| Myocardial infarction is the fundamental cause of death in cardiovascular diseases.Persistent myocardial ischemia after myocardial infarction leads to myocardial apoptosis and myocardial hypertrophy,which triggers a series of inflammatory and immune responses,leading to myocardial pathological remodeling,heart failure and sudden death.Myocardial apoptosis is considered to be one of the main causes of cardiac remodeling after myocardial infarction(MI).Circular RNA(circRNA)are a class of endogenous noncoding RNAs with covalently closed-loop structure,which are an important epigenetic regulation way and participate in the occurrence and development of a variety of cardiovascular diseases.A large number of studies have found that circRNA plays a key role in a variety of biological functions.However,The funtion of circ＿0068655 in myocardial infarction and commercial human induced pluripotent stem-derived cardiomyocytes(HCMs)remains unclear.Therefore,in this study,adult rat MI model was used to observe the expression of circ＿0068655 in myocardial infarction cells.Overexpression and down-regulation the expression of circ＿0068655 in HCMs to detect its function on hypoxia-induced cardiomyocyte,focusing on the effect on cardiomyocyte apoptosis,and preliminarily explore the mechanisms of circ＿0068566 on regulation of Cardiomyocytic apoptosis.Part One circ＿0068655 promotes hypoxia-induced cardiomyocyte injuryObjective: The expression of circ＿0068655,miR-498 and PAWR in hypoxia-induced HCMs were observed to investigate the correlation between circ＿0068655 and hypoxia-induced cardiomyocytic apoptosisMethods:1.MI model was conducted by ligating the LAD of the coronary artery。After a week of MI,heart tisses were harvested to detected the expression of circ＿0068655,miR-498 and PAWR by real-time fluorescence quantitative PCR(qRT-PCR).2.HCMs were cultured in hypoxic cell incubator for 3,6 and 12 hours,respectively.The expression levels of circ＿0068655,miR-498 and PAWR of HCMs cultured for 3,6 and 12 hours were detected by qRT-PCR.PAWR protein level was detected by Western blot3.RNA interference technology shRNA was used to knock down the expression of circ＿0068655 in HCMs and cultured in hypoxic cell incubator for 12 hours.Cell viability,caspase-3 activity,DNA fragmentation,cell migration and cell invasion were evaluated by MTT assay,caspase-3 activity assay,cell death ELISA(POD)assay,and trans-well assay in HCMs transfected with shRNA against circ＿0068655 or empty vector(sh-Ctrl),respectively.Results:1.The expression of circ＿0068655,miR-498 and PAWR in myocardial tissue of MI mice was detected by qRT-PCR.The results showed that the expression of circ＿0068655 and PAWR in MI tissues were significantly higher than that in the control group(P < 0.05),the expression of miR-498 was significantly lower than that in the control group(P < 0.05).Western blot results showed that the protein expression of PAWR was significantly increased compared with the control group(P < 0.05).2.qRT-PCR was used to detect the expression of circ＿0068655,miR-498 and PAWR in HCMs.The results showed that the expression of circ＿0068655and PAWR in HCMs cultured under hypoxia for 3,6 and 12 hours was significantly higher than that under conventional culture(P < 0.05),while the expression of miR-498 was significantly lower and time-dependent.Western blot showed that the protein expression of PAWR increased in a time-dependent manner(P < 0.05).3.The results of qRT-PCR showed that the expression of circ＿0068655in HCMs transfected with sh-circ＿0068655 was significantly decreased(P <0.01).MTT assay showed that cell viability was significantly decreased under hypoxia(P < 0.01).the cell viability was alleviated after knocking down the expression of circ＿0068655 by transfected sh-circ＿0068655 in HCMs(P <0.01);the detection of capspase-3 activity by capspase-3 activity assay showed that capspase-3 activity increased significantly than the control group;Capspase-3 activity of the groups with transfected decreased significantly compared to hypoxia group;The results of cell death detection by cell death ELISA(POD)assay,showed the number of dead cells in the sh-circ＿0068655transfected group was significantly lower than that in the control group(P <0.01).Trans-well assay showed that the ability of cell migration and invasion in the sh-circ＿0068655 transfected group was significantly higher than that in the control group(P < 0.01).Conclusions:1.The expression of circ＿0068655 and PAWR increased in myocardial infarction tissue,while the expression of miR-498 decreased in myocardial infarction tissue.2.The expression of circ＿0068655 and PAWR were increased in hypoxic-induced HCMs,while the expression of miR-498 was decreased in hypoxic-induced HCMs.And the expression of circ＿0068655 and PAWR increased with the prolongation of hypoxia time,while the expression of miR-498 showed the opposite phenomenon.3.Knock-down the expression of circ＿0068655 can ameliorate hypoxiainduced cardiomyocytic apoptosis,so circ＿0068655 can promote hypoxiainduced cardiomyocytic apoptosis.Part Two Circ＿0068655 promotes cardiomyocytic apoptosis through sponge miR-498Objective: To investigate the effect of expression of miR-498 on the exp-ression of circ＿0068655;To confirm that circ＿0068655 contains miR-498 recognition and binding sites.To explore circ＿0068655 promotes hypoxic cardiomyocytic apoptosis through sponge miR-498Methods:1.The location of circ＿0068655 was determined by nucleocytoplasmic separation and qRT-PCR2.Bioinformation analysis : Circular RNA Interactome database predicting binding site of miR-498 in circ＿0068655 sequence.3.Double luciferase reporter gene test to verify Whether there is a complementary binding relationship between circ＿0068655 and miR-498.We constructed the wild-type and mutant dual-fluorescent reporter vectors psi CHECK-2-circ0068655 WT and psi CHECK-2-circ＿0068655MT,co-transfected with miR-498 mimic and negative control(miR-NC)in HCMs and HEK-293 T cells respectively,luciferase activity of each group was observed to confirm the circ＿0068655 region contains miR-498 recognition and binding sites.4.Regulation of miR-498 by circ＿0068655: we constructed the circ＿0068655 overexpression vector PLCDH-ci R-circ＿0068655,PLCDH-ci Rcirc＿0068655 and sh-circ＿0068655 were transfected into HCMs then detected the expression level of miR-498。Results:1.qRT-PCR results showed that the expression of circ＿0068655 in the cytoplasm of HCMs was significantly higher than that in the nucleus;The circ＿0068655 transcript is located in the cytoplasm.2.Dual-luciferase reporting experiment showed that compared with the control group,the luciferase activity of the wild-type circ＿0068655 and miR-498 mimic co-transfection was significantly reduced(P < 0.01).While there was no significant difference in luciferase activity between co-transfection of mutant-type circ＿0068655 and miR-498 mimic.The results indicate that circ＿0068655 can directly interact with miR-498.3.qRT-PCR results showed that compared with the control group,the expression of miR-498 was significantly reduced in HCMs with transfected circ＿0068655 overexpression plasmid PLCDH-ci R-circ＿0068655;While the expression of miR-498 in HCMs transfected sh-circ＿0068655 was enhanced compared to control group(P < 0.01).Conclusions:1.The Circ＿0068655 transcript is located in the cytoplasm.2.Circ＿0068655 can directly bind to miR-498,which is circ＿0068655’s target miRNA.3.Circ＿0068655 can negatively regulate the expression of miR-498.Part Three Circ＿0068655 acted as a competitive Endogenous RNA to Promote PAWR expressionObjective: The regulation of miR-498 on the expression of its potential target molecule PAWR was observed in vitro,to explore the downstream mechanism of circ＿0068655 involved in hypoxia-induced cardiomyocytic apoptosis.Methods:1.Bioinformation analysis:Targetscan database predicting the target genes of miR-498.(http://www.targetscan.org/vert＿71)2.Cell transfection: HCMs were transfected with miR-498 mimic,miR-498-inhibitor and corresponding control by pofectamine 2000 kit,and the transfection effects of miR-498 mimic and miR-498 inhibitor were verified by qRT-PCR.3.Double luciferase reporter gene test to verify whether miR-498 and3’UTR of PAWR have complementary binding relationship.We constructed the wild-type and mutant dual-fluorescent reporter vectors psi CHECK-2-PAWR-WT(PAWR 3’UTR-WT)and psi CHECK-2-PAWR-MT(PAWR 3’ UTR-MT),co-transfected with miR-498 mimic and negative control(miR-NC)in HCMs and HEK-293 T cells respectively,luciferase activity of each group was observed to confirm whether PAWR is a target gene of miR-498.4.Confirmation of target genes of miR-498: the binding relationship between miR-498 and circ＿0068655,miR-498 and PAWR3’UTR were verified by Biotin-RNA pull down experiment.5.Regulation of PAWR by miR-498:The regulation of PAWR expression by miR-498 and its inhibitor was observed by qRT-PCR,and the regulation of expression level of PAWR protein by miR-498 was detected by Western blot analysis.6.Regulation of PAWR by circ＿0068655: we constructed the circ＿0068655 overexpression vector PLCDH-ci R-circ＿0068655,PLCDH-ci Rcirc＿0068655 and sh-circ＿0068655 were transfected into HCMs then detected the expression level of PAWR by qRT-PCR and the regulation of expression level of PAWR protein by circ＿0068655 was detected by Western blot analysis.Results:1.Targetscan predicted that PAWR 3’UTR had a binding site for miR-498.2.Dual-luciferase reporting experiment showed that compared with the control group,the luciferase activity of the wild-type PAWR and miR-498 mimic co-transfection was significantly reduced(P < 0.01).While there was no significant difference in luciferase activity between co-trans-fection of mutant-type PAWR and miR-498 mimic.The results indicate that PAWR can directly interact with miR-498.3.Biotinylated labeled RNA-pull down assay showed that both circ＿0068655 and PAWR were pulled down by the biotin-miR-498.4.The results of qRT-PCR showed that the expression of miR-498 was significantly increased in HCMs transfected with miR-498 mimic,while the expression of PAWR was significantly decreased in HCMs compared with the control group.Compared with the control group,the expression of miR-498 was significantly decreased in HCMs transfected with miR-498 inhibitor(P <0.01),but the level of PAWR was significantly increased in HCMs cells.Western blot results showed that the level of PAWR translation protein was significantly increased after transfection of HCMs cells with miR-498 inhibitor compared with the control(P < 0.01).5.qRT-PCR results showed that transfection of circ＿0068655 overexpression plasmid PLCDH-ci R-circ＿0068655 promotes the expression of PAWR in HCMs,while the expression of PAWR was reduced with transfected sh-circ＿0068655 into HCMs(P < 0.01).Conclusions:1.miR-498 directly binds to the 3’UTR of the PAWR sequence.2.miR-498 have both circ＿0068655 and PAWR binding sites3.miR-498 negatively regulates the expression of PAWR in cardiomyocytes.4.circ＿0068655 positive regulates the expression of PAWR in cardiomyocytes Part Four Circ＿0068655 promotes apoptosis and inhibiting cell activity through regulating the miR-498/PAWR signaling pathwayObjective: Using vitro experiment to observe whether the upregulation of circ＿0068655 by mediated hypoxia can cause promotes cardiomyocytic apoptosis through upregulate the expression of PAWR by sponge miR-498.Methods:1.Sh-circ＿0068655 and its control group were co-transfected with miR-498 inhibitor(miR-498-inhibitor)and its control group(NC-inhibitor)in HCMs,respectively,and the expression level of PAWR was detected by qRT-PCR and Western blot.2.Sh-circ＿0068655 and its control group were co-transfected with miR-498 inhibitor(miR-498-inhibitor)and its control grope(NC-inhibitor)respectively.MTT assay was used to observe cell viability,Capspase-3activity assay was used to detect Capspase-3 activity,cell death ELISA(POD)assay was used to detect cell death,and Trans-well technique was used to detect the migration and invasion ability of cardiomyocytes after hypoxia treatment.Results:1.The results of qRT-PCR and Western blot showed that hypoxia induced a significant increase in PAWR expression and protein level in HCMs(P< 0.01);Co-transfection of sh-circ＿0068655 and NC-inhibitor could reduce the increase of PAWR expression induced by hypoxia;the expression of PAWR in HCMs with co-transfect sh-circ＿0068655 and miR-498 inhibitor group was higher than co-transfected with sh-circ＿0068655 and NC-inhibitor group。2.Compared with the control group,HCMs were co-transfected with shcirc＿0068655 and NC-inhibitor,MTT assay showed that cell viability was significantly increased.Capspase-3 assay and cell death ELISA assay showed that the activity of Capspase-3 in cardiomyocyte was significantly decreased and the number of dead cells was significantly decreased(P<0.01).Trans-well assay showed that the ability of cell migration and invasion was significantly increased(P<0.01).Compared to the group with co-transfection shcirc＿0068655 and NC-inhibitor,the cell viability of the group with shcirc＿oo68655 and miR-498-inhibitor was significantly decreased,the caspase activity was significantly increased,the cardiomyocyte apoptosis was significantly increased,and the cell migration and invasion ability were significantly decreased(P < 0.01).Conclusions:1.Circ＿0068655 regulates the expression of PAWR by sponge miR-498.2.The regulation of the expression of PAWR by sh-circ＿0068655 was inhibited by miR-498-inhibitor.3.Circ＿0068655 promotes apoptosis through upgulating PAWR by sponge miR-498. |
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