Screening Of Biomarkers For Ischemia Injury Of Donor Liver And Study On The Function And Clinical Predictive Value Of MiR-23b-5p

Objective:Since 2016,DCD has become a mainstream source for liver donors in China.Ischemia-reperfusion injury(IRI)of the donor liver is an independent risk factor for the prognosis of liver transplant recipients.In addition to the obvious warm ischemia time of organ acquisition,the blood flow perfusion process in the donor body is the main determinant factor for the induction of IRI in DCD livers,which in turn depends on the etiology of donors,treatment time in the intensive care unit,and use of vasoactive drugs.These factors are uncontrollable and vary among donors.Under the current standards of the donor liver acquisition principle,most indicators have been successfully controlled,except for ischemic injury.However,it is precisely because of the hidden characteristics of DCD liver ischemia injury in donors that it is difficult to accurately evaluate the quality of DCD donor livers using known indicators.We will conduct a systematic search for biomarkers,but also excavate as deep as possible for molecular biomarkers that can be used to determine IRI of DCD donor liver.Materials and Methods:Part One:1.IHC was used to observe the expression of common injury markers in DCD donor liver at different ischemia time,and to preliminarily determine the basic pathological condition of the sample.2.Establish 100% IRI model in C57 mice.3.WB was used to detect the changes of apoptosis related molecules at different stages of ischemia and reperfusion in 100% IRI model.4.Whole transcriptome sequencing was used to search for the main signal pathway in early ischemic injury and transmission electron microscopy was used to explore the subcellular structure in early ischemic injury.Part two:1.In order to further study the early ischemic injury of mitochondria,we extracted mitochondria and detected their purity and structural integrity by transmission electron microscopy.2.Mitochondrial extraction combined with RT q PCR was used to detect the expression of 13 kinds of m RNA in the mitochondria.3.The results of full transcriptome sequencing at different ischemia time points combined with the results of miRNA microarray detection of mitochondrial purified samples preliminarily screened out micro RNAs(miRNAs)that are related to warm ischemia changes and highly enriched in mitochondria4.In situ hybridization probe was constructed to verify the mitochondrial localization of mitomirna in primary C57 Mouse Liver Cells and human He La cells,and RT q PCR was used to verify the positive correlation between the expression of mitomirna and hypoxia.Part three:1.In vitro study: lentivirus was constructed to knock down and overexpress mitomiR-23b-5p in 293 T cells.After down-regulation and over expression under hypoxia induction in vitro,apoptosis level was detected by flow cytometry and WB.2.In vivo study: Adeno associated virus(AAV)was constructed,and the apoptosis level of mir-23b-5p knock-down liver tissue in wild-type mice was detected by TUNEL and WB after 100% IRI.In order to further observe the effect of mir-23b-5p alone,we constructed AAV CRE lox P Dicer-/-conditional knockout mice.Dicer knockout will significantly knock out all miRNAs.Then,we reinfusion the mature agomir-mir-23b-5p to observe the effect of its up regulation in 100% IRI,and detected the level of apoptosis by TUNEL and WB.3.After knockdown and overexpression of mitomir-23b-5p by lentivirus in vitro,the whole transcriptome sequencing was used to search for the changes of the pathways.The potential mechanism was explored by bioinformatics,and the mechanism was verified by seahorse mitochondrial function test.Part four:1.From July 2017 to July 2019,the donor liver samples were collected from the organ transplantation center of West China Hospital of Sichuan University,and the general situation,pathological characteristics and clinical diagnosis and treatment data were collected.A total of 148 cases met the inclusion and exclusion criteria,and used RT q PCR to detect the relative expression level of target biomarkers.2.The clinical follow-up was conducted in all the recipients and the data of general condition,pathological characteristics,clinical diagnosis and treatment were also collected.3.ALT value at 3 days after operation and 90 GSR of liver transplant recipients were used as endpoint.The control study was composed of high and low expression levels of miR-23b-5p,and the cut-off value between high and low expression was median and cut-off value calculated from 90% survival rate.The PSM method was used to further analyze whether the new molecular biomarkers have clinical predictive value.Results:Part One:There was no significant difference in morphology and IHC of SOD,caspase 3,TUNEL,p62,IL-6,IL-10,TNF-α and Ki-67 between ischemia 30 min group and normal group in DCD liver.Compared with normal group,SOD staining and TUNEL in 60 min ischemia group showed positive changes in hepatic lobular portal area and positive changes in TNF-α,IL-10 and Ki-67 in hepatic lobule area.The TUNEL results of DCD donor liver were significantly inconsistent among different individuals even at the same ischemia time.Using mice 100% IRI,there was no significant difference in the above IHC results between 15 min ischemia and 30 min ischemia.Full transcriptome sequencing showed that the main enrichment pathway was apoptotic pathway.The apoptosis related molecules Caspase-3,Caspase-9,Bax and Bcl-2 were detected by WB,and the results showed that there was no significant change.After reperfusion following ischemia 30 and 60 min,we found that the Caspase-3,Caspase-9,Bax were significant up-regulated,while Bcl-2 was significantly down regulated.However,only Bcl-2 were significant down regulated after reperfusion following 15 min ischemia.By focusing on the apoptotic pathway through transcriptome sequencing,we found that the changes of apoptotic pathway were all early indicators,which were related to the changes of apoptotic mitochondrial pathway.Part two : The mitochondria with high purity and complete structure were successfully extracted.The expression levels of 13 kinds of MT-m RNA in normal group,15 min,30 min and 60 min after ischemia were still lack of trend.Only MT-ND5 showed a continuous increasing trend from 15 to 30 min.Full transcriptome sequencing showed that there were 25 miRNAs up-regulated in 15 min,44 miRNAs up-regulated in 30 min and 43 miRNAs up-regulated in 60 min.Among them,there were 14 cases with consistent trend.The miRNA chip is used to detect mitochondrial enrichment of miRNA by comparing miRNA sequencing results with samples after mitochondrial purification.Only 3 cases in 14 cases were consistent with miRNA microarray results.Only miR-23b-5p is homologous to human and mouse.It was confirmed that miR-23b-5p was located in the mitochondria of mice and human,and the expression level was positively correlated with hypoxia culture time.Part three:In vitro studies confirmed that after down-regulation of mitomiR-23b-5p,the degree of apoptosis of 293 T cultured under hypoxia will increase.In wild-type mice that down-regulated mitomiR-23b-5p,there was no significant difference between the warm ischemia injury and the control group in the apoptosis-related TUNEL results.But the WB results showed that the Bcl-2 protein expression level was significantly down-regulated.In order to further observe the effect of miR-23b-5p separately,we constructed AAV-CRE-Loxp Dicer-/-conditionally induced gene knockout mice.After all miRNAs were significantly down-regulated,we reinjected the miRNA mature body realized mitomiR-23b-5p up-regulation effect.The results showed that in the up-regulated group of mitomiR-23b-5p,a lower level of apoptosis was observed in the 100% IRI model by TUNEL.In addition,the level of Caspase-3 in the experimental group was significantly down-regulated compared to the control group.And Bcl-2 were significantly up-regulated.It is suggested that mitomiR-23b-5p has a certain protective effect in warm ischemia.Furthermore,it was found in transcriptome sequencing that lowering mitomiR-23b-5p will increase the expression of genes related to the key complexes of the mitochondrial respiratory chain,and at the same time increase the expression level of mt-RNR1 and mtRNR2 in mitochondria and overexpressing mitomiR-23b-5p will inhibit the expression level of mt-RNR1 and mt-RNR2.Seahorse mitochondrial respiratory function test confirmed that up-regulation of mitomiR-23b-5p inhibits ATP production,while down-regulation will increase the maximum mitochondrial respiratory capacity.Part four:We finally collected a total of 246 cases of donor liver,and finally 148 cases of donor liver samples that met the inclusion and exclusion criteria.Among the 148 cases,the mitomiR-23b-5p high expression group had a higher 3-day ALT level after surgery,and the low expression group had a higher 90-day GSR,and the cutoff value of the relative expression was 1.9.The ROC curve for miR-23b-5P’s 90-day GSR showed that the AUC(C statistic)of mitomiR-23b-5p = 0.698.Conclusion:miR-23b-5p is a kind of mitomiRNA,which can increase its expression level in cells under the induction of hypoxia.Under normoxia,overexpression of miR-23b-5p will reduce mitochondrial respiration,while knocking down miR-23b-5p will increase its mitochondrial respiratory function.Under hypoxia,down-regulation of miR-23b-5p will increase its apoptosis level.The expression level and function of miR-23b-5p are closely related to IRI.Clinical data confirms that miR-23b-5p is an effective biomarker for ischemic injury of DCD donor liver.The results of cell and mouse studies It is confirmed that miR-23b-5p is a protective regulation mode of the body under IRI.