LncRNA AK054386 Regulates The Endoplasmic Reticulum Stress In The Ischemia-Reperfusion Injury Of Hepatocyte:by Acting As Endogenous Sponge Of MiR-199a-5p

Background and AimsHepatic ischemia-reperfusion injury(IRI)in the liver surgery and liver transplantation is not only the most common and most serious problem,but also the most difficult problem to treat.Excessive endoplasmic reticulum stress is one of the crucial reasons,which happened in IRI.MicroRNAs(miRNA)are gens of non-coding RNA,after can regulate gene transcription.They can specialy combind the mRNAs 3 'noncoding region(3' UTR),cause the degradation of mRNA,inhibition of transcription.Indeed the list of long noncoding RNAs(LncRNAs),which are functionally defined as transcripts >200nt in length with no protein-coding potential,number in the tens of thousands,more than 98.5% of whole long noncoding genes may transcrip as LncRNAs.On multiple levels,LncRNA involved in cell differentiation,individual development and other important life process control,also related to human major diseases closely.Our previous study found out that miR-199a-5p by inhibiting excessive endoplasmic reticulum stress played a positive role in the protection of the liver.We also found that: when the IRI happened in the liver,the LncRNA AK054386 levels were elevated rapidly,the mature miR-199a-5p levels were significantly decreased and the pri-miR-199a-5p levels remained stable.Based on the results of bioinformatics analysis,we believed that there is a close link between them.However,how could this "close link" be the mechanism of endoplasmic reticulum stress is still need to be discussed.Therefore,by the use of the mouse liver IRI model and BNL CL.2 cells hypoxia-reoxygenation model,we intend to find out the relationship between LncRNA AK054386,miR-199a-5p and endoplasmic reticulum stress-related molecules;to find out the mechanism of sustaining endoplasmic reticulum stress and apoptosis of liver,which are regulated by LncRNA AK054386.After all,this study may carry out new ideas and theoretical basis not only to clarify the mechanism of IRI,but also to prevent from IRI.We focus on the following three aspects:1.The impact and effect of the miR-199a-5p on IRI of hepar.2.The impact and effect of LncRNA AK 054386 on IRI of hepar.3.The mechanism between miR-199a-5p and LncRNA AK 054386.Methods1.Analysis by bioinformatics analysis to investigate which kind of LncRNA may regulate miR-199a-5p.2.We establish a mouse hepatic ischemia/reperfusion injury model.3.We detect the levels of LncRNA AK054386 and miR-199a-5p gene transcription in mice hepatic ischemia/reperfusion injury model by Real-time PCR.4.We establish a embryonic hepatocytes(BNL CL.2 cells)hypoxia-reoxygenation model.5.We over-express or interfere the express of LncRNA AK054386 or miR-199a-5p by transfected BNL CL.2 cell lines6.We detect the apoptosis by flow cytometry of the transfected BNL CL.2 cell lines.7.We detect the levels of endoplasmic reticulum stress-related protein by western blot of the transfected BNL CL.2 cell lines.8.We detect the levels of LncRNA AK054386,mature-miR-199a-5p and pri-miR-199a-5p of the transfected BNL CL.2 cell lines.9.We Co-transfected BNL CL.2 cell with LncRNA AK054386 and luciferase plasmid vector.Then we detect the combination of LncRNA AK054386 with 3'UTR of target miR-199a-5p by dual luciferase reporter assay.Results1.Bioinformatics analysis revealed that,there may be binding sites between LncRNA AK054386 and miR-199a-5p sequence fragments,which explains that LncRNA AK054386 may be involved in transcription and regulation of miR-199a-5p.2.The mouse liver HE staining explained that the liver IRI model was successful;3.After IRI treatment,LncRNA AK054386 express increasly,and miR-199a-5p expression did not change significantly compared to the previous when ischemia happened;but LncRNA AK054386 express significantly high,miR-199a-5p expression express increasly when reperfusion happened.4.Flow cytometry confirmed that mouse embryonic hepatocytes(BNL CL.2 cells)hypoxia-reoxygenation model were envisaged in line with expectations.5.In the course of ischemia-reperfusion,miR-199a-5p may protect the BNL CL.2 cells.6.In the course of ischemia-reperfusion,LncRNA AK054386 may block the protection of BNL CL.2 cells indirectly,by regulate the levels of miR-199a-5p.7.The transfected cells after cultured by normal conditions dectected that,LncRNA AK054386 played as the adsorption sponge of mature-miR-199a-5p,while LncRNA AK054386 almost had no effect on pri-miR-199a-5p.8.The dual luciferase reporter gene assay showed that,LncRNA AK054386 can specifically bind to miR-199a-5p ConclusionIn the course of IRI happened in hapar,miR-199a-5p may reduce the intensity of endoplasmic reticulum stress,then protect the hepatocyte consequently.Due to the adsorption which LncRNA AK054386 played on mature miR-199a-5p,the endoplasmic reticulum stress level was significantly enhanced,endoplasmic reticulum stress related protein expression elevated,and the apoptosis increased significantly,the hepatic injury happened eventually.