环状RNA-circ-VCAN在胶质瘤细胞中功能及作用机制研究

Study on the function and mechanism of circular RNA-circ-VCAN in glioma

目的探索环状RNA-circ-VCAN在胶质瘤中功能及作用机制。方法荧光定量PCR验证环状RNA-circ-VCAN在正常胶质细胞以及胶质瘤细胞中的表达情况;核质分离实验验证circ-VCAN在细胞核质分布情况;RNase R消化线性RNA实验验证circ-VCAN对RNase R消化的抵抗能力;构建敲低环状RNA-circ-VCAN的U373和T98G瞬时转染细胞株;CCK8实验检测敲低环状RNA-circ-VCAN后细胞的增殖能力;Western blot检测NF-κB通路蛋白P-P65表达水平。结果与正常胶质细胞(HA1800)相比,环状RNA-circ-VCAN在胶质瘤细胞(尤其是在U373和T98G)中高表达,差异均有统计学意义(P<0.05);细胞核质分离实验证明circ-VCAN主要存在细胞质中;circ-VCAN能抵抗RNase R的消化;CCK8实验证明敲低circ-VCAN能抑制U373和T98G的细胞增殖(P<0.05);Westernblot检测敲低circ-VCAN后的U373和T98G细胞显示NF-κB通路蛋白P-P65表达水平减少。结论在胶质瘤细胞中敲低circ-VCAN能减慢胶质瘤细胞的生长,提示circ-VCAN可能成为一个新的治疗靶点。

Objective To explore the function and mechanism of circular RNA circ-VCAN in glioma. Methods The expression of circular RNA circ-VCAN in normal glial cells and glioma cells was confirmed by fluorescence quantitative PCR. Nucleocytoplasmic separationexperiments verified the distribution of circ-VCAN in the nuclear or cytoplasmic. RNase R linear RNA digestion experiment was used to examine the circ-VCAN resistance to digestion of RNase R. Circ-VCAN knockdown transfected cell lines were constructed. CCK8 was used to detect the cell proliferation ability after knockdown the circVCAN.Western blot was used to detect the expression levels of the NF-κB pathway proteins. Results When compared to the normal glial cells(HA1800)Circular RNA circ-VCAN was highly expressed in glioma cells,the differences were statistically significant(P<0.05). Nucleocytoplasmic separation experiments demonstrated that circ-VCAN mainly existed in the cytoplasm. Circ-VCAN could resist digestion of RNase R. Knock down circ-VCAN could inhibit the proliferation of U373 and T98 G cells(P<0.05).Western blot analysis showed that knockdown of circ-VCAN in U373 and T98 G cells decreased expression of NF-κB pathway protein P-P65. Conclusion Knockdown circ-VCAN in the glioma cells can slow down the growth of glioma cells,suggesting that circ-VCAN may become a new therapeutic target.