Preparation Of Endotoxin-Free SINE RNA And Its Molecular Mechanism Affecting ROS In Human Lens Epithelial Cells

Part 1 Preparation of Endotoxin-free SINE RNAObjectives: Genetically engineered RNA is a important tool for studying gene expression,but RNA produced using genetically engineered E.coli bacteria is limited by endotoxin contamination.Therefore,this study explores a technical method to remove endotoxin from genetically engineered RNA and maintain a high RNA yield.Methods: The plasmid containing the short interspersed nuclear element(SINE B1 element)in the mouse genome was transformed into Escherichia coli,and the RNA yield and endotoxin content of four RNA extraction methods(SDS-Na Cl centrifugation,SDS-Na Cl filtration,Trizol purification and SDS-thermophenol extraction)were evaluated.Then it was found that SDS-Na Cl filtration method could effectively remove endotoxin and obtain good RNA yield.Therefore,Triton X-114 was used on the basis of SDS-Na Cl filtration to further remove endotoxin.During this period,different concentrations of sodium acetate(Na AC),different concentrations of phosphate buffer(PB)and different p H were used to evaluate the effect of phosphate buffer on the removal efficiency of endotoxin.Then,using the final RNA extraction method,the genetic engineering RNA was prepared,and the rabbit pyrogen experiment was conducted to observe whether the genetic engineering RNA produced caused the fever of the animals.Results: The content of RNA endotoxin extracted by SDS-Na Cl filtration was the lowest(P<0.05),which was 1/100 of that extracted by SDS-hot phenol,1/10 of that extracted by SDS-Na Cl centrifugation,and 1/6 of that extracted by Trizol purification.The content of B1 RNA extracted by SDS-Na Cl filtration method was 3.44 times that of Trizol purification method.Then Triton X-114 was used on the basis of SDS-Na Cl filtration method,and it was found that 0.15 M phosphate buffer(p H 6.5)as medium had lower endotoxin content and higher RNA yield than Na AC as medium.In addition,the engineered RNA produced by this method did not cause fever in New Zealand rabbits.Conclusions: SDS-Na Cl filtration combined with Triton X-114 phase separation using p H 6.5 0.1M PB as medium can obviously remove the endotoxin in SINE RNA of genetically engineered E.coli,and ensure a high RNA yield.At the same time,the endotoxin content in RNA prepared by this method meets the specification of the pyrogen test method(rabbit method)in Chinese Pharmacopoeia(2010 edition).Part 2 Molecular Mechanism of Alu RNA Affecting ROS in Human Lens Epithelial CellsObjective: The lens chronological aging,oxidation stress and crystallin protein modifications are main factors for inducing cataracts.Alu RNA increases during oxidative stress,and in vitro transfection of reverse oligonucleotides targeting Alu RNA can prevent age-related macular degeneration.Therefore,in this study,the effect of Alu RNA transfection in vitro on cataract cell model was investigated.Human lens epithelial cells(HLECs)treated with methylglyglyoxal(MGO)is a commonly used cell model of cataract.According to the study in the first part of this project,SDS-Na Cl filtration combined with Triton X-114 phase separation method with 0.1M PB(p H 6.5)as the medium to prepare the humanized SINE RNA(Alu RNA),including Alu RNA and Alu antisense RNA(Aluas RNA),then transfected into cells to study the molecular mechanism of Alu RNA and Aluas RNA affecting reactive oxygen species in HLECs induced by MGO.Methods: HLECs cells were transfected with Aluas RNA,Alu RNA,t RNA(unrelated RNA control),or calcium phosphate transfection reagent(CPT reagent),respectively.After transfection,the HLECs cells were treated with varying concentrations of MGO.At the end of the experiment,the cells were harvested.The cell viability was detected using cell counting kit-8(CCK-8)assay method;cell viability/death was detected using calcein-AM/PI double stain kits;the intracellular reactive oxygen species(ROS)levels were assayed using reactive species assay kit;real-time quantitative reverse transcription PCR analysis was performed to detect the m RNA expression of nuclear-factor-erythroid-2-related factor(Nrf2)and kelch-like ECH-associated protein 1(Keap1),and the experimental data were analyzed by the-△△CT method.Results: The experimental results showed that MGO treatment increased cell death and significant ROS production with the concerntration increasing in HLECs.Aluas RNA rescued HLECs death and inhibited ROS production induced by MGO compared with Alu RNA,t RNA and CPT reagent.At the same time,Aluas RNA increased Nrf2 m RNA expression level and decreased the Keap1 m RNA expression HLECs.Conclusion: Aluas RNA reduces ROS in HLECs cells induced by MGO and alleviated cell death by activating the Nrf2/Keap1 pathway associated with cataract.