条纹斑竹鲨肝脏环形RNA的筛选、鉴定及功能分析

Identification and functional characterization of circular RNAs in the liver of whitespotted bamboo shark (Chiloscyllium plagiosum)

摘要:

为筛选和验证条纹斑竹鲨肝脏中环形RNA (circRNA)及探究其在肝癌细胞HepG2中过表达对肝癌细胞增殖、迁移能力的影响，本研究主要进行了两项实验：对条纹斑竹鲨肝脏circRNA进行高通量测序和预测，随后设计正、反向引物验证其真实性；构建circRNA过表达载体，将其瞬时转染进肝癌细胞HepG2，进行CCK-8和划痕实验来评价其对肝癌细胞增殖和迁移能力的影响。结果显示：预测到有4 558条circRNAs，并确认了14条circRNAs的真实性；qRT-PCR实验表明在肝癌细胞HepG2中能瞬时过表达circRNA 13-566、circRNA 4-475、circRNA 5-402、circRNA 294-177、circRNA 30-219；且CCK-8和划痕实验显示，这5条circRNAs过表达后，均能不同程度地抑制肝癌细胞的增殖和迁移能力，其中circRNA 4-475、circRNA 294-177作用尤为显著。上述结果为深入研究条纹斑竹鲨肝脏中circRNA及其在肝再生、肝癌治疗方面的功能提供了新思路和基础。

Abstract:

This study aims to identify the circular RNAs (circRNAs) in the liver of whitespotted bamboo shark (Chiloscyllium plagiosum) and to explore the effect of the overexpression of circRNAs on the proliferation and migration of hepatocellular carcinoma HepG2 cells. We conducted high-throughput sequencing for prediction of the circRNAs and then designed forward and reverse primers to verify them. Further, we constructed overexpression vectors for transient transfection of circRNAs into HepG2 cells. Finally, we employed CCK-8 assay and scratch assay to measure the proliferation and migration of the treated HepG2 cells. A total of 4 558 circRNAs were predicted, among which 14 circRNAs were confirmed. The qRT-PCR showed that circRNA 13-566, circRNA 4-475, circRNA 5-402, circRNA 294-177, and circRNA 30-219 were transiently overexpressed in HepG2 cells. The overexpression of these five circRNAs inhibited the proliferation and migration of HepG2 cells to varying degrees, and circRNA 4-475 and circRNA 294-177 had especially notable effect. This study provided a basic database of circRNA genes that particularly active in whitespotted bamboo shark liver and demonstrated with functional studies of these circRNAs potentially involved in normal and malignant liver cells.