Relationship between the Expression of miRNA181a-5p and the Imbalance of Treg/Th17 in Primary Immune Thrombocytopenia / 中国实验血液学杂志

OBJECTIVE@#To investigate the role of relationship between the expression of miRNA181a-5p and imbalance of Treg/Th17 in the pathogenesis of primary immune thrombocytopenia(ITP), which contributes to clarify the mechanism of T cell immune imbalance in ITP patients.@*METHODS@#Peripheral blood was collected from 37 ITP patients, concluding 21 untreated patients and 16 effectively treated patients, and 19 healthy controls; Peripheral blood mononuclear cells (PBMC) were isolated and the expression of miRNA181a-5p and Notch1 was analyzed by RT-PCR. The proportion of Th17 subsets and Treg cells in the peripheral circulation was detected by flow cytometer (FCM). Clinical data of ITP group was collected, including age, platelet count and disease course.@*RESULTS@#The expression of miR-181a-5p was significantly decreased in ITP group than that of healthy control group (P＜0.01). After effective treatment, the expression of miR-181a-5p was significantly higher than that of ITP group (P＜0.05), but still significantly lower than that of healthy control group (P＜0.01); The expression of Notch1 was significantly increased in ITP group and effectively treated group than that of healthy control group (P＜0.01). There was no significant difference in proportion of Treg cells in ITP group, effectively treated group and healthy control group (P＞0.05). The proportion of Th17 subsets in ITP group was significantly increased than that of healthy control group (P＜0.05), while the ratio of Treg/Th17 was significantly decreased (P＜0.05). There was a positive correlation between the expression of miR-181a-5p and ratio of Treg/Th17 in ITP group (r=0.555).@*CONCLUSION@#The expression of miR-181a-5p is significantly decreased in ITP patients, which is closely related to the imbalance of Treg/Th17 cells. After effective treatment, the expression of miR-181a-5p can be significantly corrected, but still failed to reach the level of healthy people. While the expression of Notch1 is significantly increased in ITP patients, and could not reach the level of healthy people after effective treatment.

Trdmt1 3'-untranslated region functions as a competing endogenous RNA in leukemia HL-60 cell differentiation

Severe blockage in myeloid differentiation is the hallmark of acute myeloid leukemia (AML). Trdmt1 plays an important role in hematopoiesis. However, little is known about the function of Trdmt1 in AML cell differentiation. In the present study, Trdmt1 was up-regulated and miR-181a was down-regulated significantly during human leukemia HL-60 cell differentiation after TAT-CT3 fusion protein treatment. Accordingly, miR-181a overexpression in HL-60 cells inhibited granulocytic maturation. In addition, our "rescue" assay demonstrated that Trdmt1 3′-untranslated region promoted myeloid differentiation of HL-60 cells by sequestering miR-181a and up-regulating C/EBPα (a critical factor for normal myelopoiesis) via its competing endogenous RNA (ceRNA) activity on miR-181a. These findings revealed an unrecognized role of Trdmt1 as a potential ceRNA for therapeutic targets in AML.

Expression characteristics and prognosis significance of miRNA-181a in acute myeloid leukemia with normal karyotype / 中华血液学杂志

Objective@#To study the expression of miRNA-181a in acute myeloid leukemia (AML) patients with normal karyotype to probe its prognosis significance.@*Methods@#The expression level of miRNA-181a in bone marrow mononuclear cells of 120 de novo AML patients with normal karyotype was detected by real time fluorescence quantitative PCR. The direct sequencing method was used to detect IDH1, IDH2, NPM1, FLT3-ITD, DNMT3A and CEBPα mutations in CN-AML patients after PCR. The relationship between miRNA-181a expression and gene mutation, the clinical parameters and prognosis were analyzed.@*Results@#The rates of overall surviva1 (OS) in high expression and low expression groups were 25.0 months and 15.0 months, respectively (P<0.05) . Relapse free survival (RFS) in high expression and low expression groups were 21.4 months and 11.2 months, respectively (P<0.05) . Significantly higher level hemoglobin, complete remission rate and proportion of wild type NPM1 expression in the high expression of miRNA-181a group were observed when compared with the lower expression of miRNA-181a group (P<0.05) . Multivariate Cox regression analysis showed miRNA-181a overexpression was an independent prognostic factor for CN-AML (HR=2.219, 95%CI 1.601~2.432, P=0.018) .@*Conclusion@#Higher expression of miRNA-181a was a good prognostic factor independent of clinical parameters and high frequency gene mutations, which implicated that the miRNA-181a expression level could be used as an important prognostic indicator of AML patients with normal karyotype.

Effects of miRNA-181a on proliferation and migration of multiple myelo-ma cells / 中国病理生理杂志

AIM:To investigate the effect of miRNA-181a inhibition on the proliferation, migration and cell cycle of the human multiple myeloma cell line RPMI 8226.METHODS:Real-time PCR was used to detect miRNA-181a expression in serum samples from multiple myeloma or healthy subjects .After transfection with miRNA-181a inhibitor, the cell viability was examined by CCK-8 assay and colony formation assay .The cell migration ability was analyzed by wound healing assay .The cell cycle was detected by flow cytometry .Moreover , the protein level of cyclin D 1 and the phosphoryla-tion of PI3K and Akt were determined by Western blot .RESULTS:The expression of miRNA-181a was significantly in-creased in the serum from multiple myeloma patients as compared with healthy group .Inhibition of miRNA-181a expression by transfection with miRNA-181a inhibitor remarkably decreased the cell viability , migratory ability, the population of G0/G1 phase and cyclin D1 protein expression in the RPMI8226 cells.However, the population of S phase and the phosphory-lation of PI3K and Akt were reduced .CONCLUSION:Down-regulation of miRNA-181a inhibits the viability and migra-tory ability in the RPMI8226 cells via inhibition of cell cycle and PI 3K/Akt signaling pathway .

Effects of human microRNA-181a-5p on proliferation and migration of gastric cancer cells / 中华消化杂志

Objective To preliminarily explore the effects of human microRNA-181a on migration of gastric cancer cells and its mechanism.Methods The expression of miRNA-181a-5p in gastric cancer cell line GC9811 and peritoneal high metastasis gastric cancer cell line GC9811-P were tested by quantitative real-time polymerase chain reaction (qRT-PCR ). GC9811 cell line was transfected byendogenous synthetic analog mimic and its homologous negative control of miRNA-181a-5p,which were considered as up-regulated group and its control group respectively;GC9811-P were transfected by miRNA inhibitor and its homologous negative control of miRNA-181a-5p,which were considered as down-regulated group and its control group respectively.After miRNA-181a-5p was up or down-regulated,cell proliferation,migration and apoptosis capabilities of gastric cells were detected by matrix thiazolyl tetrazollium (MTT)assay,cloning-forming assay,Transwell migration test,wound healing assays and apoptosis test.After miRNA-181a-5p was up or down regulated,the changes of matrix metalloproteinase (MMP)14 expression were determined by Western blot.Independent sample t test was performed for mean comparison between samples and chi square test was used for rate comparison.Results The results of qRT-PCR showed the relative expression quantity of miRNA-181a-5p in GC9811 was 1 .000 00 ± 0.021 26 and in GC9811-P was 3.175 61 ±0.106 76,and the difference was statistically significant (t =34.620,P <0.01 ).The results of MTT assay indicated that the cell proliferation rate of up-regulated group was higher than that of up-regulated control group,and that of down-regulated group was lower than down-regulated control group.The cloning-forming assay demonstrated that the number of clone forming and clone forming rate of up-regulated group,up-regulated control group,down-regulated group and down-regulated control group were 234.00±10.12 and 46.8%,93.00±9.61 and 18.6%,51 .00 ±7.96 and 10.2%,99.00±8.05 and 19.8%,respectively.The differences between up-regulated group,down-regulated group and their control group were statistically significant (t = 17.500,7.344,χ2 = 12.27, 9.51 ,all P <0.01).The results of Transwell migration experiment showed the number of cells migrated through membrane hole of up-regulated group was 164.00±19.31 ,and which was higher than that of up-regulated control group (87.00±23.04,t=4.436,P <0.05);that of down-regulated group was 157.00± 11 .50,and which was lower than that of down-regulated control group (234.00 ±12.12,t =7.982,P <0.05).The result of wound healing assays indicated that the rate of migration distance of up-regulated group,up-regulated control group,down-regulated group and down-regulated control group were 2.09 ± 0.18,1 .27 ±0.23,1 .15 ±0.15 and 1 .67 ±0.12,respectively.The differences between up-regulated group,down-regulated group and their control group were statistically significant (t =4.863 and 4.689, both P <0.05).The results of apoptosis experiments demonstrated that the apoptosis rate of up-regulated group,up-regulated control group,down-regulated group and down-regulated control group were (6.10± 1 .02 )%,(9.10 ± 2.13 )%,(12.70 ± 1 .23 )%,(8.70 ± 2.54 )%,respectively,and there was no statistically significant difference between up-regulated group,down-regulated group and their control group (both P *0.05).The results of Western blot showed that the grey value of up-regulated group was 561 .881 ±35 .740,which was higher than that of up-regulated control group (275 .784±23.520);that of down-regulated group was 579.565 ±37.950,which was lower than that of down-regulated control group (1 312.760±51 .270),and the differnces were statistically significant (t =11 .580 and 19.910,both P <0.01).Conclusion miRNA-181a-5p highly expresses in peritoneal high metastasis gastric cancer cell line GC9811-P and promoted the proliferation and migration of gastric cancer cell line GC9811 and GC9811-P with a tendency to suppress apoptosis.

Suppression of miR-181 a attenuates H2O2-induced death of mesenchymal stem cells by maintaining hexokinase II expression

BACKGROUND: Low survival rate of transplanted cells compromises the efficacy of cell therapy. Hexokinase II (HKII) is known to have anti-apoptotic activity through its interaction with mitochondria. The objective was to identify miRNAs targeting HKII and investigate whether miRNA-mediated modulation of HKII could improve the survival of mesenchymal stem cells (MSCs) exposed to H2O2. The expression of HKII in MSCs exposed to H2O2 was evaluated, and HKII-targeting miRNA was screened based on miRNA-target prediction databases. The effect of H2O2 on the expression of the selected HKII-targeting miRNA was examined and the effect of modulation of the selected HKII-targeting miRNA using anti-miRNA on H2O2-induced apoptosis of MSC was evaluated. RESULTS: H2O2 (600 µM) induced cell death of MSCs and decreased mitochondrial HKII expression. We have identified miR-181a as a HKII-targeting miRNA and H2O2 increased the expression of miR-181a in MSCs. Delivery of anti-miR-181a, which neutralizes endogenous miR-181a, significantly attenuated H2O2-induced decrease of HKII expression and disruption of mitochondrial membrane potential, improving the survival of MSCs exposed to H2O2. CONCLUSIONS: These findings suggest that H2O2-induced up-regulation of miR-181a contributes to the cell death of MSCs by down-regulating HKII. Neutralizing miR-181a can be an effective way to prime MSCs for transplantation into ischemic tissues.

Regulatory effect of miR-181 a on breast cancer resistance protein / 中国药理学通报

Aim ToinvestigatetheeffectsofmiRNA-181 a on breast cancer resistance protein ( BCRP ) . Methods Bioinformaticspredictedbindingsitesof BCRP mRNA-3ˊUTR region and miR-181 a;further lu-ciferase reporter gene analysis confirmed that miR-181 a could combine with BCRP mRNA-3ˊUTR; qRT-PCR and Western blot detected related mRNA and protein expressionlevels.Results Comparedwithnegative transfection group, after the miR-181a mimic and PGL3-BCRP 3ˊUTR were co-transfected, luciferase ac-tivity was significantly decreased ( P 0. 05 ); after transfecting miR-181 a inhibitor for 48h, compared to the negative group, the expression of miR-181 a in MCF-7 cells was reduced (P0.05).Conclusion miR-181acanregu-late BCRP expression by targeting the BCRP mRNA-3ˊUTR region.