e. modulation of FN-1, ACTA2, Smad2/3 phosphorylation, collagen deposition) of plasma-derived EVs from HDs, T0 and T6, we performed structural analysis of EVs. We found consistent differences in terms of both miRNA and protein cargos: (i) antifibrogenic miR204-5p, miR181a-5p, miR143-3p, miR93-5p and miR122-5p were statistically underrepresented in T0 EVs compared to HD EVs, while miR204-5p and miR143-3p were statistically underrepresented in T6 EVs compared to HD EVs (p <0.05); (ii) proteomic analysis highlighted, in both T0 and T6, the modulation of several proteins with respect to HDs; among them, the fibrogenic protein DIAPH1 was upregulated (Log2 fold change of 4.4). CONCLUSIONS: Taken together, these results highlight structural EV modifications that are conceivably causal for long-term liver disease progression in patients with HCV despite DAA-mediated SVR. LAY SUMMARY: Direct-acting antivirals lead to virological cure in the majority of patients with chronic hepatitis C virus infection. However, the risk of liver disease progression or complications in patients with fibrosis and cirrhosis remains in some patients even after virological cure. Herein, we show that extracellular vesicle modifications could be linked to long-term liver disease progression in patients who have achieved virological cure; these modifications could potentially be used as biomarkers or treatment targets in such patients.

MicroRNA-204-5p reduction in rat hippocampus contributes to stress-induced pathology via targeting RGS12 signaling pathway.

BACKGROUND: Neuroinflammation occupies a pivotal position in the pathogenesis of most nervous system diseases, including depression. However, the underlying molecular mechanisms of neuroinflammation associated with neuronal injury in depression remain largely uncharacterized. Therefore, identifying potential molecular mechanisms and therapeutic targets would serve to better understand the progression of this condition. METHODS: Chronic unpredictable stress (CUS) was used to induce depression-like behaviors in rats. RNA-sequencing was used to detect the differentially expressed microRNAs. Stereotactic injection of AAV virus to overexpress or knockdown the miR-204-5p. The oxidative markers and inflammatory related proteins were verified by immunoblotting or immunofluorescence assay. The oxidative stress enzyme and products were verified using enzyme-linked assay kit. Electron microscopy analysis was used to observe the synapse and ultrastructural pathology. Finally, electrophysiological recording was used to analyze the synaptic transmission. RESULTS: Here, we found that the expression of miR-204-5p within the hippocampal dentate gyrus (DG) region of rats was significantly down-regulated after chronic unpredicted stress (CUS), accompanied with the oxidative stress-induced neuronal damage within DG region of these rats. In contrast, overexpression of miR-204-5p within the DG region of CUS rats alleviated oxidative stress and neuroinflammation by directly targeting the regulator of G protein signaling 12 (RGS12), effects which were accompanied with amelioration of depressive-like behaviors in these CUS rats. In addition, down-regulation of miR-204-5p induced neuronal deterioration in DG regions and depressive-like behaviors in rats. CONCLUSION: Taken together, these results suggest that miR-204-5p plays a key role in regulating oxidative stress damage in CUS-induced pathological processes of depression. Such findings provide evidence of the involvement of miR-204-5p in mechanisms underlying oxidative stress associated with depressive phenotype.

High-mobility group AT-hook 2 promotes growth and metastasis and is regulated by miR-204-5p in oesophageal squamous cell carcinoma.

BACKGROUND: To investigate the expression of high-mobility group AT-hook 2 (HMGA2) and miR-204-5p in oesophageal squamous cell carcinoma (ESCC) and their biological roles in ESCC development and progression. METHODS: HMGA2 and miR-204-5p expression levels in ESCC tissues and cell lines were detected by qRT-PCR, Western blotting and immunohistochemical staining. ESCC cell lines were transfected with a small interfering RNA for HMGA2 and miR-204-5p mimic to downregulate and upregulate the expression levels of HMGA2 and miR-204-5p, respectively. The growth, migration and invasion abilities of ESCC cells were assessed by MTT, colony formation, wound-healing and Transwell assays, respectively. A luciferase reporter gene assay was used to determine whether the 3'-untranslated coding regions of HMGA2 could be directly bound by miR-204-5p. RESULTS: HMGA2 expression was markedly upregulated (P < .001), while miR-204-5p expression was markedly downregulated (P = .003) in ESCC tissues compared with adjacent normal tissues. HMGA2 expression was correlated with tumour size, invasion depth, lymph node metastasis and tumour-node-metastasis stage (all P < .05) and was identified as an independent prognostic factor for ESCC patients. The expression levels of HMGA2 and miR-204-5p were negatively correlated (r2  = 0.609, P < .001). HMGA2 knockdown or miR-204-5p overexpression markedly inhibited ESCC cell growth, migration and invasion (P < .05). In addition, restoration of HMGA2 expression partly reversed the inhibitory effects of miR-204-5p overexpression on migration and invasion (P < .05). The luciferase reporter gene assay suggested that HMGA2 is a direct downstream target of miR-204-5p. CONCLUSION: HMGA2 functions as an oncogene in the growth and metastasis of ESCC and is negatively regulated by miR-204-5p.