Endothelial-Derived miR-17∼92 Promotes Angiogenesis to Protect against Renal Ischemia-Reperfusion Injury.

BACKGROUND: Damage to the renal microvasculature is a hallmark of renal ischemia-reperfusion injury (IRI)-mediated AKI. The miR-17∼92 miRNA cluster (encoding miR-17, -18a, -19a, -20a, -19b-1, and -92a-1) regulates angiogenesis in multiple settings, but no definitive role in renal endothelium during AKI pathogenesis has been established. METHODS: Antibodies bound to magnetic beads were utilized to selectively enrich for renal endothelial cells from mice. Endothelial-specific miR-17∼92 knockout (miR-17∼92endo-/- ) mice were generated and given renal IRI. Mice were monitored for the development of AKI using serum chemistries and histology and for renal blood flow using magnetic resonance imaging (MRI) and laser Doppler imaging. Mice were treated with miRNA mimics during renal IRI, and therapeutic efficacies were evaluated. RESULTS: miR-17, -18a, -20a, -19b, and pri-miR-17∼92 are dynamically regulated in renal endothelial cells after renal IRI. miR-17∼92endo-/- exacerbates renal IRI in male and female mice. Specifically, miR-17∼92endo-/- promotes renal tubular injury, reduces renal blood flow, promotes microvascular rarefaction, increases renal oxidative stress, and promotes macrophage infiltration to injured kidneys. The potent antiangiogenic factor thrombospondin 1 (TSP1) is highly expressed in renal endothelium in miR-17∼92endo-/- after renal IRI and is a target of miR-18a and miR-19a/b. miR-17∼92 is critical in the angiogenic response after renal IRI, which treatment with miR-18a and miR-19b mimics can mitigate. CONCLUSIONS: These data suggest that endothelial-derived miR-17∼92 stimulates a reparative response in damaged renal vasculature during renal IRI by regulating angiogenic pathways.

Apelin enhances biological functions in lung cancer A549 cells by downregulating exosomal miR-15a-5p.

Apelin acts as a tumor promoter in multiple malignant tumors; however, its regulatory mechanism remains unclear. Previous studies have indicated that exosomes are pivotal to mediating tumor progression and metastasis. This study examined whether apelin enhances proliferation and invasion ability of lung cancer cells via exosomal microRNA (miRNA). Lung cancer A549 cells overexpressing apelin and control vector were generated by lentiviral transfection. Exosomes were isolated from the culture supernatant of each cell group and characterized. A-exo and V-exo were, respectively, cocultured with A549 cells, and assays of proliferation, apoptosis, colony formation and invasion were conducted. Exosomal miRNA sequencing (miRNA-seq) was performed on A-exo and V-exo to select a candidate miRNA. It was found that A549 cells absorbed more A-exo than V-exo, and A-exo could promote proliferation, colony formation, migration and invasion of A549 cells more than V-exo. Exosomal miRNA-seq data revealed that miR-15a-5p was markedly lower in A-exo compared with V-exo. Low expression of miR-15a-5p was also found in lung cancer tissues and cell lines, suggesting that miR-15a-5p may have an anti-tumor role. Overexpression of miR-15a-5p in A549 cells was associated with less cell proliferation, migration, invasion and suppressed cell cycle, and lower amounts of CDCA4 (cell division cycle-associated protein 4) indicated that it may be a potential target for miR-15a-5p. This study elucidated a novel regulatory mechanism that apelin may promote proliferation and invasion of lung cancer cells by inhibiting miR-15a-5p encapsulated in exosomes.

The promoting effects of hsa_circ_0050102 in pancreatic cancer and the molecular mechanism by targeting miR-1182/NPSR1.

Pancreatic cancer is one of the most lethal tumors across the world with an overall 5-year survival rate of 9%, and great efforts have been devoted in early diagnosis and treatment in the past decades. Competing endogenous RNAs are novel and specific regulatory mechanisms of gene expression, and researches have indicated its important roles in tumor regulation. In this study, we explored the circ-0050102 expression in pancreatic cancer and its impacts on tumor malignant phenotypes and further investigated the correlations among circ-0050102, miR-1182 and NPSR1. Results of real-time quantitative PCR showed that circ-0050102 expressed higher in pancreatic cancers compared with that in adjacent normal tissues. In cell functional experiment, downregulation of circ-0050102 could suppress cell proliferation, migration and invasion ability, boost cell apoptosis and arrest cell cycle in both PANC-1 and CFPAC-1 cells. Furthermore, allogeneic transplantation in nude mice was performed and results showed that the inhibition of circ-0050102 could slow down tumor formation in vivo. Mechanism research suggested that circ-0050102 could downregulate miR-1182, while miR-1182 could not influence the expression of circ-0050102, and miR-1182 could directly target at NPSR1 and suppress it. Moreover, circ-0050102 could reverse the effects of si-NPSR1 on pancreatic cancer cells. In conclusion, we identified that circ-0050102 played an important role in promoting pancreatic cancer by regulating the miR-1182/NPSR1 pathway.

ARID1A deficiency in EBV-positive gastric cancer is partially regulated by EBV-encoded miRNAs, but not by DNA promotor hypermethylation.

AT-rich interactive domain 1A (ARID1A), which is a tumor suppressor gene, is frequently mutated in Epstein-Barr virus-positive gastric cancer [EBV (+) GC]. While most ARID1A mutations in GC are truncating mutations, leading to loss of ARID1A protein expression, epigenetic modifications appear to contribute to ARID1A deficiency in EBV (+) GC harboring wild-type ARID1A. Based on the significant role of epigenetic modifications in EBV (+) GC that contributes to ARID1A deficiency, the methylation status of ARID1A was evaluated in EBV-infected cells and GC patients using a publicly available microarray and the Cancer Genome Atlas (TCGA) database. EBV-encoded miRNAs that potentially target ARID1A were identified as an additional epigenetic modulator by computational prediction. In vitro experiments were conducted to evaluate how EBV-encoded miRNAs affected ARID1A mRNA and protein levels. In clinical GC samples, the expression of predicted miRNAs and ARID1A and the mutation status of ARID1A was evaluated. As results, ARID1A was not hypermethylated in EBV (+) GC samples or EBV-infected GC cells. EBV infection did not alter ARID1A mRNA levels, suggesting that ARID1A protein deficiency was caused by post-transcriptional gene silencing in ARID1A-WT EBV (+) GC. Overexpression of miR-BART11-3p and miR-BART12, which were identified as miRNAs that potentially bind ARID1A, suppressed ARID1A protein expression in MKN7 and NCI-N87 cells. Highly expressed miR-BART11-3p and miR-BART12 were correlated with decreased ARID1A levels in GC tumors which did not harbor ARID1A mutations. The present findings revealed that ARID1A expression was epigenetically regulated by miR-BART11-3p and miR-BART12 in EBV (+) GC.

miR-210 Participates in Hepatic Ischemia Reperfusion Injury by Forming a Negative Feedback Loop With SMAD4.

BACKGROUND AND AIMS: Hepatic ischemia-reperfusion (IR) injury is a major complication of liver transplantation, resection, and hemorrhagic shock. Hypoxia is a key pathological event associated with IR injury. MicroRNA-210 (miR-210) has been characterized as a micromanager of hypoxia pathway. However, its function and mechanism in hepatic IR injury is unknown. APPROACH AND RESULTS: In this study, we found miR-210 was induced in liver tissues from patients subjected to IR-related surgeries. In a murine model of hepatic IR, the level of miR-210 was increased in hepatocytes but not in nonparenchymal cells. miR-210 deficiency remarkably alleviated liver injury, cell inflammatory responses, and cell death in a mouse hepatic IR model. In vitro, inhibition of miR-210 decreased hypoxia/reoxygenation (HR)-induced cell apoptosis of primary hepatocytes and LO2 cells, whereas overexpression of miR-210 increased cells apoptosis during HR. Mechanistically, miR-210 directly suppressed mothers against decapentaplegic homolog 4 (SMAD4) expression under normoxia and hypoxia condition by directly binding to the 3' UTR of SMAD4. The pro-apoptotic effect of miR-210 was alleviated by SMAD4, whereas short hairpin SMAD4 abrogated the anti-apoptotic role of miR-210 inhibition in primary hepatocytes. Further studies demonstrated that hypoxia-induced SMAD4 transported into nucleus, in which SMAD4 directly bound to the promoter of miR-210 and transcriptionally induced miR-210, thus forming a negative feedback loop with miR-210. CONCLUSIONS: Our study implicates a crucial role of miR-210-SMAD4 interaction in hepatic IR-induced cell death and provides a promising therapeutic approach for liver IR injury.

Circ_0003266 sponges miR-503-5p to suppress colorectal cancer progression via regulating PDCD4 expression.

BACKGROUND: Circular RNAs (circRNAs) feature prominently in tumor progression. However, the biological function and molecular mechanism of circ_0003266 in colorectal cancer (CRC) require further investigation. METHODS: Circ_0003266 expression in 46 pairs CRC tissues / adjacent tissues, and CRC cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR); after circ_0003266 was overexpressed or knocked down in CRC cells, cell proliferation, apoptosis, migration, and invasion were evaluated by the cell counting kit-8 (CCK-8), flow cytometry, and Transwell assays, respectively; the interaction among circ_0003266, miR-503-5p, and programmed cell death 4 (PDCD4) was confirmed using bioinformatics analysis and dual-luciferase reporter assay; PDCD4 protein expression in CRC cells was quantified using Western blot. RESULTS: Circ_0003266 was significantly lowly expressed in CRC tissues and cell lines. Circ_0003266 overexpression markedly repressed CRC cell proliferation, migration, and invasion, and accelerated the cell apoptosis, but its overexpression promoted the malignant phenotypes of CRC cells. PDCD4 was a direct target of miR-503-5p and circ_0003266 promoted PDCD4 expression by competitively sponging miR-503-5p. CONCLUSION: Circ_0003266 suppresses the CRC progression via sponging miR-503-5p and regulating PDCD4 expressions, which suggests that circ_0003266 may serve as a novel target for the treatment of CRC.

miR-671-5p Attenuates Neuroinflammation via Suppressing NF-κB Expression in an Acute Ischemic Stroke Model.

This study was designed to investigate the role of miR-671-5p in in vitro and in vivo models of ischemic stroke (IS). Middle cerebral artery occlusion and reperfusion (MCAO/R) in C57BL/6 mice as well as oxygen-glucose deprivation and reoxygenation (OGD/R) in a mouse hippocampal HT22 neuron line were used as in vivo and in vitro models of IS injury, respectively. miR-671-5p agomir, miR-671-5p antagomir, pcDNA3.1-NF-κB, and negative controls were transfected into cells using riboFECT CP reagent. miR-671-5p agomir, pcDNA3.1-NF-κB, and negative vectors were administered into MCAO/R mice via intracerebroventricular injection. The results showed that miR-671-5p was significantly downregulated and that miR-671-5p agomir alleviated injury and neuroinflammation induced by ischemic reperfusion. A dual-luciferase reporter assay confirmed that NF-κB is a direct target of miR-671-5p. Reverse experiments showed that miR-671-5p agomir reduced neuroinflammation via suppression of NF-κB expression in both in vitro and in vivo models of IS. Our data suggest that miR-671-5p may be a viable therapeutic target for diminishing neuroinflammation in patients with IS.

MSC-AS1 knockdown inhibits cell growth and temozolomide resistance by regulating miR-373-3p/CPEB4 axis in glioma through PI3K/Akt pathway.

Long non-coding RNAs (lncRNAs) have been widely reported to regulate the development and chemoresistance of a variety of tumors. Temozolomide (TMZ) is a first-line chemotherapy for treatment of glioma. However, the effect and the regulatory mechanism of lncRNA MSC-AS1 (MSC-AS1) in TMZ-resistant glioma remain unrevealed. Levels of MSC-AS1, microRNA-373-3p (miR-373-3p), and cytoplasmic polyadenylation element binding protein 4 (CPEB4) were determined by quantitative real-time polymerase chain reaction (qRT-PCR). All protein expression was detected by western blot. Cell viability and the half maximal inhibitory concentration (IC50) value of TMZ was assessed by cell counting kit-8 (CCK-8) assay. Cell cloning ability and apoptosis were examined by colony formation and flow cytometry assays, respectively. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were performed to verify the correlation between miR-373-3p and MSC-AS1 or CPEB4. The xenograft models were established to determine the effect of MSC-AS1 in vivo. MSC-AS1 was up-regulated in TMZ-resistant glioma tissues and cells, and glioma patients with high MSC-AS1 expression tend to have lower overall survival rate. MSC-AS1 suppression reduced the IC50 value of TMZ and proliferation, promoted apoptosis and TMZ sensitivity, and affected PI3K/Akt pathway in TMZ-resistant glioma cells. MSC-AS1 acted as miR-373-3p sponge, and miR-373-3p directly targeted CPEB4. Silencing miR-373-3p reversed the promoting effect of MSC-AS1 or CPEB4 knockdown on TMZ sensitivity. Furthermore, MSC-AS1 knockdown inhibited TMZ-resistant glioma growth in vivo by regulating miR-373-3p/CPEB4 axis through PI3K/Akt pathway. Collectively, MSC-AS1 knockdown suppressed cell growth and the chemoresistance of glioma cells to TMZ by regulating miR-373-3p/CPEB4 axis in vitro and in vivo through activating PI3K/Akt pathway.

Blocking SNHG14 Antagonizes Lipopolysaccharides-Induced Acute Lung Injury via SNHG14/miR-124-3p Axis.

BACKGROUND: Emerging evidence show that long noncoding RNAs (lncRNAs) are crucial regulators in pathophysiology of acute lung injury (ALI). Small nucleolar RNA host gene 14 (SNHG14) is a novel oncogenic lncRNA, and has been associated with inflammation-related cell injuries. Thus, we wondered the role and mechanism of SNHG14 in lipopolysaccharides (LPS)-induced ALI cell model. METHODS: Expression of SNHG14, miRNA (miR)-124-3p, and transforming growth factor ß type 2 receptor (TGFBR2) was detected by RT-qPCR and western blotting. Cell apoptosis was determined by methyl thiazolyl tetrazolium assay, flow cytometry, western blotting, and lactate dehydrogenase activity kit. Inflammation was measured by enzyme-linked immunosorbent assay. The interaction among SNHG14, miR-124-3p, and TGFBR2 was validated by dual-luciferase reporter assay and RNA immunoprecipitation. RESULTS: LPS administration attenuated human lung epithelial cell viability and B-cell lymphoma-2 expression, but augmented apoptosis rate, cleaved-caspase-3 expression, lactate dehydrogenase activity, and secretions of tumor necrosis factor-α, interleukin-1ß, and IL-6 in A549 cells. Thus, LPS induced A549 cells apoptosis and inflammation, wherein SNHG14 was upregulated and miR-124-3p was downregulated. However, silencing SNHG14 could suppress LPS-induced apoptosis and inflammation depending on upregulating miR-124-3p via target binding. Similarly, overexpressing miR-124-3p attenuated LPS-induced A549 cells injury through inhibiting its downstream target TGFBR2. Furthermore, SNHG14 knockdown could also affect TGFBR2 expression via miR-124-3p. CONCLUSIONS: SNHG14 knockdown prevents A549 cells from LPS-induced apoptosis and inflammation through regulating miR-124-3p and TGFBR2, suggesting a novel SNHG14/miR-124-3p/TGFBR2 circuit in alveolar epithelial cells on the set of ALI.

Dysregulation of LncRNA ANRIL mediated by miR-411-3p inhibits the malignant proliferation and tumor stem cell like property of multiple myeloma via hypoxia-inducible factor 1α.

Long non-coding RNA (lncRNA) ANRIL has been reported to be closely related to the relapse of multiple myeloma patients. However, the functional role and underlying mechanism of lncRNA ANRIL in multiple myeloma are not known. This study aims to investigate the biological function of lncRNA ANRIL in multiple myeloma. In this study, compared with normal tissues from healthy donors, lncRNA ANRIL and HIF-1α expressions were up-regulated in tumor tissues from multiple myeloma patients. miR-411-3p expression was down-regulated in tumor tissues from multiple myeloma patients. Besides, lncRNA ANRIL can interact with miR-411-3p. HIF-1α was confirmed to be a target of miR-411-3p. Correlation analysis showed that lncRNA ANRIL expression was negatively correlated with miR-411-3p expression. HIF-1α expression was negatively correlated with miR-411-3p expression. Further transfection experiments showed that knockdown of ANRIL or overexpression of miR-411-3p significantly inhibited cell proliferation, tumor formation ability and tumor stem cell like property, promoted cell apoptosis in vitro. Finally, miR-411-3p mimic reduced tumor volume, improved survival rate, suppressed malignant proliferation and tumor stem cell like property in U266 xenograft model. Our results demonstrate that lncRNA ANRIL mediated by miR-411-3p promotes the malignant proliferation and tumor stem cell like property of multiple myeloma through regulating HIF-1α.

MicroRNA-302 represses epithelial-mesenchymal transition and cisplatin resistance by regulating ATAD2 in ovarian carcinoma.

Epithelial-mesenchymal transition (EMT) is an important contributor to drug resistance in ovarian cancer. The aims of this study were to explore the potential role of the miR-302 cluster in modulating EMT and cisplatin resistance in ovarian cancer. We used qRT-PCR and western blotting to show that miR-302 expression was lower in chemoresistant than in chemosensitive cells, and miR-302 was upregulated in chemosensitive, but not chemoresistant ovarian cancer cells in response to cisplatin treatment. We identified ATAD2 as a target of miR-302 and showed that ectopic expression of miR-302 increased cisplatin sensitivity and inhibited EMT and the invasiveness of cisplatin-resistant cells in vitro by targeting ATAD2. Knockdown of ATAD2 restored cisplatin sensitivity and reversed EMT/metastasis in cisplatin-resistant cells, as shown by western blotting and invasion/migration assays. The effect of miR-302 overexpression on EMT and invasiveness was mediated by the modulation of ß-catenin nuclear expression. Immunofluorescence analysis showed that ATAD2 overexpression reversed the miR-302-induced downregulation of nuclear ß-catenin in cisplatin resistant cells. A xenograft tumor model was used to show that miR-302 increases the antitumor effect of cisplatin in vivo. Taken together, these results identify a potential regulatory axis involving miR-302 and ATAD2 with a role in chemoresistance, indicating that activation of miR-302 or inactivation of ATAD2 could serve as a novel approach to reverse cisplatin resistance in ovarian cancer.

MiR-34c promotes hepatic stellate cell activation and Liver Fibrogenesis by suppressing ACSL1 expression.

Normally, there are multiple microRNAs involved in the pathogenesis of liver fibrosis. In our work, we aimed at identifying the role of miR-34c in the hepatic stellate cell (HSC) activation and liver fibrosis and its potential mechanism. Our results have shown that during natural activation of HSC, the level of miR-34c was increased significantly whereas acyl-CoA synthetase long-chain family member-1(ACSL1), which is a key enzyme can affect fatty acid(FA) synthesis, was decreased. A double fluorescence reporter assay further confirmed that ACSL1 is a direct target gene of miR-34c. Moreover, the inhibition of miR-34C can attenuate the synthesis of collagen in HSC-T6. In our rescue assay, ACSL1 expression was 1.49-fold higher compared to normal control cells which were transfected with the miR-34c inhibitor in a stable low expression ACSL1 cell line. While at the same time, α-SMA and Col1α expression decreased by 18.22% and 2.58%, respectively. Moreover, we performed an in vivo model using dimethylnitrosamine (DMN) in conjunction with the miR-34c agomir, combined with the treatment of DMN and the miR-34c agomir can increase liver fibrosis. Meanwhile, the degree of hepatic fibrosis was increased and lipid droplets reduced dramatically in rats and HSC-T6 cell treated with miR-34c mimics alone compared to untreated groups. Our results indicate that miR-34c plays an essential role in liver fibrosis by targeting ACSL1 closely associated with lipid droplets, and it might be used as a potential therapeutic target.

Neuropeptide Y mediates cardiac hypertrophy through microRNA-216b/FoxO4 signaling pathway.

Cardiac hypertrophy (CH) is a major risk factor for heart failure accompanied by maladaptive cardiac remodeling. The role and potential mechanism of neuropeptide Y (NPY) in CH are still unclear. We will explore the role and the mechanism of NPY inactivation (NPY-I) in CH caused by pressure overload. Abdominal aortic constriction (AAC) was used to induce CH model in rats. NPY or angiotensin II (Ang II) was used to trigger CH model in vitro in neonatal rat ventricular myocytes (NRVMs). We found that NPY was increased in the heart and plasma of hypertrophic rats. However, Ang II did not increase NPY expression in cardiomyocytes. NPY-I attenuated CH as decreasing CH-related markers (ANP, BNP and ß-MHC mRNA) level, reducing cell surface area, and restoring cardiac function. NPY inactivation increased miR-216b and decreased FoxO4 expression in CH heart. Moreover, NPY decreased miR-216b and increased FoxO4 expression in NRVMs which were reversed by NPY type 1 receptor (NPY1R) antagonist BIBO3304. MiR-216b mimic and FoxO4 siRNA (small interfering RNA) inhibited NPY/Ang II-induced myocardial hypertrophy in vitro. Meanwhile, BIBO3304 reversed the pro-hypertrophy effect of NPY in vitro. Collectively, NPY deficiency attenuated CH by NPY1R-miR-216b-FoxO4 axis. These findings suggested that NPY would be a potential therapeutic target for the prevention and treatment of cardiac hypertrophy.

MiR-125b promotes migration and invasion by targeting the vitamin D receptor in renal cell carcinoma.

Purpose: To investigate the expression of miR-125b and vitamin D receptor (VDR) in renal cell carcinoma (RCC) and assess the biological function of miR-125b in RCC. Methods: We used quantitative real-time polymerase chain reaction (RT-PCR) to detect the expression of nucleic acids and western blotting to analyze the protein abundance in RCC cell lines. MiR-125b mimic and inhibitor were employed to investigate the function and behavior of miR-125b in RCC cell lines. The relationship between miR-125 and VDR was verified using luciferase assays. Results: Overexpression of miR-125b promoted migration and invasion and prevent cell apoptosis in ACHN cells. In contrast, miR-125b deficiency suppressed migration and invasion and induced cell apoptosis in 786-O cells. Luciferase assays indicated the interaction between miR-125b and VDR. In collected samples, miR-125b was significantly higher in RCC tissues and negatively correlated to VDR (r=-0.444, p=0.04). Conclusion: MiR-125b displays an oncogene profile in RCC, patients with high expression of miR-125b should be a more frequent follow-up. MiR-125B may be a potential therapeutic target for RCC.

Systems biology-based investigation of cooperating microRNAs as monotherapy or adjuvant therapy in cancer.

MicroRNAs (miRNAs) are short, noncoding RNAs that regulate gene expression by suppressing mRNA translation and reducing mRNA stability. A miRNA can potentially bind many mRNAs, thereby affecting the expression of oncogenes and tumor suppressor genes as well as the activity of whole pathways. The promise of miRNA therapeutics in cancer is to harness this evolutionarily conserved mechanism for the coordinated regulation of gene expression, and thus restoring a normal cell phenotype. However, the promiscuous binding of miRNAs can provoke unwanted off-target effects, which are usually caused by high-dose single-miRNA treatments. Thus, it is desirable to develop miRNA therapeutics with increased specificity and efficacy. To achieve that, we propose the concept of miRNA cooperativity in order to exert synergistic repression on target genes, thus lowering the required total amount of miRNAs. We first review miRNA therapies in clinical application. Next, we summarize the knowledge on the molecular mechanism and biological function of miRNA cooperativity and discuss its application in cancer therapies. We then propose and discuss a systems biology approach to investigate miRNA cooperativity for the clinical setting. Altogether, we point out the potential of miRNA cooperativity to reduce off-target effects and to complement conventional, targeted, or immune-based therapies for cancer.

miR-34b-5p promotes renal cell inflammation and apoptosis by inhibiting aquaporin-2 in sepsis-induced acute kidney injury.

OBJECTIVE: This study was designed to uncover the mechanism of miR-34b-5p-mediated aquaporin-2 (AQP2) in sepsis-induced injury using human renal tubular epithelial cells (HK-2). METHODS: Serum levels of miR-34b-5p, TNF-α, IL-1ß, IL-6, serum creatinine (SCr), and blood urea nitrogen (BUN) in septic patients with acute kidney injury (AKI) and healthy controls were detected. Lipopolysaccharide (LPS) was used to induce sepsis in HK-2 cells. LPS-induced HK-2 cells were transfected with miR-34b-5p inhibitor, miR-34b-5p mimic, pcDNA3.1-AQP2, si-AQP2, miR-34b-5p inhibitor + si-NC, or miR-34b-5p inhibitor + si-AQP2. The expressions of miR-34b-5p, AQP2, Bax, Bcl-2, cleaved caspase-3, TNF-α, IL-1ß, and IL-6 in HK-2 cells were detected. TUNEL staining revealed the apoptosis of HK-2 cells. Dual-luciferase reporter assay verified the binding between miR-34b-5p and AQP2. RESULTS: The expression of miR-34b-5p and the inflammatory responses were augmented in septic AKI patients. miR-34b-5p was up-regulated and AQP2 was down-regulated in LPS-induced HK-2 cells. miR-34b-5p inhibition or AQP2 overexpression ameliorated apoptosis and inflammation in LPS-induced HK-2 cells. In contrast, overexpressing miR-34b-5p deteriorated LPS-induced injury in HK-2 cells. AQP2 was a downstream target of miR-34b-5p. AQP2 silencing abolished the suppressive effects of miR-34b-5p inhibition on LPS-induced apoptosis and inflammatory response in HK-2 cells. CONCLUSION: miR-34b-5p inhibits AQP2 to promote LPS-induced injury in HK-2 cells.

Long, Noncoding RNA SRA Induces Apoptosis of ß-Cells by Promoting the IRAK1/LDHA/Lactate Pathway.

Long non-coding RNA steroid receptor RNA activators (LncRNA SRAs) are implicated in the ß-cell destruction of Type 1 diabetes mellitus (T1D), but functional association remains poorly understood. Here, we aimed to verify the role of LncRNA SRA regulation in ß-cells. LncRNA SRAs were highly expressed in plasma samples and peripheral blood mononuclear cells (PBMCs) from T1D patients. LncRNA SRA was strongly upregulated by high-glucose treatment. LncRNA SRA acts as a microRNA (miR)-146b sponge through direct sequence-structure interactions. Silencing of lncRNA SRA increased the functional genes of Tregs, resulting in metabolic reprogramming, such as decreased lactate levels, repressed lactate dehydrogenase A (LDHA)/phosphorylated LDHA (pLDHA at Tyr10) expression, decreased reactive oxygen species (ROS) production, increased ATP production, and finally, decreased ß-cell apoptosis in vitro. There was a positive association between lactate level and hemoglobin A1c (HbA1c) level in the plasma from patients with T1D. Recombinant human interleukin (IL)-2 treatment repressed lncRNA SRA expression and activity in ß-cells. Higher levels of lncRNA-SRA/lactate in the plasma are associated with poor regulation in T1D patients. LncRNA SRA contributed to T1D pathogenesis through the inhibition of miR-146b in ß-cells, with activating signaling transduction of interleukin-1 receptor-associated kinase 1 (IRAK1)/LDHA/pLDHA. Taken together, LncRNA SRA plays a critical role in the function of ß-cells.

Extracellular vesicle miRNA-21 is a potential biomarker for predicting chronic lung disease in premature infants.

Premature infants are often exposed to positive pressure ventilation and supplemental oxygen, which leads to the development of chronic lung disease (CLD). There are currently no standard serum biomarkers used for prediction or early detection of patients who go on to develop CLD. MicroRNAs (miRNAs) are a novel class of naturally occurring, short, noncoding substances that regulate gene expression at the posttranscriptional level and cause translational inhibition and/or mRNA degradation and present in body fluids packaged in extracellular vesicles (EVs), rendering them remarkably stable. Our aim was to evaluate miRNAs identified in serum EVs of premature infants as potential biomarkers for CLD. Serum EVs were extracted from premature infants at birth and on the 28th day of life (DOL). Using a human miRNA array, we identified 62 miRNAs that were universally expressed in CLD patients and non-CLD patients. Of the 62 miRNAs, 59 miRNAs and 44 miRNAs were differentially expressed on DOL0 and DOL28 in CLD and non-CLD patients, respectively. Of these miRNAs, serum EV miR-21 was upregulated in CLD patients on DOL28 compared with levels at birth and downregulated in non-CLD patients on DOL28 compared with levels at birth. In neonatal mice exposed to hyperoxia for 7days, as a model of CLD, five miRNAs (miR-34a, miR-21, miR-712, miR-682, and miR-221) were upregulated, and 7 miRNAs (miR-542-5p, miR-449a, miR-322, miR-190b, miR-153, miR-335-3p, miR-377) were downregulated. MiR-21 was detected as a common miRNA that changed in CLD patients and in the hyperoxia exposed mice. We conclude that EV miR-21 may be a biomarker of CLD.

miR-29a-3p suppresses hepatic fibrosis pathogenesis by modulating hepatic stellate cell proliferation via targeting PIK3R3 gene expression.

OBJECTIVE: Hepatic stellate cells (HSC) activation and proliferation mediated the pathogenic development of hepatic fibrosis (HF). However, the underlying mechanisms remain poorly understood. In this study, we aimed to investigate the miR-29a-3p and its effects on PIK3R3 expression in HF pathogenesis. METHODS: LX-2 cells treated with TGF-ß1 was used as the in vitro HF model. The expression of microRNAs and proteins in LX-2 cells were detected by quantitative RT-PCR and western blotting. Then, miR-29a-3p expression in LX-2 cells were altered via transfection with specific mimics or inhibitors, followed by cell proliferation measured through CCK-8, Edu staining and colony formation. The dual luciferase reporter assay was done to assess binding of miR-29a-3p with PIK3R3 gene sequences. Moreover, PIK3R3 gene overexpression in LX-2 cell was realized through transfection with recombinant pcDNA3.0-PIK3R3 plasmids. RESULTS: Successful establishment of cellular HF model was validated through the increased Col-I and a-SMA expression in TGF-ß1-treated LX-2 cells shown by qRT-PCR and Western blot. In such model, miR-29a-3p expression in LX-2 cells showed the greatest decrease among four candidate microRNAs in response to TGF-ß1 treatment. Also, miR-29a-3p directly binds with the 3' UTR region of the PIK3R3 gene to suppress its expression in LX-2 cells. Furthermore, PIK3R3 gene overexpression effectively abrogated the changes of LX-2 cell proliferation, AKT phosphorylation and Col-I and a-SMA expression caused by miR-29a-3p mimics. CONCLUSION: MiR-29a-3p regulates hepatic stellate cell proliferation and hepatic fibrosis pathogenesis by targeting PIK3R3 expression and modulating the PI-3K/AKT signaling.

Restoration of microRNA-197 expression suppresses oncogenicity in fibrosarcoma through negative regulation of RAN.

MicroRNAs (miRNAs) act as crucial regulators of biological pathways/processes by reinforcing transcriptional programs and moderating transcripts. Emerging evidences have shown the involvement of dysregulated miRNAs in pathophysiology of human diseases including several cancer types. Recently, miR-197-3p has been reported to play different roles in different cancers; however, its role in fibrosarcoma, a highly aggressive and malignant soft tissue sarcoma originated from the mesenchymal tissues, has not yet been studied. Therefore, this study aims to investigate the possible regulatory roles of miR-197-3p in the oncogenicity of fibrosarcoma. For this, we initially performed qRT-PCR of miR-197-3p, which we found to be downregulated in HT1080 human fibrosarcoma cells compared with IMR90-tert normal fibroblast cells. Subsequently, we performed gain-of-function study by employing several methods such as MTT assay, clonogenic assay, wound healing, flow cytometry cell cycle analysis, and acridine orange staining after transfecting HT1080 cells with miR-197-3p mimic. From these assays, we observed that miR-197-3p significantly inhibits viability, colony forming, and migration ability as well as triggers G2/M phase cell cycle arrest and autophagy in fibrosarcoma cells. To understand the mechanism through which miRNA performs these functions, we predicted its targets using TargetScan and performed pathway enrichment analysis after screening them by their expression in fibrosarcoma. Among the enriched targets, we found RAN (ras-related nuclear protein) to be a crucial target through which miR-197-3p represses tumorigenesis by binding to its 3´ UTR, validated by luciferase reporter assay. The tumor suppressive role of the miRNA was further confirmed by transfecting its mimic in RAN-overexpressed cells which showed significant attenuation in tumorigenic effect of RAN in fibrosarcoma as seen in different assays. Taken together, our study unveiled that miR-197-3p acts as an oncosuppressor in fibrosarcoma through G2/M phase arrest and induction of autophagy, and raises the possibility to act as a novel therapeutic intervention for the malignancy.