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ABSTRACT Background: While sarcopenia is an important clinical finding in individuals diagnosed with chronic heart failure (CHF), efforts to identify a reliable biomarker capable of predicting the overall muscular and functional decline in CHF patients have been unsuccessful to date. Objectives: The objectives of this study were to study the diagnostic utility of MicroRNA (miRNA)-1-3p as a predictor of sarcopenia status in individuals diagnosed with CHF. Methods: In total, 80 individuals with heart failure exhibiting a left ventricular ejection fraction < 50% were enrolled in this study. All patients were analyzed to assess miR-1-3p expression levels, with body composition being evaluated through dual-energy X-ray absorptiometry and sarcopenia being defined based on the sum of appendicular lean muscle mass (ALM) divided by height in meters squared and handgrip strength (HGS). In addition, the activation of the Akt/mTOR signaling pathway was evaluated in these individuals. Results: In total, 40 of the enrolled patients (50%) exhibited sarcopenia. Sarcopenic patients presented with increased miR-1-3p expression levels as compared to non-sarcopenic individuals (1.69 ± 0.132 vs. 1.22 ± 0.106; p < 0.05). With respect to sarcopenic indices, appendicular skeletal mass index was most strongly correlated with miR-1-3p expression, which was also strongly correlated with HGS. High levels of Akt/mTOR signaling pathway components were expressed in sarcopenic individuals, highlighting a significant relationship between miR-1-3p activity and signaling through this pathway. Moreover, miR-1-3p was identified as a specific marker for sarcopenia in individuals with CHF. Conclusion: These results suggest that circulating miR-1-3p levels are related to Akt/mTOR pathway activation and can offer valuable insight into the overall physical capacity and muscular integrity of CHF patients as a predictor of sarcopenia.
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@#[摘 要] 目的：通过生物信息学方法探索并实验验证胃癌相关标志物miR-1-3p对胃癌细胞增殖的作用及其分子机制。方法：收集TCGA数据库中胃癌（n=375）及癌旁组织（n=45）的转录组数据，构建胃癌特异性mRNA-miRNA网络，筛选潜在的miRNA类标志物，利用TargetScan预测标志物的下游靶基因且分析它们的功能。选取人正常胃上皮细胞GES-1及胃癌细胞AGS、MKN45、NCI-N87，用qPCR法检测细胞中miR-1-3p和心肌蛋白（MYOCD）的表达，用lipofectamine 2000将miR-1-3p模拟物转染至胃癌细胞中，CCK-8法测定轨染后细胞的增殖能力，WB法测定MYOCD的表达量，双荧光素酶报告基因实验验证miR-1-3p与MYOCD之间的靶向结合关系。结果：通过数据库数据分析得到差异表达的259个miRNA和7 545个mRNA，构建胃癌特异性mRNA-miRNA调节网络，分析网络中脆弱结构后确定miR-1-3p为潜在的胃癌标志物，ROC曲线和Kaplan-Meier分析显示其对胃癌的诊断和预后评估有重要意义。细胞实验显示miR-1-3p在胃癌细胞中呈低表达（P<0.05），过表达miR-1-3p可抑制胃癌细胞AGS和MKN-45的增殖能力（P<0.05或P<0.01），且可抑制MYOCD的表达（P<0.01）。TargetScan数据库预测到MYOCD的3'UTR区域中有两个与miR-1-3p结合的位点，双荧光素酶报告基因实验证实miR-1-3p与MYOCD靶向结合且负调控MYOCD的表达（P<0.01）。结论: miR-1-3p可能是胃癌诊断和预后相关潜在的标志物，且miR-1-3p可能是通过靶向MYOCD来影响胃癌细胞的增殖。
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OBJECTIVES@#The pathogenesis of androgenetic alopecia (AGA) is related to the level of androgen and its metabolic pathways. The binding of androgen and androgen receptor (AR) depends on the assistance of heat shock protein 27 (HSP27). HSP27 combined with microRNAs (miR)-1 can regulate AR levels. However, it is not clear whether HSP27 and miR-1 jointly participate in the pathogenesis of AGA. This study aims to investigate the role of AR up-regulation in the pathogenesis of AGA and underlying mechanisms.@*METHODS@#A total of 46 male AGA patients (AGA group), who admitted to the First Affiliated Hospital of Guangzhou Medical University from September 2019 to February 2020, and 52 healthy controls admitted to the same period were enrolled in this study. Serum levels of dihydrotestosterone (DHT) and HSP27 in patients and healthy controls were measured by ELISA. Western blotting was used to detect the protein expression of HSP27 and AR in scalp tissues of patients and the healthy controls. The levels of HSP27, AR, and miR-1 were analyzed using real-time PCR. Human dermal papilla cells were transfected with HSP27 siRNA to inhibit the expression of HSP27. MiR-1 and miR-1 inhibitors were transfected simultaneously or separately into cells and then the changes in AR protein expression were detected.@*RESULTS@#The levels of DHT and HSP27 in the AGA group were (361.4±187.7) pg/mL and (89.4±21.8) ng/mL, respectively, which were higher than those in the control group [(281.8±176.6) pg/mL and (41.2±13.7) ng/mL, both P<0.05]. However, there was no significant difference in serum HSP27 and AR levels among AGA patients with different degrees of hair loss (P>0.05). Correlation analysis showed that there was a positive correlation between HSP27 level and DHT level in the AGA patients (P<0.05). The level of HSP27 mRNA in scalp tissue was negatively correlated with that of miR-1 mRNA (P<0.05). Compared with the control group, the levels of HSP27 protein, AR protein, HSP27 mRNA, and AR mRNA in scalp tissues of AGA group were significantly increased (P<0.05). The up-regulation of HSP27 in scalp tissues of AGA patients was closely related to the increased levels of AR. However, the level of miR-1 in scalp tissues of AGA patients was significantly down-regulated, contrary to the expression of AR (P<0.05). Further in cell studies showed that inhibition of HSP27 or miR-1 expression in human dermal papilla cells could inhibit the expression of AR, and inhibition of both HSP27 and miR-1 expression was found to have an accumulative effect on AR, with statistically significant differences (all P<0.05).@*CONCLUSIONS@#HSP27 could combine with miR-1 to up-regulate AR levels, which is closely related to the development of AGA.
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Humans , Male , Alopecia/pathology , HSP27 Heat-Shock Proteins/metabolism , MicroRNAs/genetics , RNA, Messenger , Receptors, Androgen/metabolism , Up-RegulationABSTRACT
Background: Colorectal cancer (CRC) is currently the third most common cancer type in males and the second most occurring in females. The role of microRNA (miRNA) in the development of colorectal cancer is notfully elucidated. Therefore, understanding the mechanistic interaction between miRNA and their target oncogenes may hold great importance as a possible target for interventional anticancer therapy Aims:To identify miRNAs that are part of the regulating pathway of Monocarboxylate Transporter-4 (MCT4) and Vascular Endothelial Growth Factor (VEGF) oncogenes.Study Design:We used publicly available prediction tools (e.g. TargetScan, MicroCosm, PicTar, and DIANA-microT-CDS) to identify the possible miRNA that target the two oncogenes. Methodology:We used the GeneMania database to visualize the network and verify gene names and remove ambiguity and duplications. Furthermore, we used miRTarBase database to identify experimentally validated targets which we used to further confirm miRNA-oncogene relationships. Finally, we utilized miR-Mfold web-tool to further visualize the circular structures and the simulated miR-1 and miR-206 targeting arrangements.Results:We found two putative miRNA (miR-1 and miR-206) that may downregulate MCT4 coded by SLC16A3gene and VEGF which is coded by VEGF gene. We found relationships between the validated target genes of miR-1 and miR-206 through GeneMania which we extracted from the literature. And we elucidated the proposed structure of these two miRNAs through miR-Mfold web-tool.Conclusion: Our results elucidated a novel regulation pathway in CRC cells and may suggest a potential therapeutic approach for CRC therapy. MiR-1 and miR-206 may help cells go to apoptosis and inhibit the angiogenesis of colorectal cancer cells by down-regulation of MCT4 and VEGF proteins in tumor tissues.
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Objective To explore the diagnostic value of miR-1 in acute myocardial infarction(AMI).Meth-ods 148 patients with chest pain in the emergency department of this hospital from February 2013 to Decem-ber 2016 were selected and divided into the AMI group(82 cases)and non-AMI group(66 cases)according to the diagnostic criteria of acute AMI.Contemporaneous 74 healthy persons undergoing physical examination were selected as the healthy control group.The levels of serum miR-1,cardiac troponin I(cTnI)and creatine kinase isoenzyme(CK-MB)were measured in 3 groups.The correlation between miR-1 level with cTnI and CK-MB levels in the AMI group.The sensitivity and specificity of miR-1,cTnI,and CK-MB in the diagnosis of acute AMI were analyzed.Results The serum miR-1,cTnI and CK-MB levels in the AMI group were signifi-cantly higher than those in the non-AMI group,while the serum miR-1,cTnI and CK-MB levels in the non-AMI group were higher than those in the healthy control group,the difference among 3 groups was statistical-ly significant(P<0.05);the miR-1 level was positively correlated with cTnI and CK-MB levels in the AMI group(r=0.733,0.779,P<0.05);the receiver operating characteristic curve analysis showed that the sensi-tivity and specificity of miR-1 in the early diagnosis of acute AMI was 90.57% and 97.53% respectively.Con-clusion miR-1 can be used as a new index for early diagnosing acute AMI and assessing severity degree,more-over its sensitivity is higher than cTnI and CK-MB.
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Objective To provide theoretical guidance for further research on the role of miR-1 99a-3p in formation and development of bladder cancer.Methods Mature sequence of miR-1 99a-3p was analyzed;target genes and transcription factors of miRNA-1 99a-3p were predicted,and the target genes were analyzed for gene ontology (GO)enrichment and Kyoto Encyclopedia of Genes and Genome (KEGG)pathway.Then TF-miRNA-mRNA network diagram was constructed.Results Sequences of miR-1 99a-3p were highly conserved in various species.In GO analysis,the target genes of miR-1 99a-3p were enriched in many biological processes,such as regulation of cellular process,regulation of macromolecule metabolic process,and regulation of biological process (P <0.01 ).In KEGG pathway,the target genes were mainly located in bacterial invasion pathway of epithelial cells,ECM-receptor interaction pathway,PI3K-Akt signaling pathway,MAPK signaling pathway,small cell lung cancer pathway,and proteoglycans pathway in the cancer (P <0.05).According to the TF-miRNA-mRNA network diagram,the important genes that might be regulated by miR-1 99a-3p were MYC,SP1,mTOR,NFκB,and NFκB1.Conclusion miR-1 99a-3p may directly target mTOR and participate in the formation and development of bladder cancer through regulating PI3K-Akt-mTOR signaling pathway.
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Objective To investigate the levels of miR-1 9a and miR-1 9b expressions in cancer tissues and serum of patients with non-small cell lung cancer (NSCLC),and evaluate the potential of miR-1 9a and miR-1 9b as biomarkers of NSCLC.Methods Real time-PCR was used to detect the expressions of miR-1 9a and miR-1 9b in serum of normal people and patients with NSCLC and in cancer tissues and their corresponding non-tumor tissues. The diagnostic value of miR-1 9a and miR-1 9b for NSCLC was assessed by receiver operator characteristic (ROC) curve.Results The expression levels of serum miR-1 9a and miR-1 9b were significantly higher in patients with non-small cell lung cancer than in normal people (P <0.05 ).The expressions of miR-1 9a and miR-1 9b in NSCLC were much higher than in the adjacent normal tissues.For miR-1 9a the area under ROC curve was 0.989,and the sensitivity and specificity were 95.1 and 94.0.The area under ROC curve for miR-1 9b was 0.983,and the sensitivity and specificity were 93.4 and 94.0.Conclusion The high expressions of miR-1 9a and miR-1 9b are found in cancer tissues and serum from NSCLC patients.Therefore,they may be used as noninvasive biomarkers for diagnosis and prognosis of NSCLC.
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ABSTRACT:Objective To explore the differential expression of microRNAs (miRNAs)in steroid-induced osteonecrosis (ON).Methods Bone tissues were selected from patients with steroid-induced ON between the necrotic zone of femoral heads and its femoral neck to analyze the miRNA expression profile using the microarray. The most differentially expressed miR-125a-3p and miR-1 7-5p in microarray analysis were further confirmed by real-time quantitative PCR.Results According to the microarray screening,8 miRNAs were upregulated and 3 miRNAs were downregulated in the necrotic zone of femoral heads samples (Fold>2,P <0.05 ).Results of real-time PCR revealed that miR-125a-3p was upregulated and miR-1 7-5p was downregulated in the necrotic zone of femoral heads samples,which were in agreement with the microarray data.Conclusion MiRNA’s differential expression profile of human steroid-induced ON samples was obtained.Among the differentially expressed miRNAs, miR-125a-3p and miR-1 7-5p are the most apparently differentially expressed miRNAs,which may be related to the pathogenesis and development of steroid-induced ON.
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Objective To investigate the regulation of miR-1 and miR-133 a on L-type calcium channel β2 subunit ( Cavβ2 ) and α1C subunit during rat cardiomyocyte hypertrophy .Methods Cardiomyocyte hypertrophy was in-duced by isoproterenol (ISO, 10μmol/L).The targets of miR-1 and miR-133a were predicted by online database microCosm and Targetscan , respectively .The 3′untranslated region sequences of Cavβ2 andα1C were respectively cloned into reporter vector and then transiently transfected into HEK 293 cells.The luciferase activities of samples were measured for demonstrating the expression of luciferase reporter vector .The protein expression of Cavβ2 andα1C were evaluated by Western blot .The expression levels of Cavβ2 andα1C were inhibited by RNAi to determine theeffectsofCavβ2andα1Concardiomyocytehypertrophy.Results 1)Cavβ2wasoneofpotentialtargetsof miR-1,α1C was the one of potential targets of miR-133a.2) The luciferase activities of HEK293 cells with the plasmid containing widetype Cavβ2 3′UTR sequence or α1 C significantly decreased ( P <0.05 , P <0.01 ) . 3 ) Upregulation of the miR-1 and miR-133 a by miR-1 mimic and miR-133 a mimic transfection suppressed pro-tein expression of Cavβ2 and α1C, respectively(P<0.01, P<0.05).4)Downregulation of Cavβ2 andα1C by RNAi could markedly inhibit the increase of cell surface area ( P<0.01 ) , mRNA expression of ANP andβ-MHC (P<0.05).Conclusions Cavβ2 is the target gene of miR-1 and α1C is the target gene of miR-133a.miR-1 and miR-133a can negatively regulate the expression of L-type calcium channel Cavβ2 andα1C subunit, inhibi-ting cardiomyocyte hypertrophy.
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Objective To investigate the effects of up?regulated miR?206/miR?1 on the proliferation of breast cancer stem cells and the effect mech?anism. Methods Breast cancer stem cells(BCSCs)were isolated from breast cancer cell line MCF?7 by fluorescence?activated cell sorting. Cells in the experiment were divided into the blank control group,the negative control group,the miR?206 group and the miR?1 group. The BCSCs were transfected by negative control mimic,hsa?miR?206mimic and hsa?miR?1mimic in all groups except the blank control group. MiR?206and miR?1 expression levels as well as the transcription factor EVI?1 gene were detected by real time PCR. The expression levels of the transcription factor EVI?1 protein were detected by Western blot. MTT method was used to detect the effects of miR?206 and miR?1 on the proliferation of BCSCs. Results The BCSCs(CD44+/CD24-/low cells)isolated from MCF?7 cell lines were successfully cultured in serum?free medium for subsequent studies. After transfection of hsa?miR?206mimic and hsa?miR?1mimic for 48 hours,miR?206and miR?1relative expression levels increased. EVI?1mRNA ex?pression levels significantly decreased. The results of Western blot and MTT showed that up?regulated expression levels of miR?206 and miR?1 could significantly reduce the expression of EVI?1 protein and inhibited the proliferation of BCSCs. The differences in levels of miR?206,miR?1 and EVI?1 protein were statistically significant(P<0.05). Conclusion Up?regulated miR?206 and miR?1 expression can inhibit the proliferation ability of BCSCs,which may be related to the down?regulation of EVI?1.
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Objective To investigate the apoptotic effect of microRNA-1 (miR-1) on hypoxemic cardiomyocytes. Methods The cultured H9C2 cells were divided into 5 groups:normal control group, negative control group, H2O2 group, miR-1 group and H2O2+miR-1 group. After verified the success of transfection by real time PCR, MTT and flow cytometry methods were used to test the cell vitality and apoptotic rate, while the mRNA and protein expression level of Bcl-2 were de-tected by real time PCR and Western blot methods. Results Compared with normal control group, there were no significant differences in all indexes in negative control group. The application of H2O2 and miR-1 respectively or together significantly increased the miR-1 level and apoptotic rate, and reduced the cell vitality and Bcl-2 expression level. Conclusion mi-croRNA-1 can induce cardiomyocyte apoptosis by downregulating anti-apoptosis factor Bcl-2.
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Objective To investigate the protective function and mechanism of microRNA-1 (miR-1) downregula-tion in H2O2 injured cardiomyocytes. Methods The experiment was divided into 4 groups:Blank group, Negative Control group (NC), H2O2 group and H2O2+AS-miR-1 group. Cells in blank group were not underwent any treatment. Cells in NC group were transfected with random miRNA fragment. H2O2 and H2O2+AS-miR-1 groups were defined as H2O2 injured car-diomyocytes without or with transfected antisense miR-1 oligonucleotide(AS-miR-1). Real time PCR was used to test miR-1 transcription level, and cell vitality and apoptosis were analyzed by MTT and flow cytometry. The target gene of miR-1 was predicted by bioinformatics, then its mRNA transcription and protein expression level of Bcl-2 were detected by real time PCR and western blot respectively. Results There is no significant difference of all index between Blank group and NC group. H2O2 can induce cardiomyocyte injury, increase miR-1 level and rise apoptosis rate, reduce cell vitality and de-crease Bcl-2 expression level. Transfection of AS-miR-1 can decrease cell apoptosis, increase cell vitality and enhance Bcl-2 expression level. Conclusion Downregulation of microRNA-1 can protect cardiomyocytes that was injured by H2O2 through increasing anti-apoptosis factor Bcl-2 expression.
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Objective To construct and identify miR-1 adenovirus vector, and to analyze its effect on cardiac hypertrophy. Methods The primers of miR-1 precursor were designed for PCR amplification, and the PCR products were cloned into adenovirus shuttle plasmid pAdTrack and linearized by enzyme Pme I: the resultant plasmid was co-transfected into E. coli BJ5183 cells with adenovirus backbone plasmid pAdEasy-1 for homologous recombination. Then the recombinant plasmid was identified, linearized and packaged into QBI-293A cells to amplify the recombinant adenovirus Ad-miR-1, which was then used to infect cardiomyocytes. Real-time quantitative PCR was used to observe the expression of miR-1 and two hypertrophic markers, the atrial natriuretic peptide ( Nppa ) and ß-myosin heavy chain ( myh7 ), in cultured primary cardiomyocytes. Cell surface area was analyzed using software AxioVision 4. 7. 1 (Carl Zeiss). Results Sequencing and enzyme digestion showed that the miR-1 recombinant plasmid was successfully constructed. Real-time quantitative PCR confirmed that adenovirus Ad-miR-1 significantly enhanced intracellular miR-1 expression in cardiomyocytes and reduced cell surface area and the expression of Nppa and myh7. Conclusion The adenovirus expressing miR-1 has been successfully constructed and it can be transfected into cardiomyocytes to increase the expression of miR-1, thus inhibiting cardiomyocyte growth.