Mechanism Of PRKAG2-AS1 In Regulating Apoptosis Of Cardiomyocytes And Endothelial Cells

BackgroundLncRNA is a group of non-coding RNAs defined as being larger than 200nt in length,occupying 4%to 9%of mammalian genome transcripts.They have a wide range of functions,primarily regulating cis and trans-acting to regulate gene expression.LncRNA is an important transcriptional regulator involved in the regulation of the development of various cardiovascular diseases such as atherosclerosis and heart failure.There are about 100,000 LncRNAs in human genes,most of them can regulate the expression of genes near their transcriptional sites.Our research team reported a family of PRKAG2 syndrome in 2007,and then carried out a series of studies on PRKAG2 syndrome and PRKAG2 gene.Previous research by our team found that mutations in the PRKAG2 gene will cause abnormal AMPK activity,and then cause disturbances in cardiac metabolism.We found that there is a LncRNA in the promoter region of PRKAG2 gene,namely PRKAG2-AS1.At the same time,PRKAG2 can form five different subtypes according to different selective transcription start sites.PRKAG2 gene is involved in the regulation of AMPK activity,and lead to a variety of cardiovascular disease phenotypes.In addition,LncRNA can regulate the expression of adjacent genes.Therefore,we raise a scientific question,whether PRKAG2-AS1 can be used as a regulatory element to participate in PRKAG2 gene regulation by splicing,and regulate cell biological functions and influence cardiac and endothelial cell apoptosis.We speculate that PRKAG2-AS1 may serve as a scaffold to recruit transcription factors,and play an important role in the selective transcription initiation process of multiple isoforms of PRKAG2 to achieve selective transcription initiation.Different PRKAG2subtype functions may play different biological functions,leading to cardiovascular disease.PRKAG2-AS1 regulates apoptosis of CardiomyocytesMethods:1.On-line bioinformatics analysis of PRKAG2 gene through NCBI website search.2.AC16 was selected as the main research object,and the expression pattern of PRKAG2-AS1 in cardiomyocytes was detected RNA nuclear fractionation and q RT-PCR.3.The agarose gel electrophoresis method was used to detect the expression patterns of five PRKAG2 isoforms in AC16,HUVEC,HCAEC,and 293T.4.The possible mechanism of PRKAG2-AS1 involved in the regulation of cardiomyocyte apoptosis:?1?AC16 was cultured in 1%O₂incubator for 12 hours to construct a model of myocardial hypoxia injury.Annexin V-FITC/PI flow cytometry was used to detect apoptosis,and q RT-PCR to detect SOD expression in the model;q RT-PCR assay was used to detect the expression levels of PRKAG2-AS1,PRKAG2b,and d isoforms in the model.?2?Si RNA and antisense oligonucleotide technology were used to knockdown the expression of PRKAG2-AS1,q RT-PCR was used to verify the knockdown efficiency,Annexin V-FITC/PI flow cytometry was used to detect apoptosis,and q RT-PCR was used.Detect PRKAG2b,d expression levels,SOD and heart failure marker expression levels in cardiomyocytes;Western blot to detect AMPK?2 protein expression levels.?3?Specific si RNA sequences knock down PRKAG2b,d expression in cells,respectively;flow cytometry to detect myocardial cell decay In the case of death,q RT-PCR was used to detect the expression levels of PRKAG2b,d,SOD and heart failure markers in cardiomyocytes.Results:1.There is an LncRNA at the promoter of PRKAG2 gene,namely PRKAG2-AS1.According to different transcription initiation sites,five different isoforms can be produced.2.PRKAG2-AS1 is expressed in the cytoplasm and nucleus of cardiomyocytes,mainly in the nucleus.3.Among the five isoforms of PRKAG2,the expression levels of PRKAG2b and PRKAG2d were relatively highest.4.Compared with the normoxic culture group,Annexin V+/PI+and Annexin V+/PI-stained cells increased in the myocardial hypoxia model?p<0.05?,and the expression of SOD1,SOD3,ANP and MYH6 decreased?p<0.05?;The expression levels of PRKAG2-AS1 and PRKAG2b and PRKAG2d were significantly reduced?p<0.05?.Compared with the NC control group,si RNA and oligo of PRKAG2-AS1can effectively reduce its expression level in myocardial cells?p<0.05?.Compared to the NC control group,Annexin V+/PI-stained cells increased in the PRKAG2-AS1-si RNA group?p<0.05?,and down-regulated the expression of SOD1 and up-regulated the expression of SOD2 and SOD3?p<0.05?.MYH7 and MYH6 gene expression levels have no significant change,but can reduce the expression level of AMPK?2 protein,but have no significant effect on the expression of PRKAG2b and d isoforms.Compared with the NC control group,Annexin V+/PI-and Annexin V+/PI+stained cells increased in PRKAG2-AS1-oligo group?p<0.05?,and decrease SOD1,SOD3 and MYH6 expression levels?p<0.05?,increase the expression level of BNP?p<0.05?,and down regulate AMPK?2 protein expression.In addition,it can reduce the expression level of PRKAG2d?p<0.01?,but there is no significant change in the expression level of PRKAG2b.Compared with the NC control group,the use of specific si RNA of PRKAG2b and d can effectively reduce their expression levels in myocardial cells?p<0.05?.Compared with the NC control group,knocking down PRKAG2b and PRKAG2d can both reduce the expression levels of MYH6 and MYH7,but there was no significant change in the expression levels of ANP,BNP and SOD,and no significant myocardial cell apoptosis was observed.Conclusions:1.PRKAG2-AS1 is expressed in the nucleus of cardiomyocyte cytoplasm,and the nucleus is mostly.PRKAG2-AS1 in the nucleus and cytoplasm may have different functions and can affect the expression levels of myocardial SOD and heart failure markers through different mechanisms.2.The nucleus of PRKAG2-AS1 may directly regulate the expression of PRKAG2d in myocardial apoptosis and participate in the process of apoptosis.3.There are five subtypes of PRKAG2,of which two subtypes of PRKAG2b and d are relatively more expressed.4.PRKAG2-AS1 may be involved in the selective transcription initiation of PRKAG2d in the nucleus.5.In addition to PRKAG2b and d subtypes,PRKAG2-AS1 may play a biological role in regulating myocardial hypoxia-induced apoptosis by regulating the expression of other genes.PRKAG2-AS1 regulates apoptosis of Endothelial CellsMethods:1.HUVEC was selected as the main research object.RNA nuclear isolation and RNA in situ hybridization were used to further verify the expression pattern of PRKAG2-AS1 in cells.2.Construct endothelial cell apoptosis model by ox-LDL treatment,q RT-PCR was used to detect the expression levels of inflammatory factors IL-6 and IL-10 in the model.3.Biological function of PRKAG2-AS1:?1?q RT-PCR method was used to detect the RNA expression levels of PRKAG2-AS1 and b and d isoforms in endothelial cell apoptosis model.?2?Using si RNA and antisense oligonucleotide technology,knock down the expression of PRKAG2-AS1 and use RNA nucleus The proposed method verifies the knockdown effect separately.?3?The biological function of PRKAG2-AS1 in endothelial cells was detected by CCK8,TUNEL,cell scratch,and tube-forming experiments;endothelial cells that knock down PRKAG2-AS1 in the nucleus,q RT-PCR was used to detect the m RNA expression levels of inflammatory factors,apoptosis-related factors and PRKAG2b and d isoforms.Western blotting was used to further verify proliferation and of apoptosis-related proteins expression;?5?After the adenovirus overexpresses PRKAG2-AS1,the nuclear RNA content to verify the overexpression efficiency,and the expression levels of PRKAG2b and PRKAG2d genes were detected;?6?Specific si RNA knockdown PRKAG2b and d,respectively,and verified the knockdown efficiency and detect IL-6,IL-10 m RNA expression levels by q RT-PCR.?7?Finally,we chose the CHIRP method to further verify the relationship between PRKAG2-AS1 and b and d isforms.Results:1.PRKAG2-AS1 is expressed in the cytoplasm and nucleus of endothelial cells,mainly in the nucleus.2.Compared with the control group,the expression levels of IL-6 and IL-10 in endothelial cell apoptosis model increased?p<0.05?,PRKAG2-AS1expression level decreased?p<0.05?,and PRKAG2d gene expression level increased?p<0.05?,but PRKAG2b gene expression did not change significantly.3.Specific si RNA mainly reduced the expression level of PRKAG2-AS1 in the cytoplasm,while oligo mainly reduced its expression in the nucleus.4.The CCK8 method was used to detect the cell proliferation ability,and it was showed that the OD value of cells in the PRKAG2-AS1-oligo group increased significantly slowly compared with the NC control group.In the tube-forming assay,it was found that the NC control group was widely connected into tubules under observation of the optical microscope,while the PRKAG2-AS1 oligo group cells were scattered and no lumen was formed.Further analysis by Imaje J that the number of nodes,crossing points,and meshes in the PRKAG2-AS1 oligo group were lower than those in the NC control group?p<0.01?.Scratch assay were observed under the microscope and statistical analysis of Imaje J software.It was found that the scratch spacing of the PRKAG2-AS1 oligo group was much higher than that of the control group after 12 and 24 hours?p<0.01?.TUNEL kit was used to detect apoptosis.It was showed that compared with the NC control group,apoptosis in the PRKAG2-AS1 oligo group was significantly increased.Flow cytometry was used to detect apoptosis in Annexin V-FITC/PI staining,and the results showed that the in the oligo group Annexin V+/PI-and Annexin V+/PI+stained cells increased significantly than the NC control group?p<0.01?.It is suggested that knockdown of PRKAG2-AS1 in the nucleus can significantly inhibit the proliferation,migration and tubule formation of endothelial cells.And promote endothelial cell apoptosis,especially early and late apoptosis.5.Compared to the control group,PRKAG2-AS1 si RNA increased the expression level of IL-6 and decreased the level of IL-10?p<0.05?,while the levels of CRP,MCP1,BCL2 and Caspase3 did not change significantly.At the same time,PRKAG2-AS1 oligo up-regutated the expression levels of IL-6,IL-10,CRP,MCP1and BCL2 significantly?p<0.05?,and the protein expression level of BAL2 and Cleaved-Caspase3 increased,but PCNA decreased.Moreover,knockdown of PRKAG2-AS1 in both the cytoplasm and the nucleus could lead to a decrease in the expression of PRKAG2d?p<0.05?.6.Compared with the GFP control group,the expression level of PRKAG2-AS1 in the nucleus was significantly increased after transfection with specific adenovirus?p<0.05?,and the expression level of PRKAG2d increased.7.The m RNA expression levels of PRKAG2b and d in the experimental group cells were significantly lower than those in the NC control group?p<0.05?.Compared with the NC group,the expression level of IL-6 decreased,while the expression of IL-10increased in PRKAG2d si RNA group?p<0.05?.But both IL-6 and IL-10 expression were not significantly changed in the PRKAG2b si RNA group.8.CHIRP results suggest that the expression level of PRKAG2d gene in the probe group with specific biotin labeling is significantly higher than that in the ctl group,which is the same pattern as that of PRKAG2-AS1,and the PRKAG2-b expression pattern was opposite,which suggested that PRKAG2-AS1 interacts with PRKAG2d.Conclusion:1.PRKAG2-AS1 is expressed in the cytoplasmic and nucleus,mostly in the nucleus.PRKAG2-AS1 in the nucleus and cytoplasm may have different functions and can affect the expression levels of inflammatory factors in endothelial cells through different mechanisms.2.PRKAG2-AS1 in the nucleus can promote endothelial cell proliferation,migration and tube forming ability,and inhibit endothelial cell apoptosis.2.PRKAG2-AS1 in the nucleus may directly regulate the expression of PRKAG2d and participate in the process of apoptosis in endothelial cells.3.Nucleus PRKAG2-AS1 may involved in the selective transcription initiation of PRKAG2d.4.PRKAG2-AS1 in the nucleus may be involved in the selective transcription initiation of PRKAG2d.Conclusion1.PRKAG2-AS1 is expressed in the cytoplasmic nucleus,mostly in the nucleus.PRKAG2-AS1 in the nucleus and cytoplasm may have different functions,which can affect the expression of myocardial SOD,heart failure markers,and expression of inflammatory factors in endothelial cells through different mechanisms.2.The nucleus PRKAG2-AS1 may directly regulate the expression of PRKAG2d in hypoxia-induced myocardial cell apoptosis and ox-LDL-induced endothelial cell apoptosis and participate in the process of apoptosis.3.There are five isoforms of PRKAG2,of which PRKAG2b and d are more abundant.4.PRKAG2-AS1 in the nucleus may be involved in the selective transcription initiation of PRKAG2d.5.In addition to PRKAG2d,PRKAG2-AS1 may also regulate the expression of other genes in cardiomyocyte apoptosis and endothelial cell apoptosis.