miR-548b-3p通过调控诱骗受体3的表达影响肝癌细胞恶性生物学性状的研究

miR-548b-3p regulates the expression of decoy receptor 3 and influences the malignant biological traits of hepatoma cells

目的探讨miR-548b-3p是否可以通过下调诱骗受体3(DcR3)的表达进而调节肝癌细胞的恶性生物学性状。方法通过实时荧光定量PCR检测Hep3b、HepG2、Huh7.5.1、PLC、97H等5种常见肝癌细胞系中miR-548b-3p的水平,实验用肝癌细胞系购自上海市肿瘤研究所。裸鼠皮下成瘤性实验包括miR-548b-3p过表达和低表达两部分:miR-548b-3p过表达实验包括Huh7.5.1-miR-548b-3p细胞组和Huh7.5.1-NC细胞组;miR-548b-3p低表达实验组包括PLC-miR-548b-3p-sponge组和PLC-NC细胞组,每组分别5只裸鼠。实验用裸鼠为雄性Balb/c品系,体重20~25 g,4~6周龄。利用Starbase软件预测miR-548b-3p与DcR3是否存在结合位点;双荧光素酶报告实验验证DcR3是否为miR-548b-3p的靶基因。通过拯救实验验证miR-548b-3p对肝癌细胞增殖、侵袭和迁移能力的影响是否通过DcR3来实现。采用Graphpad Prism 9进行统计分析,符合正态分布的计量资料以均数±标准差(x^(-)±s)表示,组间比较采用t检验。结果实时荧光定量PCR以2-ΔΔCt相对表达量为参数进行比较,假设Hep3b相对表达量为1,HepG2相对表达量为0.902,Huh7.5.1相对表达量为0.712,PLC相对表达量为1.293,97H相对表达量为0.818,最后结果表明,miR-548b-3p表达最高的是PLC细胞,表达最低的是Huh7.5.1细胞;裸鼠皮下成瘤性实验表明,实验4周后,Huh7.5.1-miR-548b-3p组成瘤体积为(444.77±142.34)mm^(3),Huh7.5.1-NC组成瘤体积为(918.80±139.21)mm^(3);PLC-miR-548b-3p-sponge组成瘤体积为(407.49±58.50)mm^(3),PLC-NC组成瘤体积为(218.62±47.55)mm^(3);利用Starbase软件预测到miR-548b-3p与DcR3存在结合位点;双荧光素酶报告实验证明DcR3是miR-548b-3p的靶基因;拯救实验验证miR-548b-3p对肝癌细胞增殖、侵袭和迁移能力的影响是通过DcR3来实现的。结论miR-548b-3p可调节肝癌细胞的增殖、侵袭和迁移等恶性生物学性状,并且是通过下调DcR3的表达水平来实现的。

Objective:To investigate whether miR-548b-3p can regulate the malignant biological traits of hepatocellular carcinoma cells by down-regulating the expression of decoy receptor 3(DcR3).Methods:The miR-548b-3p levels in 5 common hepatoma cell lines Hep3b,HepG2,Huh7.5.1,PLC and 97H were detected by real-time fluorescence quantitative PCR.The experimental hepatoma cell lines were purchased from Shanghai Cancer Institute.The subcutaneous tumor formation experiment in nude mice included two parts:miR-548b-3p over expression group and down expression group.miR-548b-3p over expression experiment included Huh7.5.1-miR-548b-3p cell group and Huh7.5.1-NC cell group.The experimental group with low expression of miR-548b-3p included PLC-miR-548b-3p-sponge group and PLC-NC cell group,with 5 nude mice in each group.The nude mice used in the experiment were male Balb/c strain,body weight 20-25 g,4-6 weeks old.Starbase software was used to predict whether there were binding sites between miR-548b-3p and DcR3.Dual luciferase reporting assay to verify whether DcR3 is the target gene of miR-548b-3p.Rescue experiments were conducted to verify whether the effects of miR-548b-3p on the proliferation,invasion and migration of liver cancer cells were realized through DcR3.Statistical analysis was performed using Graphpad Prism 9.Measurement data with normal distribution were expressed as mean±standard deviation(x^(-)±s),and t-test was adopted for comparison between general measurement data groups.Results:The relative expression level of 2-ΔΔCt was used as the parameter for real-time fluorescence quantitative PCR comparison.Hep3b expression level was 1,HepG2 expression level was 0.902,Huh7.5.1 expression level was 0.712,PLC expression level was 1.293,and 97H expression level was 0.818.The final results showed that,The highest expression of miR-548b-3p was in PLC cells,and the lowest expression was in Huh7.5.1 cells.The subcutaneous tumor formation experiment of nude mice showed that after 4 weeks,the tumor volume of Huh7.5.1-miR-548b-3p was(444.77±142.34)mm 3,and that of Huh7.5.1-NC was(918.80±139.21)mm 3.The tumor volume of PLC-miR-548b-3p-sponge was(407.49±58.50)mm 3,and that of PLC-NC was(218.62±47.55)mm 3.The binding sites of miR-548b-3p and DcR3 were predicted by Starbase software.Dual luciferase assay showed that DcR3 was the target gene of miR-548b-3p.Rescue experiments verified that the effects of miR-548b-3p on the proliferation,invasion and migration of liver cancer cells were realized through DcR3.Conclusion:miR-548b-3p can regulate the proliferation,invasion,migration and other malignant biological traits of liver cancer cells,and it is achieved by down-regulating the expression level of DcR3.