The Role And Mechanism Of LncRNA ERLNC1 In Neonatal Parenteral Nutrition Associated Liver Disease

Background:The clinical application of parenteral nutrition(PN)has provided a life-saving support for preterm infants,especially for those very low and ultra-low birth weight preterm newborns.However,it does have a series of therapy-related complications,the most common one is parenteral nutrition associated liver disease(PNALD).Main features of PNALD include abnormal liver function and hepatocyte damage,such as hepatic steatosis,cholestasis,elevated transaminase level,and even liver cirrhosis in severe cases.It has been reported that premature infants develop PNALD earlier,have a faster decline in liver function,and a higher mortality rate than other age groups.PNALD of premature infants is currently considered relating to premature,low birthweight,long-lasting use of PN,lipid emulsion composition,intestinal flora shift and endotoxemia,while the exact etiology and pathogenesis still remain unclear.LncRNA has recently become a hot spot in molecular biology research as it plays a key role in many liver pathological processes.For example,LncRNA LALR1 promotes hepatocyte proliferation by activating Wnt/β-Catenin signaling pathway during liver regeneration.Therefore,this study intends to explore the role and mechanism of lncRNA in the pathogenesis of PNALD,in order to provide potential targets for the treatment of PNALD.Part Ⅰ:The expression analysis of lncRNA and mRNA in PNALD of premature infants and the predictive value of lncRNA ERLNC1Objective:To analyze the expressions of lncRNA and mRNA in the peripheral blood of premature infants with PNALD,and further analyze and verify the differential lncRNAs,then determine the predictive value in extrauterine growth retardation.Methods:The blood samples from three PNALD children and three control group children were collected for high-throughput chip sequencing,and the screening results were analyzed by bioinformatics to find the differentially expressed lncRNAs and mRNAs.According to the results of chip screening and the standard of fold change≥2 and P<0.05,the five lncRNAs with the largest differences were then selected,real-time fluorescent quantitative PCR(qRT-PCR)was used to verify the expression.Expand the sample size,the PNALD preterm infants hospitalized in the**Hospital from January 2019 to December 2020 were selected as the PNALD group of 34 cases,and the non-PNALD preterm infants hospitalized in the same period with similar gestational age and birth weight were selected as the control group,A total of 42 cases.The birth conditions of the two groups of children were compared,including gestational age,birth weight,gender composition,cesarean section,amniotic fluid contamination,premature rupture of membranes,and the proportion of small for gestational age(SGA).The blood biochemical indicators included direct bilirubin(DBIL),albumin(ALB),alkaline phosphatase(ALP),alanine aminotransferase(ALT),glutamyltransferase(GGT),triglycerides(TG),total bile acid(TBA).Treatment and complications during hospitalization include:milking time,breastfeeding ratio,PN time,time to reach half of enteral nutrition,time of fat emulsion,time of invasive ventilation;invasive ventilation,hemodynamically significant patent ductus arteriosus(hsPDA),paraventricular leukomalacia(PVL),bronchopulmonary dysplasia(BPD),necrotizing enterocolitis(NEC),Peripherally inserted central venous catheters(PICC),PICC complications,The proportion of late onset sepsis,meningitis,intracranial hemorrhage(IVH)and the rate of weight gain.Discharge conditions include the incidence of extrauterine growth retardation(EUGR),discharge weight,time of stay in hospital and rate of cure and improvement.Collect blood samples,use qRT-PCR method for further verification,detect the expression of these 5 lncRNAs,and analyze the correlation between the relative expression of 5 lncRNAs in the PNALD group and DBIL.According to the mean of the relative expression of lncRNA ERLNC1 in the PNALD group,the children were divided into two subgroups:low ERLNC1 group and high ERLNC1 group.The two subgroups were compared in birth,hospitalization and complications,and blood biochemical at discharge and discharge conditions.ROC curve used to evaluate the predictive value of relevant indicators in the EUGR,and calculate the area under the curve.Results:The results of high-throughput sequencing showed that compared with the control group,according to the fold change≥2 and P<0.05 standards,there were a total of 203 differentially expressed lncRNAs in peripheral blood of PNALD patients,containing 44 up-regulated ones,the first four were lncRNAs ERLNC1,CTA-29F11.1,G034337 and RP11-372K14.2 respectively,and the other 159 down-regulated ones,lncRNA uc.189 was tested to have most significantly difference.Meanwhile,the total number of differentially expressed mRNAs were 143,including 38 up-regulated and 105 down-regulated ones.qRT-PCR showed that the expression of 4 lncRNAs that were significantly up-regulated in chip screening was significantly up-regulated(P<0.001),and the expression of lncRNA uc.189 in peripheral blood was significantly down-regulated,and the difference was statistically significant(P<0.001).Expand the sample size for further verification,compared with the control group,children in the PNALD group had gestational age,gender composition,birth weight,delivery method,premature rupture of membranes,amniotic fluid contamination,SGA ratio,ALB,ALP,milk feeding time,breastfeeding ratio,and half-dose enteral nutrition time,invasive ventilation,hsPDA,PVL,BPD,NEC,PICC,PICC complications,late-onset sepsis,purulent meningitis,IVH ratio,discharge weight,and cure improvement rate were not significantly different(P>0.05),The DBIL,ALT,GGT,TG,TBA,PN time,time of fat emulsion,the time of invasive ventilation,the incidence of EUGR and the time of stay in the hospital of PNALD group were higher than those in the control group,and the difference was statistically significant(P<0.01).The body weight growth rate was lower than that of the control group,and the difference was statistically significant(P<0.05).The above 4 significantly up-regulated and 1 significantly down-regulated lncRNA are consistent with the chip screening results,Correlation analysis showed that 5 lncRNAs in the peripheral blood of the PNALD group were all correlated with DBIL,and the correlation between lncRNA ERLNC1 and DBIL was the highest,r=0.912(P<0.0001).The average value of relative expression of ERLNC1 in the PNALD group was 4.83.According to this value,the children in the PNALD group were divided into a high ERLNC1 group with 17 cases and a low ERLNC1 group with 17 cases.The time of fat emulsion,PN time,EUGR rate,TBIL,DBIL,ALT and time of stay in hospital were significantly higher in the high ERLNCI group than those in the low ERLNC1 group,and the rate of weight gain was lower than that in the low ERLNC1 group(P<0.05).There was no statistically significant difference between the two groups of children at birth,the incidence of IVH,hsPDA,PVL and other complications,weight at discharge,and cure improvement rate(P>0.05).ROC curve analysis showed that the area under the ROC curve predicted by lncRNA ERLNC1,PN use time,and fat emulsion use time for EUGR were 0.78,0.804,0.811(P<0.05),and the combined predicted area under the curve was 0.882,(P<0.01).Conclusions:The expressions of lncRNA and mRNA in peripheral blood of premature children with PNALD have indeed changed.LncRNA ERLNC1 has also been proved to contribute to the changes of the level of DBIL,which can be used to predict the occurrence of EUGR.Part Ⅱ:The effect of regulating lncRNA ERLNC1 on hepatocyte functionObjective:To explore the construct methods of PNALD cell model and the effect of overexpressed and interfered lncRNA ERLNC1 on hepatocyte function.Methods:We applied different concentrations of lipid emulsion and different periods of time to construct cell model of PNALD,and later on hematoxylin and red oil staining were used to judge the effect of induction.Their effects on the viability of hepatocytes were finally detected by CCK-8 to obtain the optimal concentration of lipid emulsion to construct the cell model.Flow cytometry was used to detect the apoptosis of hepatocytes after induced.In order to get stable transgenic strains,we constructed some specific shRNA interfered sequences and repeated sequences of lncRNA ERLNC1,which were then used to transfect hepatocytes respectively through lentivirus vectors.Additionally,we observed the effects of lipid emulsion on the expressions of lncRNA ERLNC1 by performing qRT-PCR and on the apoptosis of hepatocytes by flow cytometry.Results:We cultured liver cells in the concentrations of 0.3%,0.6%,0.9%,1.5%and 4%of lipid emulsion for 24,48 and 72 hours respectively.The results of hematoxylin and red oil staining experiments suggested that the degree of accumulation of lipid particles in liver cells and the changes of cell morphology were both positively associated with the concentrations of lipid emulsion and the periods of culture time.CCK-8 tests demonstrated that the OD value of 24-hour cell proliferation activity had no significant difference between the experimental group and the control group after culturing liver cells in 0.3%and 0.6%lipid emulsion separately(P>0.05),while the OD value showed a significant difference immediately(P<0.05)when the culture time was extended to 48 and 72 hours.the cells proliferated much slower than before.Having been cultured in the concentrations of 0.9%,1.5%and 4%of lipid emulsion for 24,48 and 72 hours,the proliferation activity of hepatocytes was much lower than the control group(P<0.01).Therefore,we finally determined to culture target liver cells in 0.6%lipid emulsion for 24 hours in the following research.LncRNA ERLNC1 overexpression and interference lentivirus were successfully constructed,then transfected into hepatocytes,and a stable transgenic strain was established.Besides,qRT-PCR verified that the number of lncRNA ERLNC1 in liver cells transfecting by lentivirus were 7.5 times more than that of the control group and the interference efficiency of lncRNA ERLNC1 was up to 74.6%.Furthermore,hepatocytes activity showed a downward trend when the expression of lncRNA ERLNC1 was interfered,whereas the activity strengthened when lncRNA ERLNC1 was overexpressed,the difference was statistically significant(P<0.05).On the other hand,qRT-PCR again indicated that the expression of lncRNA ERLNC1 improved gradually as the concentration of lipid emulsion increased,which was always higher than the control group(P<0.05).What had surprised us was that the expression in the 4%group was lower than the 1.5%group.Last but not the least,according to flow cytometry tests,either to interfere the expression of lncRNA ERLNC1 or to induce steatosis of liver cells,they could both accelerate the apoptosis of hepatocytes.Conclusions:PNALD cell model and stable transgenic hepatocyte strains with overexpressed and interfered lncRNA ERLNC1 have all been constructed successfully.Also,we have learned that interfering the expression of lncRNA ERLNC1 can reduce the hepatocyte activity and increase its apoptosis.Part Ⅲ:The mechanism of lncRNA ERLNC1 and differentially expressed mRNAs in PNALDObjective:To investigate the mechanisms of lncRNA ERLNC1 in PNALD and differentially expressed mRNAs in hepatocyte apoptosis.Methods:CHIRP-MS test was first used to detect the protein molecules conjugating with lncRNA ERLNC1.In order to build a protein-protein interaction(PPI)network and locate the hub genes,we next applied GO and KEGG method to analyze the conjugated molecules of the CHIRP-MS test.According to the score,the protein molecule with the highest score in the hub genes was then selected for in situ hybridization and immunofluorescence experiments in the cell model,exploring its relationship with lncRNA ERLNC1,especially with overexpressed and interfered lncRNA ERLNC1.High-fat diet was used to construct a PNALD animal model,normal diet was used as a control,and oil red staining was used to determine the construction effect.Westernblot and qRT-PCR techniques were used to detect the expression of the protein and its mRNA in cell models and animal models,and immunohistochemical techniques were used to detect the expression of the protein in an animal model.Also,in order to build a gene disease network,we applied the same methods as before:GO and KEGG was used one more time to screen for differentially expressed mRNAs,the hub gene was then located in the PPI network.After the gene disease network was successfully built,it enabled us to choose the central node protein and its mRNA,whose expressions in different models were tested again by westernblot and qRT-PCR technology,especially in the cell models,overexpressed and interfered lncRNA ERLNC1.Results:The number of conjugated lncRNA ERLNC1 proteins from CHIRP-MS test were 197.GO and KEGG method showed that the top 20 of these proteins,mostly gathering in spliceosomes and estrogen signal pathways,RNA conjugated,structural molecular activity and cytoskeleton structures.After a PPI network with 76 nodes and 109 edges was constructed successfully,we found the SRSF3 protein according to the cytohubba calculation system.Red oil staining of liver slices of high-fat diet mice showed that the PNALD animal model was successfully constructed.Meanwhile,compared with the control group,westernblot and qRT-PCR both suggested that SRSF3 protein and its mRNAs were all down regulated in cell and animal models,and so were the results of immunohistochemistry from animal models.We observed from in situ hybridization and cellular immunofluorescence tests,lncRNA ERLNC1 existed both in human hepatocyte nucleus and cytoplasm,mainly in hepatocyte cytoplasm,while SRSF3 protein only existed in human hepatocyte cytoplasm.Also,their expression positions in human hepatocyte cytoplasm were proved to be highly coincident.The fluorescence intensity of SRSF3 protein from overexpressed lncRNA ERLNC1 group was stronger and the expression was higher than normal and interfered group,which was consistent with the results of westernblot.As to differentially expressed mRNAs,GO analysis demonstrated that the up-regulated DEGs were mainly found bounding with a Subunits in G protein,DNA transcription factors and RNA polymerase Ⅱ,while the down-regulated ones were mainly in chemokine factors.The results from KEGG pathway indicated that the up-regulated DEGs were mostly enriched in MAPK signaling pathways,whereas the opposite ones were mainly in Toll like receptor signaling pathways.We constructed another PPI network with 27 nodes and 45 edges,including the most significant cluster,which constituted by seven nodes:MAPK1 0,MAPK 8,MAPK 9,JU-N,ATF3,FOS and FOSB.The genes from the gene disease network relating with liver cirrhosis,juvenile arthritis,myocardial ischemia and lung tumor were JUN,MAP3K8,ID1,METRNL and CD83 respectively.The expressions of MAPK10 and c-Jun protein and their mRNAs all significantly increased in different cell models(P<0.01).Conclusions:The down-regulated expression of the SRSF3 protein resulting from the accumulation of lncRNA ERLNC1 might have caused PNALD,while its deterioration and interfered expression of lncRNA ERLNC1 are considered to associate with hepatocyte apoptosis through MAPK10/c-Jun signaling pathway.