Effect of ATAD3A gene silencing on the proliferation, invasion and migration of A375 human melanoma cells and its mechanisms
- VernacularTitle:沉默ATAD3A对黑素瘤A375细胞增殖、侵袭和迁移的影响及机制研究
- Author:
Mao LUO
1
;
Zhuofu LUO
;
Jianjun BI
;
Yang XU
Author Information
- From: Chinese Journal of Dermatology 2020;53(7):539-545
- CountryChina
- Language:Chinese
- Abstract: Objective:To evaluate the effect of ATPase family AAA-domain containing protein 3A (ATAD3A) gene silencing on the proliferation, invasion and migration of A375 human melanoma cells.Methods:From August to December in 2019, melanoma and paracancerous tissues were collected from 3 patients with pathologically diagnosed melanoma in People′s Hospital of Chongqing Yubei District, and Western blot analysis was performed to measure the protein expression of ATAD3A in the above tissues. Cultured A375 human melanoma cells were divided into 2 groups to be infected with a lentiviral vector carrying shATAD3A (shATAD3A group) and an empty vector (shCtrl group) respectively, and real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis were performed to verify the interference efficiency. Cell counting kit-8 (CCK8) assay and colony formation assay were performed to compare cell proliferative ability and colony-formation ability respectively between the 2 groups, and Transwell invasion assay and wound healing assay to compare invasive and migratory abilities respectively between the above 2 groups. Western blot analysis was performed to determine the expression of cell self-renewal-related proteins (NANOG, SRY-related high-mobility-group box protein SOX2, octamer-binding protein 4[OCT4]) and invasion- and migration-related proteins (matrix metalloproteinase 2[MMP2], vimentin, zinc-finger transcription factor SLUG) in the 2 groups. Two-independent-sample t test was used to compare the experimental indices between the 2 groups. Results:Western blot analysis showed that ATAD3A was significantly highly expressed in the 3 melanoma tissues compared with the paracancerous tissues ( t = 10.825, P < 0.001) . qRT-PCR and Western blot analysis showed that the mRNA and protein expression of ATAD3A in A375 cells was significantly lower in the shATAD3A group (0.230 ± 0.073, 0.279 ± 0.267, respectively) than in the shCtrl group (1.000 ± 0.244, 0.867 ± 0.115, respectively; t = 9.461, 8.595, respectively; P < 0.001 or = 0.002) , indicating that the ATAD3A gene-silenced A375 cell line was successfully constructed. Colony formation assay revealed that the colony-formation rate was significantly lower in the shATAD3A group than in the shCtrl group (22.667% ± 2.510% vs. 43.667% ± 5.030%, t = 6.464, P = 0.003) , and CCK-8 assay showed that the cellular proliferative activity significantly decreased from day 2 to day 4 in the shATAD3A group compared with the shCtrl group. Wound healing assay showed significantly slower wound healing and decreased wound healing rate from the 12 th hour (32.920% ± 4.642% vs. 49.302% ± 1.448%, t = 5.835, P = 0.004) to the 24 th hour in the shATAD3A group compared with the shCtrl group, and Transwell invasion assay revealed significantly decreased number of invasive cells in the lower Transwell chambers in the shATAD3A group compared with the shCtrl group (68.330 ± 13.050 vs. 234.330 ± 19.139, t = 12.411, P < 0.001) . Western blot analysis showed that the protein expression of NANOG, SOX2, OCT4, MMP2, vimentin, SLUG was significantly lower in the shATAD3A group than in the shCtrl group ( P < 0.05 or 0.001) . Conclusion:ATAD3A is highly expressed in melanoma tissues, and ATAD3A gene silencing can inhibit the proliferation, invasion and migration abilities of melanoma A375 cells.
