| Micro RNAs(mi RNAs), a class of 18 to 25 noncoding nucleotides, are capable of regulating gene expression through messenger RNA degradation or translational repression and are involved in various biological processes, such as proliferation, differentiation, development, and apoptosis. Recently, the presence of mi RNAs in the blood has been reported. Interestingly, deregulation of circulating mi RNAs has been associated with a variety of human diseases, including cancer and cardiovascular diseases, indicating that mi RNAs could be used as biomarkers for cancer and other diseases.Pulmonary arterial hypertension(PAH), a life-threatening disease, is known as the "malignant tumour of the cardiovascular system". The pathogenesis of PAH is characterized by abnormal proliferation and migration of pulmonary arterial smooth muscle cells(PASMCs).PDGF-BB is well known as a potent factor for smooth muscle cell proliferation and migration. A huge number of experimental data have shown that mi RNAs play important roles in mediating the PDGF-BB induced dysfunction of PASMCs. However, the function and the underlying molecular mechanism need further explored.As a tiny amount of RNAs is present in the circulating system,the first step of detecting circulating mi RNA is the total RNA separation and purification. The existing method had low RNA content and.a high price. We developed a sensitivity of circulating mi RNA detection by S/P mi RSOL, a modified method of total RNA isolation from serum/plasma, in which 15 ?g/ml glycogen was used to increase the RNA yield. This new method—S-Poly(T) Plus mi RNA assay is based on our previously developed S-Poly(T) method, in which a unique S-Poly(T) primer and universial probe are used during reverse-transcription and real-time PCR to increase sensitivity and specificity. Further increased sensitivity and simplicity of S-Poly(T) Plus,in comparison with the S-Poly(T) method, were achieved by a single-step, multiple-stage reaction,where RNAs were polyadenylated and reverse-transcribed at the same time.It's simple and time-saving.Testing mi RNAs in HEK293 A and healthy human serum,we found that the sensitivity of S-Poly(T) Plus assay was about 2~8 fold higher than S-Poly(T). The correlation coefficients R2 produced by S-Poly(T) Plus and S-Poly(T) methods were 0.9933 ~ 0.9991 and 0.9745 ~ 0.9968,respectively,indicating the increased sensitivity of S-Poly(T) Plus.The dynamic range of S-Poly(T) Plus was further evaluated with human serum samples and observed an excellent linear relationship between total RNA inputs and Ct values with R2 ranging between 0.9536 and 0.9972. We validated our methods by quantifying mi RNA expression profiles in the sera of the patients with pulmonary arterial hypertension associated with congenital heart disease(CHD-PAH). Our results revealed the up-regulation of mi R-20a-5p and mi R-451 a,and down-regulation of mi R-204-5p, mi R-424-5p,mi R-126-3p, mi R-26a-5p,mi R-9-5p and mi R-328-3p in the sera of CHD-PAH patients. These mi RNAs may be of potential in use as biomarkers for CHD-PAH early detection.We developed a simple,sensitive, and specific method for detecting circulating mi RNAs that probably allows the measurement of 266 mi RNAs from 100 ?l of serum or plasma. This method presents a promising tool for basic mi RNA research and clinical diagnosis of human diseases based on mi RNA biomarkers.We profiled the expression of mi RNAs in HPASMCs stimulated with PDGF-BB by S-Poly(T) Plus real-time PCR. Fifteen mi RNAs were screened out and verified to be significantly altered in response to PDGF-BB, The downregulation of mi R-328 was further confirmed in rat PASMC treated by PDGF-BB, displaying a time- and dose-dependent manner. Interestingly, the expression of mi R-328 was specifically inhibited by the treatment of PDGF-BB rather than other growth factors.We found that overexpression of mi R-328 significantly inhibited the PASMC proliferation and migration.Target gene prediction and dual-luciferase assay indicated that PIM-1 is a target gene of mi R-328,and we futher confirm this at both m RNA and protein levels.Rescue experiments were performed indicating that PIM-1 could rescue the effect of mi R-328 on PASMC proliferation. The decrease of mi R-328 expression in response to PDGF-BB was completely recovered by the pretreatment of demethylating agent 5-aza-2'-deoxycytidine, a inhibitor of DNA methylation transferase. The expression of DNMT1 was increased by the treatment of PDGF-BB, while the knockdown of DNMT1 significantly inhibited the PDGF-BB-induced downregulation of mi R-328. The expression of circulating mi R-328 decreased remarkably in PAH associated with congenital heart disease(CHD-PAH) patients, which suggests a potential therapeutic target of mi R-328 for preventing and treating CHD-PAH caused by activation of the PDGF-BB pathway. |
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