The microRNA miR-17 regulates lung FoxA1 expression during lipopolysaccharide-induced acute lung injury

Acute lung injury (ALI) is a severe pulmonary disease that causes a high number of fatalities worldwide. Studies have shown that FoxA1 expression is upregulated during ALI and may play an important role in ALI by promoting the apoptosis of alveolar type II epithelial cells. However, the mechanism of FoxA1 overexpression in ALI is unclear. In this study, an in vivo murine model of ALI and alveolar type II epithelial cells injury was induced using lipopolysaccharide (LPS). LPS upregulated FoxA1 in the lung tissue of the in vivo ALI model and in LPS-challenged type II epithelial cells. In contrast, miR-17 was significantly downregulated in these models. After miR-17 antagomir injection, the expression of FoxA1 was significantly increased in ALI mice. MiR-17 mimics could significantly inhibit FoxA1 mRNA and protein expression, whereas the miR-17 inhibitor could significantly increase FoxA1 mRNA and protein expression in LPS-induced type II epithelial cells. Thus, our results suggest that the downregulation of miR-17 expression could lead to FoxA1 overexpression in ALI.     

miR-27a is up regulated and promotes inflammatory response in sepsis

MicroRNAs (miRNAs) are short, non-coding RNAs that regulate the expression of multiple target genes. Dysregulation of miRNAs is common in sepsis. Through microRNA microarray and qRT-PCR we found that the levels of miR-27a, miR-153 and miR-143 are up regulated, while let-7a, miR-218 and miR-129-5p are down regulated in lungs of septic mice. Knocking down of miR-27a down regulates expression levels of TNF-α and IL-6 significantly via reducing the phosphorylation level of NF-κB p65 and inhibiting its DNA binding activity. Furthermore, neutralisation of miR-27a up regulates PPARγ level, down regulates TNF-α expression, relieves pulmonary inflammation and promotes survival of septic mice, which demonstrates that miR-27a plays an important role in regulating inflammatory response in sepsis and provides a potential target for clinical sepsis research and treatment.     

MiR-27靶基因的分析及鉴定

MicroRNAs(miRNAs)是一类约20～25nt的小分子核苷酸,在细胞内的多种生物学过程,如细胞增殖、凋亡、生长、分化和代谢等过程中具有重要的功能。已知miR-27在脂肪细胞和肌肉细胞的发育过程中起了重要作用,其在神经细胞中的表达调节至今仍不清楚。在本研究中,通过miRBase和TargetScan数据库分析了miR-27的靶基因,构建了miR-27的真核表达载体,改造了萤火虫荧光素酶和海肾荧光素酶报告载体,将miR-27的靶基因Bmi1的3′-UTR融合到报告载体中,转染神经胶质瘤细胞,利用双荧光素酶检测系统分析荧光素酶的活性。研究发现miR-27a和miR-27b共同的靶基因主要调节发育过程。MiR-27真核表达载体能产生成熟态的miR-27。MiR-27a、miR-27b或miR-27a和miR-27b联合与Bmi1的3′-UTR的正义序列共转染U343细胞能明显降低萤火虫荧光素酶的活性(分别P0.05,P0.05,P0.01),这提示了Bmi1可能为miR-27的靶基因。     

Effects of the dioxin-like PCB 126 on larval summer flounder (Paralichthys dentatus)

Little is known about the sensitivity of teleost post-embryonic developmental stages (larval and metamorphic) to dioxin-like compounds. Larval and metamorphosing summer flounder (Paralichthys dentatus) were exposed to the dioxin-like polychlorinated biphenyl congener PCB 126, to compare their sensitivity to other fish species early life stages, and to document effects on metamorphic development, including degree of eye migration and gastric maturation. Median lethal doses (LD 50 s) ranged between 30 and 220 ng/g wet mass, indicating that pre- and early-metamorphic stages of summer flounder are equally sensitive to the embryos of some of the most vulnerable fish species tested. Consistent with the presence of a functional aryl hydrocarbon receptor pathway, dose-dependent induction of cytochrome P-4501A (CYP1A) at four days post-exposure was observed in liver, stomach, intestine, and kidney of metamorphosing larvae. Stage-dependent differences in the epithelial distribution of CYP1A immunoreactivity were observed in the developing stomach of fish exposed to relatively high PCB 126 doses. A single sublethal dose (15 ng/g) delayed metamorphic progress (determined by the degree of eye migration), and resulted in abnormally high levels of cell proliferation and abnormal gastric gland morphology in late metamorphic stages. These results suggest that the post-embryonic larval and metamorphic stages of summer flounder, and potentially other fish species with complex life histories, are vulnerable to the effects of dioxin-like compounds, including lethality, developmental delay, and malformations.     

miR-34基因家族的分子进化

根据miRNA基因在进化中高度保守的特点,利用生物信息学方法在目前已测序的动物物种中搜寻参与哺乳动物早期发育调控的mir-34基因的同源序列,在33个不同的动物物种中获得了miR-34基因的54条同源序列,其中18条为新发现的序列。表明miR-34是高度保守的,广泛存在于后生动物中。目前发现的mir-34基因80%位于基因间隔区,少数位于蛋白编码基因的内含子区和3′UTR上。不同动物中,mir-34基因成熟序列的同源性为68%,前体序列为38.89%。在无脊椎动物中只有一个mir-34,而在几乎所有的脊椎动物中都有mir-34a,mir-34b,mir-34c,形成miR-34基因家族。系统进化分析表明,脊椎动物中miR-34基因家族是通过基因的串联和局部重复形成的,这个过程中伴随着个别碱基的变异。     

Comparative analysis of methods to determine DNA methylation levels of a tumor-related microRNA gene

Quantifying levels of DNA methylation in tumors is a useful approach for the identification of potential tumor suppressors and to find biomarkers that can be used as prognostic or therapeutic indicators. In the current study, we compared three methods commonly used for quantifying DNA methylation—bisulfite pyrosequencing, quantitative methylation-specific PCR (Q-MSP), and MethyLight—by focusing on the CpG island of the gene encoding the microRNA-34b and microRNA-34c (miR-34b/c); aberrant regulation of this miR is associated with various human malignancies, including gastric cancer. Standard curve analysis using control DNA samples demonstrated the highest quantitative accuracy in Q-MSP analysis. We also carried out methylation analysis using gastric mucosa specimens obtained from gastric cancer patients. We found a high correlation between methylation levels determined by Q-MSP and those determined by MethyLight (R² = 0.952), whereas the results of bisulfite pyrosequencing and the other two methods were less well correlated (R² = 0.864 and R² = 0.804 for Q-MSP and MethyLight, respectively). This may reflect possible PCR bias in the pyrosequencing technique, which we show can be corrected for by applying a cubic approximate equation to the original data. Thus, although results obtained by the different DNA methylation analysis techniques are largely comparable, an appropriate correction may be necessary for stringent comparison.     

A Solute Carrier Family 22 Member 3 Variant rs3088442 G→A Associated with Coronary Heart Disease Inhibits Lipopolysaccharide-induced Inflammatory Response

Recent genome-wide association studies have identified single-nucleotide polymorphism (SNPs) within the SLC22A3 (solute carrier family 22 member 3) gene associated with coronary heart disease (CHD) in the Caucasian population. We performed molecular analysis to investigate the potential role of SLC22A3 variants in CHD. Our study showed that the common polymorphism rs3088442 G→A, which is localized in the 3′ UTR of the SLC22A3 gene, was associated with a decreased risk of CHD in the Chinese population by a case control study. In silico analysis indicated that G→A substitution of SNP rs3088442 created a putative binding site for miR-147 in the SLC22A3 mRNA. By overexpressing miR-147 or inhibiting endogenous miR-147, we demonstrated that SNP rs3088442 G→A recruited miR-147 to inhibit SLC22A3 expression. Moreover, SLC22A3 deficiency significantly decreased LPS-induced monocytic inflammatory response by interrupting NF-κB and MAPK signaling cascades in a histamine-dependent manner. Notably, the expression of SLC22A3^A was also suppressed by LPS stimulus. Our findings might indicate a negative feedback mechanism against inflammatory response by which SLC22A3 polymorphisms decreased the risk of CHD.