Novel role of the clustered miR-23b-3p and miR-27b-3p in enhanced expression of fibrosis-associated genes by targeting TGFBR3 in atrial fibroblasts.

Atrial fibrillation (AF) is the most common type of arrhythmia in cardiovascular diseases. Atrial fibrosis is an important pathophysiological contributor to AF. This study aimed to investigate the role of the clustered miR-23b-3p and miR-27b-3p in atrial fibrosis. Human atrial fibroblasts (HAFs) were isolated from atrial appendage tissue of patients with sinus rhythm. A cell model of atrial fibrosis was achieved in Ang-II-induced HAFs. Cell proliferation and migration were detected. We found that miR-23b-3p and miR-27b-3p were markedly increased in atrial appendage tissues of AF patients and in Ang-II-treated HAFs. Overexpression of miR-23b-3p and miR-27b-3p enhanced the expression of collagen, type I, alpha 1 (COL1A1), COL3A1 and ACTA2 in HAFs without significant effects on their proliferation and migration. Luciferase assay showed that miR-23b-3p and miR-27b-3p targeted two different sites in 3'-UTR of transforming growth factor (TGF)-ß1 receptor 3 (TGFBR3) respectively. Consistently, TGFBR3 siRNA could increase fibrosis-related genes expression, along with the Smad1 inactivation and Smad3 activation in HAFs. Additionally, overexpression of TGFBR3 could alleviate the increase of COL1A1, COL3A1 and ACTA2 in HAFs after transfection with miR-23b-3p and miR-27b-3p respectively. Moreover, Smad3 was activated in HAFs in response to Ang-II treatment and inactivation of Smad3 attenuated up-regulation of miR-23b-3p and miR-27b-3p in Ang-II-treated HAFs. Taken together, these results suggest that the clustered miR-23b-3p and miR-27b-3p consistently promote atrial fibrosis by targeting TGFBR3 to activate Smad3 signalling in HAFs, suggesting that miR-23b-3p and miR-27b-3p are potential therapeutic targets for atrial fibrosis.

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.

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.

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.

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.

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-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.

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.