Overexpression Of LncRNA H19 Inhibits Cardiomyocyte Apoptosis In Neonatal Rats With Hypoxic-Ischemic Brain Damage By Activating PI3K/AKT Signaling Pathway Through MiR-149-5p/LIF Axis

Part 1.Expression and Function Research of long non-coding RNA H19 in the myocardium of neonatal rats with HIBDObjective To study the expression and function of long non-coding RNA(LncRNA)H19 in the myocardium of neonatal rats with hypoxic-ischemic brain damage(HIBD).Methods Healthy CD(SD)IGS rats aged 7 days were randomly divided into sham group,HIBD group,HIBD + Lv-H19 group,HIBD + Lv-NC group.HIBD model was established by ligation of carotid artery and hypoxia.Cerebral infarct volume of neonatal rats in sham group and HIBD group was measured by TTC method at 24 hours after hypoxic ischemia operation.Myocardial apoptosis in sham group and HIBD group was detected by TUNEL staining at 2h,12 h,24h,48 h,72h and 120 h after operation;The expression of LncRNA H19 was detected by q RT-PCR at above times.At 48 hours before hypoxic ischemia operation,neonatal rats in HIBD + Lv-H19 group and HIBD + Lv-NC group were injected with Lv-H19 and virus without anti-Smad in tail vein respectively.Detected myocardial cell apoptosis at 48 hours after hypoxic ischemia operation on sham group,HIBD group,HIBD + Lv-H19 group and HIBD + Lv-NC group by TUNEL,and the expression of anti-apoptosis protein B-cell lymphoma-2(Bcl-2),Bcl-2-associated X(Bax)and Caspase 3(cleaved)were detected by Western blot at 48 h after operation;The expression of inflammatory factors,including tumor necrosis factor-α(TNF-α),interleukin-1 β(IL-1β),interleukin-6(IL-6),were detected by ELISA kit;Oxidative stress related proteins such as malondialdehyde(MDA),glutathione peroxidase(GSH-Px)and superoxide dismutase(SOD)were detected by oxidative stress test kit.Results1.Compared with sham group,the infarct volume on the right side of neonatal rats in HIBD group increased significantly at 24 hours after operation(P<0.001),which indicated that the model of neonatal rats with HIBD was successfully established.Compared with sham group,the apoptosis index of myocardial cells in HIBD group increased significantly at all time points(all P<0.05),while the expression of LncRNA H19 in myocardial tissue decreased significantly at all time points(all P<0.05).2.Compared with HIBD + Lv-NC group,HIBD + Lv-H19 group after Lv-H19 was injected into the tail vein of rats,the expression of H19 in myocardium increased significantly(P<0.05)and the apoptosis index of myocardial cells decreased significantly(P<0.05)at 48 h after operation;Compared with sham group,Caspase-3(cleared)and Bax were significantly increased in the myocardium of HIBD rats,while anti-apoptotic protein Bcl-2 was significantly decreased.While after overexpression of H19,the levels of apoptosis-related proteins showed an opposite trend.3.Compared with HIBD+ Lv-NC group,TNF-α,IL-1β and IL-6 in myocardium of HIBD + Lv-H19 group were significantly decreased after overexpression H19(P<0.05);MDA decreased significantly(P<0.05),while SOD and GSH-Px activities increased significantly(both P<0.05).Conclusion The expression of LncRNA H19 was down-regulated in the myocardium of neonatal HIBD rats.Overexpression of H19 inhibited the apoptosis of neonatal HIBD rats,and reduced the level of inflammation and oxidative stress in the myocardium of neonatal HIBD rats.Part 2.LncRNA H19 Competitively Binds miR-149-5p to Upregulate LIF ExpressionObjective To verify the targeting relationship between LncRNA H19 and miR-149-5p and LIF.Methods The bioinformatics online website predicted the relationships between LncRNA H19/miR-149-5p and miR-149-5p/LIF through Star Base,and used HEK293 T cells to transfect miR-149-5p enhancer(miR-149-5p mimics)and no-load control(NC-mimics),and verified their targeting relationships by double luciferase gene reporting experiment.Animal grouping is the same as before: In sham group,HIBD group,HIBD + Lv-H19 group,HIBD + Lv-N group,the expression levels of miR-149-5p and LIF were detected by q RT-PCR,and the expression level of LIF protein was detected by Western blot.Results1.The results of cell experiment showed that there was a binding relationship between LncRNA H19 and miR-149-5p.Compared with NC-mimics,the fluorescence intensity of wild-type(wt)H19(H19-wt)treated by miR-149-5p mimic was significantly decreased(P<0.05),while the fluorescence intensity of mutant H19(H19-mut)was significantly decreased(P<0.05).There was no significant difference in intensity of H19-mut.Meanwhile,compared with NC-mimics,the fluorescence intensity of LIF-wt treated by miR-149-5p mimic decreased significantly(P<0.05),while the fluorescence intensity of mutant LIF(LIF-mut)had no significant difference.2.The results of animal experiments showed that,compared with sham group,the expression level of miR-149-5p in neonatal rats of HIBD group increased significantly(P<0.05),while the expression level of LIF m RNA and protein decreased significantly(P<0.05).In contrast,the expression of miR-149-5p in HIBD+ Lv-H19 group was significantly lower than that in HIBD + Lv-NC group(P<0.05),while the expression of LIF m RNA and protein were significantly higher than that in HIBD + Lv-H19 group(P<0.05).Conclusion LncRNAH19 competitive binding miR-149-5p upregulates LIF expressionPart 3: LncRNA H19/miR-149-5P/LIF axis regulates Cardiomyocyte Apoptosis in Neonatal Rats with Hypoxic-Ischemic Brain Damage by activating PI3K/AKT signaling pathwayObjective To explore the effect and mechanism of LncRNA H19 on cardiomyocyte apoptosis in neonatal rats with HIBD by regulating PI3K/AKT pathway.Methods First,the animals were divided into sham group,HIBD group,HIBD+ Lv-H19 group,HIBD+Lv-NC group;Western blot was used to detect the expression of p-PI3 K,PI3K,p-AKT and AKT in PI3K/AKT signaling pathway.Secondly,the animals were divided into HIBD+Lv-H19+saline group(control group)and HIBD+Lv-H19+Wortmannin group(intervention group).Wortmannin(PI3K/AKT pathway inhibitor)was administered at a dose of 15μg/kg via tail vein after HIBD.The expression levels of p-PI3 K,PI3K,p-AKT,AKT,Bcl-2,Bax and Caspase 3(cleaved)were detected by Western blot;TUNEL was used to detect apoptosis in myocardial tissue.Results1.Compared with sham group,HIBD group,their p-PI3K/PI3 K and p-AKT/AKT ratio decreased significantly(P<0.01);Compared with HIBD + Lv-NC group,the ratios of p-PI3K/PI3 K and p-AKT/AKT were significantly increased in neonatal rat myocardial tissue in HIBD+Lv-H19 overexpressing H19 group(P<0.01).2.Compared with the normal saline injection control group,after Wortmannin injection,the myocardial tissue p-PI3K/PI3 K and p-AKT/AKT ratios in the HIBD +Lv-H19 group were significantly decreased(P<0.05);Caspase-3(cleaved)and Bax increased significantly in rat myocardium,while anti-apoptotic protein Bcl-2 decreased significantly(all P<0.05);TUNEL staining showed a significant increase in apoptosis index(P<0.05).Conclusion LncRNA H19 inhibits cardiomyocyte apoptosis in neonatal rats with HIBD through PI3K/AKT pathway.