MicroRNA-122 Aggravates Hypoxia-induced H9C2 Cardiomyocyte Apoptosis Via PI3K/AKT Pathway

Objective:In this study,we investigated the effect of MiR-122 on hypoxia-induced myocardial apoptosis and its possible pathway.To provide the basic experimental theoretical basis for MiR-122 in the clinical diagnosis and treatment of AMI.Methods:（1）Experimental Subgroup:control group:normal medium culture,the condition is 37℃5%CO₂culture;Hypoxia group:normal medium culture,when the degree of cell fusion reached 60-70%,the serum-free medium was placed under anoxic condition（O₂content<1%）for 24 hours.（2）QRT-PCR to detect MiR-122 expression levels:using QRT-PCR techniques to detect MiR-122 expression levels.Collect normal oxygen and hypoxia cultured cells and extract the total RNA.Use fastking one-step reverse transcription-fluorescence quantitative kit to detect MiR-122 expression levels;（3）Annexin V/PI double flow cytometry to detect myocardial apoptosis:Annexinv FITC/PE apoptosis kit was purchased to evaluate the apoptosis rate of H9c2cardiomyocytes.Normal oxygen and hypoxia cultured cells were collected,washed with kit buffer,and then stained with double dyes under dark condition for 15 min.the samples were filtered through cell sieve and then put into flow cytometry to detect the apoptosis rate of each group of cells;（4）Western blot assay for protein expression:normal and anoxic cells were collected and cultured.After the total protein was separated from H9C2 cells by cell lysis buffer,the BCA method was used to quantify the protein concentration of the two groups of cells.Anti-PI3K,anti-AKT,anti-p-p I3k,anti-p-AKT and anti-GAPDH were diluted in 5%sealing solution by 1:1000.The polyvinylidene fluoride membrane was blocked with 5%blocking solution,the primary antibody was injected overnight at 4°C,and the goat anti-rabbit second antibody was injected for 2 hours in the dark at room temperature.Then,the PVDF membrane was coated with ECL luminescent liquid and put into the machine to record the protein signal.The band strength was calculated by image software.The experiment was repeated 3 times;（5）Statistical analysis:all data were analyzed using Graph Pad PRISM8.0 statistical software.The data obtained from the experiment were all in the form of mean±standard deviation.The difference between the two groups was calculated by one-way Anova.P<0.05 was defined as the statistical difference between the two groups.Results:（1）The expression level of MiR-122 was measured by QRT-PCR.The results showed that the level of MiR-122 in cultured cardiomyocytes under hypoxia was significantly higher than that under normal oxygen（P<0.01）;（2）Flow cytometry was used to measure the ratio of apoptosis in rat cardiomyocytes.The results showed that the ratio of apoptosis in hypoxic cardiomyocytes was higher than that in control group（P<0.01）;（3）The Anti-PI3K,AKT,p-PI3K,p-AKT protein expression was detected by Western blotting.Compared with control group,the expression of p-PI3K,p-AKT protein was significantly decreased in hypoxia group（P<0.01）,but the expression of PI3k,AKT protein was not significantly changed in the two groups（P>0.05）.Conclusion:（1）Hypoxia could increase the expression of mi R-122in rat cardiomyocytes;（2）Hypoxia could increase the ratio of apoptosis in rat cardiac myocytes;（3）MiR-122 aggravates hypoxia-induced H9C2cardiomyocyte apoptosis via PI3K/AKT pathway.