Abstract:
Objective To investigate and explorethe function of RNA methylase Wilms' 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 by Western blot.Hepatoblastoma cell lines(HuH6 & HepG2 cells)were stably knocked down with WTAP-siRNA, and were infected with a control lentivirus or a WTAP overexpression lentivirus.Using qPCR and Western 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 by WTAP stable knockdown cell line (701±82.31) mm3 was significantly smaller than the control group (200.2±31.59)mm3, 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