Bone marrow mesenchymal stem cell-derived exosomal miR-34c-5p ameliorates RIF by inhibiting the core fucosylation of multiple proteins

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Knocking-out of H1F1α gene by CRISPR/cas9 inhibits proliferation and invasiveness of prostate cancer DU145 cellsCSCD

Objective To explore the role of H1F1α gene in prostate cancer cell line DU145 by knocking it out with a novel gene-editing tool CRISPR/cas9 system. Methods A CRISPR/cas9 system with two sgRNAs targeting exon 1 of the H1F1α gene was constructed for the knock out experiment. CCK8 assay and transwell experiment were carried out to assess the effect of the knock out on the proliferation, migration and invasiveness of DU145 cells. Results The efficiency of gene-targeting was measured through a T7E1 assaying and sequence analysis, which confirmed that the partial knock out was successful and has led to a significant decrease in the expression of H1F1α and inhibition of cell proliferation, migration and invasiveness. Conclusion A CRISPR/cas9 system for the knock out of H1F1α has been successfully constructed, which could inhibit the proliferation and migration of DU145 cells. The system can facilitate further studies of the H1F1α gene and its roles in tumorigenesis. © 2018 West China University of Medical Sciences. All rights reserved.     

Overexpression of tissue microRNA10b may help predict glioma prognosis

We investigated the relationship between microRNA-10b (miR-10b) expression and prognosis in human glioma patients. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to characterize the expression patterns of miR-10b in 128 glioma and 20 normal brain tissues. Clinical information – age, sex, Karnofsky Performance Status (KPS) and World Health Organization (WHO) grade – were also collected. The associations between miR-10b expression and the clinicopathological factors and outcome of glioma patients were statistically analyzed. Expression levels of miR-10b in glioma tissue were significantly higher than in normal brain tissue (P < 0.001). High-grade glioma (WHO grade III and IV) had much higher miR-10b expression levels than low-grade tumors (WHO grade I and II). Additionally, the increased miR-10b expression in the glioma tissues was significantly associated with a low KPS (P = 0.03). Kaplan–Meier survival curves and Cox regression analyses showed that overexpression of miR-10b (P = 0.01) and high grade (P = 0.02) were independent factors predicting poor outcome for glioma patients. Furthermore, subgroup analyses showed that the miR-10b expression level was significantly associated with poor overall survival in glioma patients with high grades (P < 0.001). Up-regulation of miR-10b may have value in predicting clinical outcome in glioma patients, particularly for those with high pathological grades.     

BDNF-AS/miR-145-5p轴对高糖诱导的肾小管上皮细胞损伤的影响CSCD

目的探讨脑源性神经营养因子反义RNA(brain-derived neurotrophic factor-antisense, BDNF-AS)对高糖诱导的肾小管上皮细胞损伤的影响和可能机制。方法体外培养肾小管上皮细胞HK-2, 分别转染BDNF-AS小干扰RNA、miR-145-5p模拟物或共转染BDNF-AS小干扰RNA和miR-145-5p抑制剂, 之后采用30 mmol/L葡萄糖干预转染后的细胞24 h, 用RT-qPCR法检测细胞中BDNF-AS和miR-145-5p的表达, 用CCK-8法检测细胞增殖, 流式细胞术检测细胞凋亡, Western印迹法检测细胞中Bcl-2和Bax蛋白的表达, 酶联免疫吸附法检测细胞培养上清中IL-1β和IL-6的水平。用双荧光素酶报告基因实验验证BDNF-AS和miR-145-5p的调控关系。结果高糖处理促进了HK-2细胞中BDNF-AS的表达(P<0.05), 而抑制了miR-145-5p的表达(P<0.05)。干扰BDNF-AS或过表达miR-145-5p降低了高糖诱导的HK-2细胞抑制率、凋亡率及Bax蛋白、IL-1β和IL-6的表达(P<0.05), 促进了Bcl-2蛋白的表达(P<0.05)。干扰miR-145-5p逆转了干扰BDNF-AS对高糖诱导的HK-2细胞增殖、凋亡及IL-1β和IL-6表达的影响。BDNF-AS可靶向结合并负调控miR-145-5p。结论干扰BDNF-AS可能通过靶向负调控miR-145-5p促进高糖诱导的肾小管上皮细胞增殖, 并抑制细胞凋亡及炎症因子表达。     

miR-145对血管平滑肌细胞生物活性及SM22α mRNA表达的作用*

目的：探究miR-145 对血管平滑肌细胞（VSMC）的生物活性及对SM22α mRNA 表达的作用。方法： VSMC分为3 组,增殖VSMC组（ 增殖组）、增殖VSMC + 转染miR-145-NC 组（ 空载体组）、增殖VSMC+转染 miR-145 组（ 增殖转染组）。采用RT-PCR 检测VSMC中SM22α mRNA、miR-145 的表达;免疫印迹检测cyclin E、p27 蛋白水平;Transwell 小室检测VSMC侵袭能力;MTT检测VSMC活力;流式细胞术检测VSMC凋亡率。 结果：增殖组VSMC中SM22α mRNA、miR-145 水平与空载体组相比数值较为接近;与增殖组及空载体组相比, 增殖转染组VSMC中miR-145、SM22α mRNA 表达升高。增殖组VSMC中cyclin E、p27 蛋白水平与空载体组相 比无差异;增殖转染组cyclin E 蛋白水平低于空载体组、增殖组,p27 蛋白水平高于空载体组、增殖组。增殖组 VSMC迁移数量与空载体组相比无差异;增殖转染组VSMC迁移数量明显低于空载体组、增殖组。增殖组与空载 体组VSMC在不同时间点的OD 值均较为接近;增殖转染组OD 值在不同时间点均低于空载体组、增殖组。增殖 组VSMC凋亡率与空载体组数值接近,组间比较差距较小;增殖转染组VSMC凋亡率较空载体组和增殖组升高。 结论：过表达miR-145 通过促进SM22α 水平增加,抑制血管平滑肌细胞的增殖,并促进其凋亡。     

miR-7靶向缺氧诱导因子1α对肝癌细胞的影响*

目的： 探究微小RNA-7（miR-7）靶向缺氧诱导因子1α（HIF-1α）对肝癌细胞迁移、凋亡及干细胞特性的影响。 方法： 采用慢病毒转染肝癌细胞MGCC97H,分为对照组、miR-7 模拟物组、miR-7 干扰组（ 转染miR-7 模拟物 组+ 干扰物）、HIF-1α 过表达组（ 转染pc-HIF1α）和共转染组（ 转染miR-7 模拟物+pc-HIF1α）,RT-PCR 检测各 组细胞miR-7、HIF-1α mRNA 表达量,免疫印迹检测各组HIF-1α 蛋白的表达量,Transwell 实验检测细胞迁移能 力,流式细胞法检测细胞凋亡情况,软琼脂克隆形成实验分析其干细胞特性,荧光素酶实验验证miR-7 与HIF-1α 靶向关系。结果： miR-7 模拟物组细胞HIF-1α 蛋白表达、迁移率、克隆形成率低于对照组,凋亡率高于对照组; HIF-1α 过表达组细胞迁移率、克隆形成率高于对照组,凋亡率低于对照组;共转染组HIF-1α 蛋白表达、细胞迁 移率、克隆形成率低于HIF-1α 过表达组,凋亡率高于HIF-1α 过表达组。结论： miR-7 靶向HIF-1α 可降低肝癌细 胞迁移能力及干细胞特性,促进细胞凋亡,miR-7 及HIF-1α 可能是肝癌的潜在治疗靶点。     

抑制miR-133b通过靶向FOXP3对PD大鼠调节性T淋巴细胞、炎性反应和神经元凋亡的影响

目的探究抑制微小RNA(microRNA,miR)-133b通过靶向叉头盒蛋白3(forkhead box protein 3,FOXP3)对帕金森病(Parkinson’s disease,PD)大鼠调节性T细胞(regulatory T cells,Treg)的影响。方法 32只PD模型大鼠随机分为PD组和PD+miR-133b antagomir组(n=16),健康大鼠16只作为对照组。尾静脉注射miR-133b antagomir(300μg)来抑制miR-133b的水平。检测和比较各组大鼠神经功能、黑质损伤、细胞凋亡、炎性反应、Treg细胞水平、miR-133b、FOXP3 mRNA和蛋白表达水平;通过双荧光素酶报告验证miR-133b和FOXP3的靶向关系。结果 PD组的逃避潜伏期、旋转速率、细胞凋亡情况、IL-6、miR-133b水平显著高于对照组,穿越次数、IL-10、FOXP3 mRNA和蛋白表达量显著低于对照组(P<0.05)。PD+miR-133b antagomir组的逃避潜伏期、旋转速率、细胞凋亡情况、IL-6、miR-133b水平显著低于PD组,穿越次...     

Genetic deletions in AML and MDS

Chromosomal deletions are common molecular events in myeloid malignancies. Heterozygous deletions may contain a tumor suppressor gene that undergoes homozygous inactivation or may contain one or more genes that alter the disease phenotype through haploinsufficiency. The most common karyotypic abnormality in myelodysplastic syndrome (MDS) is deletion of chromosome 5q. A subset of patients with del(5q) as a sole cytogenetic abnormality has a consistent set of clinical features, termed the 5q- syndrome. While no tumor suppressor genes have been identified on 5q that are homozygously inactivated, recent studies have highlighted several genes and micro RNAs (miRNAs) that cause the phenotype of the 5q- syndrome through allelic insufficiency. For example, deletion of one allele of the RPS14 gene causes a severe defect in erythropoiesis, analogous to the congenital syndrome Diamond Blackfan anemia, which is itself caused by mutations that inactivate one allele of a ribosomal gene. Loss of one allele of miR-145 and miR-146a causes an increase in megakaryocyte production and may contribute to the clonal advantage of cells with del(5q). The functional approaches used to dissect the molecular basis of the 5q deletion in MDS have the potential to identify key genes and therapeutic targets within other chromosomal deletions in hematologic malignancies.     

MicroRNA155通过下调清道夫受体表达抑制巨噬细胞泡沫化形成

 探讨microRNA-155(miR-155)对巨噬细胞泡沫化过程的影响及机制。【方法】 实时定量PCR检测miR-155的表达,Western Blot方法检测巨噬细胞A类清道夫受体SR-A和B型清道夫受体CD36的表达,激光共聚焦显微镜观察miR-155对THP-1结合、摄取DiI标记氧化型低密度脂蛋白(DiI-oxLDL)能力的影响。【结果】 80μg/mL的氧化型低密度脂蛋白(ox-LDL)时间依赖性地诱导巨噬细胞miR-155表达上调。过表达miR-155抑制SR-A和CD36的表达,同时巨噬细胞结合、摄取DiI-oxLDL的能力明显降低。而反义-miR-155则明显上调SR-A和CD36的表达,同时巨噬细胞结合、摄取DiI-oxLDL的能力明显增强。【结论】 MiR-155通过降低巨噬细胞SR-A和CD36的表达抑制巨噬泡沫细胞的形成。