Effect of miR-27b-3p and Nrf2 in human retinal pigment epithelial cell induced by high-glucose.

AIM: To determine whether the microRNA-27b-3p (miR-27b-3p)/NF-E2-related factor 2 (Nrf2) pathway plays a role in human retinal pigment epithelial (hRPE) cell response to high glucose, how miR-27b-3p and Nrf2 expression are regulated, and whether this pathway could be specifically targeted. METHODS: hRPE cells were cultured in normal glucose or high glucose for 1, 3, or 6d before measuring cellular proliferation rates using cell counting kit-8 and reactive oxygen species (ROS) levels using a dihydroethidium kit. miR-27b-3p, Nrf2, NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) mRNA and protein levels were analyzed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunocytofluorescence (ICF), respectively. Western blot analyses were performed to determine nuclear and total Nrf2 protein levels. Nrf2, NQO1, and HO-1 expression levels by RT-qPCR, ICF, or Western blot were further tested after miR-27b-3p overexpression or inhibitor lentiviral transfection. Finally, the expression level of those target genes was analyzed after treating hRPE cells with pyridoxamine. RESULTS: Persistent exposure to high glucose gradually suppressed hRPE Nrf2, NQO1, and HO-1 mRNA and protein levels and increased miR-27b-3p mRNA levels. High glucose also promoted ROS release and inhibited cellular proliferation. Nrf2, NQO1, and HO-1 mRNA levels decreased after miR-27b-3p overexpression and, conversely, both mRNA and protein levels increased after expressing a miR-27b-3p inhibitor. After treating hRPE cells exposed to high glucose with pyridoxamine, ROS levels tended to decreased, proliferation rate increased, Nrf2, NQO1, and HO-1 mRNA and protein levels were upregulated, and miR-27b-3p mRNA levels were suppressed. CONCLUSION: Nrf2 is a downstream target of miR-27b-3p. Furthermore, the miR-27b-3p inhibitor pyridoxamine can alleviate high glucose injury by regulating the miR-27b-3p/Nrf2 axis.

MicroRNA-27b Impairs Nrf2-Mediated Angiogenesis in the Progression of Diabetic Foot Ulcer.

Nuclear factor erythroid-2-related factor 2 (Nrf2) is a stress-activated transcription factor regulating antioxidant genes, and a deficiency thereof, slowing lymphangiogenesis, has been reported in diabetic foot ulcer (DFU). The mode of Nrf2 regulation in DFU has been less explored. Emerging studies on miRNA-mediated target regulation show miRNA to be the leading player in the pathogenesis of the disease. In the present study, we demonstrated the role of miR-27b in regulating Nrf2-mediated angiogenesis in DFU. A lower expression of mRNA targets, such as Nrf2, HO-1, SDF-1α, and VEGF, was observed in tissue biopsied from chronic DFU subjects, which was in line with miR-27b, signifying a positive correlation with Nrf2. Similarly, we found significantly reduced expression of miR-27b and target mRNAs Nrf2, HO-1, SDF-1α, and VEGF in endothelial cells under a hyperglycemic microenvironment (HGM). To confirm the association of miR-27b on regulating Nrf2-mediated angiogenesis, we inhibited its expression through RNA interference-mediated knockdown and observed disturbances in angiogenic signaling with reduced endothelial cell migration. In addition, to explore the role of miR-27b and angiogenesis in the activation of Nrf2, we pretreated the endothelial cells with two well-known pharmacological compounds-pterostilbene and resveratrol. We observed that activation of Nrf2 through these compounds ameliorates impaired angiogenesis on HGM-induced endothelial cells. This study suggests a positive role of miR-27b in regulating Nrf2, which seems to be decreased in DFU and improves on treatment with pterostilbene and resveratrol.

Inhibitory effect of miR-27b-3p and Nrf2 regulation on metabolic memory formation in human RPE cells / 中华实验眼科杂志

Objective:To investigate the effect of microRNA-27b-3p (miR-27b-3p)/nuclear factor-E2-related factor 2 (Nrf2) on metabolic memory impairment of human retinal pigment epithelial (RPE) cells and to explore its regulatory mechanism.Methods:ARPE-19 cells were divided into normal control group, metabolic memory group, miR-27b-3p control group, miR-27b-3p inhibitor group, and liraglutide group.Cells in normal control group were cultured in 5.5 mmol/L normal glucose medium for 6 days.Cells in metabolic memory group were cultured in 30 mmol/L glucose for 3 days and changed to 5.5 mmol/L for 3 days.Cells in miR-27b-3p inhibitor group were added with puromycin after lentiviral transfection to select the successfully transfected cells, and were cultured in 30 mmol/L glucose for 3 days then 5.5 mmol/L glucose for 3 days.Cells in liraglutide group were cultured in 30 mmol/L glucose with liraglutide for 3 days then 5.5 mmol/L glucose for 3 days.The regulatory relationship between miR-27b-3p and Nrf2 was verified by lentiviral transfection.Expressions of miR-27b-3p, Nrf2, NAD(P)H dehydrogenase[quinone]1 (NQO1), heme oxygenase-1 (HO-1) mRNA and protein levels were analyzed by real-time quantitative PCR.Total and nuclear Nrf2 protein expressions were detected by Western blot.The cell proliferation rates of various groups were determined by cell counting kit-8 (CCK-8).The reactive oxygen species (ROS) level was detected by the DHE kit.Results:The miR-27b-3p mRNA relative expression of normal control group, metabolic memory group, miR-27b-3p control group, miR-27b-3p inhibitor group was 1.000±0.000, 1.881±0.034, 1.683±0.088 and 0.111±0.008, respectively, with a statistically significant difference ( F=850.815, P<0.001).The miR-27b-3p mRNA relative expression level was lower in normal control group than in metabolic memory group, lower in miR-27b-3p inhibitor group than in normal control group, and the differences were statistically significant (both at P<0.01).The expression levels of Nrf2 mRNA, total protein, and nuclear protein were decreased in metabolic memory group in comparison with normal control group and were significantly increased in miR-27b-3p inhibitor group in comparison with miR-27b-3p control group, showing statistically significant differences (all at P<0.01).The NQO1 and HO-1 mRNA expressions were decreased in metabolic memory group in comparison with normal control group, and were significantly higher in miR-27b-3p inhibitor group compared with miR-27b-3p control group, showing statistically significant differences (all at P<0.01).The fluorescence intensity of Nrf2, NQO1, and HO-1 was lower in metabolic memory group than in normal control group, and was higher in miR-27b-3p inhibitor group than in miR-27b-3p control group, showing statistically significant differences (all at P<0.01).Compared with metabolic memory group, the relative expression of miR-27b-3p mRNA declined in liraglutide group, with a statistically significant difference ( P<0.05).The relative expression levels of Nrf2 mRNA, NQO1 mRNA, HO-1 mRNA, total and nuclear Nrf2 protein of liraglutide group were enhanced in comparison with metabolic memory group, with statistically significant differences (all at P<0.05).The fluorescence intensity of Nrf2, NQO1, and HO-1 was enhanced in liraglutide group in comparison with metabolic memory group, and the differences were statistically significant (all at P<0.05).Compared with normal control group and liraglutide group, the cell proliferation viability was decreased in metabolic memory group, and the differences were statistically significant (both at P<0.01).The relative content of ROS was higher in metabolic memory group than in normal control group and liraglutide group, and the difference was significant (all at P<0.01). Conclusions:Liraglutide reverses the inhibition of metabolic memory on Nrf2, NQO1, and HO-1 by downregulating miR-27b-3p.

Chrysophanol exerts a protective effect against sepsis-induced acute myocardial injury through modulating the microRNA-27b-3p/Peroxisomal proliferating-activated receptor gamma axis.

Sepsis, a leading contributor to the death of inpatients, results in severe organ dysfunction as complications. The heart is one of the major organs attacked by sepsis, and the effective control of the inflammatory cascade reaction in sepsis is of great significance in alleviating sepsis-associated acute myocardial injury (S-AMI). Chrysophanol, a natural anthraquinone, has been discovered to carry anti-inflammatory effects. The aim of this paper is to probe the impact of Chrysophanol on S-AMI. An S-AMI model was engineered in rats via CLP. Pathological alterations in the myocardial tissues of rats were monitored. qRT-PCR, ELISA, and western blot measured the profiles of miR-27b-3p, Peroxisomal proliferating-activated receptor gamma (PPARG), inflammatory cytokines (TNF-α, IL-1ß, IL-6, IL-8), and inflammatory response proteins (NF-κB-p65, MAPK-p38, JNK1/2). Besides, miR-27b-3p mimics were transfected into cardiomyocytes, and the proliferation and apoptosis of cardiomyocytes were examined through MTT and flow cytometry. As evidenced by the experimental outcomes, chrysophanol suppressed sepsis-mediated acute myocardial injury and LPS-mediated apoptosis in myocardial cells and lessened the release of pro-inflammatory cytokines and inflammatory response proteins. Moreover, chrysophanol cramped miR-27b-3p expression and heightened PPARG expression. miR-27b-3p targeted PPARG and restrained its expression. On the other hand, the PPARG agonist (RGZ) partially eliminated the apoptosis and pro-inflammatory responses of myocardial cells elicited by LPS. Therefore, this study revealed that Chrysophanol guarded against sepsis-mediated acute myocardial injury through dampening inflammation and apoptosis via the miR-27b-3p-PPARG axis, adding to the references for treating sepsis-AMI.

LINC01234 regulates microRNA-27b-5p to induce the migration, invasion and self-renewal of ovarian cancer stem cells through targeting SIRT5.

LINC01234 has been suggested to correlate with the survival of ovarian cancer (OS), but its role in the properties of OC stem cells (OCSCs) has been rarely described. We aim to investigate the effect of LINC01234 on the differentiation and self-renewal of OCSCs through adsorption of microRNA (miR)-27b-5p to target sirtuins 5 (SIRT5). Expression of LINC01234 and SIRT5 in OC and normal samples included in TCGA and GTEx was searched through the GEPIA2 database. Bioinformatics analysis was conducted to predict the relation of LINC01234, miR-27b-5p and SIRT5. Expression of LINC01234, miR-27b-5p and SIRT5 in OC tissues and cells was detected. OCSCs were cultured and identified. CD133+ OCSCs were introduced with related oligonucleotides or vectors of LINC01234 or miR-27b-5p and SIRT5 to figure out their roles in OCSCs progression and tumorigenesis in vivo. The interaction of miR-27b-5p with LINC01234 or SIRT5 was analyzed. Bioinformatics analysis suggested that LINC01234 was very likely to influence SIRT5 and regulate the development of OC through miR-27b-5p. Up-regulated LINC01234 exhibited in OC tissues and cells. Down-regulated LINC01234 or elevated miR-27b-5p suppressed OCSCs progression and tumorigenesis in vivo. LINC01234 could restore SIRT5 expression by binding to miR-27b-5p. Down-regulated miR-27b-5p reversed the effect of silenced LINC01234 on OCSCs development and tumorigenesis in vivo. Up-regulation of SIRT5 reduced the effects of elevated miR-27b-5p on OCSCs progression and tumorigenesis in vivo. LINC01234 regulates miR-27b-5p to induce the migration, invasion and self-renewal of OCSCs through targeting SIRT5.

Relationship between peripheral blood microRNA-27b expression and left ventricular hypertrophy in patients with hypertensive / 中国医师进修杂志

Objective:To investigate the correlation between peripheral blood microRNA (miR)-27b expression and left ventricular hypertrophy (LVH) in patients with hypertensive.Methods:The clinical data of 120 patients with hypertension from February 2019 to February 2021 in Zhuzhou Hospital Affiliated to Xiangya School of Medicine, Central South University were retrospectively analyzed. Among them, LVH occurred in 70 cases (LVH group), none in 50 cases (NLVH group). The ventricular septal thickness (VST), left ventricular posterior diastolic wall thickness (LVPWTd) and left ventricular end diastolic diameter (LVEDD) were measured by echocardiography, the left ventricular mass (LVM) and left ventricular mass index (LVMI) were calculated. The peripheral blood expression level of miR-27b was detected by real-time quantitative polymerase chain reaction. Correlation was analyzed by Pearson correlation analysis. Receiver operating characteristic (ROC) curve was drawn to evaluate the efficacy of peripheral blood miR-27b in diagnosing LVH in patients with hypertensive.Results:The peripheral blood expression level of miR-27b in LVH group was significantly higher than that in NLVH group (6.37 ± 0.23 vs. 3.42 ± 0.18), and there was statistical difference ( t = 9.58, P<0.01). Pearson correlation analysis result showed that the peripheral blood expression of miR-27b was positively correlated with LVMI, VST, LVEDD and LVPWTd in hypertensive LVH patients ( r = 0.71, 0.63, 0.75 and 0.68; P<0.01). After eliminating risk factors (duration of hypertension, systolic blood pressure, diastolic blood pressure, different medications, uric acid), multiple linear regression analysis result showed that the peripheral blood expression of miR-27b was positively correlated with LVMI, VST, LVEDD and LVPWTd in hypertensive LVH patients ( β = 0.07, 0.63, 0.42 and 0.48; P<0.01). ROC curve analysis result showed that the area under the curve of peripheral blood expression of miR-27b in predicted LVH in patients with hypertension was 0.905 (95% CI 0.854 to 0.957), the optimal cut-off value was 4.45, the sensitivity was 84.0%, and the specificity was 82.9%. Conclusions:The high peripheral blood expression of miR-27b in hypertension LVH patients is positively correlated with LVMI and other echocardiographic parameters, which has a good predictive value of LVH in patients with hypertension.

MicroRNA-27b-3p inhibits the proliferation and invasion of cutaneous squamous cell carcinoma by targeting EGFR and MMP-13.

Cutaneous squamous cell carcinoma is a common malignant tumor. The aim of the present study was to examine the biological function of microRNA (miR)-27b-3p in cutaneous squamous cell carcinoma (CSCC) and its underlying mechanism. The relative expression levels of miR-27b-3p were determined in A-431, Colo-16 and NHEK/SVTERT3-5 cell lines. The regulatory effects of miR-27b-3p on the proliferation of CSCC cells were evaluated using MTT and colony formation assays. Transwell assays were conducted to examine the role of miR-27b-5p in the migratory and invasive abilities of CSCC cells. The levels of EGFR, MMP-13, Akt, phosphorylated (p)-Akt, cyclin D1, N-cadherin (CAD) and E-CAD were detected in CSCC cells using reverse transcription-quantitative PCR and western blot analysis. Binding between miR-27b-3p and the 3'-untranslated region (UTR) of EGFR or MMP-13 was assessed using a dual-luciferase reporter assay. miR-27b-3p was significantly downregulated in CSCC cell lines, compared with the skin keratinocyte cell line. Transfection with a miR-27b-3p mimic significantly reduced the proliferative, migratory and invasive abilities of CSCC cells in vitro. Moreover, miR-27b-3p mimic transfection downregulated the mRNA and protein levels of EGFR, MMP-13, cyclin D1, p-Akt and N-CAD, whilst upregulating E-CAD levels in CSCC cells. miR-27b-3p was found to target the EGFR and MMP-13 3'-UTRs, thus downregulating the expression of these molecules. The inhibition of CSCC proliferation by miR-27b-3p was effectively reversed by EGFR overexpression. Moreover, the inhibitory effect of miR-27b-3p on the migratory and invasive abilities of CSCC cells was abolished by MMP-13 overexpression. In conclusion, miR-27b-3p inhibits the proliferation, migration and invasion of CSCC cells by downregulating the expression of EGFR and MMP-13 and may represent a potential diagnostic marker and therapeutic option for CSCC.

[Retracted] Upregulation of microRNA­27b contributes to the migration and invasion of gastric cancer cells via the inhibition of sprouty2­mediated ERK signaling.

Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that the Transwell cell migration assay data shown in Fig. 4C were strikingly similar to data appearing in different form in other articles by different authors. Owing to the fact that the contentious data in the above article had already been published elsewhere, or were already under consideration for publication, prior to its submission to Molecular Medicine Reports, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive any reply. The Editor apologizes to the readership for any inconvenience caused. [the original article was published in Molecular Medicine Reports 13: 2267­2272, 2016; DOI: 10.3892/mmr.2016.4779].

MicroRNA-27b expression in rats submitted to physical exercise and experimental cerebral ischemia / Expresión de MicroRNA-27b en ratas sometidas a ejercicio físico e isquemia cerebral experimental

SUMMARY: Cerebral ischemia has not only a high mortality rate, which is the second leading cause of death worldwide, but is also responsible for severe disabilities in working age individuals, generating enormous public expending for treatment and rehabilitation of the affected individuals. The role of microRNAs in the pathophysiology of cerebral ischemia has been highlighted in current investigations. In addition, recent studies have also highlighted physical exercise as a possible protective factor both in the prevention and in the effects of cerebral ischemia, placing it as an important study resource. Thus, we investigated the role of physical exercise in experimental cerebral ischemia associated with the expression of microRNA-27b. 16 animals were used, divided into four experimental groups: Control, Physical Exercise, Cerebral Ischemia and Cerebral Ischemia associated with Physical Exercise. The real-time PCR methodology was used to analyze the expression of microRNA-27b. Although there were no statistically significant differences in the expression of microRNA-27b between the groups studied, the increased expression of microRNA-27b in the Physical Exercise group indicates its neuroprotective role in the pathophysiology of cerebral ischemia.

RESUMEN: La isquemia cerebral no solo tiene una alta tasa de mortalidad y es la segunda causa principal de muerte en todo el mundo, sino también es la causa de enfermedades invalidantes en personas en edad laboral, lo que genera un gasto público enorme para el tratamiento y la rehabilitación de las personas afectadas. El papel de los microARN en la fisiopatología de la isquemia cerebral se ha destacado en las investigaciones actuales. Además, estudios recientes también han destacado el ejercicio físico como un posible factor protector tanto en la prevención como en los efectos de la isquemia cerebral, situándolo como un importante recurso de estudio. Por lo tanto, investigamos el papel del ejercicio físico en la isquemia cerebral experimental asociada con la expresión del microARN-27b. Se utilizaron 16 animales, divididos en cuatro grupos experimentales: Control, Ejercicio Físico, Isquemia Cerebral e Isquemia Cerebral asociada al Ejercicio Físico. Se utili- zó la metodología de PCR en tiempo real para analizar la expresión de microARN-27b. Aunque no se observaron diferencias estadísticamente significativas en la expresión de microARN-27b entre los grupos estudiados, la mayor expresión de microARN-27b en el grupo de Ejercicio Físico indica su papel neuroprotector en la fisiopatología de la isquemia cerebral.

MicroRNA-27b inhibits the development of melanoma by targeting MYC.

Cutaneous malignant melanoma is a malignancy with one of the fastest increasing incidence rates worldwide; however, the mechanism underlying the occurrence and development of melanoma remains unclear. The aim of the present study was to identify novel biomarkers for the occurrence and development of melanoma. The results of the present study demonstrated that the expression levels of microRNA (miR)-27b were decreased in melanoma tissue samples compared with those in adjacent noncancerous tissue samples and cells according to online and experimental data. By contrast, MYC expression levels were upregulated in melanoma compared with those in adjacent noncancerous tissue samples. miR-27b overexpression significantly inhibited A375 and A2085 melanoma cell DNA synthesis, viability and invasive ability. Dual-luciferase reporter assay results demonstrated that miR-27b inhibited MYC expression through binding to the 3'-untranslated region of MYC mRNA. MYC knockdown in melanoma cells exerted similar effects to those of miR-27b overexpression on DNA synthesis, cell viability and invasive ability; the effects of miR-27b inhibition were significantly reversed by MYC knockdown. In conclusion, the miR-27b/MYC axis may modulate malignant melanoma cell biological behaviors and may be a potential target for melanoma treatment.

Long non-coding RNA OIP5-AS1 contributes to cisplatin resistance of oral squamous cell carcinoma through the miR-27b-3p/TRIM14 axis.

Oral squamous cell carcinoma (OSCC) accounts for 90% of oral cavity cancer types, but the overall prognosis for patients with OSCC remains unfavorable. Cisplatin (DDP) is an effective drug in OSCC treatment, but DDP resistance weakens its therapeutic effect. Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) can trigger DDP resistance. The purpose of the current study was to explore the role and mechanism ofOIP5-AS1 in OSCC DDP resistance. In the present study, the expression levels of OIP5-AS1, microRNA (miR)-27b-3p and tripartite motif-containing 14 (TRIM14) were detected by reverse transcription-quantitative PCR. DDP resistance was measured using an MTT assay. Moreover, cell proliferation, migration and invasion were assessed by MTT, Transwell, and Matrigel assays. Protein expression levels of TRIM14, E-cadherin, N-cadherin and Vimentin were detected by western blot analysis. Putative binding sites between miR-27b-3p andOIP5-AS1 or TRIM14werepredicted with starBase and verified using a dual-luciferase reporter assay. The role of OIP5-AS1 in DDP resistance of OSCC in vivo was measured using a xenograft tumor model. It was observed that OIP5-AS1 was upregulated in DDP-resistant OSCC cells, and the knockdown of OIP5-AS1 improved DDP sensitivity in DDP-resistant OSCC cells. The present study identified that miR-27b-3p was a target of OIP5-AS1. Furthermore, miR-27b-3p silencing reversed the effect of OIP5-AS1 knockdown on DDP sensitivity in DDP-resistant OSCC cells. TRIM14was shown to be a direct target of miR-27b-3p, and TRIM14 overexpression abolished the effect of miR-27b-3p on DDP sensitivity in DDP-resistant OSCC cells. The results suggested that OIP5-AS1 increased TRIM14 expression by sponging miR-27b-3p. In addition, OIP5-AS1 knockdown enhanced DDP sensitivity of OSCC in vivo. Data from the present study indicated that OIP5-AS1 may improve DDP resistance through theupregulationTRIM14 mediated bymiR-27b-3p, providing a possible therapeutic strategy for OSCC treatment.

Serum from patients with hypertension promotes endothelial dysfunction to induce trophoblast invasion through the miR­27b­3p/ATPase plasma membrane Ca2+ transporting 1 axis.

Pregnancy­induced hypertension is often accompanied by preeclampsia. The present study investigated whether microRNA (miR)­27b­3p affected the occurrence of preeclampsia by regulating the function of endothelial cells. Expressions levels of miR­27b­3p and ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) were determined using reverse­transcription quantitative PCR. miR­27b­3p targeting ATP2B1 was predicted using bioinformatics and further confirmed by dual­luciferase reporter assays. Cell Counting Kit­8, Transwell and Matrigel tube formation assays were performed to detect the effects of miR­27b­3p on proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs), respectively. Moreover, HTR8/SVneos cells were co­cultured with HUVECs to detect the invasion of trophoblast cells, and the expression levels of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)­2 and MMP­9 of HUVECs and HTR8/SVneos were detected by western blotting. Expression levels of miR­27b­3p were upregulated in the serum of patients with hypertension and preeclampsia, which could target and regulate the expression of ATP2B1. The expression levels of miR­27b­3p were increased and those of ATP2B1 were reduced in HUVECs from hypertensive serums. Moreover, miR­27b­3p mimics reduced the expression level of ATP2B1, and miR­27b­3p inhibitor reversed the effect of hypertensive serum on ATP2B1 expression. Furthermore, patients with hypertension showed increased endothelial dysfunction, reduced trophoblastic invasion and the expressions of VEGF, MMP­2 and MMP­9, and miR­27b­3p mimics and silencing of ATP2B1 produced similar results to HUVECs. The miR­27b­3p inhibitor reversed the effect of hypertensive serum, and silencing of ATP2B1 inhibited the improvement of miR­27b­3p inhibitor to HUVECs and HTR­8/SVneo cells in proliferation, migration and tube formation. The current findings suggested that miR­27b­3p promoted proliferation, migration and tube formation of HUVECs and enhanced invasion of trophoblast cells, via regulation of ATP2B1. Thus, miR­27b­3p could be considered as a molecular risk factor in the pathogenesis and development of preeclampsia.

microRNA-27b shuttled by mesenchymal stem cell-derived exosomes prevents sepsis by targeting JMJD3 and downregulating NF-κB signaling pathway.

BACKGROUND: Exosomal microRNAs (miRs) derived from mesenchymal stem cells (MSCs) have been shown to play roles in the pathophysiological processes of sepsis. Moreover, miR-27b is highly enriched in MSC-derived exosomes. Herein, we aimed to investigate the potential role and downstream molecular mechanism of exosomal miR-27b in sepsis. METHODS: Inflammation was induced in bone marrow-derived macrophages (BMDMs) by lipopolysaccharide (LPS), and mice were made septic by cecal ligation and puncture (CLP). The expression pattern of miR-27b in MSC-derived exosomes was characterized using RT-qPCR, and its downstream gene was predicted by in silico analysis. The binding affinity between miR-27b, Jumonji D3 (JMJD3), or nuclear factor κB (NF-κB) was characterized to identify the underlying mechanism. We induced miR-27b overexpression or downregulation, along with silencing of JMJD3 or NF-κB to examine their effects on sepsis. The production of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 was detected by ELISA. RESULTS: miR-27b was highly expressed in MSC-derived exosomes. Mechanistic investigations showed that miR-27b targeted JMJD3. miR-27b decreased expression of pro-inflammatory genes by inhibiting the recruitment of JMJD3 and NF-κB at gene promoter region. Through this, MSC-derived exosomal miR-27b diminished production of pro-inflammatory cytokines in LPS-treated BMDMs and septic mice, which could be rescued by upregulation of JMJD3 and NF-κB. Besides, in vitro findings were reproduced by in vivo findings. CONCLUSION: These data demonstrated that exosomal miR-27b derived from MSCs inhibited the development of sepsis by downregulating JMJD3 and inactivating the NF-κB signaling pathway.

Downregulating long non-coding RNA PVT1 expression inhibited the viability, migration and phenotypic switch of PDGF-BB-treated human aortic smooth muscle cells via targeting miR-27b-3p.

Long non-coding RNA Plasmacytoma Variant Translocation 1 (LncRNA PVT1) was involved in various human diseases, but its role in aortic dissection (AD) remained to be fully examined. In this study, the viability and migration of human aortic smooth muscle cells (HASMCs) were respectively measured by MTT assay and wound-healing assay. Relative phenotypic switch-related protein expressions were measured with quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot as needed. An AD model was established in animals and hematoxylin-eosin (H&E) staining was used for pathological examination. We found that, in HASMCs, microRNA (miR)-27b-3p could competitively bind with PVT1. In AD, PVT1 expression was upregulated, yet that of miR-27b-3p was downregulated. Downregulating PVT1 reversed the effects of growth factor-BB (PDGF-BB) treatment on PVT1, miR-27b-3p and expressions of phenotypic switch-related markers, and cell viability and migration, while downregulating miR-27b-3p reversed the effects of downregulating PVT1. Moreover, downregulating PVT1 suppressed the effects of upregulated PVT1 and downregulated miR-27b-3p induced by AD as well as media degeneration in vivo. In conclusion, downregulating PVT1 expression suppressed the proliferation, migration and phenotypic switch of HASMCs treated by PDGF-BB via targeting miR-27b-3p.

Downregulation of miR-27b promotes skin wound healing in a rat model of scald burn by promoting fibroblast proliferation.

The aim of the present study was to investigate the effect and mechanism of action of microRNA (miR)-27b on skin wound healing in rats with deep second-degree scald burns and in BJ human skin fibroblast cells. Rat models with deep second-degree scald burns were constructed and injected with miR-27b mimics and inhibitors at the wound site daily for 21 days. Healing of burned skin tissues was observed at 0, 3, 7, 14 and 21 days following modeling. H&E and Masson staining were used to observe the pathological structure and degree of collagen fibers in the burned skin tissues. The effects of miR-27b on BJ cell proliferation and migration were determined by MTT and scratch assays. Matrix metalloproteinase-1 (MMP-1), α-smooth muscle actin (α-SMA), collagen I and collagen III expression in rat skin tissues and BJ cells were measured via reverse transcription-quantitative PCR and western blot analysis. The results of the in vivo experiments demonstrated that miR-27b inhibition accelerated scalded skin healing and induced fibroblast growth. Furthermore, the in vitro experiments revealed that miR-27b inhibition increased BJ cell proliferation and migration. Furthermore, miR-27b inhibition upregulated MMP-1, α-SMA, collagen I and collagen III expression in the skin tissues and cells, while the overexpression of miR-27b demonstrated the opposite effect. In conclusion, the results of the present study revealed that miR-27b inhibition increased fibroblast proliferation, thereby accelerating scald wound healing in rats.

Impaired mitochondrial function of alveolar macrophages in carbon nanotube-induced chronic pulmonary granulomatous disease.

Human exposure to carbon nanotubes (CNT) has been associated with the development of pulmonary sarcoid-like granulomatous disease. Our previous studies demonstrated that multi-walled carbon nanotubes (MWCNT) induced chronic pulmonary granulomatous inflammation in mice. Granuloma formation was accompanied by decreased peroxisome proliferator-activated receptor gamma (PPARγ) and disrupted intracellular lipid homeostasis in alveolar macrophages. Others have shown that PPARγ activation increases mitochondrial fatty acid oxidation (FAO) to reduce free fatty acid accumulation. Hence, we hypothesized that the disrupted lipid metabolism suppresses mitochondrial FAO. To test our hypothesis, C57BL/6 J mice were instilled by an oropharyngeal route with 100 µg MWCNT freshly suspended in 35 % Infasurf. Control sham mice received vehicle alone. Sixty days following instillation, mitochondrial FAO was measured in permeabilized bronchoalveolar lavage (BAL) cells. MWCNT instillation reduced the mitochondrial oxygen consumption rate of BAL cells in the presence of palmitoyl-carnitine as mitochondrial fuel. MWCNT also reduced mRNA expression of mitochondrial genes regulating FAO, carnitine palmitoyl transferase-1 (CPT1), carnitine palmitoyl transferase-2 (CPT2), hydroxyacyl-CoA dehydrogenase subunit beta (HADHB), and PPARγ coactivator 1 alpha (PPARGC1A). Importantly, both oxidative stress and apoptosis in alveolar macrophages and lung tissues of MWCNT-instilled mice were increased. Because macrophage PPARγ expression has been reported to be controlled by miR-27b which is known to induce oxidative stress and apoptosis, we measured the expression of miR-27b. Results indicated elevated levels in alveolar macrophages from MWCNT-instilled mice compared to controls. Given that inhibition of FAO and apoptosis are linked to M1 and M2 macrophage activation, respectively, the expression of both M1 and M2 key indicator genes were measured. Interestingly, results showed that both M1 and M2 phenotypes of alveolar macrophages were activated in MWCNT-instilled mice. In conclusion, alveolar macrophages of MWCNT-instilled mice had increased miR-27b expression, which may reduce the expression of PPARγ resulting in attenuation of FAO. This reduction in FAO may lead to activation of M1 macrophages. The upregulation of miR-27b may also induce apoptosis, which in turn can cause M2 activation of alveolar macrophages. These observations indicate a possible role of miR-27b in impaired mitochondrial function in the chronic activation of alveolar macrophages by MWCNT and the development of chronic pulmonary granulomatous inflammation.

Inhibition of microRNA-27b-3p relieves osteoarthritis pain via regulation of KDM4B-dependent DLX5.

Osteoarthritis (OA) represents a progressive degenerative disorder that predominantly affects the synovial membranes of joints. Recent studies have highlighted the significant role played by microRNAs (miRNAs) in OA development. The current study aimed to elucidate the underlying modulatory role of miR-27b-3p in the development of OA. The expression of miR-27b-3p in the OA patients and rat models post anterior cruciate ligament transection operation was measured using reverse transcription quantitative polymerase chain reaction, through which overexpressed miR-27b-3p was found in both of the samples. To further explore the miR-27b-3p functions in OA, western blot analysis, enzyme-linked immunosorbent assay, and ß-galactosidase activity assay were conducted with the results showing that knockdown of miR-27b-3p promoted expression of the osteogenic differentiation markers while inhibiting expression of the adipogenic differentiation markers, inflammatory factors, and cellular senescence of bone marrow mesenchymal stem cells (BMSCs). After that, the interactions between miR-27b-3p, lysine Demethylase 4B (KDM4B), and Distal-Less Homeobox 5 (DLX5) identified using dual-luciferase reporter gene assay and ChIP assay revealed that miR-27b-3p inhibited KDM4B and further reduced expression of DLX5. Finally, the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were assessed in rat models, and increased PWT and PWL were detected after miR-27b-3p silencing. In conclusion, suppression of miR-27b-3p could enhance KDM4B and DLX5 to alleviate OA pain, shedding light on a new potential therapeutic target for OA.

Extracellular vesicle-carried microRNA-27b derived from mesenchymal stem cells accelerates cutaneous wound healing via E3 ubiquitin ligase ITCH.

Mesenchymal stem cells (MSCs) have been highlighted as promising candidate cells in relation to cutaneous wound healing. The current study aimed to investigate whether MSC-derived extracellular vesicles (EVs) could transfer microRNA-27b (miR-27b) to influence cutaneous wound healing. The miR-27b expression was examined in the established cutaneous wound mouse model, and its correlation with the wound healing rate was evaluated by Pearson's correlation analysis. The identified human umbilical cord MSC-derived EVs were co-cultured with human immortal keratinocyte line HaCaT and human skin fibroblasts (HSFs). The mice with cutaneous wound received injections of MSC-derived EVs. The effects of EVs or miR-27b loaded on wound healing and cellular functions were analysed via gain- and loss-of-function approaches in the co-culture system. Dual-luciferase reporter gene assay was employed to verify the relationship between miR-27b and Itchy E3 ubiquitin protein ligase (ITCH). Rescue experiments were conducted to investigate the underlying mechanisms associated with the ITCH/JUNB/inositol-requiring enzyme 1α (IRE1α) axis. miR-27b was down-regulated in the mouse model, with its expression found to be positively correlated with the wound healing rate. Abundant miR-27b was detected in the MSC-derived EVs, while EV-transferred miR-27b improved cutaneous wound healing in mice and improved proliferation and migration of HaCaT cells and HSFs in vitro. As a target of miR-27b, ITCH was found to repress cell proliferation and migration. ITCH enhanced the JUNB ubiquitination and degradation, ultimately inhibiting JUNB and IRE1α expressions and restraining wound healing. Collectively, MSC-derived EVs transferring miR-27b can promote cutaneous wound healing via ITCH/JUNB/IRE1α signalling, providing insight with clinical implications.

MicroRNA-27b-3p Promotes Tumor Progression and Metastasis by Inhibiting Peroxisome Proliferator-Activated Receptor Gamma in Triple-Negative Breast Cancer.

Introduction: The role and underlying mechanisms of miR-27b-3p in triple-negative breast cancer (TNBC) remains unclear. Methods: miR-27b-3p expression level was evaluated in 99 TNBC patients with a median follow-up time of 133 months. The biological functions of miR-27b-3p by targeting PPARG were assessed by luciferase reporter assay, CCK-8 assay, Transwell assay, wound healing assay, western blot analysis and xenograft models. Results: High level of miR-27b-3p expression was found to confer poor prognosis in TNBC patients. MiR-27b-3p overexpression increased TNBC cell proliferation, migration, invasion, and metastasis. Our data suggested peroxisome proliferator-activated receptor gamma (PPARG) was a target of miR-27b-3p. The capacity of miR-27b-3p to induce TNBC progression and metastasis depended on its inhibition of the PPARG expression. Furthermore, restoring PPARG expression reversed the effect of miR-27b-3p overexpression. Mechanistically, miR-27b-3p regulated metastasis-related pathways through PPARG by promoting epithelial-mesenchymal transition. By suppressing PPARG, miR-27b-3p could also activate transcription factors Snail and NF-κB, thereby promoting metastasis. Conclusions: miR-27b-3p promotes TNBC progression and metastasis by inhibiting PPARG. MiR-27b-3p may be a potential prognostic marker of TNBC, and PPARG may be a potential molecular therapeutic target of TNBC.

Vascular smooth muscle cell phenotypic transition regulates gap junctions of cardiomyocyte.

Atrial fibrillation (AF) is one of the most prevalent arrhythmias. Myocardial sleeves of the pulmonary vein are critical in the occurrence of AF. Our study aims to investigate the effect of synthetic vascular smooth muscle cells (SMCs) on gap junction proteins in cardiomyocytes. (1) Extraction of vascular SMCs from the pulmonary veins of Norway rats. TGF-ß1 was used to induce the vascular SMCs switching to the synthetic phenotype and 18-α-GA was used to inhibit gap junctions of SMCs. The contractile and synthetic phenotype vascular SMCs were cocultured with HL-1 cells; (2) Western blotting was used to detect the expression of Cx43, Cx40 and Cx45 in HL-1 cells, and RT-PCR to test microRNA 27b in vascular SMCs or in HL-1 cells; (3) Lucifer yellow dye transfer experiment was used to detect the function of gap junctions. (1) TGF- ß1 induced the vascular SMCs switching to synthetic phenotype; (2) Cx43 was significantly increased, and Cx40 and Cx45 were decreased in HL-1 cocultured with synthetic SMCs; (3) The fluorescence intensity of Lucifer yellow was higher in HL-1 cocultured with synthetic SMCs than that in the cells cocultured with contractile SMCs, which was inhibited by18-α-GA; (4) the expression of microRNA 27b was increased in HL-1 cocultured with synthetic SMCs, which was attenuated markedly by 18-α-GA. (5) the expression of ZFHX3 was decreased in HL-1 cocultured with synthetic SMCs, which was reversed by 18-α-GA. The gap junction proteins of HL-1 were regulated by pulmonary venous SMCs undergoing phenotypic transition in this study, accompanied with the up-regulation of microRNA 27b and the down-regulation of ZFHX3 in HL-1 cells, which was associated with heterocellular gap junctions between HL-1 and pulmonary venous SMCs.