Expression Of MicroRNA-30e In Sepsis-induced Acute Lung Injury And The Mechanism Investigation In Inflammation

BackgroundSepsis is a complex condition that occurs as systemic inflammatory response syndrome (SIRS) caused by infection, which is the Systemic Inflammatory Response Syndrom caused or autoimmunity injury caused by microorganism or any other immunogenicity. It may cause severe sepsis, septic shock and MODS with a high clinical morbidity and mortality. Severe sepsis and septic shock are among the most important causes of morbidity and mortality in patients admitted to PICU. Severe sepsis can occur sequential multiple organ injury and ultimately develop into MODS, which bear the brunt of the lungs, and cause acute lung injury/acute respiratory distress syndrome (ALI/ARDS) eventually. ALI/ARDS is based on diffuse lung cell injury, the pathology characteristics are pulmonary edema and inflammatory cells infiltrated into the lung tissue which is caused by pulmonary vascular injury. They show as severe hypoxemia, diffused pulmonary infiltrates and pulmonary edema in clinical. ALI/ARDS was still among the most important causes of morbidity and mortality in patients admitted to PICU with mortality as high as 25%-40% at the moment. To explore the biomarker of sepsis-induced acute lung injury is of considerable realistic significance in the diagnosis, therapy and prognosis evaluation of sepsis-induced acute lung injury.miRNAs, a newly discovered kind of non-coding single chain small molecule, can cause degradation of the target mRNA, or inhibit its translation by complementary pairing with the nucleotide, then regulate the gene expression in a post-transcriptional way. Many publications reported that miRNAs have a critical role in regulating cellular events, including gene expression, cell differentiation, proliferation, and apoptosis, as well as in the pathogenesis of many diseases such as inflammation and tumor. Studies have shown that miRNA involved in the regulation of inflammation reaction in sepsis, which has become a hot topic in recent years. Related researches also attached a great importance in academic. A large number of miRNAs have been confirmed to play important roles of modulation in the progession of sepsis, such as miR-147, miR-98, let-7, miR-346, miR-9, miR-155.miRNA-30e(microRNA-30e-5p) belongs to miR-30 family, involved in several cellular processes, including tumor invasion and the metastasis of liver cancer cells, inhibition of cardiomyocyte apoptosis, the apoptosis and self-renewal disruption of breast tumor-initiating cells, tumor cell autophagy, pronephros development in adipogenesis, cellular senescence, and the epithelial-to-mesenchymal transition of hepatocytes. Despite a small amount of studies in immunological inflammation and sepsis, the functions of miR-30s in this field can not be ignored. The study found that expression of miR-30 family was significantly down regulated in LPS stimulated podocyte cells. Among so many target proteins of miR-30 family, Notch 1 can amplifies macrophages inflammatory response through enhancing NF-κ B signaling. Besides, knockdown of Notch 1 with either genetic or pharmacological inhibition exhibited a negative regulation in LPS-induced macrophages inflammation.Objectives1. To investigate the differentially expression of microRNA-30e in sepsis-induced acute lung injury and its correlation with Inflammatory cytokines IL-1 3 and TNF-α from two aspects of in vivo and in vitro.2. To investigate the role of the up- or down-regulated miR-30e in macrophage activation upon LPS stimulation.3. To reveal the mechanism of miR-30e as a negative regulator for LPS-induced expression of inflammatory cytokines.Methods1. Establishment of rat model of sepsis, rats were observed general, lung tissue samples collected, analyzed changes in lung tissue structure to determine whether the model was successfully constructed. RT-qPCR method was used to measure the expression of miR-30e and inflammatory cytokines in lung tissue of septic rats, so as to explore their correlation.2. Building sepsis model in LPS-stimulated NR8383 cells with Oh,3h,6h,12h, 24h five time changes, the detection of expression levels of miR-30e and inflammatory cytokines were completed by using RT-qPCR, so as to explore their correlation.3. Using microRNA target gene prediction software TargetScan and RNAhybrid database on miRNA-30e-5p target genes were predicted, with two or more co-prediction software as its candidate target genes. According to bioinformatics analysis, the binding sites of miR-30e and its target gene as well as related signaling pathways can be forecasted. The target gene and related signaling pathways were detected by Western Blot in LPS-stimulated NR8383 cells.4. The miR-30e mimics/inhibitor were transfected into LPS-stimulated NR8383 cells, changes of inflammatory factors can be detected at mRNA level by RT-qPCR and at protein level by ELISA method, protein expression of target gene can be detected by Western Blot.5. Statistical Methods:SPSS 19.0 statistical software were used to analyze the data, the results were showed as "mean ± standard deviation". Multiple groups were compared by ANOVA analysis, the comparison between any two means were completed by LSD-t. Referring to correlation test, using the Pearson correlation analysis when the double variables were subject to normal distribution, besides, the Spearman correlation analysis was chosen when the double variables were not obey to normal distribution. P< 0.05 for the differences were statistically significant.Results1. Establishment of rat model of sepsis and pathological changes of lung tissue. Rats in normal control group were in good condition generally, free activity, smooth breathing, no abnormalities in diet, normal response to external stimuli; Referring to LPS treatment group,30 minutes after LPS injection, rats began to appear restless, heart rate, breathing rate significantly accelerated; 3 hours after LPS injection, endotoxemia occurred characterized by shortness of breath symptoms, limbs, lips and nose cyanosis, elevation of temperature, and poor mental reaction, curled up lazy move, decreased muscle tone, no appetite, lethargy, piloerection, diarrhea, weakened response to external stimuli, in addition, some rats turned more serious with overflow of red foam liquid from mouth and nose. Lung tissue HE staining showed structural damage, alveolar atelectasis, alveolar integration or collapse, alveolar exudate, alveolar septal thickening, seen a large number of inflammatory cell infiltration, lung tissue capillary dilation, congestion, tube cavity filled with red blood cells, even with thrombosis. As the extension of LPS-stimulated time, the damage of lung tissue turned aggravated.2. The expression of miR-30e and inflammatory cytokines in lung tissue of septic rats and their correlation. The relative expression of miR-30e in lung tissues of septic rats were significantly lower than those in normal control groups, and decreased most obviously at 24 h post stimulation, the difference had statistical significance (P＜0.01). The levels of IL-1β and TNF-α in lung tissues of rats in sepsis groups were obviously up-regulated when compared with that in normal control groups(both P＜0.01), and they both lowered after reaching the peak value at 3 hours post-LPS-injection. The expression of miR-30e in lung tissues of groups showed significantly negative correlations with that of IL-1 β and TNF-α (IL-1 β: r=-0.417, P=0.022; TNF-a:r=-0.437, P=0.016).3. The expression of miR-30e and inflammatory cytokines in LPS-stimulated NR8383 cells and their correlation. The relative expression of miR-30e in LPS stimulated NR8383 cells at different time point were obviously down-regulated when compared with that in blank control groups(P<0.01), and decreased most obviously at 3 h post stimulation. The levels of IL-1β and TNF-α in LPS stimulated NR8383 cells at different time point were obviously up-regulated when compared with that in blank control groups(both P<0.01), and they both lowered after reaching the peak value at 3 hours post-LPS-injection with the same trend as lung tissues of rats. The expression of miR-30e in LPS stimulated NR8383 cells of groups also showed significantly negative correlations with that of IL-13 and TNF-α (IL-1β:r=-0.713,P=0.003;TNF-α:r=-0.712, P=0.002).4. The prediction and validation of the target gene of miR-30e-5p. Bioinformatics analysis suggested that Notch 1 was predicted to have a putative miR-30e-5p binding site within its 3’UTR, which indicated a probable direct interaction between miR-30e-5p and Notchl, providing a theoretical basis for subsequent experiments. The protein expression of Notchl in LPS-stimulated 6,12, 24 hours groups were significantly higher than those in LPS Oh and LPS 3h group, along with the extension of time of stimulation, the protein expression of Notchl gradually increased, the difference also both had statistical significance(P<0.05). The protein expression of Notchl was greatly decreased with transfection of miR-30e mimics, compared to that of control microRNA mimics transfection(t= 33.767, P< 0.01), transfection of miR-30e inhibitors substantially increased the protein expression of Notchl in NR8383, compared to transfection of control miRNA inhibitors(t=-11.371, P< 0.01). These results indicated that the endogenous Notchl was directly regulated by miR-30e-5p in macrophage.5. miR-30e as a negative regulator for LPS-induced expression of inflammatory cytokines. The expression of TNF-a and IL-1β were greatly decreased with transfection of miR-30e mimics, compared to that of control microRNA mimics transfection, transfection of miR-30e inhibitors substantially increased the expression of TNF-α and IL-1βin NR8383, compared to transfection of control miRNA inhibitors, the difference both had statistical significance(P<0.05).Conclusion1. The expression level of miR-30e was significantly down-regulated in sepsis-induced acute lung injury, and had a significantly negative correlation with IL-1β and TNF-a from two aspects of in vivo and in vitro.2. miR-30e could reduce the secretion of TNF-α and IL-1β in LPS-induced NR8383 may through regulating Notchl signal pathway, while how does it works is still subject to further exploration.