肝癌干细胞差异表达miRNA的筛选及鉴定

目的培养卵圆细胞、肝癌干细胞样细胞,筛选肝癌干细胞差异表达的microRNA并进行验证。方法体外分离培养卵圆细胞,肝癌干细胞样细胞,通过microRNA芯片检测差异表达的microRNA,对所得到的差异表达显著的microRNA进行Northern验证。结果表达差异显著的microRNA共有17个,经Northem杂交验证,其中6个差异显著的microRNA,发现hsa—let-7f-2、hsa—miR-199a-3p、hsa-miR-122在肝癌干细胞中的表达较卵圆细胞明显升高。结论hsa—let一7f-2、hsa—miR-199a一3p、hsa—miR-122在肝癌干细胞中的表达较卵圆细胞明显升高,可以进一步验证其在肝癌于细胞中的功能。     

Investigating VEGF. miR-145-3p,and miR-101-3p Expression in Patients with Cholangiocarcinoma

Introduction: Cholangiocarcinoma (CCA) is the second most common type of primary liver cancer. Several factors, such as epigenetic changes in promoter genes, gene expression, and microRNAs (miR), can contribute to genomic instability in cancer. This study aimed at evaluating the expression of VEGF, miRs 145-3p, and 101-3p in patients with CCA and their potential as biomarkers for diagnosis and prognosis of CCA. Material and methods : Sixty two patients were studied. Out of these 62 patients, 41 cases had confirm CCA and 21 cases had hepatopathies complications. The RNA was extracted from a paraffined tissue block, and then the synthesis of cDNA was performed. The analysis of the expression of VEGF, miR-145-3p, and miR-101-3p was carried out by polymerase chain reaction in real time.  Results: The findings revealed that miRs 145-3p and 101-3p were under expressed in the case group compared to the control group (0.46; 0.17; P = 0.0001, respectively). VEGF was overexpressed in the case group compared to the control group (11.8; P = 0.0001). An increase in miR-145-3p expression level was observed in patients with perihilar CCA compared to those with distal CCA (0.51 ± 0.41; 0.17 ± 0.13; P = 0.0698). Survival rate analysis showed that 41.9% of patients with intrahepatic CCA and 31.5% of patients with extrahepatic CCA were free from death within 11 months, leading to a significant difference (P> 0.05). Conclusion: The underexpression of miRNAs, tumor suppressors, the overexpression of VEGF, smoking, and aging were associated with CCA based on our findings. It seems that the reduced expression of the studies miRNAs and increased expression of VEGF can contribute to a decrease in survival rate of patients with tumor in their intrahepatic bile ducts.     

Sustained increase of microRNA-21 abundance drives aristolochic acid-induced acute kidney injury to renal tubulointerstitial fibrosis

Objective To investigate the role of increased microRNA-21 (miR-21) in the development of renal tubulointerstitial fibrosis secondary to aristolochic acid induced acute kidney injury. Methods C57BL/6J male mice were intraperitoneally injected with aristolochic acid at a dose of 10 mg/kg. Blood samples and kidneys were harvested at day 1, 3, 7, 14, 28 after aristolochic acid treatment. To assess the role of miR-21 in aristolochic acid induced acute kidney injury to chronic kidney disease progression, mice were intravenously injected with anti-miR-21 or anti-scramble (10 mg/kg) at 1 h before aristolochic acid dosing, as well as d5 and d10 after aristolochic acid dosing. Results Increased serum creatinine and severe kidney injury were found at d3 after aristolochic acid treatment. Renal tubulointerstitial fibrosis was developed at d14 after aristolochic acid treatment. Protein expression of α-SMA, vimentin and collagen I were significantly up-regulated at d7 and peaked at d14 (P＜0.01), while protein abundance of E-Cadherin decreased at d14 and lasted until d28 (P＜0.01). The abundance of miR-21 increased at d7 after aristolochic acid dosing, peaking at d14 and thereafter maintaining at a high level. Anti-miR-21 intervention relieved renal injury with reduced serum creatinine (P＜0.05) and attenuation of renal tubulointerstitial fibrosis. Besides, the protein expression of α-SMA, vimentin, and collagen I/IV was all down-regulated after anti-miR-21 treatment (P＜0.05). PTEN was up-regulated and the ratio of its downstream genes p-AKT/AKT was decreased. (P＜0.05) Conclusions A single high dose of aristolochic acid leads to acute kidney injury and the development of renal tubulointerstitial fibrosis secondary to AKI. Renal tubulointerstitial fibrosis could be partially reversed by inhibiting miR-21 via PTEN/ p-AKT pathway.     

微小RNA-3619-5p通过靶向作用于p21基因对卵巢癌细胞生长的影响

目的:探讨微小RNA-3619-5p(miR-3619-5p)过表达对卵巢癌细胞系A2780和SKOV3中p21基因的上调作用及对卵巢癌细胞生长的影响。方法:使用Lipofectamine 3000分别向卵巢癌细胞系A2780和SKOV3瞬时转染miR-3619-5p(实验组)或者dsControl(对照组)。通过实时荧光定量聚合酶链反应(q RT-PCR)检测p21、细胞周期依赖性激酶4(CDK4)和细胞周期蛋白D1(Cyclin D1)mRNA的表达情况。蛋白质印迹(Western blotting)检测p21、CDK4和Cyclin D1蛋白的表达情况。流式细胞术检测对照组和实验组细胞周期分布差异和细胞凋亡情况。EdU增殖实验和集落形成实验检测细胞增殖能力。结果:与对照组相比,转染miR-3619-5p后2种细胞系中p21 mRNA均显著升高(P0.01),而CDK4和Cyclin D1 mRNA的表达均明显降低(P0.01)。Western blotting实验结果与qRT-PCR结果一致。与对照组相比,转染miR-3619-5p后,位于S期和G_2/M期的细胞比例明显下降,位于G_0/G_1期的细胞比例明显增大,细胞凋亡率明显升高。EdU增殖实验和集落形成实验均显示,与对照组相比,转染miR-3619-5p的卵巢癌细胞的增殖能力明显下降(P0.05)。结论:miR-3619-5p可通过激活卵巢癌细胞中p21蛋白的表达抑制卵巢癌细胞的生长。     

Differential expression of microRNAs related to apoptosis in human osteoblasts induced by sodium fluoride北大核心

BACKGROUND: Long-term excessive intake of fluoride, especially through drinking water, can cause chronic fluorosis of bone. The disease can lead to bone damage and deformity, and is difficult to recover. Unfortunately, we have not developed a noninvasive or minimally invasive method for its early diagnosis. OBJECTIVE: To observe the expression of apoptosis-related miRNAs under the action of excessive fluorine in human osteoblasts. METHODS: The fluorine model was established in the human osteoblasts by cultured with 20 and 40 mg/L sodium fluoride for 24 and 48 hours, respectively. The expression levels of apoptosis-related miRNAs were determined by PCR array. RESULTS AND CONCLUSION: After 24-hour treatment of sodium fluoride, 48 kinds of miRNAs were upregulated and 4 ones were down-regulated in the osteoblasts. After 48-hour treatment of sodium fluoride, 21 kinds of miRNAs were upregulated and 2 ones were down-regulated. It showed that nine up-regulated miRNAs and one down-regulated miRNA were same in two periods. The 10 miRNAs are selected for target gene analysis on bioinformatics software that refer to the effect of anti-apoptosis and pro-apoptosis, which is of great significance for the early identification of skeletal fluorosis. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

急性脑梗死早期血中miRNAs水平与脑侧支循环建立的关系

目的探讨急性脑梗死早期外周血清中miRNAs水平与侧支循环建立的关系及临床意义。方法选择2014年1月-2015年11月在我院住院的急性脑梗死患者,根据其脑侧支循环状况,分为侧支循环良好组(42例)与不良组(28例),另设31例健康体检者为对照。比较各组间一般临床资料及梗死早期血清中一组miRNAs的分子变化,并进一步用Logistic回归分析miRNAs水平与梗死患者侧支循环建立的关系。结果血清中抑制血管新生的MIR-15b、MIR-92a及促血管新生的MIR-126、MIR-132和MIR-210水平在侧支循环良好、不良组及对照组间存在差异表达,部分特异性miRNAs分子组间比较有显著性差异(P0.05,P0.01);Logistic回归显示,这些抑制或促血管新生的miRNAs,特别是促血管新生的MIR-126、MIR-132和MIR-210是急性脑梗死侧支循环建立好坏的影响因素。结论早期血清中一组特异性miRNAs分子可能作为一种预测急性脑梗死患者脑内侧支循环状况的便捷指标。     

循环 microRNA-21对冠心病不稳定斑块的预测意义

目的：探讨冠心病（CAD）患者循环 microRNA-21（miRNA-21）相对表达量与冠状动脉内不稳定斑块的关系。方法连续收集2012年1月至2014年12月在广州市第一人民医院心内科住院并接受冠状动脉造影（CAG）及血管内超声（IVUS）检查的 CAD 患者100例,根据 IVUS检查患者冠脉内是否具有不稳定斑块分为稳定斑块组（SP 组）45例、不稳定斑块组（UP 组）55例,并选择同期在本院体检中心进行健康体检的50例健康对照病例作为对照组。采用实时荧光定量聚合酶链式反应技术（qRT-PCR）测定入选病例空腹静脉血浆 miRNA-21的相对表达量,分析血浆 miRNA-21水平与 CAD 患者不稳定斑块的关系。结果不稳定斑块组患者血浆 miRNA-21相对表达量显著高于稳定斑块组和对照组[（0.87±0.10） vs.（0.78±0.11） vs.（0.67±0.08）,P ＜0.05],将 CAD 患者血浆 miRNA-21水平与患者是否存在不稳定斑块做出受试者工作曲线（ROC）,血浆 miRNA-21的曲线下面积（AUC）为0.869（95％CI：0.797～0.940）。多因素 Logistic 回归分析显示血浆 miRNA-21是 CAD 患者存在不稳定斑块的独立预测因子（P ＜0.05）。结论血浆miRNA-21水平升高预示 CAD 患者冠状动脉内斑块的不稳定状态,是预测 CAD 患者存在不稳定斑块的重要生物标志物之一。