Molecular Mechanism Of CircRASA2 Targeting MiR-877-3p/AK4 And Binding DNMT1 To Regulate The Proliferation Of Pulmonary Smooth Muscle Cells In Pulmonary Hypertension

This study aims to explore the relationship between circRASA2 expression and pulmonary hypertension through clinical samples,cell function and molecular mechanism.1.Clinical sample detection:collect peripheral blood of children with congenital heart disease and pulmonary hypertension,and children with congenital heart disease without pulmonary hypertension,screen and verify the significantly differentially expressed circular RNA,and determine the research target circRASA2;2.Cell function experiment:small hairpin RNA interference technology mediated by lentivirus was used to silence circRASA2 in human pulmonary artery smooth muscle cells（HPASMCS）,to detect the changes of cell proliferation,and to further explore its role in pulmonary hypertension;3.Molecular mechanism experiments:（1）the localization of circRASA2 in cells was detected by fluorescence in situ hybridization;（2）RNA antisense purification technology was used to screen miRNAs and proteins that may bind to circRASA2,and to mine the gene network regulated by circRASA2;（3）The double luciferase experiment verified the targeted regulation relationship between circRNA-miRNA and miRNA-mRNA,and combined with the results of RAP and FISH to analyze the mechanism of circRASA2 in the proliferation of pulmonary artery smooth muscle cells.Part Ⅰ Screening and validation of circRNAs associated with pulmonary hypertensionObjective The differential expression profile of circRNAs associated with pulmonary hypertension in children with congenital heart disease was constructed,and the circRNAs with obvious differential expression were screened and verified.Methods Eight children with congenital heart disease combined with pulmonary hypertension（pulmonary hypertension group）and five children with congenital heart disease without pulmonary hypertension（control group）were selected.Mean pulmonary artery pressure was measured by right heart catheterization and plasma was collected.CircRNAs expression profile was analyzed using Array star Human circRNA Array.qRT-PCR was used to detect the relative expression levels of 5 differentially expressed circrnas in plasma of 16 patients with pulmonary hypertension and 16 patients with control group.Bioinformatics methods were used to analyze the possible molecular mechanism of differential expression of circRNA in the regulation of pulmonary hypertension.Results 1.We found that 27 circRNAs（3 up-regulated and 24 down-regulated）were differentially expressed between the pulmonary hypertension group and the control group（multiple of difference≥1.5 and P<0.05）.2.Compared with the control group,the expression of circ₀03416 was significantly down-regulated in pulmonary hypertension group（P<0.05）,while the expression of circ₀05372 was significantly up-regulated（P<0.05）.3.Bioinformatics analysis:KEGG analysis showed that circ₀05372 significantly enriched signaling pathway was related to MAPK signaling pathway and oxidative phosphorylation.Conclusion 1.There were many differentially expressed circRNAs in the plasma of children with congenital heart disease associated with pulmonary hypertension,among which the expression of circ₀05372 was significantly up-regulated and that of circ₀03416 was significantly down-regulated.2.Bioinformatics analysis showed that abnormal expression of circ₀05372 may be related to the occurrence and regulation of pulmonary hypertension.Part Ⅱ Regulation of circRAS A2 on proliferation of hypoxia pulmonary artery smooth muscle cellsObjective To study the expression level of circRASA2 in hypoxia pulmonary artery smooth muscle cells and to explore the effect of circRASA2 on the proliferation of pulmonary artery smooth muscle cells.Methods HPASMCs were divided into normoxia group,hypoxia 24h group,hypoxia 48h group and hypoxia 72h group.The expression of circRASA2 in HPASMCs was verified by qRT-PCR.The cyclization sites and cyclization of circRASA2 were verified by generation sequencing,agarose gel electrophoresis and RNase R digestion experiments.Nuclear localization and fluorescence in situ hybridization were used to identify the localization of circRASA2 in the cells.The expression of circRASA2 was interfered by lentivirus technology in PASMCs,and the interference efficiency was verified by qRT-PCR.The effect of circRASA2 silencing on the proliferation of PASMCs was studied by detecting the cells proliferation activity of CCK8 and EdU.Results 1.Compared with the normal control group,the expression of circRASA2 in PASMCs in hypoxia 24h group and hypoxia 48h group was significantly higher,and the difference was statistically significant（P=0.000,P=0.004,all<0.05）;There was no significant difference between normal control group and hypoxia 72 h group（P=0.70）.2.The qPCR product of circRASA2 was confirmed by Sanger sequencing,the cyclization site is CTGCAT.3.In RNase enzyme digestion experiment,compared with RNase R-group,there was no significant difference in the expression of circRASA2 in RNase R+group（P=0.503）,but the expression of RASA2 mRNA、β-actin decreased significantly（P=0.002,P=0.000,all<0.05）.4.Verification of cyclicity:when electrophoresis the PCR products,it was found that the products of cDNA and gDNA could be amplified by convergent primers,while only cDNA could be amplified by divergent primers,and the products of circRASA2 were not amplified by gDNA.5.Nucleocytoplasmic separation experiment showed that circRASA2 was mainly distributed in the cytoplasm,and the results of fish experiment were consistent with those of nucleocytoplasmic separation.6.There was no significant difference in the expression of RASA2 mRNA between LV-RNAi-42,LV-RNAi-43,LV-RNAi-44 and the negative control vector group（P=0.22,P=0.11,P=0.55）;The expression of circRASA2 decreased significantly compared with the negative control vector group（P=0.000,P=0.000,P=0.002,all<0.05）.7.circRASA2 function study:CCK method showed that there was no significant difference in PASMCs proliferation activity among the three groups 24 hours after inoculation（P=0.27）.At 48,72 and 96 hours after inoculation,the PASMCs proliferation activity in LV-circRASA2-RNAi transfection group decreased significantly compared with hypoxia group and negative control vector group（P<0.05）;EdU method showed that 24 hours after inoculation,the proportion of PASMCs proliferating cells in LV-circRASA2-RNAi transfection group decreased significantly compared with hypoxia group and negative control vector group（P<0.05）;There was no significant difference in PASMCs proliferation activity between hypoxia group and negative control carrier group（P=0.69）.Conclusion 1.circRASA2 is mainly located in the cytoplasm.2.circRASA2 is highly expressed in hypoxia human PASMCs.3.circRASA2 promotes the proliferation of pulmonary artery smooth muscle cells.Part Ⅲ Molecular mechanism of circRASA2 targeting miR-877-3p/AK4 and directly binding DNMT1 to regulate pulmonary artery smooth muscle cell proliferationObjective To explore the molecular mechanism of circRASA2 promoting the proliferation of pulmonary artery smooth muscle cells.Methods The biotin probe group was designed,and the miRNAs and proteins bound to circRASA2 were screened and verified by RNA antisense purification technology combined with qRT-PCR and proteomics LC-MS;Lentivirus transfection experiment verified the correlation between circRASA2 and miR-877-3p,circRASA2 and DNMT1;The double luciferase reporter vectors and mutants of circRASA2 and AK4 were constructed.The double luciferase was used to verify the binding between circRASA2 and miR-877-3p,miR-877-3p and AK4;The expression levels of AK4 and DNMT1 proteins were verified by Western blot;Overexpression/inhibition of miR-877-3p,CCK8 and EdU were used to detect cell proliferation;The rescue experiment verified that circRASA2 regulated cell proliferation through miR-877-3p/AK4.Results 1.Compared with LacZ control probe group,the%input and enrichment multiple of miR-877-3p,miR-365a-5p and miR-216a-5p in circRASA2 probe group were significantly higher（P<0.05）.2.RAP protein products were analyzed by LC-MS,there were 51 differentially expressed proteins between the circRASA2 probe group and the control group.3.The relative expressions of miR-873-5p and miR-877-3p in LV-circRASA2-RNAi transfection PASMCs group were significantly higher than those in negative control virus transfection group（P<0.05）,but there was no significant difference in the relative expressions of miR-365a-5p,miR-384 and miR-216a-5p between the two groups（P=0.07,P=0.66,P-0.06）.4.Study on the function of miR-877-3p:CCK8 method:48h,72h and 96h after inoculation,the proliferative activity of PASMCs in miR-877-3p mimics group was significantly lower than that in miR-877-3p mimics NC group（P<0.05）;48h,72h and 96h after inoculation,the proliferative activity of miR-877-3p inhibitor group was significantly higher than that of miR-877-3p inhibitor NC group（P<0.05）;EdU method:48h after inoculation,the proportion of proliferating cells of PASMCs in miR-877-3p mimics group was significantly lower than that in miR-877-3p mimics NC group（P<0.05）;The proportion of proliferating cells in miR-877-3p inhibitor group was significantly higher than that in miR-877-3p inhibitor NC group（P<0.05）.5.Rescue experiment,CCK8 method,48h,72h and 96h after inoculation,the proliferation activity of PASMCs in LV-circRASA2-RNAi+mimics group was significantly lower than that in LV-circRASA2-RNAi+mimics NC group（P<0.05）.At 48h,72h and 96h after inoculation,the proliferative activity of LV-circRASA2-RNAi+inhibitor group was significantly higher than that of LV-circRASA2-RNAi+inhibitor NC group（P<0.05）;EdU method:48 hours after inoculation,the proportion of PASMCs proliferating cells in LV-circRASA2-RNAi+mimics group was significantly lower than that LV-circRASA2-RNAi+mimics NC group（P<0.05）.The proportion of proliferating cells in LV-circRASA2-RNAi+inhibitor group was significantly higher than that in LV-circRASA2-RNAi+inhibitor NC group（P<0.05）.6.The relative expression of AK4 in PASMCs in LV-circRASA2-RNAi transfection group was significantly lower than that in PASMCs in negative control virus transfection group（P<0.05）;The relative expression of AK4 in PASMCs of miR-877-3p mimics transfection group was significantly lower than that of negative control vector group,while the relative expression of AK4 in PASMCs of miR-877-3p inhibitor transfection group was significantly higher than that of negative control vector group（P<0.05）.7.Double Luciferase Report showed that after overexpression of miR-877-3p,the luciferase activity of circRASA2 and AK4 wild-type groups decreased significantly,while after inhibition of miR-877-3p,the luciferase activity of circRASA2 and AK4 wild-type groups increased significantly（P<0.05）,and there was no significant difference between mutant luciferase activity and control group.8.Compared with hypoxia group and lentivirus negative control vector group,the expression of AK4 protein in PASMCs in circRASA2 lentivirus interference group was significantly decreased（P<0.05）;There was no significant difference between hypoxia group and lentivirus negative control vector group（P=0.89）;Compared with circRASA2 lentivirus interference group and circRASA2 lentivirus interference+miR-877-3p inhibitor NC,the expression of AK4 protein in PASMCs of circRASA2 lentivirus interference+miR-877-3p inhibitor was significantly up-regulated（P<0.05）;There was no significant difference between lentivirus group and negative control vector group（P=0.49）.9.Compared with hypoxia group and lentivirus negative control vector group,the expression of DNMT1 protein in PASMCs in circRASA2 lentivirus interference group was significantly down regulated（P<0.05）.Conclusion 1.circRASA2 targeted regulation of miR-877-3p/AK4 axis to promote the proliferation of PASMCs.2.circRASA2 may regulate the proliferation of PASMCs by directly binding to DNMT1.