WTAP在肝母细胞瘤增殖和进展中的作用

Role of WTAP in the development and progression of hepatoblastoma

目的:探讨RNA甲基化酶WTAP在肝母细胞瘤细胞中的生物学功能。方法:收集2016年8月至2020年6月同济大学附属第十人民医院手术治疗的肝母细胞瘤患者的肿瘤组织和正常对照组织13对,采用Western blot检测组织中WTAP的表达,在肝母细胞瘤细胞系中转染siRNA敲减WTAP,慢病毒包装WTAP过表达载体WTAP过表达,使用q PCR和Western blot检测敲减和过表达效率,采用CCK8实验检测细胞增殖活性,克隆形成实验检测细胞克隆形成能力,流式细胞术检测细胞凋亡。裸鼠皮下成瘤实验检测细胞在体内的成瘤能力。结果:WTAP在肝母细胞瘤组织中高表达(P=0.0007);Huh6细胞(P=0.0002)和HepG2细胞(P=0.0030)中的WTAP表达水平与QSG-7701细胞相比显著升高,Huh6细胞(P<0.0001)和HepG2细胞(P=0.0035)中的WTAP表达水平与HL-7702细胞相比也显著升高。在肝母细胞瘤细胞中敲减WTAP后,细胞增殖活性和克隆形成能力受到显著抑制,而过表达WTAP则显著增强了细胞增殖活性和克隆形成能力。流式细胞术检测结果显示,Huh6细胞MOCK组的凋亡细胞百分比(10.93±0.2603)%显著低于siWTAP-1组[(16.43±0.6333)%,P=0.0013]和siWTAP-2组[(20.27±0.7535)%,P=0.0003],HepG2细胞MOCK组的凋亡细胞百分比(4.733±0.1764)%显著低于siWTAP-1组[(14.33±0.2728)%,P<0.0001]和siWTAP-2组[(15.33±0.2728)%,P<0.0001]。WTAP稳定敲减细胞株形成的移植瘤的体积(701±82.31)mm^(3)显著小于对照组[(200.2±31.59)mm^(3),P=0.0001];WTAP稳定敲减细胞株形成的移植瘤的重量(0.6368±0.08391)g显著小于对照组[(0.1356±0.03329)g,P<0.0001];WTAP稳定敲减细胞株形成的移植瘤的Ki-67阳性细胞数(108±13.05)显著少于对照组[(406±14.73),P=0.0001]。结论:WTAP在肝母细胞瘤细胞中发挥着促进增殖抑制凋亡的重要生物学功能,可能是肝母细胞瘤诊疗的一个潜在靶点。

Objective:To investigate and explorethe function of RNA methylaseWilms'tumor 1-associating protein(WTAP)in hepatoblastoma tumor development.Methods:From August 2016 to June 2020,13 pairs of tumor tissues and normal control tissues were collected from patients with hepatoblastoma underwent surgery in Shanghai Tenth People's Hospital of Tongji University.The expression of WTAP was detected byWestern blot.Hepatoblastoma cell lines(HuH6&HepG2 cells)were stably knocked down withWTAP-siRNA,and were infected with a control lentivirus or a WTAP overexpression lentivirus.Using qPCR andWestern blot methods to detect knockdown and overexpression efficiency.Similarly,the effect on the cell proliferation and colony formation was determined using the CCK8 and colony formation assays respectively.And apoptosis was detected by flow cytometry assays.To further evaluate the effect of WTAP on tumorigenesis we performed the in vivo xenograft tumorigenicity test on BALB/C nude mice using the stably WTAP knockdown HB cell lines.Results:WTAP is highly expressed in hepatoblastoma tissues(P=0.0007).The WTAP expression level in Huh6 cells(P=0.0002)and HepG2 cells(P=0.0030)was significantly higher than that in QSG-7701 cells,and the WTAP expression level in Huh6 cells(P<0.0001)and HepG2 cells(P=0.0035)was also significantly higher compared with HL-7702 cells.After knocking down WTAP in hepatoblastoma cells,cell proliferation activity and clone formation ability were significantly inhibited,and the overexpression of WTAP significantly enhanced the cell proliferation and clone formation ability.The results of flow cytometry showed that the percentage of apoptotic cells in the MOCK group of Huh6 cells(10.93±0.2603)%was significantly lower than that of the siWTAP-1 group[(16.43±0.6333)%,P=0.0013]and siWTAP-2 group[(20.27±0.7535)%,P=0.0003],and the percentage of apoptotic cells in the MOCK group of HepG2 cells(4.733±0.1764)%was significantly lower than that of the siWTAP-1 group[(14.33±0.2728)%,P<0.0001]and siWTAP-2 group[(15.33±0.2728)%,P<0.0001].The volume of transplanted tumor formed byWTAP stable knockdown cell line(701±82.31)mm^(3)was significantly smaller than the control group[(200.2±31.59)mm^(3),P=0.0001],and the weight of xenografts formed by the WTAP stable knockdown cell line(0.6368±0.08391)g was significantly less than that of the control group[(0.1356±0.03329)g,P<0.0001].The number of Ki-67-positive cells(108±13.05)in transplanted tumors formed by WTAP stable knockdown cell lines was significantly less than that of the control group[(406±14.73),P=0.0001].Conclusions:WTAP plays a crucial role in hepatoblastoma development and progression via regulating the biological and molecular function of cell.Thus,WTAP may be potential biological target in diagnosis and treatment of hepatoblastoma patients.