The Research On Circulating MicroRNAs As Novel Biomarker Of Acute Pulmonary Embolism

Cardiovascular disease is used to be a great threat to the public health. People pay high attention to the coronary artery disease and cerebral vascular incidence, because of the high morbidity and mortality they cause. At the same time, we are lack of recognition and attention to acute pulmonary embolism which is the third cause to death among cardiovascular disease. Pulmonary embolism is caused by thrombosis, fat, amniotic fluid, air and some other embolus, the most common is thromboembolism. The thrombus is derived from the venous system or the right heart, mostly lower extremity deep vein. After the embolus blocking the pulmonary artery and its branches, it will soon cause dysfunction of circulation and respiration. When the lung is widely blocked, it may cause pulmonary hypertension, if the situation gets worse it may leads to right heart decompensate, enlargement and then acute right heart failure, which eventually lead to death. Pulmonary Angiography is now the gold standard for the diagnosis of APE, but it is an invasive examination which has the possibility of fatal or severe complication. Computed Tomographic Pulmonary Angiography is by far the most common used method for APE confirmed, but it is not suitable for patients with renal insufficiency and contrast allergy. D-dimer is a biomarker in the blood which has certain reference to diagnosis APE, while it is an index with high specificity and poor sensitivity, mainly used for excluding APE diagnosis and risk stratification. At present, we need a method that is noninvasive and of high benefit, can be widely used, can quickly get the results. If we can find out a biomarker which has both high specificity and sensitivity in the blood, it will bring new hope to early and quick diagnosis of APE.Micro RNAs are small, non-coding RNA which regulate gene expression at post-transcriptional level. It is confirmed that mi RNAs express in specific tissue and cell type and can be released to blood in particular cases. Extracellular mi RNAs are remarkably stable in the circulation by bind to proteins or micro-vesicles to avoid the degradation of RNA enzymes. These features make people associate the expression level of mi RNAs in the circulation with some diseases. The increased concentration- 5-of tissue-specific blood mi RNAs may play a role in the process of disease occurrence and development, or they are just directly released by the damaged tissues of patients. The researchers began to explore whether circulating mi RNAs have a certain diagnostic value for different diseases. Nowadays related studies have been rapidly expending to multiple disease areas from tumor. In the field of cardiovascular, most studies explore the relationship between circulating mi RNAs and acute myocardial infarction or heart failure. Several studies have suggested that mi RNAs are of great potential in the diagnosis of myocardial infarction. The purpose of this study is to explore if the circulating mi RNAs can became the biomarker for APE diagnosis.I. Establishment of animal model of acute pulmonary embolism.Objective: To establishment rat animal model of acute pulmonary embolism.Methods: 1. These SD rats were randomly allocated to six groups: control group, APE 1h, 3h, 6h, 12 h, 24 h group(n=10). The autologous blood clots were injected through jugular vein. 2. Collect blood samples and lung tissue samples of each rat. 3. The model was validated by two methods, one was to observe and contrast the general situation of the rats before and after modeling, the other was to compare the lung tissue HE staining slice of APE model groups and control group.Results: The anesthesia success rate was 96.55%(56/58) and the embolism success rate was 89.29%(50/56). As soon as the clots were injected into jugular vein the rats became dyspnea, heart rate elevated, lips and skin turned cyanosis. When the rats recovered from anesthesia, reduced physical activity, poor appetite and slow response were observed. Histology of the control group was normal. There was thrombus stretched to the distal vessels in the APE group, simultaneously different degrees of edema and inflammatory cell infiltration, destroyed alveoli and hemorrhage could be seen occasionally.Conclusion: Choose the appropriate experimental animal and method at the same time pay attention to the details is the key to successfully model building.II. Detect the concentration of the candidate circulating mi RNAs in the acute pulmonary embolism animal.Objective: Profiling the expression of candidate circulating mi RNAs in APE animal models to find out the ones that have diagnostic value.Methods: 1. Select the candidate mi RNAs through consulting the literature in connection with our previous studies and establish the endogenous control for data normalization.2. Analysis of mi RNAs expression by quantitative Real-Time PCR is performed by SYBR Green methodology. Profile expression of the candidate mi RNAs in the APE model and describe the trend of dynamic change over time. 3. ELISA is applied to detect the D-dimer level and its dynamic change over time in the plasma. 4. The value of mi R-195, mi R-486, D-dimer to diagnosis APE animal model was evaluated by drawing the receiver operating curve(ROC curve).Results: 1.We identified 13 circulating mi RNAs as the candidate mi RNAs including mi R-134, mi R-410, mi R-195, mi R-200 c, mi R-486, mi R-146 b, mi R-34 b, mi R-192, mi R-363, mi R-328, mi R-26, mi R-101 and let-7α. Mi R-16 is elected as endogenous control. 2. The signals of mi R-134, mi R-410, mi R-200 c, mi R-34 b, mi R-363 and mi R-101 were too low to be detected. The concentration of mi R-195 and mi R-192 evaluated after APE, mi R-486 and let-7α decreased, meanwhile mi R-328, mi R-26, mi R-146 were with no change. Among these the change of mi R-195 was statistically different(P<0.01). Further we have tested the trend of target mi RNAs along with the change of time, then we found that the expression amount of mi R-195 increased significantly at 1 h(P<0.05), peaked at 6 ~ 12 h and decreased at 24 h after pulmonary embolism. The expression level of mi R-486 decreased at 6 h(P<0.05) with statistical significance, looking at the lowest number at 12 h and began to rise at 24 h after pulmonary embolism. The change of the concentration of the rest mi RNAs along with time after pulmonary embolism saw no statistically significant(P>0.05). 3. D-dimer content elevated at 3 h(P<0.05), then gradually died down. 4. Mi R-195 reflected strong separation between APE groups and control group with an area under curve(AUC) of 0.956(P<0.01), while mi R-486 with an AUC of 0.740(P>0.05), D-dimer of 0.861(P<0.05). Furthermore, plasma level of mi R-195 could detect individuals of APE with 80% sensitivity at 100% specificity, when the relative expression of mi R-195 was 1.706. The AUC of mi R-195 combined with D-dimer(APE 6h) was 0.964(P<0.01), when the specificity was 100%, the sensitivity was 85.7%. The AUC of mi R-195 combined with D-dimer(APE 3h) was 0.813(P<0.05), when the specificity was 100%, the sensitivity was 62.5%.Conclusion: Circulating level of mi R-195 increased in the APE model at the early stage and followed a time-dependent pattern. Considering the high value of circulating mi R-195 for APE model diagnosis, especially when combined with D-dimer, it may bring new ideas for diagnosing APE in clinical.