高通量miRNA活性谱检测发现BHK21细胞中miR-206高活性

High activity level of miR-206 discovered in BHK21 cells by high-throughput miRNA activity profiling

摘要:

用我们建立的活细胞miRNA活性谱检测技术测定BHK21、HEK293和Vero三种肾来源细胞系中58种miRNA活性谱，发现miR-206在BHK21细胞中具有特征性高活性。鉴于miR-206是典型的横纹肌特征性miRNA，进一步以小鼠成肌细胞C2C12及人胚肾细胞HEK293为对照，检测了BHK21细胞中miR-206的活性及表达水平。之后，用马血清诱导培养BHK21细胞，检测诱导前后BHK21细胞中骨骼肌肌球蛋白重链 (Slow skeletal myosin heavy chain，MHC) 表达情况，miR-206活性和表达水平以及受miR-206负调控的Connexin43(Cx43) 的表达水平。结果发现，miR-206在BHK21细胞中活性和表达水平都明显高于C2C12细胞；马血清诱导后，BHK21细胞中MHC表达水平升高，miR-206活性和表达水平都升高，而Cx43表达水平下降。结果提示BHK21细胞具有成肌细胞特性。本研究首次发现BHK21细胞中miR-206的高活性，从miRNA角度证实了BHK21细胞来源于肾间质细胞，而不是肾实质细胞。研究结果还提示BHK21细胞有可能作为一种体外模型用于miR-206的功能研究

Abstract:

Activities of 58 miRNAs for BHK21, HEK293, and Vero cell lines were screened using the high-throughput miRNA activity profiling method. miR-206 activity was found specifically high in BHK21. Considering miR-206 was recognized as a muscle-specific miRNA, we further detected the expression and activity level of miR-206 in BHK21 cells, with myoblast cells C2C12 as positive control and HEK293 cells as negative control. Then, we induced BHK21 by culturing with medium containing 2% horse serum (HS) and tested expression level of slow skeletal myosin heavy chain (MHC), activity and expression levels of miR-206, and expression level of Connexin43 (Cx43) which was reported negatively regulated by miR-206. Results demonstrated that activity and expression levels of miR-206 were both higher in BHK21 cells than in C2C12 cells. After induction of HS, MHC expression level was increased in BHK21 cells. The activity and expression levels of miR-206 were further enhanced. The Cx43 expression level was decreased. These results suggested that BHK21 had the characters of myoblast cells. In conclusion, we firstly discovered that miR-206 activity was specifically high in BHK21 cells, suggesting that BHK21 cells were derived from interstitial cells other than parenchyma cells of kidney from miRNA point of view. Our study also indicated that BHK21 cells were able to be used as models in vitro for research of miR-206 function