筛选血清miRNA作为类固醇兴奋剂检测新指标的探索性研究

Nowadays, steroid is the drug that mostly abused in competitive sports, however, the analyzing technology of steroid still has flaw. At first, the testosterone over epitestosterone ratio (T/E) is much lower in Asian people than Caucasian. Secondly, it is hardly to detect some new synthetic endogenous steroid by IRMS technology of which the C13/C12 ratio is nearly similar with ratio in human body. Thirdly, the new synthetic exogenous steroid which is only provided in black market cannot be detected, for the reason there is no standard sample. Above all, the technology of steroid detection needs to be improved necessarily...MiRNAs are a recently discovered class of small non-coding RNAs that regulate gene expression and have a critical role in many biological and pathological processes. Since it is found that miRNAs are existed in body fluids, circulant miRNAs, have been extensively applied in clinical diagnosis and treatment monitoring. It has been reported that miRNAs were under research on bio-markers for doping detection, such as EPO and rhGH, from which target miRNAs were obtained. ..This program aims at the protein synthesis in human skeletal muscle, focuses on the variation of miRNAs concerned with steroid-induced anabolic signal pathway, such as androgen receptor (AR) pathway, mammalian target of rapamycin (mTOR) pathway, mitogen-activated protein kinases (MAPK) pathway. Target miRNAs will be anticipated to find as new bio-markers for doping detection of steroid...At first step of research, Testosterone undecaoate excretion with healthy people will be conducted, both blood and urine samples will be collected. MiRNAs will be isolated from serum. The target miRNAs will be selected using microarray for screening, RNA22 or TargetScan database for prediction of target mRNA, KEGG pathway database for signal pathway location, real time RT-PCR for confirmation. Besides that, the target mRNAs of selected miRNAs will be validated by experiments. MiRNAs will be over expression in human skeletal muscular myobalsts by transfection, target miRNAs and relevant mRNAs will be detected by real time RT-PCR, western blot will be used to detect relevant proteins. The same technologies will also be used to validate the variation of miRNAs in vitro, which human skeletal muscular myoblasts will be treated with testosterone or dihydrotestosterone. Combined the validation of miRNAs, the target miRNAs will be determined. Finally, the urine samples will be analyzed by official methods from WADA, the steroid profile will be analyzed for athlete biological passport. Comparing the stability, sensitivity, positive rate and time window of detection, different methods will be contrasted. ..This will be the first time, taking miRNAs as the bio-markers, of detecting the misuse of steroid from the gene expression level. This program will produce the creative idea for the development of doping detection of steroid, which will make up the limitation of current technology.

类固醇是竞技体育中滥用程度最高的一类药物，其检测方法的改进一直是兴奋剂检测工作的关注焦点。体液miRNA自发现以来，广泛应用于临床诊断与治疗监控，目前也正在作为EPO、rhGH的兴奋剂检测指标开展研究，并已得到相关特异miRNA指标。本研究拟从类固醇在人体骨骼肌内蛋白同化作用机制入手，重点研究类固醇使用后与骨骼肌合成代谢相关的血清miRNA的变化情况。研究将开展内源性类固醇药物受试，利用微阵列芯片、实时定量RT-PCR、生物信息学等技术筛选特异变化的miRNA；并通过miRNA转染过表达、实时定量RT-PCR和western blot技术开展生物学验证；同时对比miRNA指标与常规检测方法，为miRNA指标在类固醇兴奋剂检测中的应用探明方向。以miRNA为指标，从基因表达水平监控类固醇滥用的研究在世界范围尚属首次，本研究拟为建立类固醇检测新方法提供创新性思路，从而弥补当前检测技术的局限性。

类固醇是目前竞技体育中滥用程度最高的一类药物，其检测方法的改进一直是兴奋剂检测工作的关注焦点。体液miRNA自发现以来，广泛应用于临床诊断与治疗监控，目前也正在被用来作为EPO、rhGH的兴奋剂检测指标开展研究，并已得到相关特异miRNA指标。本研究从类固醇在人体骨骼肌内蛋白同化作用机制入手，筛选使用十一酸睾酮后特异性变化的血清miRNA，从药物在体内的作用机制着手筛选新型兴奋剂检测生物学指标。.本研究招募6男6女共12名受试者参加十一酸睾酮受试，服用剂量分别为男性每天160mg，女性每天80mg，连续服药14天。分别在第1（用药前）、4、8、15、22、29天采集血样，并于每天收集晨尿。特异性血清miRNA的筛选分别采用384孔板PCR array和实时定量RT-PCR 技术作为指标的初筛和验证方法，同时对收集的尿样进行内源性类固醇的兴奋剂检测。.研究结果显示，使用十一酸睾酮后，女性受试者hsa-miR-122-5p和hsa-miR-206显著升高，男性受试者hsa-miR-122-5p和hsa-miR-183-5p显著升高，通过miRTarBase V7.0，三个血清miRNA指标均已有实验证据表明作用于骨骼肌合成通路。此外，与现有的内源性类固醇兴奋剂检测方法对比，停药后血清miRNA 指标检测时间窗口较现有兴奋剂检测方法长达14天，因此该指标可作为类固醇兴奋剂检测的新型指标开展进一步研究。.本研究以血清miRNA为指标，从基因表达水平监控类固醇滥用的研究在世界范围尚属首次，筛选得到的特异性血清miRNA指标为建立类固醇新型检测方法提供创新性思路，从而弥补当前兴奋剂检测技术的局限性。