| Object:In recent years with the rapid rise in incidence of respiratory diseases,studies have shown that just only the change of the gene pool to explain the occurrence of diseases is unable to complete mechanism to reveal the occurrence of diseases,one of the many hazard factors is environmental pollution affectting human health,the pollution of the environment factors on the influence on the development of the respiratory system related to the occurrence of diseases is more and more attention.At present,the scientific view holds that gene-environment interaction is the common pathogenesis of most human diseases,which is closely related to the pathogenesis of respiratory system.Human organs of the respiratory system as a direct contact with outside air,and Fine particulate Matter?Fine Particular Matter?PM2.5.5 and the main pollutants in the air,it can cause the urban air quality deteriorated sharply and the formation of dust haze weather,plus its aerodynamic characteristics of small diameter are more likely to enter the respiratory tract,lung respiratory system deep induce respiratory related diseases.And because PM2.5.5 specific surface area is big,easy to carry a large number of harmful organic pollutants,heavy metals,acidic oxides and composition of bacteria,viruses and other poisonous and harmful material and then enter the human body cell cause serious harm to human body health.Existing research shows that the crowd respiratory symptoms?cough,sputum,breathing?the incidence of associated with grey haze pollution level,the ash haze pollution increased the risk of respiratory system,cardiovascular system diseases,also increase the risk of the residents died of heart system disease.PM2.5.5 of inflammation of the respiratory system effect is the main toxic effect,most of the domestic and foreign scholars think PM2.5cause inflammation of the lungs is the basis of cause other series of toxicology effects,including induced inflammation may further damage of equilibrium state of the sympathetic and parasympathetic nerve,causing a series of nervous system diseases.With the deep understanding of and research on epigenetic RNA high-throughput detection technology development,long chain noncoding RNA?long non-coding RNA?as new regulatory gene in the genome research field wide attention by the researchers.Long chain non-coding rnas are a class of RNA molecules that do not encode proteins and have transcripts that are longer than 200nt.More and more about lncRNA in immune inflammation research has shown that lncRNA can interact through a variety of ways such as combined with certain cell factors of STAT,regulate immune inflammation related gene expression and affect the growth of certain immune cells differentiation and development process involved in regulating the immune inflammatory disease.So does lncRNA play a regulatory role in the respiratory inflammation caused by PM2.5?In this study,we hypothesize that lncrnas play an important role in environmental exposure induced inflammatory response.We induce cells to produce inflammatory response by haze.The lncRNA gene expression profile was analyzed by high-throughput sequencing and found that many lncRNA expression changes were changed after PM2.5.5 exposure.Then using rt-pcr validation key lncRNA and at the same time using RNA interference and cell transfection research system,establish the function analysis of lncRNA function in inflammatory effect,finally found the lncRNA LOC101927514abnormally high expression in the process of PM2.5.5 inflammatory function significance;Pull through high-throughput sequencing gene screening and RNA dwon methods confirm the lncRNA LOC101927514 regulating target genes,revealing the lncRNA LOC101927514 gray haze PM2.5.5 respiratory inflammation effect in the regulatory mechanism,from the view of the epigenetics of non-coding RNA regulation to clarify the molecular mechanisms of inflammation.Methods1.PM2.5.5 sampling and processingIn downtown guangzhou yuexiu,tianhe,Choose three representative point as PM2.5.5 sampling points,including 2.5?mdiameter of glass fiber membrane filter?USA?for sample collection.The collection time is for the winter of 2015 and the winter of 2016.After collection,the particles in PM2.5.5 were collected by ultrasonic washing and freeze-drying.The samples were divided into two parts.Some samples were used for chemical composition analysis,and some samples were used for subsequent cytotoxicity studies.2.CCK8 method was used to detect the effect of PM2.5.5 on cell survivalIn order to ensure a certain cell survival rate,we first used CCK8 method to detect the effect of PM2.5.5 on cell vitality.Meanwhile,the changes of cell morphology after exposure to PM2.5.5 were observed under electron microscope.3.In vitro cell inflammatory model experimentSaid take 0.1g dry weight good PM2.5.5 particles dissolve in 1ml PBS liquid mixture of 0.1g/ml mother liquor stock solution,Using the PBS diluted to different concentrations of PM2.5.5 dye eventually poison to deal with the exposed human bronchial epithelial cells,Using ELISA to detect cells that inflammatory factors in the supernatant fluid IL6 and IL8 production;The changes in transcription level of IL6 and IL8 were verified by qRT-PCR.4.The changes of lncRNA spectrum after high throughput sequencing analysis of PM2.5.5 treated cellsAfter PM2.5.5 exposure to the infected cells,the total RNA of the cells was extracted by lncRNA sequencing samples to be sent to guangzhou ruibo biological co.,ltd.for sequencing detection and data analysis of lncRNA.5.Real-time fluorescence quantitative PCR detection gene expressionUsing GoTaq?qPCR Master Mix?Promega USA?strictly according to the instruction manual operation steps for real-time fluorescent quantitative PCR,lncRNA detection using fluorescent dyes?SYBR Green II?qRT-PCR detection method.6.In vitro cell function experimentChoose jamming efficiency is more than 50%of lncRNA LOC101927514SiRNA sequences,in strict accordance with the experiment instruction with LipofectaminetransfectionreagentwillLOC101927514-2000SiRNA instantaneous low transfection 16HBE cells 6h to knock lncRNA LOC101927514 expression level,then add the PM2.5.5 exposed infected fluid after48h ELISA method to detect cells that secretion of the inflammatory factors in supernatant fluid.7.LOC101927514 subcellular localizationAccording to PARIS?Kit instructions cell cytoplasm nucleus separation and extraction of RNA,use qRT-PCR detection LOC101927514 and internal genes?GAPDH,U6?in cytoplasm relative expression in the nucleus.8.RNA Pull downBy suzhou-acquisitive biotechnology co.,LTD.,gene synthesis to pcDNA3.1plasmid as RNA probe RNA template preparation Pull down the probe,the probe to degeneration,formation of RNA secondary structure after the compound preparation of probe,magnetic beads,Pull down the experiment using RNA technology from normal human bronchial epithelial cells.9.Western Blot experimentsProtein extract cells,mixed 10%sds-page separation and 6%concentrated adhesive protein electrophoresis,after the transfer film with specific resistance and marked with fluorescent markers of incubation,Finally,the PDVF film was scanned by the ODYSSEY dual color infrared imaging system,and the data were saved and analyzed.The software Odyssey V3.0 was used to analyze the data,and the beta-actin was used as the internal reference.10.Statistical analysis.SPSS 16.0 was used for statistical analysis and the comparison between the two groups was tested.The data of each experimental group were measured with a mean of plus or minus standard deviation.Results1.Analysis of PM2.5.5 chemical compositionWe analyzed the composition of atmospheric PM2.5.5 collected by this study,the results showed that heavy metal elements include Si,K and Na was significantly higher than that of other elements,and the concentration was 3.65±2.36?g/m3?1.73±0.9?g/m3 and 1.62±1.23?g/m3.Atmospheric particulate matter in the main water soluble ion SO42-,NO3-,Cl-,F-,NH4+,K+,Na+,Ca2+,such as magnesium SO42-,NO3-and NH4+is a major water soluble ion of PM2.5 in this study,accounted for 18%,7%,7%of PM2.5.5 quality.PM2.5.5 in 16 kinds of polycyclic aromatic hydrocarbons?PAHs?total concentration of about 9.05±6.82 ng/m3,the average concentration of the BaP is about 0.97±0.82 ng/m3,the mean concentration does not exceed the standard limit?1ng/m3??GB3095-1996?and the standard values of the world health organization?WHO??1ng/m3?.PM2.5Carbon composition analysis found that the average concentration of OC and EC was 8.76±4.81g/m3,and 3.91±2.67g/m3.The ratio of OC/EC in PM2.5.5 was greater than 2.2,and the organic carbon content was significantly higher than that of inorganic carbon.2.Effect of PM2.5.5 on cell morphology and activityDifferent concentrations of PM2.5.5 processing cells after 48 hours,with the concentration increased,cell morphology is affected,clean and clear contour background compared to the control group,contour increases with the concentration of PM2.5.5 becomes unclear boundaries,cell death also increases with the concentration of PM2.5.5 exposure happened.CCK8 test cell activity decreased with increasing concentration of PM2.5.?*P<0.05,**P<0.01?3.The effect of PM2.5.5 on the secretion of inflammatory cytokinesAt the same concentration,the cytokine interleukin-6 secreted by the cells was increased with the increase of exposure time.There was no obvious time-effect relationship between cytokine interleukin-8,but the secretion of PM2.5.5 treatment group increased and the difference was statistically significant compared with the control group without PM2.5.In different concentrations of PM2.5.5 processing after 48h 16HBE cells,decreased with the increase of concentration,cause cells to secrete interleukin 6 and interleukin 8,and a certain dose dependent.?*P<0.05,**P<0.01?.4.PM2.5.5 caused the change of lncRNA and the screening of the major lncRNAsCompared with the control group,the concentration group of 50?g/mL and 100?g/mL was increased by 58 and 308 LncRNAs,and 13 and 15 LncRNAs were down-regulated.The expression of lncRNA LOC101927514 was expressed in50?g/mL,100?g/mL treatment group,and it had a dose-response relationship with PM2.5.5.Functional verification of target lncRNA LOC101927514 molecule SiRNACompared with the control group,the expression levels of mRNA in the cells were significantly decreased in the treatment group(siRNA-2+PM2.5),and the IL-6 and IL-8's mRNA expression levels were significantly reduced in the treatment group(SiRNA-2+PM2.5)after the LOC101927514 RNAi.6.Interaction between lncRNA LOC101927514 and STAT3 protein moleculesThe binding protein STAT3 and phosphorylation of Western Blot test for RNA-pull-down experiment were tested.The results showed that lncRNA LOC101927514 was combined with STAT3 and the phosphorylated STAT3 was also combined.Low lncRNA expression was used to detect the expression of STAT3 protein and the expression level of phosphorylation.It was found that the expression of STAT3 was unchanged,while the phosphorylated p-STAT3 protein changed.Conclusion1.The cell morphology of PM2.5.5 was affected and the cell activity decreased.2.Inflammatory response of cells caused by PM2.5.3.The expression of lncRNAs after the treatment of cells by PM2.5;Long-chain non-coding RNA LOC101927514 is up-regulated and has a dose-effect relationship with PM2.5.5 treatment.4.Long chain non-coding RNA LOC101927514 plays the role of pro-inflammatory gene in the development of cell inflammation induced by PM2.5.5.Long chain non-coding RNALOC101927514 is involved in the process of inflammatory reaction by inducing the phosphorylation of STAT3 protein. |
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