Target Gene Of miR-21 Research Articles

Elevated Serum and Urine MiR-429 Contributes to the Progression of Gestational Diabetes Mellitus.

The current study aims to explore a noninvasive molecular marker that can evaluate the therapeutic effect and prognosis of gestational diabetes mellitus (GDM). Differentially expressed miRs were identified using microarray assay and the findings were verified using RT-PCR. Receiver operating characteristic (ROC) was performed to explore the diagnostic value of serum and urine miR-429 in GDM women. The relationship between miR-429 and homeostatic model assessment-insulin resistance (HOMA-IR) was analyzed using Pearson's correlation assay. Dual luciferase assay and western blot were carried out to explore the possible target gene of miR-429. Microarray assay and RT-PCR showed that miR-429 was found to be significantly increased in both serum and urine samples of GDM women compared with those of normal blood glucose pregnancy group (NGT). Also, the expression of miR-429 in serum and urine was positively correlated with HOMA-IR. ROC analysis showed that both serum and urine miR-429 could screen GDM from NGT. Furthermore, dual luciferase reporter and western blot assay indicated that insulin receptor substrate- (IRS-) 1 was a target gene of miR-429. In summary, the increase of serum and urine miR-429 level is a risk factor of insulin resistance in pregnant women with GDM.

MiR-155 Inhibits Malignant Biological Behavior of Human Liver Cancer Cells by Regulating SRPK1.

Although the treatment of liver cancer has made great progress, the mechanism of its occurrence is not completely clear. miR-155 plays an important regulatory role in tumorigenesis and development, including survival, proliferation, migration and invasion. However, the role and regulatory mechanism of miR-155 in liver cancer has rarely been reported. We analyzed miR-155 expression in liver cancer tissue samples and cell lines by qRT-PCR. The expression of miR-155 was measured by qRT-PCR before and after miR-155-mimic and sh-miR-155 transfection. CCK-8 and clonogenic assays were used to detect the proliferation of liver cancer cells. Cell scratch and invasion assays were used to detect migration and invasion. RNA-seq was used to detect the difference in RNA expression in liver cancer cells. SRPK1 expression was detected in liver cancer cells before and after transfection by qRT-PCR and western blotting. We observed that miR-155 was downregulated in liver cancer tissues compared with normal tissues. Furthermore, we demonstrated that liver cancer cell proliferation, migration and invasion are markedly suppressed by miR-155. Importantly, we also demonstrated that SRPK1 is directly regulated by miR-155 during the process of liver cancer cell proliferation and metastasis. Finally, the overexpression of miR-155 inhibits malignant biological behavior of human liver cancer cells. We report the abnormal expression of the miR-155 cluster in liver cancer cells, which inhibits cancer cell proliferation and metastasis. In addition, we identified SRPK1 as a target gene of miR-155 during the process of liver cancer cell proliferation and metastasis.

Down-regulation of miR-155 after treatment with narrow-band UVB and methotrexate associates with apoptosis of keratinocytes in psoriasis.

Psoriasis is a chronic inflammatory skin disease arising from a complex interaction between genetics, epigenetics, the host's immune system and the environment. Recent accumulated data revealed the dysregulation of various microRNAs (miRNAs) in several diseases including psoriasis. We explored the functional role and regulation of hsa-miR-155-5p (miR-155) in an immortalized keratinocyte cell line (HaCaT), in relation to the pathogenesis and treatment of psoriasis. miR-155 expression in normal skin and psoriatic skin lesion before and after treatment with methotrexate (MTX) and narrow-band ultraviolet B phototherapy (NB-UVB) were analyzed using quantitative reverse transcription PCR (qRT-PCR). Apoptotic activity, cell cycle and viable cells of miR-155 transfected HaCaT were measured using flow cytometry and MTS assay. Since, caspase-3 (CASP3) gene was predicted as a target gene of miR-155, the expression of CASP3 was detected in transfected HaCaT using western blot. We discovered that both MTX and NB-UVB significantly down-regulated miR-155 expression in psoriatic skin lesions. We also found that overexpression of miR-155 in HaCaT led to suppression of cell apoptosis and induced cell arrest at G0/G1 phase. Moreover, CASP3 expression was down-regulated in miR-155 transfected HaCaT. This study demonstrates down-regulation of miR155 after treatment with MTX and NB-UVB in psoriatic skin lesion. miR155 plays significant role in apoptosis on HaCaT via CASP3. This finding provides a better understanding of the pathogenesis of psoriasis and might aid on developing the new monitoring tool or therapy for psoriasis in the future.

Circ01592 regulates unsaturated fatty acid metabolism through adsorbing miR-218 in bovine mammary epithelial cells.

The composition of fatty acids plays a key role in regulating milk flavor and quality. Therefore, to improve the quality of milk, it is particularly important to study the regulatory mechanism of fatty acid metabolism in dairy cows. In this study, the expression profiles at non-lactation, peak-lactation, mid-lactation and late-lactation were constructed by high-throughput sequencing. Considering non-lactation as the control group and the other points as the experimental groups, the differentially expressed genes were screened. ELOVL5 was significantly upregulated and was selected for subsequent analyses. Bioinformatics prediction, a dual-luciferase assay, qPCR analysis and western blot analysis were used for verification. The results showed that ELOVL5 was a downstream target gene of miR-218 that regulated milk fat metabolism. A dual-luciferase assay and expression level analysis showed that circ01592 can directly bind to miR-218 and that overexpression of circ01592 (pcDNA-circ01592) significantly reduced the expression of miR-218 and enhanced the expression of ELOVL5, the target gene of miR-218 in BMECs. A functional study of BMECs showed that circ01592 promoted the synthesis of TAG and increased the content of UFA. The function of miR-218 was opposite to that of circ01592. Overall, the data showed that circ01592 promoted TAG synthesis and fatty acid composition by binding miR-218, alleviating the inhibitory effect of miR-218 on ELOVL5 expression. These mechanisms provide a new research approach and theoretical basis for improving milk quality.

MicroRNA-96 is Downregulated in Sepsis Neonates and Attenuates LPSInduced Inflammatory Response by Inhibiting IL-16 in Monocytes.

Neonatal sepsis (NS) remains one of the leading causes of mortality among newborns. This study found the deregulated microRNA-96 (miR-96) in NS neonates, and aimed to evaluate the clinical significance of miR-96, as well as its effect on LPS-induced inflammatory response in monocytes. In addition, the relationship of interleukin-16 (IL-16) and miR-96 was investigated to understand the underlying mechanisms. Expression of miR-96 was examined using real-time quantitative PCR. Monocytes stimulated by LPS was used to mimic excessive inflammation in the pathogenesis of NS. The enzyme-linked immunosorbent assay was applied to evaluate pro-inflammatory cytokine levels. A luciferase reporter assay was used to confirm the interaction between miR-96 and IL-16. Serum miR-96 expression was decreased in NS newborns and had considerable diagnostic value for NS screening. LPS inhibited miR-96 expression in monocytes, and the overexpression of miR-96 could reverse the effects of LPS on the inflammation of monocytes. IL-16 was a target gene of miR-96 and negatively correlated with miR-96 levels in NS neonates. The inhibited inflammatory responses induced by miR-96 overexpression was abolished by the elevated IL-16 in monocytes. All the data reveal that serum decreased miR-96 may serve as a candidate noninvasive biomarker for NS diagnosis. In addition, miR-96 inhibits LPS-induced inflammatory responses by targeting IL-16 in monocytes. The miR-96/IL-16 axis may provide novel therapeutic targets for NS treatment.

Oncogenic role of early growth response-1 in liver cancer through the regulation of the microRNA-675/sestrin 3 and the Wnt/β-catenin signaling pathway

ABSTRACT Early growth response-1 (EGR1) is a multi-domain protein and an immediate early transcription factor that is induced during liver injury and controls the expression of a variety of genes implicated in metabolism, cell proliferation, and tumorigenesis. Liver cancer (LC) is a highly malignant disease with high mortality worldwide. This study focused on the function of EGR1 in LC development and the mechanism of action. Two LC-related datasets GSE101728 and GSE138178 downloaded from the Gene Expression Omnibus (GEO) database were used for identification of key genes involved in cancer progression. A microarray analysis was conducted to identify differentially expressed microRNAs (miRNAs) after EGR1 knockdown. The target gene of miR-675 was identified by integrated analysis. EGR1 and miR-675 were highly expressed, whereas sestrin 3 (SESN3) was poorly expressed in LC tissues and cells. High EGR1 expression was associated with poor liver function and disease severity in patients with LC. Knockdown of EGR1 weakened proliferation and invasiveness of LC cells. EGR1 bound to the miR-675 promoter and increased its transcription, and miR-675 bound to SESN3 mRNA to induce its downregulation. miR-675 upregulation promoted the malignance of LC cells, but further upregulation of SESN3 reduced invasiveness of cells. SESN3 was enriched in the Wnt/β-catenin signaling. EGR1 and miR-675 activated the Wnt/β-catenin through downregulating SESN3. This study demonstrated that EGR1 promotes the malignant behaviors of LC cells through mediating the miRNA-675/SESN3/Wnt/β-catenin axis.

MiR-141 is negatively correlated with TLR4 in neonatal sepsis and regulates LPS-induced inflammatory responses in monocytes.

Neonatal sepsis is an inflammatory system syndrome and a main cause of neonatal mortality. However, there is a lack of ideal biomarkers for early neonatal sepsis diagnosis. The aim of this study was to evaluate the clinical significance of miR-141 in sepsis in neonates, and explore the regulatory effects of miR-141 on inflammation in monocytes. This study used qRT-PCR to calculate the expression of miR-141 in the serum of septic neonates. The diagnostic values of procalcitonin (PCT) and serum miR-141 were evaluated by receiver operating characteristic (ROC) curves. The relationship between miR-141 and TLR4 was determined using luciferase reporter assay. An inflammation model was established using monocytes with lipopolysaccharide (LPS) treatment. ELISA assay was used to analyze the levels of pro-inflammatory cytokines. The expression of miR-141 in neonatal sepsis was significantly lower than healthy controls. ROC curves showed that miR-141 had diagnostic accuracy. LPS stimulation in monocytes led to a decrease in the expression of miR-141. A luciferase reporter assay proved that miR-141 targeted TLR4, and a negative correlation of miR-141 with TLR4 was found in septic neonates. ELISA results demonstrated that the overexpression of miR-141 inhibited LPS-induced inflammation in monocytes. In conclusion, serum decreased miR-141 expression served as a candidate diagnostic biomarker of neonatal sepsis. TLR4 is a target gene of miR-141, which may mediate the inhibitory effects of miR-141 overexpression on LPS-induced inflammation in monocytes. Therefore, miR-141 is expected to be a potential diagnostic biomarker and a therapeutic target in neonatal sepsis.

Downregulation of miR-375 contributes to ERBB2-mediated VEGFA overexpression in esophageal cancer.

Esophageal cancer (EC) is a lethal cancer with an extremely aggressive nature and poor survival rate. However, the molecular mechanisms driving the occurrence and progression of EC are not well understood. MicroRNAs (miRNAs) are small RNA molecules that regulate the expression of protein-coding genes. miRNA-mediated gene regulation plays an important role in EC. By cross-referencing studies from NCBI, we found that microRNA-375 (miR-375) is one of the most frequently downregulated miRNAs in EC. We assessed expression of miR-375 in EC cell lines and primary EC tissues and their matched normal tissues. We found significant downregulation of miR-375 in both cell lines and EC tissues. Forced expression of miR-375 attenuated EC cell proliferation and invasion. Human epidermal growth factor receptor 2 (HER2, ERBB2), a known proto-oncogene, was identified here as one of the potential target genes of miR-375. Ectopic expression of miR-375 significantly suppressed the expression of ERBB2 and subsequently downregulated one of its target genes, vascular endothelial growth factor A (VEGFA), which is related to cancer invasion and metastasis. These findings suggest that miR-375 acts as a tumor suppressor by blocking the ERBB2/VEGFA pathway with the potential to modulate the occurrence and/ or progression of EC.

MiR-144-3p Targets FoxO1 to Reduce Its Regulation of Adiponectin and Promote Adipogenesis.

MicroRNAs (miRNAs), as a series of important short-chain non-coding RNAs, play an important post-transcriptional role in many biological activities, including adipogenesis. miR-144 is significantly upregulated in type II diabetes (T2D), and is considered to be an important biomarker for T2D. However, although the occurrence of T2D is inextricably linked to adipogenesis, whether miR-144 directly regulates adipogenesis remains to be further explored. In this paper, we demonstrate that miR-144 has a higher expression level in a porcine high backfat group, and it has a significant positive effect on promoting the differentiation of pre-adipocytes. FoxO1 is a target gene of miR-144, and inhibits the differentiation of pre-adipocytes. On the other hand, we demonstrate that FoxO1 can bind to the AdipoQ gene promoter, then regulate the AdipoQ expression by binding to the FoxO1 binding site in the AdipoQ promoter -1,499 to -1,489 bp and -1,238 to -1,228 bp regions, especially the -1,499 to -1,489 bp region. Meanwhile, miR-144 and FoxO1 co-expressional research has also shown that both factors regulate adipogenesis. To sum up, our research indicates that miR-144 targets FoxO1, thus reducing its expression and inhibiting its promotional effect on adiponectin, thereby alleviating the inhibitory effect of adiponectin on adipogenesis.

Bioinformatics analysis to determine the prognostic value and prospective pathway signaling of miR-126 in non-small cell lung cancer.

BackgroundMicroRNAs (miRNAs) have been demonstrated to play crucial roles in the initiation and development of non-small cell lung cancer (NSCLC). However, further investigation of the specific role of miR-126 in NSCLC is still required.MethodsAn analysis of miR-126 expression in NSCLC was carried out using the Gene Expression Omnibus (GEO) database, and a literature review was also performed. The differentially expressed genes (DEGs) in three mRNA datasets, GSE18842, GSE19804, and GSE101929, from GEO were identified. Following the prediction of hsa-miR-126-5p target genes by TargetScan, the overlap of miR-126 target genes with DEGs in NSCLC was examined. After that, Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed. Finally, an analysis to identify the impact of hub genes on the prognosis of NSCLC was carried out on the basis of a protein-protein interaction (PPI) network constructed using STRING and Cytoscape.ResultsThe data in the literature review revealed a trend that miR126 was downregulated in NSCLC. The number of both NSCLC-related and miR-126-related DEGs was 187. Dozens of DEGs were significantly enriched in biological regulation, cell membrane binding, and signal receptor binding. In the PPI network analysis, 3 of 10 identified hub genes, namely NCAPG, MELK, and KIAA0101, were obviously related to poor prognosis in NSCLC; the survival rate was low among patients with high expression levels of these genes. Furthermore, through network analysis, TPX2, HMMR, and ANLN were identified as recessive miR-126-related genes that may be involved in NSCLC.ConclusionsMiR-126 plays an essential role in the biological processes of NSCLC through binding to target genes and influences the prognosis of patients with the disease.

RETRACTED ARTICLE: microRNA-375 released from extracellular vesicles of bone marrow mesenchymal stem cells exerts anti-oncogenic effects against cervical cancer

BackgroundCervical cancer is the most prevalent gynecological malignancies accompanied by high mortality, where finding a more effective therapeutic option for cervical cancer is necessary. The inhibitory role of microRNAs (miRNAs) derived from the extracellular vesicles (EVs) of the bone marrow mesenchymal stem cells (BMSCs) was analyzed in cervical cancer.MethodsExpression of miR-375 was examined by RT-qPCR in cervical cancer cell lines. The targeting relation between miR-375 and maternal embryonic leucine zipper kinase (MELK) was predicted by bioinformatics analysis and verified by dual-luciferase reporter gene assay. Isolated BMSCs were transfected with lentivirus-mediated vectors, followed by EV extraction. The morphology of EVs was then identified using a NanoSight particle size analyzer and transmission electron microscope (TEM). The biological properties of cervical cancer cells were evaluated using Transwell, EdU, and TUNEL assays, respectively. Xenograft tumors in nude mice were observed to assess cervical tumorigenesis in vivo.ResultsLow expression of miR-375 and high expression of MELK were detected in cervical cancer samples. MELK was identified as the target gene of miR-375, which was negatively correlated with miR-375 levels. Overexpression of miR-375 suppressed proliferation, migration, and invasion of cervical cancer cells, but enhanced cell apoptosis by cooperating with downregulated MELK expression. miR-375 transferred from BMSC-derived EVs exerted the same effects on cell biological activities. Xenograft assays in vivo proved that miR-375 from BMSC-derived EVs inhibited tumor growth.ConclusionThe present study highlighted the role of miR-375 from BMSC-derived EVs in suppressing the progression of cervical cancer, which may contribute to the discovery of novel potential biomarkers for cervical cancer therapy.

Exosomal miR-21 secreted by IL-1β-primed-mesenchymal stem cells induces macrophage M2 polarization and ameliorates sepsis

Sepsis occurs due to a damaging host response to infection and is the chief cause of death in most intensive care units. Mesenchymal stem cells (MSCs) exhibit immunomodulatory properties and can modulate key cells of the innate and adaptive immune systems through various effector mechanisms, such as exosomes. Exosomes and their microRNA (miRNA or miR) cargo including miR-21 can initiate profound phenotypic changes in the tumor microenvironment due to their intercellular communication transmitting the pleiotropic messages between different cell types, tissues, and body fluids. Here, we aimed to characterize the effect of miR-21 delivered from MSC-derived exosomes on the polarization of macrophages in a mouse sepsis model. First, we isolated exosomes from interleukin-1β (IL-1β)-pretreated murine MSCs (βMSCs) and injected them into cecal ligation and puncture (CLP) septic models. We found that βMSCs-derived exosomes could more effectively induce M2-like polarization of macrophages in vitro and in vivo. Administration of βMSCs-derived exosomes attenuated the symptoms in septic mice more effectively and increased their survival rate as compared to exosomes released by naïve MSCs. Importantly, we found that miR-21 was abundantly upregulated in MSCs upon IL-1β stimulation and packaged into exosomes. This exosomal miR-21 was transferred to macrophages, leading to M2 polarization in vitro and in vivo. The therapeutic efficacy of βMSC-derived exosomes was partially lost upon miR-21 inhibition by its specific inhibitors. More specifically, we demonstrated βMSCs-derived exosomes inhibited the effects of PDCD4, the target gene of miR-21, on macrophage polarization and sepsis. In conclusion, exosomal miR-21 emerged as a key mediator of IL-1β pretreatment induced immunomodulatory properties of MSCs. The study indicated a novel basis for therapeutic application of MSCs in sepsis.

MiR-122 regulates the JAK-STAT signalling pathway by down-regulating EPO in the mammary gland during Streptococcus agalactiae-induced mastitis

MicroRNA (miRNA) are single-stranded non-coding RNA of approximately 22 nucleotides estimated to regulate 60% of human and animal genes some of which are associated with cell differentiation, signal transduction and immune processes. Streptococcus agalactiae (S. agalactiae)-induced bovine mastitis is associated with up-regulation of miR-122 and down-regulation of erythropoietin (EPO) abundance in mammary gland tissue. TargetScan analysis revealed that EPO is a predicted target gene of miR-122 and this regulatory relationship was verified by dual-luciferase reporter assay. Overexpression of miR-122 in primary bovine mammary cells led to down-regulation of EPO and also the JAK-STAT signalling pathway genes EPOR, JAK2, STAT5A and STAT5B. Accordingly, the mRNA abundance of BCL-2, a downstream gene in the JAK-STAT signalling pathway was significantly down-regulated. Under conditions of miR-122 inhibition, EPO, EPOR, JAK2, STAT5A and STAT5B were up-regulated. These results demonstrated that, through its action on EPO, miR-122 may play an important role in S. agalactiae-induced mastitis by regulating the JAK-STAT signalling pathway. Highlights Understanding the relationship among miR-122, EPO and the JAK-STAT pathway would be helpful in developing new strategies for controlling mastitis.

MicroRNA‑138 modulates glioma cell growth, apoptosis and invasion through the suppression of the AKT/mTOR signalling pathway by targeting CREB1.

Alterations in the expression of microRNA (miR)-138 have been demonstrated to result in the development of several malignant tumours. However, the possible function of miR-138 in human glioma cells remains unclear. The present study demonstrated that miR-138 was significantly downregulated in 48 human glioma specimens by quantitative PCR analysis. The upregulation of miR-138 exerted significant antiproliferative and anti-invasive effects on glioma cells and promoted their apoptosis. In addition, cAMP response element-binding protein 1 (CREB1) was confirmed as a direct target gene of miR-138 by luciferase gene reporter assay, and the antitumour effect of miR-138 on glioma cells was significantly reversed by CREB1 overexpression. Moreover, the molecular mechanisms underlying the tumour-suppressive role of miR-138 in malignant glioma may be associated with the dephosphorylation of AKT/mTOR caused by the miR-138 upregulation-induced decrease in CREB1 expression in glioma cells. The results of the present study indicated that miR-138 may affect CREB1/AKT/mTOR signalling to regulate the proliferation, apoptosis and invasion of glioma cells and the malignant progression of glioma, thereby suggesting that miR-138 may be a potential target for the treatment of gliomas.

MALAT1-mediated recruitment of the histone methyltransferase EZH2 to the microRNA-22 promoter leads to cardiomyocyte apoptosis in diabetic cardiomyopathy

Diabetic patients often have a heightened risk of cardiomyopathy, even in the absence of traditional risk factors such as hypertension and atherosclerotic coronary artery disease. Diabetic cardiomyopathy is characterized by a typical cardiomyopathy specific to diabetes, the pathogenesis of which has yet to be fully elucidated. As a well-documented oncogenic long noncoding RNA (lncRNA), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been implicated in a variety of pathological processes, including diabetic complications. This study aimed to evaluate the functional roles of MALAT1 in the pathogenesis of diabetic cardiomyopathy. Spontaneously diabetic (db/db) C57BL/Ks mice were employed to establish diabetic cardiomyopathy models in vivo and high glucose (HG)-cultured mouse cardiomyocytes for myocardial damage models in vitro. Mouse left ventricular volume and function were evaluated by echocardiography, while the myocyte cross-sectional area was calculated to evaluate the degree of myocardial hypertrophy. TUNEL staining and flow cytometric analysis were performed to evaluate myocardial damage and cardiomyocyte apoptosis. Silencing of MALAT1 was found to attenuate cardiac dysfunction and inhibit cardiomyocyte apoptosis in db/db mice and HG-cultured mouse cardiomyocytes. MALAT1 recruited the histone methyltransferase EZH2 to the miR-22 promoter region and inhibited its expression. EZH2 induced an increased in the expression of ATP-binding cassette transporter A1 (ABCA1), which was identified to be a target gene of miR-22. Silencing of EZH2 was found to improve cardiac function and prevent cardiomyocyte apoptosis in db/db mice and HG-cultured mouse cardiomyocytes in the presence of MALAT1, suggesting that MALAT1 mediated myocardial damage by recruiting EZH2 to the miR-22 promoter. Taken together, this study's findings provide evidence confirming our hypothesis, suggesting the involvement of MALAT1 in the processes of cardiac function and cardiomyocyte apoptosis via the EZH2/miR-22/ABCA1 signaling cascade, which has potential therapeutic implications for the understanding of diabetic cardiomyopathy.

Fuzheng Huayu recipe, a traditional Chinese compound herbal medicine, attenuates renal interstitial fibrosis via targeting the miR-21/PTEN/AKT axis

ObjectiveMicroRNAs (miRNAs) may be viable targets for treating renal interstitial fibrosis (RIF). Fuzheng Huayu recipe (FZHY), a traditional Chinese compound herbal medicine, is often used in China to treat fibrosis. This study sought to assess the mechanisms through which FZHY influences miRNAs to treat RIF. MethodsRIF was induced in rats by mercury chloride and treated with FZHY. Hydroxyproline content, Masson’s staining and type I collagen expression were used to evaluate renal collagen deposition. Renal miRNA profiles were evaluated using a miRNA microarray. Those miRNAs that were differentially expressed following FZHY treatment were identified and subjected to bioinformatic analyses. The miR-21 target gene phosphatase and tensin homolog (PTEN) expression and AKT phosphorylation in kidney tissues were assessed via Western blotting. In addition, HK-2 human proximal tubule epithelial cells were treated using angiotensin II (Ang-II) to induce epithelial-to-mesenchymal transition (EMT), followed by FZHY exposure. miR-21 and PTEN expressions were evaluated via quantitative reverse transcription-polymerase chain reaction (qRT-PCR), while E-cadherin and α-smooth muscle actin (α-SMA) expressions were assessed by immunofluorescent staining and qRT-PCR. Western blotting was used to assess PTEN and AKT phosphorylation. ResultsFZHY significantly decreased kidney collagen deposition, hydroxyproline content and type I collagen level. The miRNA microarray identified 20 miRNAs that were differentially expressed in response to FZHY treatment. Subsequent bioinformatic analyses found that miR-21 was the key fibrosis-related miRNA regulated by FZHY. FZHY also decreased PTEN expression and AKT phosphorylation in fibrotic kidneys. Results from in vitro tests also suggested that FZHY promoted E-cadherin upregulation and inhibited α-SMA expression in Ang-II-treated HK-2 cells, effectively reversing Ang-II-mediated EMT. We also determined that FZHY reduced miR-21 expression, increased PTEN expression and decreased AKT phosphorylation in these cells. ConclusionmiR-21 is the key fibrosis-related miRNA regulated by FZHY. The ability of FZHY to modulate miR-21/PTEN/AKT signaling may be a viable approach for treating RIF.

MiR-122 is involved in immune response by regulating Interleukin-15 in the orange-spotted grouper (Epinephelus coioides)

Epinephelus coioides is an important economic culture marine fish and is susceptible to various pathogenic diseases. Increasingly evidences showed that miRNAs participated in the regulation of the cell proliferation, differentiation and immune response. MiR-122 has been reported to play an essential role in immune response by triggering an inflammatory reaction. However, the function of miR-122 in response to bacterial infection is unclear in Epinephelus coioides. Herein, we report that miR-122 is involved in response to Aeromonas hydrophila infection of grouper spleen cells (GS). IL-15, IL-6 and IL-1β are inhibited in overexpression miR-122 GS cells, while induced in silence miR-122 GS cells. In addition, IL-15 is predicted to be the target gene of miR-122, which is further confirmed by LUC. Taken together, we propose that miR-122 regulates the immune response to bacterial infection by triggering IL-15.

Abstract 1486: Exosomal miR-141 promoted tumor angiogenesis via KLF12 in small cell lung cancer

Abstract Background: Angiogenesis, the basic requirement for the survival of tumor cells, has been considered to be one of the malignant characteristics of small cell lung cancer (SCLC) and was closely related to the poor outcomes of SCLC patients. MiR-141 has been found to play a pro- or anti-angiogenetic role in different cancers while its role in SCLC angiogenesis has never been explored. Methods: We isolate total RNA from plasm exosomes and serum samples from two cohorts of SCLC patients respectively to detect the expression of circulating miR-141 in SCLC patients using qRT-PCR. miR-141 was overexpressed in SCLC cell-secreted exosomes to investigate its effects on cell migration, tube formation and microvessel sprouting capabilities of human umbilical vein vascular endothelial cells (HUVECs) both in vitro and in vivo. A dual-luciferase assay was conducted to validate the direct target gene of miR-141. Results: We showed that the level of circulating miR-141 was significantly upregulated in a total of 116 SCLC patients compared with normal volunteers, and the increased miR-141 was revealed to be associated with advanced patient TNM stages which implying the potential oncogenic role of miR-141 in SCLC malignances. In vitro experiments indicated that miR-141 was packaged into SCLC cell-secreted exosomes and was delivered to HUVECs via exosomes to strengthen the migration, invasion, tube formation and microvessel sprouting capabilities of HUVECs. Moreover, mouse models demonstrated that exosomal miR-141 derived from SCLC cells induced neo-angiogenesis in vivo and subcutaneous tumor nodules developed from SCLC cells with miR-141 overexpression showed higher microvessel densities and faster tumor growth than which developed from negative control cells. Combining miRNA target prediction tools with a dual-luciferase assay, we identified that KLF12 was the direct target gene of miR-141 and the angiogenetic promotion effect of miR-141 on HUVECs was abrogated by overexpressing KLF12. Conclusions: Our results elaborated the specific function of exosomal miR-141/KLF12 pathway in SCLC angiogenesis for the first time and provided potential novel targets for anti-angiogenetic therapies in SCLC patients. Our study highlighted the critical role of exosomal miRNAs in SCLC microenvironments and cell communications which offered novel clues for the future exploration of molecular mechanisms of SCLC malignances. Citation Format: Shuangshuang Mao, Zhiliang Lu, Sufei Zheng, Guochao Zhang, Jianbing Huang, Nan Sun, Jie He. Exosomal miR-141 promoted tumor angiogenesis via KLF12 in small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1486.

MiR-21-5p but not miR-1-3p expression is modulated by preconditioning in a rat model of myocardial infarction

Isoflurane (Iso) preconditioning (PC) is known to be cardioprotective against ischemia/reperfusion (I/R) injury. It was previously shown that microRNA-21-5p (miR-21-5p) is regulated by Iso-PC. It is unclear, if expression of cardiac enriched miR-1-3p is also affected by Iso-PC, and associated with activation of HIF1α (hypoxia-inducible factor 1-alpha). Male Wistar rats (n = 6–8) were randomly assigned to treatment with or without 1 MAC Iso for 30 min, followed by 25 min of regional myocardial ischemia, with 120 min reperfusion. At the end of reperfusion, myocardial expression of miR-1-3p, miR-21-5p and mRNAs of two HIF-1α-dependent genes, VEGF (vascular endothelial growth factor) and HO-1 (heme oxygenase-1), were determined by quantitative PCR. Protein expression of a miR-21 target gene, PDCD4 (programmed cell death protein 4), was assessed by western blot analysis. Infarct sizes were analyzed with triphenyltetrazoliumchloride staining. MiR-21-5p expression was increased by Iso, whereas expression of miR-1-3p was not altered. The expression of VEGF but not HO-1 was induced by Iso. Iso-PC reduced infarct sizes compared to untreated controls. No regulation of miRNA and mRNA expression was detected after I/R. PDCD4 protein expression was not affected after Iso exposure. Expression of miR-21-5p, in contrast to miR-1-3p, is altered during this early time point of Iso-PC. HIF1α signaling seems to be involved in miR-21-5p regulation.

Cold-induced Yes-associated-protein expression through miR-429 mediates the browning of white adipose tissue.

Targeting the white-to-brown fat conversion is important for developing potential strategies to counteract metabolic diseases; yet the mechanisms are not fully understood. Yes-associated-protein (YAP), a transcription co-activator, was demonstrated to regulate adipose tissue functions; however, its effects on browning of subcutaneous white adipose tissue (sWAT) are unclear. We demonstrated that YAP was highly expressed in cold-induced beige fat. Mechanistically, YAP was found as a target gene of miR-429, which downregulated YAP expression in vivo and in vitro. In addition, miR-429 level was decreased in cold-induced beige fat. Additionally, pharmacological inhibition of the interaction between YAP and transcriptional enhanced associate domains by verteporfin dampened the browning of sWAT. Although adipose tissue-specific YAP overexpression increased energy expenditure with increased basal uncoupling protein 1 expression, it had no additional effects on the browning of sWAT in young mice. However, we found age-related impairment of sWAT browning along with decreased YAP expression. Under these circumstances, YAP overexpression significantly improved the impaired WAT browning in middle-aged mice. In conclusion, YAP as a regulator of sWAT browning, was upregulated by lowering miR-429 level in cold-induced beige fat. Targeting the miR-429-YAP pathway could be exploited for therapeutic strategies for age-related impairment of sWAT browning.