骨骼肌circRNA在死亡时间推断中的初步筛选及验证研究

Preliminary Screening and Verification of Skeletal Muscle circRNA in PMI Esitimation

目的通过构建骨骼肌中环状RNA(circular RNA,circRNA)表达谱,筛选出人体骨骼肌中与死亡时间具有时序性变化的circRNAs,并对筛选出的circRNAs进行验证,进而探讨其相对表达量与死亡时间的相关性。方法芯片实验选取2例外界温度、湿度相对一致,已知死亡时间在24 h内的个体,分别于尸检即刻(12 h以内)、3、5、7、10 d各取50 g组织进行总RNA提取,并进行逆转录、探针标记、基因芯片检测等实验,初步得到circRNA表达谱;对筛选出的circRNAs在后续收集的18例检材中进行验证,对所得数据进行统计分析。结果骨骼肌组织中共检测出13156个circRNAs,但在10 d内整体呈下降趋势的circRNAs只有hsa_circ_0001727、hsa_circ_0001136、hsa_circ_0001871、hsa_circ_0005251、hsa_circ_0000284、hsa_circ_0000520、hsa_circ_00019717个。对所筛选出的7个circRNAs在18例检材中进行验证,发现只有hsa_circ_0001727、hsa_circ_0001136、hsa_circ_0001871、hsa_circ_0005251、hsa_circ_00002845个circRNAs随着死亡时间增加,其相对表达量显著下降,具有统计学意义(P<0.05)。而hsa_circ_0000520、hsa_circ_00019712个circRNAs的表达为阴性。结论circRNA是一种较为稳定的生物标记物,部分可用于死亡时间推断的研究。

Objective CircRNAs in human skeletal muscle with temporal changes in death time were screened by constructing circular RNA(circRNA)expression profiles in skeletal muscle.The selected circRNAs were further verified,and then the correlation between the relative expression and the time of death was investigated.Methods T In the chip experiment,2 individuals with relatively uniform external temperature and humidity,and known to have a death time of less than 24 hours,were taken for 50 g each at the autopsy(within 12 hours),3 days,5 days,7 days,and 10 days.The RNA was extracted,and then reverse transcription,probe labeling,and gene chip detection were performed to obtain the expression profile of circRNA.The selected circRNAs were verified in 18 samples collected in the subsequent samples,and the obtained data were statistically analyzed.Results A total of 13156 circRNAs were detected in skeletal muscle tissue,but the total number of circRNAs that showed a downward trend within 10 days was only hsa_circ_0001727,hsa_circ_0001136,hsa_circ_0001871,hsa_circ_0005251,hsa_circ_0000284,hsa_circ_0000520,hsa_circ_0001971 and so on.The 7 circRNAs screened were verified in 18 samples,and only 5 circRNAs such as hsa_circ_0001727,hsa_circ_0001136,hsa_circ_0001871,hsa_circ_0005251,hsa_circ_0000284,etc.,with the increase of death time,the relative expression decreased significantly and statistically(P<0.05).The expression of two circRNAs,hsa_circ_0000520 and hsa_circ_0001971,was negative.Conclusion circRNA can be used as a more stable biomarker for the study of death time inference.