| Background and objectiveCardiovascular disease has been widely concerned by all walks of life because of its increasing incidence and mortality rate. Despite the widely available diagnostic and therapeutic approaches, the prevalence, mortality, and treatment cost of cardiovascular disease in developed and developing countries are still increasing.Acute myocardial infarction(AMI) is one of the most common cardiovascular diseases. It is necessary to seek new diagnostic and therapeutic targets and methods to control the occurrence and development of AMI effectively.Recently, a number of studies have indicated that mi RNA may play a role in the development of disease through regulating its downstream target genes. mi RNA has received extensive attention in many research fields, including cancer, cardiovascular disease and so on. The expression of many mi RNA in the blood and plasma of patients with AMI has significant deference, such as mi R-1, mi R-133 a, mi R-208 b,mi R-499, and mi R-328, suggesting that circulating mi RNA make effects in the early diagnosis of AMI.The purpose of this study is to screen the abnormal expressed mi RNA in theblood circulation when AMI occurs, analysis the abnormal expression of mi RNA, and evaluate its auxiliary diagnostic value, explore possible ways in which mi RNA play an important role in AMI.Part ? Screening and identification of abnormal expression of circulating mi RNA in patients with AMIMethods1. The plasma samples were collected from 30 pairs of AMI patients and healthy adult volunteers2. Agilent mi RNA chip was used to detect the expression of mi RNA in the plasma of 3 AMI patients and 3 healthy adult volunteers respectively, and to screen the mi RNA with significant difference.3. q RT-PCR was used to detect the expression of mi RNA in plasma samples from 30 AMI patients and 30 healthy adult volunteers, and the results were used to verify the mi RNA chip results.4. SPSS21.0 software was used to process statistical analysis with inspection level ?=0.05.Results1. 36 kinds of mi RNA were detected in the plasma of AMI patients by mi RNA chip, among them, 23 kinds of mi RNA expression levels were significantly increased(P<0.05), and 13 kinds of mi RNA expression levels were significantly decreased(P<0.05).2. The q RT-PCR results demonstrated that the expression of four mi RNA,mi R-361-5p, mi R-182-5p, mi R-497-5p, mi R-20a-5p showed no significant difference in the 30 cases of AMI patients compared with the normal control group(P>0.05). And the expression level of the remaining 32 mi RNA showed no significant difference(P<0.05), among them, 20 kinds of mi RNA expression levels were increased, 12 kinds of mi RNA expression levels were decreased.Part ? The analysis of expression and auxiliary diagnostic value of mi R-486,mi R-150 and mi R-26 a in AMIMethods1. The plasma samples were collected from 110 pairs of AMI patients and healthy adult volunteers. The 110 cases of AMI patients contained 65 cases of STEMI patients and 45 cases of NSTEMI patients. The first plasma samples were collected immediately from AMI patients after admission within 4 h of symptom onset, and subsequent blood samples were obtained at admission <4, 24, 48, and 72 h after hospitalization.2. q RT-PCR was used to quantify mi R-486, mi R-150 and mi R-26 a expression in AMI samples.3. Receiver operating characteristic curve(ROC) was applied to reflect the sensitivity and specificity, the area under the ROC curve(AUCROC) was used to compare the value of mi R-486, mi R-150 and mi R-26 a in the diagnosis of AMI.4. SPSS21.0 software was used to process statistical analysis with inspection level ?=0.05.Results1. In AMI cases within 4 h, the relative expression of mi R-486 and mi R-150 were 9.655±1.427 and 7.420±0.911 respectively, which showed a significantly increased trend compared with the normal healthy cases(P<0.05), the relative expression of mi R-26 a was 0.092±0.104, which showed a significantly decreased trend compared with the normal healthy cases(P<0.05)2. In AMI cases <4h, 24 h, 48 h and 72 h, the expression levels of mi R-486 and mi R-150 were lower and lower, and in 72 h, AMI patients reverted to normal level(P> 0.05); the expression levels of mi R-26 a was higher and higher, and in 72 h, AMI patients reverted to normal level(P> 0.05).3. The AUC of mi R-486,mi R-150 and mi R-26 a in AMI patients within 4h were 0.731(P<0.05),0.678(P<0.05),0.763(P<0.05)respectively, which showed that the expression level of the three mi RNA has a certain value for the detection of AMI. The combination of the three mi RNA resulted in a higher AUC value of 0.792(P<0.05)which suggested that the combination of circulating mi R-486,mi R-150 and mi R-26 a might be more suitable than mi R-486 or mi R-150 or mi R-26 a alone for diagnosing.4. There was distinct difference among the level of plasma mi R-486, mi R-150 and mi R-26 a between STEMI patients and NSTEMI patients within 4h.ROC curve analysis of mi RNA showed the AUC of plasma mi R-486,mi R-150 and mi R-26 a in STEMI patients to be 0.695( P<0.05),0.639(P<0.05),0.750(P<0.05), which showed that the expression level of the three mi RNA has a certain value for the detection of STEMI. The AUC of plasma mi R-486, mi R-150 and mi R-26 a in NSTEMI patients to be 0.782(P<0.05),0.734(P<0.05),0.782(P<0.05), which showed that the expression level of the three mi RNA has a certain value for the detection of NSTEMI. The combination of circulating mi R-486 mi R-150 and mi R-26 a resulted in a higher AUC value of 0.765(P<0.05),0.833(P<0.05)in STEMI and NSTEMI patients respectively, which had both higher sensitivity and specificity, might be more suitable than mi R-486 or mi R-150 or mi R-26 a alone for diagnosing, especially in NSTEMI patients.Part ? : Preliminary study exploration on the mechanisms of mi R-150 and mi R-486 in the occurrence of AMIMethods1. Predict the potential target genes of mi R-150 and mi R-486 using bioinformation software Target Scan and mi Randa.2. The mutant type of CDKN1 B 3'UTR fragments and mutant type of ALDH23'UTR fragments were amplified by Overlap PCR, The wild type of CDKN1 B 3'UTR fragments and wild type of ALDH2 3'UTR fragments were amplified by regular PCR.Then combined the fragments above with the pmir GLO vector, as a result, further constructed mutant and wild type of CDKN1 B 3'UTR and ALDH2 3'UTR dual luciferase reporter vectors.3. Co-transfected mutant or wild type CDKN1 B 3'UTR vectors with either mi R-150 mimics or mi R-159 scramble into HEK 293 T cells to confirm whether CDKN1 B was one of the targets of mi R-150; co-transfected mutant or wild type ALDH2 3'UTR vectors with either mi R-486 mimics or mi R-486 scramble into HEK 293 T cells to confirm whether ALDH2 was one of the targets of mi R-486.4. SPSS21.0 software was used to process statistical analysis with inspection level ?=0.05.Results1. According to bioinformation, CDKN1 B may be one of the targets of mi R-150, ALDH2 may be one of the targets of mi R-486.2. The mutant and wild type of CDKN1 B 3'UTR and ALDH2 3'UTR dual luciferase reporter vectors were successfully constructed verified by enzyme digestion, PCR and sequencing results.3. Dual luciferase reporter experiments showed that the luciferase activity in cells co-transfected mi R-150 mimics and WT- pmir GLO- CDKN1 B was0.54±0.065, which was significantly lower than the other three groups(P<0.05), indicating that CDKN1 B is one of the target genes of mi R-150.The luciferase activity in cells co-transfected mi R-486 mimics and WTpmir GLO- ALDH2 was 0.62±0.045, which was significantly lower than the other three groups(P<0.05), indicating that ALDH2 is one of the target genes of mi R-486.Conclusions1. A total of 32 mi RNAs in AMI patients have abnormal expression, including20 up-regulation and 12 down-regulation.2. In AMI cases within 4 h, the relative expression of mi R-486 and mi R-150 were significantly upregulated; the relative expression of mi R-26 a was significantly downregulated. And the change was becoming not obvious over time. The combined detection of mi R-486, mi R-150 and mi R-26 a is expected to be an auxiliary diagnostic index for AMI, especially for NSTEMI.3. mi R-150 and mi R-486 negatively regulate the expression of CDKN1 B and ALDH2 respectively, which may play a role in the occurrence and development of AMI. |
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