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Objective:To explore the effect of miR-181a on chondrosarcoma cell growth through phosphatase and tensin homolog(PTEN) and its possible regulatory mechanism.Methods:From January to December 2022, 10 fresh chondrosarcoma and 10 chondroma tissues from orthopedic patients of Hunan Provincial People′s Hospital were collected, and the expression of miR-181a in chondrosarcoma and chondroma tissues was detected using real-time fluorescence quantitative polymerase chain reaction (qRT-PCR); Chondrosarcoma cell SW1353 was cultured in vitro and transfected with miR-181a inhibitor (miR-181a inhibition group) and control (miR-NC, control group), respectively. The effects of miR-181a on the growth and proliferation of SW1353 cells were detected by cell counting kit (CCK-8) and clone formation test, respectively; The binding sites between miR-181a and PTEN were predicted through the Target Scan database, and verified using dual luciferase reporter gene experiments; The mimetic miR-181a (miR-181a group) and its control (miR-NC, control group) were transfected into chondrosarcoma cell SW1353, respectively. The adenosine triphosphate (ATP) content, glucose consumption, and lactic acid production in the cells were measured, and the effect of miR-181a on glycolysis of SW1353 cells was analyzed. Results:The expression of miR-181a in chondrosarcoma tissues was significantly higher than that in chondroma tissues ( P<0.05). The cell growth and clonogenesis ability of miR-181a inhibition group were significantly lower than those of control group (all P<0.05). It was predicted by Target Scan online website that there might be binding sites between miR-181a and PTEN, and co-transfection of wild-type PTEN and miR-181a could significantly reduce luciferase activity by double luciferase reporter assay ( P<0.05). The ATP content, glucose consumption and lactic acid production of miR-181a group were significantly higher than those of miR-NC group (all P<0.05). Conclusions:MiR-181a promotes the growth of chondrosarcoma cells through PTEN-mediated glycolysis.
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Regulatory T cells (Tregs) play critical roles in restricting inflammatory pathogenesis and limiting undesirable Th2 response to environmental allergens. However, the role of miR-181a in regulating acute gouty arthritis (AGA) and Treg function remains unclear. This study aimed to investigate the potential roles of miR-181a in Treg immunity and the associated signaling pathway in the AGA mouse model. A solution with monosodium urate (MSU) crystals was injected into the joint tissue of mice to induce AGA. ELISA was used to examine inflammatory factors in blood samples, and flow cytometry was used to analyze Treg profile in mice with MSU-induced AGA. Cell proliferation and viability were assessed by CCK-8 assay. TGF-β1/Smad signaling activation was detected by western blot. We found that miR-181a expression showed a positive correlation with the changes of splenic Tregs percentage in AGA mice. miR-181a regulated the TGF-β1/Smad axis, since the transfection of miR-181a mimic increased the level of TGF-β1 and the phosphorylation of Smad2/3 in Tregs in AGA mice. Additionally, miR-181a mimic also promoted responses of Tregs via TGF-β1 in vitro and in vivo. Our work uncovered a vital role of miR-181a in the immune function of Treg cells by mediating the activity of the TGF-β1/Smad pathway in the AGA mouse model induced by MSU.
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Abstract Objectives Accumulating research have reported that microRNAs (miRNAs) play important roles in Retinoblastoma (RB). Nonetheless, the function and underlying mechanism of miR-181a-5p in RB remain ambiguous. Methods The relative expression levels of miR-181a-5p and NRAS mRNA were detected by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). RB cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) and 5′-Bromo-2′-deoxyuridine (BrdU) assays. Transwell assays and flow cytometry were performed to detect the migration, invasion, and apoptosis of RB cells. The interaction between miR-181a-5p and NRAS was explored using luciferase experiments, western blotting, and qRT-PCR. Results miR-181a-5p expression was found to be decreased in RB tissues and cell lines, and its expression was correlated with unfavorable pathological features of the patients. In vitro experiments revealed that miR-181a-5p reduced RB cell proliferation, migration, and invasion while enhancing apoptosis. Further research confirmed that NRAS is a direct target of miR-181a-5p. miR-181a-5p inhibited NRAS expression at both the mRNA and protein levels. Co-transfection of pcDNA-NRAS or NRAS small interfering RNA (siRNA) reversed the effects of miR-181a-5p mimics or miR-181a-5p inhibitors on RB cells. Conclusion miR-181a-5p was significantly downregulated during the development of RB, and it suppressed the malignant behaviors of RB cells by targeting NRAS.
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Objective:To investigate the clinical significance of MAPT-IT1 in breast cancer and its biological effect in vitro.Methods:The expression of MAPT-IT1 in breast cancer was analyzed by TCGA database. 64 cases of breast cancer and normal adjacent tissues were collected. Human breast cancer MDA-MB-231 and MCF-7 cells were cultured and overexpressed MAPT-IT1 or rescue miR-181a-5p by cell transfection. MDA-MB-231 cells were divided into blank group A, overexpression group A and recovery group A; MCF-7 cells were divided into blank group B, overexpression group B and recovery group B. Real time quantitative PCR (RT-qPCR) was used to detect the expression of MAPT-IT1, miR-181a-5p and MAPT mRNA. Western blot was used to detect the expression of MAPT protein. CCK-8 assay was used to detect cell proliferation, and Transwell invasion assay was used to detect cell invasion.Results:The expression of MAPT-IT1 in normal paracancerous tissues and breast cancer tissues was 0.011±0.002 and 0.028±0.003 respectively. The expressions of MAPT-IT1 in breast cancer tissues were significantly higher than those in adjacent normal tissues, and high expression of MAPT-IT1 was correlated with early tumor progression, ER positive and prolonged prognosis ( P<0.05) . In blank group A, overexpression group A and recovery group A, the expressions of MAPT-IT1 were 1.000±0.078, 8.597±0.320 and 8.540±0.177, miR-181a-5p were 1.000±0.027, 0.263±0.024, 4.433±0.239, MAPT were 1.000±0.071, 3.297±0.243, 0.497±0.029. In blank group B, overexpression group B and recovery group B, the expressions of MAPT-IT1 were 1.000± 0.081, 5.716±0.309, 5.288±0.176, miR-181a-5p were 1.000±0.024, 0.291±0.022, 3.648±0.073, and MAPT were 1.000±0.054, 3.309±0.177, 0.883±0.075. After overexpression of MAPT-IT1, the expression of miR-181a-5p was down-regulated, while the expression of MAPT was significantly increased ( P<0.001) , and the proliferation and invasion ability of MDA-MB-231 and MCF-7 cells were significantly decreased ( P<0.05) . Re-expression of miR-181a-5p down-regulated MAPT and promoted cell proliferation and invasion ( P<0.05) . Conclusion:Overexpression of MAPT-IT1 can significantly down-regulate the expression of miR-181a-5p and enhance MAPT and inhibit the malignant phenotype of breast cancer cells in vitro.
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@#[Abstract] Objective: To explore the effect of exosome-derived miR-181a on angiogenesis and tumor progression in gliomas. Methods: 83 cases of glioma tissues and 13 cases of peritumoral tissues resected in the Second Affiliated Hospital of Hainan Medical University from August 2017 to December 2019, glioma cells U87, A172, U251, LN229, U373 and microglial cell line HM, were selected to detect the expression of miR-181a in tumor tissues and cells by qPCR method. Glioma U373 cells with miR-181a over-expression or knockdown were constructed, and exosomes were isolated and identified. The effects of exsome-derived miR-181a on angiogenesis of HUVEC cells were investigated by tubule formation and chicken chorioallantoic membrane assay in vitro. Nude mice bearing U373 cell transplanted xenograft was constructed to observe the effect of exsome-derived miR-181a on angiogenesis and tumor growth in vivo. Results: The expression of miR-181a in glioma tissues and cells was significantly higher than that in normal tissues and normal glial HM cells (all P<0.01). The exsome-derived miR-181a could significantly promote the tubule formation of HUVEC cells (P<0.01) and the angiogenesis of chicken chorioallantoic membrane (all P<0.01). In vivo experiments showed that the growth of xenografts was promoted (P<0.05) and the amount of angiogenesis in the tumor tissues was increased in the nude mice after being transfused with exsome-derived miR-181a (P<0.01). Conclusion: miR-181a plays an important role in promoting angiogenesis of gliomas and may be a potential target for diagnosis and treatment of gliomas.
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Objective To explore the effect and mechanism of miR-181a on sepsis-induced acute lung injury in mice by targeting silent information regulator 1 (SIRT1). Methods Lipopolysaccharide was used to induce the A549 cell model of acute lung injury. miR-181 inhibitor and inhibitor negative control (inhibitor NC) were transfected in the cells. Cecal ligation and puncture (CLP) was used to establish the mouse model of sepsis-induced acute lung injury. The mice were intravenously infused with miR-181a antagomir and antagomir NC. Then survival rate and wet-to-dry ratio of the lungs were examined, the targeting relationship between miR-181a and SIRT1 was tested by luciferase reporter assay, RT-PCR was used to detect the gene expression of miR-181a and SIRT1 in vitro and vivo. Western blotting was used to detect the protein level of SIRT1 in vitro and vivo, as well as the levels of apoptosis-related protein B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), and cleave caspase-3 (cl-CASP3). Cell apoptosis was quantified by flow cytometry, cell viability was measured by CCK-8, CASP3 activity was detected by kit. HE staining was used to observe the histopathological changes in the lungs; TUNNEL staining was used to detected the cell apoptosis in the lungs. The concentrations of inflammatory cytokines were measured by ELISA. Results In cellular experiments, compared with those in control group, the expression of miR-181a was increased and the expression of SIRT1 was decreased in LPS group (P<0.01). Meanwhile the cell apoptosis rate was increased, CASP3 activity was increased, and cell viability was decreased. Treatment by miR-181a inhibitor could reverse the changes of the above indicators (P<0.01). In animal experiments, compared with those in sham group, in CLP group the mice's survival rate was decreased, wet-to-dry ratio of the lungs was increased, significant pathological changes and cell apoptosis in the lungs could be observed. Meanwhile, the concentrations of inflammatory cytokines were increased, the protein levels of miR-181a, Bax and cl-CASP3 were increased, and the protein levels of SIRT1 and Bcl-2 were decreased (P<0.01). Treatment by miR-181a antagomir could reverse the changes of the above indicators (P<0.05, P<0.01). Conclusion miR-181a can be targeted to SIRT1, and inhibition of miR-181a expression can increase SIRT1 expression, there by protecting against acute lung injury induced by sepsis in mice.
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Objective: To evaluate the clinical efficacy and molecular mechanism of Shenfu combined with azithromycin on infantile mycoplasma pneumonia. Methods: Totally 80 children with mycoplasma pneumonia treated in Children's Hospital Affiliated to Soochow University from June 2016 to June 2017 were selected and randomly divided into two groups with 40 in each. Azithromycin was provided in both groups. Shenfu was administered in the observation group. The clinical efficacy, immunological functions and miR-181a level, and related molecular markers of all the subjects were observed. Lentivirus was used to transfer miR-181a into human T lymphocytes to observe its effects on lymphocyte proliferation, cytokine secretion level and related signaling pathways. Results: Compared with the normal group after treatment, the clinical efficacy, T lymphocyte subset proportion and inflammation cytokines were significantly increased (P<0.05), and the time of clinical symptoms remission was significantly decreased in the observation group after treatment (P<0.05). In addition, the content of miR-181a (9.3±5.3) in lymphocytes of the observation group after treatment was significantly lower than that (12.2±4.5) of the control group after treatment (P<0.05). In vitro assay revealed that miR-181a was significantly decreased lymphocyte proliferation and the ability of secretory cytokine. Luciferase reporter assay demonstrated that miR-181a could inhibit KRAS, NRAS and MAPK1 expressions, thus down-regulating P-AKT and P-MEK phosphorylation. Conclusion: Shenfu combined with azithromycin can effectively improve immunity functions and its mechanism might be related to the level of miR-181a in lymphocytes.
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Objective To investigate the expression and correlation of microRNA-181a (miR-181a) and CA12S in human types I and II endometrial carcinomas. Methods A total of 78 formalin-fixed and paraffin-embedded endometrium tissue specimens were used in the present study, and they were supplied by Xiaolan People's Hospital affiliated to Southern Medical University, Southern Hospital and Zhongshan Hospital affiliated to Zhongshan University during Jan. 2011 to Dec. 2013. Among them, 13 were determined as normal endothelium by pathological and examination with immunohistochemical staining, 18 were endometrial hyperplasia, and 47 of them diagnosed were as endometrial carcinoma (type I 37 and type E 10). Total RNA was extracted from each of the specimens, and then the expression of miR-181a was determined by real-time PCR, and the expression of CA125 was detected by immunohistochemical staining. Results The expression levels of both miR-181a and CA125 were obviously higher in type I and II endometrial carcinoma tissues than in normal tissue (P<0.05). In addition, the expression of miR-181a was significantly higher in type II endometrial carcinoma than in type I endometrial carcinoma and endometrial hyperplasia tissues (P<0.05). Moreover, it was found that the expression of miR-181a increased gradually from that of normal endometrium to endometrial hyperplasia and then endometrial carcinoma, with statistically significant difference among them (P<0.05). In addition, there was also a significant difference in the expression of CA12S in different types of endometrial carcinoma (P<0.05). Correlation analysis showed that the expressions of miR-181a and CA12S were negatively correlated with each other in the process of carcinogenesis of endometrium (P<0.05). Conclusion There is a difference in expression of miR-181a between type I and type II endometrial carcinoma, and it may be related to the development and progression of endometrial carcinoma. The mechanism may be related to the expression of CA12S as regulated by miR-181a in the process of endometrial carcinogenesis, but the specific mechanism still needs further validation with experiments on the cellular level.
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Objective To investigate whether the expression of sterile alpha motif and histidine/aspartic acid domain containing protein 1 ( SAMHD1 ) could be induced by IFN-α and mediated by microRNA-181a (miR-181a). Methods THP-1 and Jurkat cells were treated with different doses of IFN-α(200 IU/ml and 1 000 IU/ml) for 24 h. The expression of miR-181a and SAMHD1 at mRNA level were de-tected by quantitative PCR. Western blot assay was performed to measure the expression of SAMHD1 at pro-tein level. THP-1 and Jurkat cells were transfected with p-181a, an over-expression vector of miR-181a, and then treated with 200 IU/ml of IFN-α. Changes in the expression of SAMHD1 in those cells were analyzed. Results IFN-α significantly enhanced the expression of SAMHD1 at mRNA and protein levels, but inhibi-ted the expression of miR-181a, especially in Jurkat cells. The expression of SAMHD induced by IFN-αwas inhibited in cells over-expressing miR-181a. Conclusion This study suggests that IFN-α could induce the expression of SAMHD1 by down-regulating the level of miR-181a.
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Objective To explore the expression and significance of hsa-miR-181a (miR-181a) in can-ceration progression of endometrial carcinoma. Methods A total of 75 formalin-fixed paraffin-embedded tissue specimens were studied in this study , of which , 13 were normal endometrium , 18 were endometrial hyperplasia , 44 were endometrial carcinoma. After total RNA had been extracted , real-time PCR was applied to detect the ex-pression level of miR-181a in endometrial tissue in each group. Results miR-181a expression in formalin-fixed paraffin-embedded tissue specimens can be detected. Expression of miR-181a in endometrial carcinoma was high-er than that in endometrial hyperplasia , its expression in endometrial hyperplasia was also higher than that in normal endometrium, and the difference was statistically significant (P < 0.05). The expression of miR-181a in endometrial carcinoma was associated with FIGO stages (P < 0.05). Conclusion The up-regulation of miR-181a expression in women with endometrial carcinoma may play the role of oncogenes. Abnormal expression of miR-181a is probably associated with the occurrence and development of endometrial carcinoma.
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ObjectiveTo construct expressing vector of microRNA with molecular cloning methods which target CD4 and electroporating the vector to the MT4 cell to determine its effect to CD4 expression.MethodsPredict a microRNA can target CD4.Linking the pre-mir-181a PCR products to T vector,then cloning into the pEGFP-N1 vector after enzyme digestion.To test the integrity through the colony PCR and sequencing analysis.Electroporating the vector to MT4 cell,using FACS to test the CD4 expression.ResultsHsa-mir-181a is able to target CD4.The sequence of the construct was correct.The hsa-mir-181a is stable expressing in MT4 after electroporating with the vector and MT4 cell CD4 was down-regulated.ConclusionThe construct can be stable expressing hsa-mir-181a in MT4 cell and down-regulating the CD4 expressing.This method can be utilized as a novel intervention to the HIV fusion,it shows potential as a gene therapy tool in vitro.
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Objective To identify and analyze the expression of microRNA miR-181a in HepG2.2.15 cell line transfected by full-length hepatitis B virus genome,and explore the potential role of miR-181a engaged in the development of HBV-related liver disease.Methods Based on the previous data from microRNA microarray,miR-181a specific probe was designed and synthesized.The expressive levels of miR-181a in HepG2.2.15 and its parent cell line HepG2 were analyzed using Northern blot analysis.Some putative targets of miR-181a were predicted by computational software RNAhybrid and confirmed by mRNA microarray.One of the targets was selected and flow cytometry analysis was used to further determine the difference of intracellular HLA-A2 level between the two cell lines.Results Northern blot analysis showed that the expressive level of miR-181a in HepG2.2.15 cell was significantly up-regulated compared with that in HepG2 cell.Some putative targets of miR-181a including C8A,IDH1 and HLA-A were predicted and miR-181a might down-regulate the expression of HLA-A gene via partial complement to the 3'-UTR of HLA-A gene.Consistency with the result of mRNA profile microarray,FCM analysis also showed a significantly lower expression of HLA-A2(43.9%)in HepG2.2.15 cell than that in HepG2 cell(96.6%).Conclusion The expressive level of miR-181a in HepG2.2.15 cell is significantly up-regulated and miR-181a might down-regulate its target gene HLA-A,which might be one of the molecular mechanisms that HBV may escape from the immune response and continue replication in hepatocytes.The knowledge is also helpful for understanding the mechanism of HBV-host microRNA interaction.