Liu X L, Sun C F, Yuan Y, Sheng Y J, Deng C L
Department of Infectious Diseases, Affiliated Hospital of Southwest Medical University, Laboratory of Infection and Immunology, Luzhou 646000, China.
Zhonghua Gan Zang Bing Za Zhi. 2022 Oct 20;30(10):1074-1081. doi: 10.3760/cma.j.cn501113-20210819-00413.
To explore the effect of MicroRNA 424-5p/Kinesin family member 23(miR-424-5p/KIF23)axis on the malignant phenotype of hepatoma cells and its sensitivity of sorafenib. Real-time quantitative reverse PCR(qRT-PCR) and/or Western blot were used to detect the expression of miR-424-5p and KIF23 in liver cancer tissues and paracancerous tissues, human hepatocellular carcinoma(HCC) cells HepG2 and normal hepatocyte LO2. HepG2 cells transfected with miR-424-5p mimic and miR-424-5p mimic NC were respectively defined as miR-424-5p mimic group and mimic NC group, HepG2 cells transfected with KIF23 overexpression vector pcDNA3.1-KIF23 or empty vector pcDNA3.1 respectively were defined as OE-KIF23 group and Vector group, and HepG2 cells co-transfected with miR-424-5p mimic and overexpression vector pcDNA3.1-KIF23 were defined as mimic+OE-KIF23 group: The KIF23-3'UTR wild-type vector (KIF23-WT) and the mutant vector (KIF23-MT) were co-transfected with miR-424-5p micic and mimic NC, respectively, and the targeting relationship between miR-424-5p and KIF23 was verified by dual-luciferase reporting experiments. The cell counting Kit-8 (CCK-8) was used to detect HepG2 cell proliferation and sensitivity to sorafenib. Flow cytometry was used to assess apoptosis in HepG2 cells. Transwell and scratch experiments were used to detect HepG2 cell migration and invasion capabilities. Intergroup data were compared using -tests or analysis of variance. Compared with the paracancerous tissue and normal hepatocytes, miR-424-5p in the HCC tissue and hepatocellular cells was significantly down-regulated (the relative expression was 0.604±0.121, 0.585±0.064), and KIF23 was significantly up-regulated (the relative expression was 5.451±1.834, 2.482±0.545), <0.05. miR-424-5p mimic can inhibit the proliferation, migration and invasion of HCC cells and promote apoptosis of HCC cells (<0.05). Overexpression of KIF23 can promote the proliferation, migration and invasion of HCC cells and inhibit apoptosis of HCC cells (<0.05). The luciferase activity of HepG2 cells in the mimic and KIF23-WT co-transfection groups was significantly reduced compared with HepG2 cells in the mimic NC and KIF23-WT co-transfection groups (the relative fluorescence intensities were 3.668±0.091 and 2.629±0.056, respectively, <0.05),however, there was no significant comparison between the luciferase activity of cells in the mimic and KIF23-MT co-transfection groups compared with those in the mimic NC and KIF23-MT co-transfection groups. miR-424-5p mimic can reverse the role of overexpression of KIF23 in promoting the ability of HCC cells to proliferate, migrate and invade (<0.05). The inhibition rates of sorafenib on HepG2 cells in the mimic+OE-KIF23 group and the OE-KIF23 group were 47.491%±3.863% and 36.246%±6.063% (=3.027, <0.05). miR-424-5p can inhibit the proliferation, migration and invasion of HCC cells and can increase the sensitivity of HCC cells to sorafenib by targeting the expression level of KIF23.
探讨微小RNA 424-5p/驱动蛋白家族成员23(miR-424-5p/KIF23)轴对肝癌细胞恶性表型及其对索拉非尼敏感性的影响。采用实时定量逆转录聚合酶链反应(qRT-PCR)和/或蛋白质免疫印迹法检测肝癌组织及癌旁组织、人肝癌(HCC)细胞HepG2和正常肝细胞LO2中miR-424-5p和KIF23的表达。将转染miR-424-5p模拟物和miR-424-5p模拟物阴性对照(NC)的HepG2细胞分别定义为miR-424-5p模拟物组和模拟物NC组,将分别转染KIF23过表达载体pcDNA3.1-KIF23或空载体pcDNA3.1的HepG2细胞定义为OE-KIF23组和载体组,将共转染miR-424-5p模拟物和过表达载体pcDNA3.1-KIF23的HepG2细胞定义为模拟物+OE-KIF23组。将KIF23-3'UTR野生型载体(KIF23-WT)和突变载体(KIF23-MT)分别与miR-424-5p模拟物和模拟物NC共转染,通过双荧光素酶报告实验验证miR-424-5p与KIF23的靶向关系。采用细胞计数试剂盒-8(CCK-8)检测HepG2细胞增殖及对索拉非尼的敏感性。采用流式细胞术评估HepG2细胞凋亡情况。采用Transwell和划痕实验检测HepG2细胞迁移和侵袭能力。组间数据比较采用t检验或方差分析。与癌旁组织和正常肝细胞相比,HCC组织和肝癌细胞中miR-424-5p显著下调(相对表达量分别为0.604±0.121、0.585±0.064),KIF23显著上调(相对表达量分别为5.451±1.834、2.482±0.545),P<0.05。miR-424-5p模拟物可抑制HCC细胞增殖、迁移和侵袭,并促进HCC细胞凋亡(P<0.05)。KIF23过表达可促进HCC细胞增殖、迁移和侵袭,并抑制HCC细胞凋亡(P<0.05)。模拟物与KIF23-WT共转染组HepG2细胞的荧光素酶活性与模拟物NC与KIF23-WT共转染组相比显著降低(相对荧光强度分别为3.668±0.091和2.629±0.056,P<0.05),然而,模拟物与KIF23-MT共转染组细胞的荧光素酶活性与模拟物NC与KIF23-MT共转染组相比无显著差异。miR-424-5p模拟物可逆转KIF23过表达对HCC细胞增殖、迁移和侵袭能力的促进作用(P<0.05)。模拟物+OE-KIF23组和OE-KIF23组中索拉非尼对HepG2细胞的抑制率分别为47.491%±3.863%和36.246%±6.063%(t=3.027,P<0.05)。miR-424-5p可通过靶向KIF23的表达水平抑制HCC细胞增殖、迁移和侵袭,并可提高HCC细胞对索拉非尼的敏感性。