Tumour necrosis factor α regulates the miR-27a-3p-Sfrp1 axis in a mouse model of osteoporosis.

Osteoporosis is a metabolic bone disease that involves gradual loss of bone density and mass, thus resulting in increased fragility and risk of fracture. Inflammatory cytokines, such as tumour necrosis factor α (TNF-α), inhibit osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), and several microRNAs are implicated in osteoporosis development. This study aimed to explore the correlation between TNF-α treatment and miR-27a-3p expression in BMSC osteogenesis and further understand their roles in osteoporosis. An osteoporosis animal model was established using ovariectomized (OVX) mice. Compared with Sham mice, the OVX mice had a significantly elevated level of serum TNF-α and decreased level of bone miR-27a-3p, and in vitro TNF-α treatment inhibited miR-27a-3p expression in BMSCs. In addition, miR-27a-3p promoted osteogenic differentiation of mouse BMSCs in vitro, as evidenced by alkaline phosphatase staining and Alizarin Red-S staining, as well as enhanced expression of the osteogenic markers Runx2 and Osterix. Subsequent bioinformatics analysis combined with experimental validation identified secreted frizzled-related protein 1 (Sfrp1) as a downstream target of miR-27a-3p. Sfrp1 overexpression significantly inhibited the osteogenic differentiation of BMSCs in vitro and additional TNF-α treatment augmented this inhibition. Moreover, Sfrp1 overexpression abrogated the promotive effect of miR-27a-3p on the osteogenic differentiation of BMSCs. Furthermore, the miR-27a-3p-Sfrp1 axis was found to exert its regulatory function in BMSC osteogenic differentiation via regulating Wnt3a-ß-catenin signalling. In summary, this study revealed that TNF-α regulated a novel miR-27a-3p-Sfrp1 axis in osteogenic differentiation of BMSCs. The data provide new insights into the development of novel therapeutic strategies for osteoporosis.

Identification and Validation of Lipid Metabolism Gene FASN-Associated miRNA in Wilms Tumor.

miRNA has been a research hotspot in recent years and its scope of action is very wide, involving the regulation of cell proliferation, differentiation, apoptosis, and other biological behaviors. This study intends to explore the role of miRNA in the lipid metabolism and development of Wilms tumor (WT) by detecting and analyzing the differences in the expression profiles of miRNAs between the tumor and adjacent normal tissue. Gene detection was performed in tumor tissues and adjacent normal tissues of three cases of WT to screen differentially expressed miRNAs (DEMs). According to our previous research, FASN, which participates in the lipid metabolism pathway, may be a target of WT. The starBase database was used to predict FASN-targeted miRNAs. The above two groups of miRNAs were intersected to obtain FASN-targeted DEMs and then GO Ontology (GO) functional enrichment analysis of FASN-targeted DEMs was performed. Finally, the FASN-targeted DEMs were compared and further verified by qRT‒PCR. Through gene sequencing and differential analysis, 287 DEMs were obtained, including 132 upregulated and 155 downregulated miRNAs. The top ten DEMs were all downregulated. Fourteen miRNAs targeted by the lipid metabolism-related gene FASN were predicted by starBase. After intersection with the DEMs, three miRNAs were finally obtained, namely, miR-107, miR-27a-3p, and miR-335-5p. GO enrichment analysis was mainly concentrated in the Parkin-FBXW7-Cul1 ubiquitin ligase complex and response to prostaglandin E. Further experimental verification showed that miR-27a-3p was significantly correlated with WT (P = 0.0018). Imbalanced expression of miRNAs may be involved in the occurrence and development of WT through lipid metabolism. The expression of miR-27a-3p is related to the malignant degree of WT, and it may become the target of diagnosis, prognosis, and treatment of WT in the later stage.

[Expression relationship and significance of NEAT1 and miR-27a-3p in serum and cerebrospinal fluid of patients with Alzheimer disease].

OBJECTIVE: To explore the expression relationship and significance of long chain non-coding RNA nuclear-enriched abundant transcript 1 (LncRNA NEAT1) and miR-27a-3p in serum and cerebrospinal fluid of patients with Alzheimer disease (AD). METHODS: Sixty-six AD patients received by the department of neurology of our hospital from October 2019 to September 2021 were gathered, according to the clinical dementia rating scale score, they were grouped into mild group (≤1 point, n=41) and moderate-to-severe group (>1 point, n=25). Another 66 cases of serum and cerebrospinal fluid samples from outpatient physical examination personnel were regarded as the control group. The general information on all subjects was recorded and cognition was assessed; real-time quantitative PCR was performed to measure the expression levels of miR-27a-3p and NEAT1 in serum and cerebrospinal fluid; enzyme-linked immunosorbent assay was performed to measure the protein levels of ß-amyloid precursor protein cleaving enzyme 1 (BACE1), ß-amyloid (Aß) 40 and Aß42 in cerebrospinal fluid; Spearman' s method was performed to analyze the correlation of serum miR-27a-3p and NEAT1 levels with mini-mental state examination (MMSE) and montreal cognitive assessment (MoCA) scores; Pearson method was performed to analyze the correlation between serum miR-27a-3p and NEAT1 levels and Aß deposition standard uptake value ratio (SUVR) and cerebrospinal fluid miR-27a-3p, NEAT1, BACE1, Aß42 and Aß40 levels. RESULTS: The MMSE score [21 (17, 25), 9(7, 11) vs. 27 (21, 34)], MoCA score [17 (12, 21), 10 (7, 13) vs. 27 (21, 31)], serum miR-27a-3p level (0.55±0.13, 0.46±0.06 vs. 0.97±0.22), cerebrospinal fluid miR-27a-3p (0.48±0.10, 0.35±0.10 vs. 1.03±0.31), Aß42 levels [(303.55±36.77) ng/L, (231.45±34.14) ng/L vs. (499.99±53.63) ng/L] and Aß42/Aß40 ratio (0.030±0.008, 0.022±0.007 vs. 0.048±0.010) of AD patients in mild group and moderate-to-severe group were all lower than those in the control group, and the moderate-to-severe group were lower than the mild group (all P < 0.05); the serum NEAT1 level (2.31±0.64, 3.13±0.76 vs. 1.05±0.20), SUVR (1.50±0.29, 1.76±0.52 vs. 0.74±0.15), and cerebrospinal fluid NEAT1 (3.51±1.24, 4.30±1.65 vs. 1.01±0.23) and BACE1 levels [(55.78±5.98) µg/L, (72.32±16.08) µg/L vs. (21.39±3.73) µg/L] were higher than those in the control group, and the moderate-to-severe group were higher than the mild group (all P < 0.05). Serum NEAT1 level in AD patients was positively correlated with SUVR, cerebrospinal fluid NEAT1 and BACE1 (r=0.350, 0.606, 0.341, P < 0.05), and negatively correlated with MMSE score and MoCA score (r=-0.473, -0.482, all P < 0.05); serum miR-27a-3p level was positively correlated with cerebrospinal fluid miR-27a-3p level, MMSE score and MoCA score (r=0.695, 0.424, 0.412, all P < 0.05), and negatively correlated with SUVR and cerebrospinal fluid BACE1 level (r=-0.521, -0.447, all P < 0.05). CONCLUSION: The expression trends of NEAT1 and miR-27a-3p in the serum and cerebrospinal fluid of AD patients are consistent, the level of NEAT1 is increased, and the level of miR-27a-3p is decreased. The levels of the two are negatively correlated, which is related to the degree of Aß deposition in the brain of AD patients and is involved in the progression of AD.

Inhibition of inflammation and infiltration of M2 macrophages in NSCLC through the ATF3/CSF1 axis: Role of miR-27a-3p.

Non-small cell lung cancer (NSCLC) imposes a significant economic burden on patients and society due to its low overall cure and survival rates. Tumour-associated macrophages (TAM) affect tumour development and may be a novel therapeutic target for cancer. We collected NSCLC and tumour-adjacent tissue samples. Compared with the tumour-adjacent tissues, the Activation Transcription Factor 3 (ATF3) and Colony Stimulating Factor 1 (CSF-1) were increased in NSCLC tissues. Levels of ATF3 and CSF-1 were identified in different cell lines (HBE, A549, SPC-A-1, NCI-H1299 and NCI-H1795). Overexpression of ATF3 in A549 cells increased the expression of CD68, CD206 and CSF-1. Moreover, levels of CD206, CD163, IL-10 and TGF-ß increased when A549 cells were co-cultured with M0 macrophages under the stimulation of CSF-1. Using the starbase online software prediction and dual-luciferase assays, we identified the targeting between miR-27a-3p and ATF3. Levels of ATF3, CSF-1, CD206, CD163, IL-10 and TGF-ß decreased in the miR-27a mimics, and the tumour growth was slowed in the miR-27a mimics compared with the mimics NC group. Overall, the study suggested that miR-27a-3p might inhibit the ATF3/CFS1 axis, regulate the M2 polarization of macrophages and ultimately hinder the progress of NSCLC. This research might provide a new therapeutic strategy for NSCLC.

Ligustrazine promotes hypoxia/reoxygenation-treated trophoblast cell proliferation and migration by regulating the microRNA-27a-3p/ATF3 axis.

OBJECTIVE: Preeclampsia (PE) is a pregnancy-specific syndrome. Ligustrazine (LSZ) is involved in hypoxia/reoxygenation (H/R)-treated trophoblast cell regulation, but its mechanism remains elusive. This study explored the mechanism of LSZ in H/R-treated trophoblast cells to provide a theoretical basis for the new treatment method development for PE. METHODS: H/R HTR8/SVneo cell model was established for PE simulation to some extent. Trophoblast cell proliferation, apoptosis rate, migration, and invasion were detected by MTT assay, flow cytometry, scratch test, and Transwell assay. miR-27a-3p expression in trophoblast cells was detected by RT-qPCR. Binding sites between miR-27a-3p and ATF3 were predicted using Starbase and verified by dual-luciferase reporter assay. Activating transcription factor 3 (ATF3), ß-catenin, Cyclin D1, and c-Myc protein levels were examined using Western blot. After LSZ treatment, H/R-induced HTR8/SVneo cells were delivered with miR-27a-3p mimic or ATF3 siRNA to verify their roles in HTR8/SVneo cells. RESULTS: LSZ facilitated the proliferation, migration, and invasion of trophoblast cells and inhibited apoptosis. miR-27a-3p was elevated in H/R-induced HTR8/SVneo cells and miR-27a-3p overexpression annulled the effect of LSZ on trophoblast cells. miR-27a-3p targeted ATF3. ATF3 silencing averted the property of LSZ on trophoblast cells. Wnt/ß-catenin pathway-related proteins were repressed in H/R-induced HTR8/SVneo cells, and LSZ activated the Wnt/ß-catenin pathway by promoting ATF3 expression. CONCLUSION: LSZ mediated the Wnt pathway by regulating the miR-27a-3p/ATF3 axis, thus promoting the proliferation and migration of trophoblast cells. The protective mechanism of LSZ showed the potential application value in the treatment of PE.

METTL3 facilitates multiple myeloma tumorigenesis by enhancing YY1 stability and pri-microRNA-27 maturation in m6A-dependent manner.

Multiple myeloma (MM) is a pernicious plasma cell disorder and has a poor prognosis. N6-methyladenosine (m6A) is an abundant epigenetic RNA modification and is important in cancer progression. Nevertheless, the function of m6A and its regulator METTL3 in MM are rarely reported. Here, we identified the m6A "writers", METTL3, was enhanced in MM and found that Yin Yang 1 (YY1) and primary-miR-27a-3p were the potential target for METTL3. METTL3 promoted primary-miR-27a-3p maturation and YY1 mRNA stability in an m6A manner. YY1 also was found to facilitate miR-27a-3p transcription. METTL3 affected the growth, apoptosis, and stemness of MM cells through accelerating the stability of YY1 mRNA and the maturation of primary-miR-27a-3p in vitro and in vivo. Our results reveal the key function of the METTL3/YY1/miR-27a-3p axis in MM and may provide fresh insights into MM therapy.

Circ_0000011 promotes cerebral ischemia/reperfusion injury via miR-27a-3p-dependent regulation of NRIP1.

BACKGROUND: Cerebral ischemia/reperfusion (I/R) can result in brain function impairments. Circular RNAs (circRNAs) have emerged as vital regulators in cerebral I/R injury. However, the functions of mmu_circ_0000011 in cerebral I/R injury are still unclear. Thus, in this study, we aimed to explore the effect of mmu_circ_0000011 on cerebral I/R injury. METHODS: Oxygen-glucose deprivation and reperfusion (OGD/R)-induced HT-22 cells were used to mimic the condition of cerebral I/R injury in vitro. Cell Counting Kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) assay, 5'-ethynyl-2'-deoxyuridine (EdU) assay and flow cytometry analysis were utilized to assess cell viability, LDH release, proliferation and apoptosis, respectively. qRT-PCR and western blot were performed to determined the levels of circ_0000011, miR-27a-3p and NRIP1. Dual-luciferase reporter assay and RNA pull-down assay were utilized to analyze the targeting relation of circ_0000011, miR-27a-3p and NRIP1. RESULTS: OGD/R treatment inhibited HT-22 cell viability and promoted LDH release, cell apoptosis and inflammation. Circ_0000011 level was increased in OGD/R-induced HT-22 cells. Silencing of circ_0000011 promoted cell proliferation and inhibited LDH release, apoptosis and inflammation in OGD/R-treated HT-22 cells. For mechanism analysis, circ_0000011 was demonstrated to sponge miR-27a-3p, which directly targeted NRIP1. MiR-27a-3p inhibition or NRIP1 overexpression ameliorated the impacts of circ_0000011 silencing on cell proliferation, LDH release, apoptosis and inflammation in OGD/R-treated HT-22 cells. CONCLUSIONS: Circ_0000011 promotes OGD/R-induced HT-22 cell impairments by elevating NRIP1 through sponging miR-27a-3p.

The miR-27a-3p/FTO axis modifies hypoxia-induced malignant behaviors of glioma cells.

Glioblastoma multiforme (GBM) is one of the most malignant types of central nervous system (CNS) tumors. N6-methyladenine (m6A) RNA modification is a main type of RNA modification in eukaryotic cells. In this study, we find that the m6A RNA methylation eraser FTO is dramatically downregulated in glioma samples and cell lines, particularly in intermediate and core regions and hypoxia-challenged glioma cells. In vitro, FTO overexpression inhibits the hypoxia-induced capacities of glioma cells to proliferate, migrate and invade, and decreases the percentage of cells with m6A RNA methylation. In vivo, FTO overexpression inhibits tumor growth in the xenograft model and decreases the protein levels of migration markers, including Vimentin and Twist. miR-27a-3p is upregulated within glioma intermediate and core regions and hypoxia-challenged glioma cells. miR-27a-3p inhibits the expression of FTO via direct binding to FTO. miR-27a-3p overexpression promotes hypoxia-challenged glioma cell aggressiveness, whereas FTO overexpression partially diminishes the oncogenic effects of miR-27a-3p overexpression. FTO overexpression promotes the nuclear translocation of FOXO3a and upregulates the expression levels of the FOXO3a downstream targets BIM, BNIP3, BCL-6, and PUMA, possibly by interacting with FOXO3a. Conclusively, FTO serves as a tumor suppressor in glioma by suppressing hypoxia-induced malignant behaviors of glioma cells, possibly by promoting the nuclear translocation of FOXO3a and upregulating FOXO3a downstream targets. miR-27a-3p is a major contributor to FTO downregulation in glioma under hypoxia.

lncRNA RMST suppresses the progression of colorectal cancer by competitively binding to miR-27a-3p/RXRα axis and inactivating Wnt signaling pathway.

Colorectal cancer (CRC) ranks the 3rd in cancer types globally. Long noncoding RNAs (lncRNAs) are related to the initiation and progression of CRC. The current study plans to reveal the action of rhabdomyosarcoma 2-associated transcript (RMST) in CRC. The results show that RMST is downregulated in CRC specimens and cell lines relative to normal specimens and a fetal normal colon cell line (FHC), respectively. Elevation of RMST represses cell proliferation and colony formation and induces cell apoptosis in CRC cells. Bioinformatic analysis reveals a binding site in RMST for miR-27a-3p. The direct association between RMST and miR-27a-3p is confirmed by dual luciferase reporter assay, RNA pull-down assay, and real time-quantitative polymerase chain reaction (RT-qPCR). miR-27a-3p is upregulated in CRC tumor specimens relative to normal specimens, and there is a negative correlation between RMST and miR-27a-3p in CRC tumor specimens. In addition, the effects of RMST overexpression are weakened by the elevation of miR-27a-3p. RMST and retinoid X receptor (RXRα) share the same complementary site with miR-27a-3p. The direct association between RXRα and miR-27a-3p is confirmed by RNA pull-down assay, RT-qPCR and western blot analysis. Overexpression of RMST induces RXRα expression and inactivates the Wnt signaling pathway by decreasing ß-catenin levels in CRC cells. Collectively, our findings reveal a pivotal role of RMST in regulating miR-27a-3p/RXRα axis and counteracting Wnt signaling pathway during the progression of CRC.

MicroRNA-27a-3p inhibits lung and skin fibrosis of systemic sclerosis by negatively regulating SPP1.

OBJECTIVE: To investigate the role and mechanism of microRNAs (miRNAs) in fibrotic processes involved in the pathology of systemic sclerosis (SSc). METHODS: R language and bioinformatics methods were used to identify differential miRNAs and mRNAs and analyze their biological functions. Transfection experiments were performed to evaluate the function and regulatory mechanism of miR-27a-3p in vitro. Levels of fibrosis-related genes, SPP1 and cell proliferation were assessed. RESULTS: MiR-27a-3p is reduced both in SSc lung and skin tissues. Overexpression of miR-27a-3p significantly inhibited fibrosis-related genes expression and protein abundance and cell proliferation, whereas inhibition of miR-27a-3p significantly enhanced these phenomena. Moreover, miR-27a-3p exerts its anti-fibrosis effect by negatively regulating SPP1 and ERK signal, more prominent in fibroblasts. CONCLUSIONS: Our findings show that miR-27a-3p regulates a common mechanism in the process of SSc skin and lung fibrosis. MiR-27a-3p/SPP1/ERK1/2 axis may be an important target for delaying the progression of SSc fibrosis.

MicroRNA-27a-3p relieves inflammation and neurologic impairment after cerebral ischaemia reperfusion via inhibiting lipopolysaccharide induced TNF factor and the TLR4/NF-κB pathway.

Cerebral ischaemia reperfusion (CIR) affects microRNA (miR) expression and causes substantial inflammation. Here, we investigated the influence and underlying mechanism of miR-27a-3p in rats with CIR. First, biliverdin treatment relieved cerebral infarction and decreased the levels of serum interleukin (IL)-1ß, IL-6, and TNF-α. Through our previous study, we found key miR-27a-3p and its targeted gene LITAF might involve in the molecular mechanism of CIR. Then, the regulation between miR-27a-3p and LITAF was verified by the temporal miR-27a-3p and LITAF expression profiles and luciferase assay. Moreover, intracerebroventricular injection of the miR-27a-3p mimic significantly decreased the LITAF, TLR4, NF-κB, and IL-6 levels at 24 h post-surgery, whereas miR-27a-3p inhibitor reversed these effects. Furthermore, miR-27a-3p mimic could relieve cerebral infarct and neurologic deficit after CIR. In addition, injection of miR-27a-3p mimic decreased neuronal damage induced by CIR. Taken together, our results suggest that miR-27a-3p protects against CIR by relieving inflammation, neuronal damage, and neurologic deficit via regulating LITAF and the TLR4/NF-κB pathway.

CircGSAP alleviates pulmonary microvascular endothelial cells dysfunction in pulmonary hypertension via regulating miR-27a-3p/BMPR2 axis.

BACKGROUND: Our previous study showed that circular RNA-gamma-secretase-activating protein (circGSAP) was down-regulated in pulmonary microvascular endothelial cells (PMECs) in response to hypoxia, and regulated the cell cycle of PMECs via miR-942-5p sponge in pulmonary hypertension (PH). However, the mechanism whether circGSAP affects the dysfunction of PEMCs through other microRNAs (miRNAs) remains largely unknown. Therefore, we aimed to demonstrate the underlying mechanisms of circGSAP regulating PMECs dysfunction by absorbing other miRNAs to regulate target genes in idiopathic pulmonary arterial hypertension (IPAH). METHODS: Quantitative real-time polymerase chain reaction, immunofluorescence staining, Cell Counting Kit-8, Calcein-AM/PI staining, Transwell assay, dual-luciferase reporter assay, and ELISA were used to elucidate the roles of circGSAP. RESULTS: Here we showed that plasma circGSAP levels were significantly decreased in patients with IPAH and associated with poor outcomes. In vivo, circGSAP overexpression improved survival, and alleviated pulmonary vascular remodeling of monocrotaline-induced PH (MCT-PH) rats. In vitro, circGSAP overexpression inhibited hypoxia-induced PMECs proliferation, migration and increased mortality by absorbing miR-27a-3p. BMPR2 was identified as a miR-27a-3p target gene. BMPR2 silencing ameliorated the effect of the miR-27a-3p inhibitor on PMECs proliferation,migration and mortality. The levels of BMPR2 were upregulated in circGSAP-overexpressed PMECs and lung tissues of MCT-PH rats. CONCLUSION: Our findings demonstrated that circGSAP alleviated the dysfunction of PMECs via the increase of BMPR2 by competitively binding with miR-27a-3p, and mitigated pulmonary vascular remodeling of MCT-PH rats, providing potential therapeutic strategies for IPAH.

Expression relationship and significance of NEAT1 and miR-27a-3p in serum and cerebrospinal fluid of patients with Alzheimer's disease.

OBJECTIVE: To explore the expression relationship and significance of long chain non-coding RNA nuclear-enriched abundant transcript 1 (LncRNA NEAT1) and miR-27a-3p in serum and cerebrospinal fluid of patients with Alzheimer's disease (AD). METHODS: Sixty-six AD patients received by the Department of Neurology of our hospital from October 2019 to September 2021 were gathered, according to the Clinical Dementia Rating Scale (CDR) score, they were grouped into mild group (≤1 point, n = 41) and moderate-to-severe group (> 1 point, n = 25). Another 32 cases of serum and cerebrospinal fluid samples from outpatient physical examination personnel were regarded as the control group. The general materials on all subjects was recorded and cognition was assessed;real-time quantitative PCR was performed to measure the expression levels of miR-27a-3p and NEAT1 in serum and cerebrospinal fluid;enzyme-linked immunosorbent assay was performed to measure the protein levels of ß-amyloid precursor protein cleaving enzyme 1 (BACE1), ß-amyloid (Aß) 40 and Aß42 in cerebrospinal fluid;Spearman's method was performed to analyze the correlation of serum miR-27a-3p and NEAT1 levels with MMSE and MoCA scores;Pearson method was performed to analyze the correlation between serum miR-27a-3p and NEAT1 levels and Aß deposition standard uptake value ratio (SUVR) and cerebrospinal fluid miR-27a-3p, NEAT1, BACE1, Aß42 and Aß40 levels. RESULTS: The MMSE score, MoCA score, serum miR-27a-3p level, cerebrospinal fluid miR-27a-3p, Aß42 levels and Aß42/Aß40 ratio of AD patients in mild group and moderate-to-severe group were all lower than those in the control group, and the moderate-to-severe group were lower than the mild group (all P < 0.05);the serum NEAT1 level, SUVR, and cerebrospinal fluid NEAT1 and BACE1 levels were higher than those in the control group, and the moderate-to-severe group were higher than the mild group (all P < 0.05). Serum NEAT1 level in AD patients was positively correlated with SUVR, cerebrospinal fluid NEAT1 and BACE1 (r = 0.350, 0.606, 0.341, all P < 0.05);serum miR-27a-3p level was positively correlated with cerebrospinal fluid miR-27a-3p level (r = 0.695, P < 0.05), and negatively correlated with SUVR and cerebrospinal fluid BACE1 level (r = - 0.521, - 0.447, both P < 0.05). CONCLUSIONS: The expression trends of NEAT1 and miR-27a-3p in the serum and cerebrospinal fluid of AD patients are consistent, the level of NEAT1 is increased, and the level of miR-27a-3p is decreased. The levels of the two are negatively correlated, which is related to the degree of Aß deposition in the brain of AD patients and is involved in the progression of AD.

Circulating miR-148b-3p and miR-27a-3p can be potential biomarkers for diagnosis of pre-diabetes and type 2 diabetes: integrating experimental and in-silico approaches.

BACKGROUND: In view of the growing global prevalence of type 2 diabetes (T2D), detection of prediabetes and type 2 diabetes in the early stages is necessary to reduce the risk of developing diabetes, prevent the progression of the disease, and dysfunction of different organs. Since miRNAs are involved in the initiation and progression of numerous pathogenic processes, including diabetes, in the present study, we aimed to investigate the expression of miR-148b-3p and miR-27a-3p in prediabetic and T2D patients and to evaluate the diagnostic potential of these miRNAs. METHODS: We evaluated the expression of miR-148b-3p and miR-27a-3p in the plasma of three groups: 20 prediabetic patients, 20 T2D patients, and 20 healthy controls. The biochemical parameters were determined by the auto-analyzer. The possible target genes of these miRNAs were identified using an in-silico approach. RESULTS: Our results showed that, as compared to the healthy controls, there was a significant up regulation and down regulation in the expression of miR-148b-3p and miR-27a-3p in the T2D patients, respectively. The results of receiver operating characteristic curve analysis also suggested that miR-148b-3p acted successfully in discriminating the prediabetic and diabetic patients from the control group. According to in-silico analysis, miRs influence biological pathways involved in T2DM development, such as insulin signaling. CONCLUSIONS: The miR148b-3p and miR-27a-3p expression levels were deregulated in diabetes and pre-diabetes. Furthermore, miR-148b-3p showed significant ability in discriminating between diabetic and healthy individuals, suggesting a potential diagnostic use of miR-148b-3p in the detection of T2D.

Involvement of miR-27a-3p in diabetic nephropathy via affecting renal fibrosis, mitochondrial dysfunction, and endoplasmic reticulum stress.

Diabetic nephropathy (DN) is acknowledged as a serious chronic complication of diabetes mellitus. Nevertheless, its pathogenesis is complicated and unclear. Thus, in this study, the role of miR-27a-3p-prohibitin/TMBIM6 signaling axis in the progression of DN was elucidated. Type 2 diabetic db/db mice and high glucose (HG)-challenged HK-2 cells were used as in vivo and in vitro models. Our results showed that miR-27a-3p was upregulated and prohibitin or transmembrane BAX inhibitor motif containing 6 (TMBIM6) was downregulated in the kidney tissues of db/db mice and HG-treated HK-2 cells. Silencing miR-27a-3p enhanced the expression of prohibitin and TMBIM6 in the kidney tissues and HK-2 cells. Inhibition of miR-27a-3p improved functional injury, as evidenced by decreased blood glucose, urinary albumin, serum creatinine, and blood urea nitrogen levels. MiR-27a-3p silencing ameliorated renal fibrosis, reflected by reduced profibrogenic genes (e.g., transforming growth factor ß1, fibronectin, collagen I and III, and α-smooth muscle actin). Furthermore, inhibition of miR-27a-3p relieved mitochondrial dysfunction in the kidney of db/db mice, including upregulation of mitochondrial membrane potential, complex I and III activities, adenosine triphosphate, and mitochondrial cytochrome C, as well as suppressing reactive oxygen species production. In addition, miR-27a-3p silencing attenuated endoplasmic reticulum (ER) stress, reflected by reduced expression of p-IRE1α, p-eIF2α, XBP1s, and CHOP. Mechanically, we identified prohibitin and TMBIM6 as direct targets of miR-27a-3p. Inhibition of miR-27a-3p protected HG-treated HK-2 cells from apoptosis, extracellular matrix accumulation, mitochondrial dysfunction, and ER stress by regulating prohibitin or TMBIM6. Taken together, we reveal that miR-27a-3p-prohibitin/TMBIM6 signaling axis regulates the progression of DN, which can be a potential therapeutic target.

MiR-27a-3p promotes the osteogenic differentiation by activating CRY2/ERK1/2 axis.

BACKGROUND: Osteoporosis seriously disturbs the life of people. Meanwhile, inhibition or weakening of osteogenic differentiation is one of the important factors in the pathogenesis of osteoporosis. It was reported that miR-27a-3p reduced the symptoms of osteoporosis. However, the mechanism by which miR-27a-3p in osteogenic differentiation remains largely unknown. METHODS: To induce the osteogenic differentiation in MC3T3-E1 cells, cells were treated with osteogenic induction medium (OIM). RT-qPCR was used to evaluate the mRNA expression of miR-27a-3p and CRY2 in cells. The protein levels of CRY2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), osteocalcin (OCN) and the phosphorylation level of extracellular regulated protein kinases (ERK) 1/2 in MC3T3-E1 cells were evaluated by western blotting. Meanwhile, calcium nodules and ALP activity were tested by alizarin red staining and ALP kit, respectively. Luciferase reporter gene assay was used to analyze the correlation between CRY2 and miR-27a-3p. RESULTS: The expression of miR-27a-3p and the phosphorylation level of ERK1/2 were increased by OIM in MC3T3-E1 cells, while CRY2 expression was decreased. In addition, OIM-induced increase of calcified nodules, ALP content and osteogenesis-related protein expression was significantly reversed by downregulation of miR-27a-3p and overexpression of CRY2. In addition, miR-27a-3p directly targeted CRY2 and negatively regulated CRY2. Meanwhile, the inhibitory effect of miR-27a-3p inhibitor on osteogenic differentiation was reversed by knockdown of CRY2 or using honokiol (ERK1/2 signal activator). Furthermore, miR-27a-3p significantly inhibited the apoptosis of MC3T3-E1 cells treated by OIM. Taken together, miR-27a-3p/CRY2/ERK axis plays an important role in osteoblast differentiation. CONCLUSIONS: MiR-27a-3p promoted osteoblast differentiation via mediation of CRY2/ERK1/2 axis. Thereby, miR-27a-3p might serve as a new target for the treatment of osteoporosis.

LINC01089 inhibits the progression of cervical cancer via inhibiting miR-27a-3p and increasing BTG2.

BACKGROUND: Increasing evidence confirms that long non-coding RNA (lncRNA) has a vital impact on the procession of cervical cancer (CC). The present study aimed to investigate the clinical significance of LINC01089 in CC, as well as explore its biological functions and potential molecular mechanisms. METHODS: A quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to investigate the expression of LINC01089 and miR-27a-3p in CC cells and tissues. Analysis of the correlation between the expression level of LINC01089 and the clinical pathological parameters of CC was then conducted. The human CC cell lines HeLa and SiHa were utilized for transfection to establish a gain-of-function model and loss-of-function models. Western blotting and a qRT-PCR were performed to detect B-cell translocation gene-2 (BTG2) expression in CC cells. Cell counting kit (CCK)-8 and 5-bromo-2-deoxyuridine (BrdU) assays were performed to detect the proliferation of CC cells. The transwell method was employed to evaluate the migration and invasion of CC cells. The interactions between LINC01089 and miR-27a-3p were verified by bioinformatics, a dual luciferase reporter gene experiment and a RNA immunoprecipitation experiment, respectively. RESULTS: The expression of LINC01089 in CC was markedly down-regulated. The low expression of LINC01089 in CC was closely associated with a larger tumor size and positive lymph node metastasis. Moreover, overexpression of LINC01089 impeded the proliferation and metastasis of CC cells, whereas knockdown of LINC01089 had the opposite biological functions. In terms of mechanism, LINC01089 could sponge miR-27a-3p and indirectly up-regulate BTG2 expression. CONCLUSIONS: LINC01089, as a tumor suppressor, impedes the development of CC by targeting miR-27a-3p to up-regulate BTG2 expression.

Exosomal miR-27a-3p derived from tumor-associated macrophage suppresses propranolol sensitivity in infantile hemangioma.

Propranolol is the first-line drug for infantile hemangioma (IH) therapy, whereas propranolol resistance is clinically observed. Tumor-associated macrophages (TAMs)-derived exosomes may deliver biological molecules to promote tumor progression. Here, we aimed to investigate the relationship between TAMs-derived exosomal miR-27a-3p and propranolol sensitivity in IH. Human peripheral blood monocytes (PBMCs) were cultured with macrophage colony-stimulating factor (M-CSF) for 7 days to get unactivated macrophages (Un-Mac), which were further treated with IL-4 and IL-13 to induce M2 polarized macrophages. Exosomes were isolated from the conditioned medium of M2 macrophage, followed by identification. Cell co-culture and/or transfection were performed to explore whether M2 polarized macrophage-derived exosomes (M2-exos) could mediate the crosstalk between TAMs-derived miR-27a-3p and hemangioma stem cells (HemSCs). In addition, nude mice were subcutaneously transplanted with HemSCs pretreated with or without M2-Exos to examine the effects of M2-Exos on IH in vivo. M2 polarized macrophages inhibited propranolol sensitivity of HemSCs, as shown by the increased cell viability and decreased apoptosis. miR-27a-3p was upregulated in M2 polarized macrophages and M2-Exos. Moreover, M2-exos delivered miR-27a-3p from macrophages to HemSCs and subsequently reduced propranolol sensitivity. Luciferase reporter and biotin-RNA pulldown assay proved that dickkopf-related protein 2 (DKK2) was the direct target of miR-27a-3p. These results demonstrate that M2-exos could deliver miR-27a-3p from macrophages to HemSCs to reduce the sensitivity of HemSCs to propranolol by down-regulating DKK2 expression, and exosomal miR-27a-3p and DKK2 in HemSCs could be considered as treatment targets.

Clinical Value and Potential Target of miR-27a-3p in Pulpitis.

INTRODUCTION: This study investigated the clinical values of miR-27a-3p for pulpitis patients, and its association with TLR4. METHODS: Sixty-six patients with pulpitis and 34 cases without pulpitis were recruited; the pulp tissue and serum samples were collected from each participant. Real-time polymerase chain reaction was used for measurement of gene expression levels. The diagnosis values were assessed by the receiver operating characteristic curve. The target gene of miR-27a-3p was confirmed by the luciferase reporter assay. RESULTS: MiR-27a-3p was downregulated in both serum and pulp tissue of pulpitis patients. MiR-27a-3p could distinguish pulpitis patients from healthy controls and might be a predictor for the development of irreversible pulpitis. A high level of TLR4 was also detected in both peripheral blood monocytes and pulp tissues from pulpitis patients and showed a negative association with the miR-27a-3p level. TLR4 was a direct target gene of miR-27a-3p. DISCUSSION/CONCLUSION: MiR-27a-3p might be a promising biomarker for the diagnosis of pulpitis and predict the development of irreversible pulpitis. MiR-27a-3p might be involved in the pathogenesis of pulpitis via targeting TLR4.

Lipopolysaccharide inhibits triglyceride synthesis in dairy cow mammary epithelial cells by upregulating miR-27a-3p, which targets the PPARG gene.

The fat content of milk determines the quality of milk, and triglycerides are the major components of milk fat. Milk fat synthesis is regulated by many factors. Lipopolysaccharide (LPS) has been shown to inhibit milk fat synthesis in bovine mammary epithelial cells, but research on the underlying mechanisms has been limited. MicroRNA (miRNA) are involved in many physiological processes, but there have been few studies on their regulation in milk fat synthesis. In this study, we aimed to investigate whether LPS upregulates miR-27a-3p, which targets PPARG, thereby inhibiting the synthesis of triglycerides in a dairy cow mammary epithelial cell line (MAC-T). After LPS stimulation of MAC-T cells, PPARG gene expression and milk fat synthesis were inhibited. TargetScan software was used to predict miRNA targeting PPARG, and miR-27a-3p was selected as a candidate. A dual luciferase reporter assay further confirmed the targeting connection between miR-27a-3p and the PPARG gene. To investigate the functions of miR-27a-3p, miR-27a-3p mimic and inhibitors were transfected into MAC-T cells. The mRNA and protein levels of PPAR-γ were negatively correlated with the expression of miR-27a-3p. Lipid droplet accumulation and triglyceride synthesis were also negatively correlated with miR-27a-3p expression. Inhibition of miR-27a-3p partially reversed the LPS-induced decreases in PPARG expression and milk fat synthesis. In summary, our results reveal that LPS can inhibit MAC-T cell milk fat synthesis by upregulating miR-27a-3p, which targets the PPARG gene.