长链非编码RNA LINC00662/微小RNA-16-5p/wee1信号通路对肝细胞癌细胞增殖及凋亡的作用研究

Effect of LncRNA LINC00662/miR-16-5p/wee1 signaling pathway on proliferation and apoptosis of hepatocellular carcinoma cells

目的探讨长链非编码RNA linc00662(LncRNA linc00662)通过微小RNA-16-5p(miR-16-5p)[/丝氨酸/苏氨酸蛋白激酶1(wee1)]信号轴在肝细胞癌(HCC)细胞增殖及凋亡中的作用。方法该研究时间为2019年8月至2020年10月,体外培养正常肝细胞株LO2和肝癌细胞株SK-HEP-1、HepG2、Huh-7和Li-7。采用实时荧光定量聚合酶链反应(RT-PCR)检测细胞中linc00662、miR-16-5p和wee1的表达。以HepG2细胞作为研究对象,设置shRNA阴性对照(sh-NC)组、linc00662-shRNA干扰(sh-linc00662)组、抑制剂阴性对照(inhibitor-NC)组、miR-16-5p抑制剂(miR-16-5p inhibitor)组、siRNA阴性对照(si-NC)组、wee1-siRNA干扰(siwee1)组、linc00662-shRNA干扰联合miR-16-5p抑制剂(sh-linc00662+miR-16-5p inhibitor)组和miR-16-5p抑制剂联合wee1-siRNA干扰(miR-16-5p inhibitor+si-wee1)组。CCK-8和克隆形成实验检测细胞增殖活性;流式细胞仪检测细胞凋亡水平;蛋白质印迹法检测凋亡相关蛋白[B细胞淋巴瘤-2基因(Bcl-2)和Bcl-2相关X蛋白基因(Bax)]表达。双萤光素酶报告基因实验检测linc00662、miR-16-5p和wee1之间的相关性。结果PCR结果显示相比较LO2细胞,linc00662在SK-HEP-1、HepG2、Huh-7和Li-7细胞中普遍高表达[0.95±0.14比2.12±0.17、3.86±0.15、2.31±0.14、1.82±0.18,P<0.001]。与sh-NC组相比,敲低linc00662能抑制肝癌细胞增殖(P<0.001)并诱导细胞凋亡(P<0.001)。双萤光素酶报告结果显示linc00662直接靶向结合miR-16-5p并负调控miR-16-5p的表达。同时,miR-16-5p直接靶向结合wee1并负调控wee1的表达。下调miR-16-5p表达可以减轻敲低linc00662对肝癌细胞生长的抑制作用。此外,linc00662可能通过miR-16-5p/wee1通路发挥促癌作用。结论Linc00662是一种在肝癌细胞中上调的LncRNA,可以通过miR-16-5p/wee1轴促进肝癌细胞增殖并抑制细胞凋亡。

Objective To investigate the role of long non-coding RNA(LncRNA)linc00662 in proliferation and apoptosis of hepatocellular carcinoma(HCC)cells through microRNA(miR)-16-5p/wee1 axis.Methods The start and end time of this research was from August 2019 to October 2020.Normal liver cell line(LO2)and HCC cell lines(SK-HEP-1,HepG2,Huh-7 and Li-7)were cultured in vitro.The expressions of linc00662,miR-16-5p and wee1 were detected by RT-PCR.HepG2 cells were used for most of the study.Cells were randomly divided into 6 groups:negative shRNA control(sh-NC),shRNA-scrambled linc00662(sh-linc00662),inhibitor control(inhibitor-NC),miR-16-5p inhibitor,negative siRNA control(si-NC),siRNA-scrambled wee1(si-wee1),shRNA-scrambled linc00662 combined with miR-16-5p(sh-linc00662+miR-16-5p inhibitor)and miR-16-5p inhibitor combined with siRNA-scrambled wee1(miR-16-5p inhibitor+si-wee1)groups.CCK-8 assay and clone formation assay were used to detect the cell proliferation activity.Flow cytometry was performed to analyze the apoptosis level,and western blotting assay was employed to measure the expression of apoptosis-related proteins[B-cell lymphoma 2(Bcl-2)and Bcl-2-associated X protein(Bax)].Dual luciferase reporter gene assay was conducted to determine the relationship among linc00662,miR-16-5p and wee1.Results PCR results showed that compared with LO2 cells,the expression of linc00662 was generally highly expressed in HCC cells(SK-HEP-1,HepG2,Huh-7 and Li-7)[(0.95±0.14)vs.(2.12±0.17)/(3.86±0.15)/(2.31±0.14)/(1.82±0.18)](P<0.001).Compared with the sh-NC group,linc00662 knockdown inhibited HepG2 cell proliferation(P<0.001)and induced cell apoptosis(P<0.001).Luciferase assay demonstrated that linc00662 directly targeted miR-16-5p and negatively regulated the expression of miR-16-5p.Moreover,miR-16-5p could directly target wee1 and negatively regulated the expression of wee1.Subsequently,downregulation of miR-16-5p alleviated the inhibitory effect of linc00662 knockdown on HepG2 cell growth.In addition,linc00662 may play a pro-cancer role through miR-16-5p/wee1 pathway.Conclusion Linc00662 is an upregulated lncRNA in liver carcinoma,which can promote the proliferation and inhibit apoptosis of HCC cells via miR-16-5p/wee1 axis.