miR-27a-3p inhibited synthesis of Col I and Col III in pulmonary fibroblasts through Wnt3a/β-catenin signaling pathway / 中国药理学通报

Aim: To explore the effects of microRNA- 27a-3p(miR-27a-3p) on collagen type I (Col I) and collagen type III (Col III) synthesis in pulmonary fibroblasts and the underlying mechanisms. Methods: Human pulmonary fibroblasts MRC-5 were cultured and then transfected with miR-27a-3p mimic or its inhibitor. qPCR and Western blot were used to detect miR- 27a-3p levels and nuclear β-catenin content, respectively. The expression of Col I, Col III and Wnt3a was measured using qPCR and Western blot. Bioinformatics predicted the potential of miR-27a-3p bound to Wnt3a 3'-untranslated region (3'-UTR). Dual luciferase reporter gene analyzed the effects of miR-27a-3p mimic transfection on luciferase activity of wild type and mutant Wnt3a 3'-UTR. MRC-5 cells were treated with the Wnt3a/β-catenin signaling pathway inhibitor Dkkl, followed by transfection with miR-27a-3p mimic or its inhibitor. Col I and Col III expression was detected by qPCR and Western blot. Results: miR-27a- 3p mimic markedly increased miR-27a-3p levels and decreased Col I, Col III, Wnt3a and β-catenin expression (P 0. 05), revealing Wnt3a as a target of miR-27a-3p. In addition, Dkkl pretreatment almost fully reversed the inductive effect of miR-27a-3p inhibitor on Col I and Col III expression in MRC-5 cells (P < 0. 05). Conclusions: miR-27a-3p inhibits the biosynthesis of Col I and Col III in pulmonary fibroblasts, which is attributed to inactivated Wnt3a/β-catenin signaling pathway.

miR-27a protects human mitral valve interstitial cell from TNF-alpha-induced inflammatory injury via up-regulation of NELL-1

MicroRNAs (miRNAs) have been reported to be associated with heart valve disease, which can be caused by inflammation. This study aimed to investigate the functional impacts of miR-27a on TNF-α-induced inflammatory injury in human mitral valve interstitial cells (hMVICs). hMVICs were subjected to 40 ng/mL TNF-α for 48 h, before which the expressions of miR-27a and NELL-1 in hMVICs were altered by stable transfection. Trypan blue staining, BrdU incorporation assay, flow cytometry detection, ELISA, and western blot assay were performed to detect cell proliferation, apoptosis, and the release of proinflammatory cytokines. We found that miR-27a was lowly expressed in response to TNF-α exposure in hMVICs. Overexpression of miR-27a rescued hMVICs from TNF-α-induced inflammatory injury, as cell viability and BrdU incorporation were increased, apoptotic cell rate was decreased, Bcl-2 was up-regulated, Bax and cleaved caspase-3/9 were down-regulated, and the release of IL-1β, IL-6, and MMP-9 were reduced. NELL-1 was positively regulated by miR-27a, and NELL-1 up-regulation exhibited protective functions during TNF-α-induced cell damage. Furthermore, miR-27a blocked JNK and Wnt/β-catenin signaling pathways, and the blockage was abolished when NELL-1 was silenced. This study demonstrated that miR-27a overexpression protected hMVICs from TNF-α-induced cell damage, which might be via up-regulation of NELL-1 and thus modulation of JNK and Wnt/β-catenin signaling pathways.

Role of miR-27a in the osteogenic differentiation of beagle maxillary sinus membrane stem cells / 口腔疾病防治

Objective@#To detect the expression level of miR-27a during the osteogenic differentiation of beagle maxillary sinus membrane stem cells (MSMSCs) and explore the role of miR-27a in the osteogenic differentiation of MSMSCs.@*Methods@#Beagle MSMSCs were cultured in vitro. The expression level of miR-27a was detected via RT-PCR after an osteogenic inductive culture was prepared. The mRNA expression levels of Runx2 and OPN were examined via RT-PCR, and the protein expression levels of Runx2 and OPN were examined via Western blot after the cells were transfected with pre-miR-27a or anti-miR-27a. Finally, osteoprogenitor cells transfected with pre-miR-27a were composited with Bio-Oss particles and subcutaneously implanted into nude mice to form ectopic bone formation models, and then the inhibition of bone formation from miR-27a was observed in vivo. @*Results@#The expression level of miR-27a in the beagle MSMSCs decreased after osteogenic inductive culturing. The relative miR-27a levels were significantly decreased at day 1 (t=3.795, P=0.023), day 3 (t=4.493, P=0.011), day 7 (t=11.591, P < 0.001), day 14 (t=12.542, P < 0.001), and day 21 (t=5.621, P=0.008) compared with day 0. In addition, the expression levels of Runx2 mRNA (t=4.923, P=0.007) and protein (t=4.425, P=0.008) were reduced after the cells were transfected with pre-miR-27a. The expression levels OPN mRNA (t=5.253, P=0.006) and protein (t=5.132, P=0.006) were also reduced. In contrast, the mRNA expression levels of Runx2 (t=3.925, P=0.013) and OPN (t=3.712, P=0.019) were increased after the cells were transfected with anti-miR-27a, and bone formation was observed after the subcutaneous implantation of beagle MSMSCs composited with Bio-Oss in nude mice. Nevertheless, ectopic bone formation was inhibited by pre-miR-27a-transfected beagle MSMSCs composited with Bio-Oss (t=7.219, P=0.0020). @* Conclusion @# MiR-27a negatively regulates the osteogenic differentiation of MSMSCs.

Effects of circulating microvesicles containing microRNA-27 a on blood brain barrier tight junction injury of ischemia stroke mice / 中国药理学通报

Aim To study the effect of circulating mi-crovesicles containing miR-27 a on blood brain barrier tight junction injury of ischemia stroke mice and its mechanism. Methods The middle cerebral artery occlusion mouse model was established, and the mi-crovesicles in the supernatant of 2 h of ischemic stroke brain tissues were separated by centrifugal ultrafiltra-tion method. The transmission electron microscopy was used to observe microvesicle morphology, and the di-ameter of microvesicles was detected. Based on the 2 h ischemia stroke mouse model, mice were injected via femoral vein with microvesicles at 5 mg · kg-1 . TTC staining was applied to detect infarction volume of is-chemia brain, while HE staining was applied to detect the expression change of tight junction protein occludin and claudin-5 . Western blot was subjected to detect occludin, claudin-5, TLR4, NF-κB and p38 protein expression. ELISA method was used to measure the contents of cytokines IL-1β and TNF-α. Results The microvesicle shape was approximately circular bi-lateral membrane structure, with an average diameter of 160 nm, which conformed to the morphological char-acteristics of microvesicles. Compared with the ische-mic stroke group, injection of microvesicles could ag-gravate the damage of brain tissues in ischemic mice, further increase the infarct volume and reduce the posi-tive staining areas of occludin and claudin-5 in ische-mia brain tissues. Meanwhile, the protein expressions of occludin and claudin-5 further decreased, and fur-ther up-regulated TLR4 and phosphorylation of NF-κB and p38 compared with the ischemia stroke group. Al-so, more content of IL-1βand TNF-αwere detected in ischemia stroke group injected with microvesicles com-pared with those in ischemia stroke group with signifi-cant difference, while the injection of antagomir-27a could alleviate brain damage and reduce the activation of TLR4, NF-κB and p38 in ischemia stroke mice. Conclusions Microvesicles containing miR-27 a could significantly attenuate brain injury in ischemia stroke mice, while aggravate the tight junction damage of ischemia brain. The mechanism might be correlated with the up-regulation of the expression of TLR4 , the phosphorylation of NF-κB, p38, and the release of cy-tokines IL-1β and TNF-α.

Expression of miR-93-5p and miR-27a-3p in rectal cancer tissues and its clinical significance / 中华放射肿瘤学杂志

Objective To investigate the miRNA expression profiles in rectal cancer tissues and their associations with clinical pathological stage, depth of tumor invasion, and lymph node metastasis, and to evaluate the potential of miRNA as diagnostic and prognostic markers of rectal cancer. Methods Human miRNA microarray was used to profile miRNA expression in rectal cancer tissues and matched adjacent normal tissues (n=71). The up-regulated miR-93-5p and down-regulated miR-27a-3p were screened out, and the top differentially expressed miRNA were validated by quantitative real-time polymerase chain reaction ( qRT-PCR) . The relationship between the expression of miRNA and clinical parameters was analyzed by ANOVA and Spearman correlation. Results The expression of miR-27a-3p was down-regulated in miRNA microarray, but was up-regulated in qRT-PCR analysis;the data were relatively discrete. The expression of miR-93-5p was up-regulated in both miRNA microarray and qRT-PCR analysis;the expression level of miR-93-5p in rectal cancer tissues was 3165 times that in adjacent normal tissues ( P=00058);the expression level was correlated with tumor volume ( P= 0004 ) , and was positively correlated with the level of carcinoembryonic antigen ( CEA) before treatment ( P=0001) and the number of lymph nodes metastases (rs=0534, P=0005). Conclusions There is a differential miRNA expression pattern between rectal cancer tissues and matched adjacent normal tissues. The miR-93-5p is highly up-regulated in rectal cancer tissues and may serve as a diagnostic and prognostic marker of rectal cancer.

The differences between miR-27a and miR-23a in patients with premature ovarian failure before and after treatmentand the feasibility of miR for assessing efficacy / 实用医学杂志

Objective To investigate the changes of microRNA(miR)-27a and miR-23a in patients with premature ovarian failure(POF)after treatment,and to analyze the feasibility of these miRsfor assessing the efficacy.Methods 77 women with POF and 50 healthy women were assigned to a study group and a control group. miR-27a and miR-23a in peripheral blood were detected,andthe diagnostic value of these miRs for POF were ana-lyzed. miRs in the study group were detected again after treatment. The correlation between these miRs and modi-fied Kupperman score was analyzed.According to the cut-off values of miRs,the study group was subdivided into a high-efficacy group and a low-efficacy group,the differences among the two groups and the control group were com-pared. Results MiR-27a and miR-23a were reliable for diagnosis of POF,the best cut-off value was 2.29 and 1.95 respectively.After treatment,the miRs decreased in the study group(P<0.05).MiR-27a and miR-23a were positively correlated with the modified Kupperman score(P < 0.001). All indexes detected in high-efficacy group were close to those in the control group(P>0.05).Conclusions MiR-27a and miR-23a obviously decreases in women with POF after treatment,these miRs are helpful for assessing the efficacy.

Expression of miR-93-5p and miR-27a-3p in rectal cancer tissues and its clinical significance / 中华放射肿瘤学杂志

Objective To investigate the miRNA expression profiles in rectal cancer tissues and their associations with clinical pathological stage, depth of tumor invasion, and lymph node metastasis, and to evaluate the potential of miRNA as diagnostic and prognostic markers of rectal cancer. Methods Human miRNA microarray was used to profile miRNA expression in rectal cancer tissues and matched adjacent normal tissues (n=71). The up-regulated miR-93-5p and down-regulated miR-27a-3p were screened out, and the top differentially expressed miRNA were validated by quantitative real-time polymerase chain reaction ( qRT-PCR) . The relationship between the expression of miRNA and clinical parameters was analyzed by ANOVA and Spearman correlation. Results The expression of miR-27a-3p was down-regulated in miRNA microarray, but was up-regulated in qRT-PCR analysis;the data were relatively discrete. The expression of miR-93-5p was up-regulated in both miRNA microarray and qRT-PCR analysis;the expression level of miR-93-5p in rectal cancer tissues was 3165 times that in adjacent normal tissues ( P=00058);the expression level was correlated with tumor volume ( P= 0004 ) , and was positively correlated with the level of carcinoembryonic antigen ( CEA) before treatment ( P=0001) and the number of lymph nodes metastases (rs=0534, P=0005). Conclusions There is a differential miRNA expression pattern between rectal cancer tissues and matched adjacent normal tissues. The miR-93-5p is highly up-regulated in rectal cancer tissues and may serve as a diagnostic and prognostic marker of rectal cancer.

Expression Difference and Its Mechanism Study of miR-27a-3p and HOXB8 Protein in Cervical Cancer Tis-sues of HPV16-positive and HPV16-negative Patients / 中国药房

OBJECTIVE:To investigate the expression difference and its mechanism of miR-27a-3p and HOXB8 protein in cer-vical cancer tissues of HPV16-positive and HPV16-negative patients. METHODS:A total of 120 patients with cervical cancer in our hospital during Jan. 2012-Jan. 2016 were divided into HPV16-positive group(60 cases)and HPV16-negative group(60 cases) according to HPV16 infection situation. The expression of miR-27a-3p mRNA and HOXB8 mRNA in cervical cancer tissue were de-tected by RT-qPCR. The expression of HOXB8 protein was detected by Western blotting assay. DNA methylation level of miR-27a-3p promoter region was detected by nested-down methylation specific PCR (nMS-PCR). Histone methylation level of miR-27a-3p promoter region was detected by chromatin immunoprecipitation PCR (CHIP-PCR). The expression of miR-27a-3p mRNA and HOXB8 mRNA were detected after transfecting miR-27a-3p mimic and inhibitor into Human cervical cancer cell line SiHa,respectively. RESULTS:The expression of miR-27a-3p in HPV16-positive group was significantly lower than HPV16-nega-tive group,while HOXB8 mRNA and protein expression,DNA and histone methylation levels of miR-27a-3p promoter region were significantly higher than HPV16-negative group,with statistical significance (P<0.05 or P<0.01). After transfecting miR-27a-3p mimic into SiHa cells,the expression of miR-27a-3p was increased significantly,while that of HOXB8 mRNA was de-creased significantly;after miR-27a-3p inhibitor transfection,the expression of miR-27a-3p was decreased significantly,while that of HOXB8 mRNA was increased significantly,with statistical significance(P<0.01). CONCLUSIONS:HPV16 may down-regu-late the expression of miR-27a-3p through DNA methylation and histone methylation of promoter region,so as to influence the gen-eration and development of cervical cancer. HOXB8 may be the target protein of miR-27a-3p.

miR-27a is negatively regulated by mTOR and inhibits liver cancer cell invasion via targeting GP73 / 基础医学与临床

Objective To explore the mechanism of mTOR-mediated liver cancer cell invasion.Methods q-PCR was used to check the expression of miR-27a and GP73;miR-27a mimics were transfected into GP73-high expressing M97H cells and miR-27a inhibitors were transfected into GP73-low expressing HepG2 cells,q-PCR and Western blot were performed to observe the expression of GP73;Dual-luciferase assay was also performed to verify the binding sites of miR-27a in GP73 3'UTR;miR-27a mimics were transfected into M97H cells and miR-27a inhibitors were transfected into HepG2 cells,Transwell assay was used to measure cell invasion.Results mTOR downregulated miR-27a and upregulated GP73;GP73 was downregulated by miR-27a and upregulated by miR-27a suppression;GP73 was a target gene of miR-27a;miR-27a inhibited the invasion of M97H cells rather than HepG2 cells.Conclusions miR-27a is negatively regulated by mTOR and inhibits liver cancer cell invasion via targeting GP73.

Cell-Free miR-27a, a Potential Diagnostic and Prognostic Biomarker for Gastric Cancer

MicroRNAs (miRNAs) have been demonstrated to play an important role in carcinogenesis. Previous studies revealed that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. In this study, we measured the plasma expression levels of three miRNAs (miR-21, miR-27a, and miR-155) to investigate the usefulness of miRNAs for gastric cancer detection. We initially examined plasma miRNA expression levels in a screening cohort consisting of 15 patients with gastric cancer and 15 healthy controls from Korean population, using TaqMan quantitative real-time polymerase chain reaction. We observed that the expression level of miR-27a was significantly higher in patients with gastric cancer than in healthy controls, whereas the miR-21 and miR-155a expression levels were not significantly higher in the patients with gastric cancer. Therefore, we further validated the miR-27a expression level in 73 paired gastric cancer tissues and in a validation plasma cohort from 35 patients with gastric cancer and 35 healthy controls. In both the gastric cancer tissues and the validation plasma cohort, the miR-27a expression levels were significantly higher in patients with gastric cancer. Receiver-operator characteristic (ROC) analysis of the validation cohort, revealed an area under the ROC curve value of 0.70 with 75% sensitivity and 56% specificity in discriminating gastric cancer. Thus, the miR-27a expression level in plasma could be a useful biomarker for the diagnosis and/or prognosis of gastric cancer.