摘要
【目的】本研究旨通过细胞学和动物实验验证甲硫氨酸亚砜还原酶A(MSRA)对肾透明细胞癌(ccRCC)增殖、凋亡、侵袭和转移的影响,揭示其潜在的生物学机制,为ccRCC的治疗提供新的分子靶点和策略。【方法】利用转染实验构建MSRA过表达细胞株;通过细胞增殖实验、平板克隆实验、凋亡实验、划痕实验、侵袭实验评估MSRA对ccRCC细胞增殖、凋亡、侵袭和转移的影响;并进一步通过ROS检测,RT-qPCR、Western blot实验探讨其作用机制及相关信号通路;随后,利用裸鼠皮下移植瘤模型在动物水平上验证MSRA对ccRCC肿瘤生长和转移的影响,并初步探索其潜在的分子机制。【结果】OS-RC-2和786-O细胞株MSRA mRNA水平和蛋白表达量较低(RT-qPCR:0.57±0.09,0.56±0.04;WB:0.26±0.13,0.24±0.09);RT-qPCR和Western blot实验提示成功构建了OS-RC-2-pLVSO2-MSRA(RT-qPCR:108.04±1.80;WB:117.01±20.19)和786-O-pLVSO2-MSRA(973.45±51.37,WB:190.34±30.13)过表达细胞株,差异具有统计学意义(P<0.001);增殖实验提示OS-RC-2-pLVSO2-MSRA(72 h:1.246±0.003)和786-O-pLVSO2-MSRA(72 h:1.468±0.001)增殖活力降低,差异具有统计学意义(P<0.001);平板克隆实验提示OS-RC-2-pLVSO2-MSRA(0.090±0.002)和786-O-pLVSO2-MSRA(0.080±0.002)克隆数减少,差异具有统计学意义(P<0.001);凋亡实验提示OS-RC-2-pLVSO2-MSRA(2.013±0.116)和786-O-pLVSO2-MSRA(4.767±0.199)凋亡率增加,差异具有统计学意义(P<0.001);划痕实验提示OS-RC-2-pLVSO2-MSRA(0.643±0.028)和786-O-pLVSO2-MSRA(0.603±0.034)迁移距离较少,差异具有统计学意义(P<0.001);Transwell实验提示OS-RC-2-pLVSO2-MSRA(16.80±2.28)和786-O-pLVSO2-MSRA(21.40±4.78)穿透细胞数减少,差异具有统计学意义(P<0.001);荧光实验显示OS-RC-2-pLVSO2-MSRA(50.59±6.24)和786-O-pLVSO2-MSRA(62.87±5.35)活性氧含量降低,差异具有统计学意义(P<0.001);Western blot实验显示OS-RC-2-pLVSO2-MSRA和786-O-pLVSO2-MSRA中N-cadherin和Vimentin的表达量下降,而E-cadherin的表达量增加,差异具有统计学意义(P<0.001);Western blot实验显示OSRC-2-pLVSO2-MSRA和786-O-pLVSO2-MSRA中p-ERK1/2和p-SMAD3的表达量下降,差异具有统计学意义(P<0.001);动物实验提示OS-RC-2-pLVSO2-MSRA肿瘤体积降低(155.00±50.46),差异具有统计学意义(P<0.001);动物组织Western blot实验提示N-cadherin和Vimentin的表达量下降,而E-cadherin的表达量增加,差异具有统计学意义(P<0.001);OS-RC-2-pLVSO2-MSRA中IHC实验提示Ki-67、N-cadherin和Vimentin的表达量下降,而E-cadherin的表达量增加,差异具有统计学意义(P<0.001)。【结论】表达MSRA抑制ccRCCROS的表达,抑制EMT过程,进而抑制ccRCC的增殖、侵袭和转移。
【Objective】This study aims to investigate the effects of methionine sulfoxide reductase A(MSRA)on the proliferation,apoptosis,invasion,and metastasis of clear cell renal cell carcinoma(ccRCC)through cellular and animal experiments to elucidate its potential biological mechanisms,providing new molecular targets and strategies for the treatment of ccRCC.【Methods】MSRA-overexpressing cell lines were constructed with transfection.The effects of MSRA on the proliferation,apoptosis,invasion,and migration of ccRCC cells were assessed through proliferation assays,colony formation assays,apoptosis assays,wound healing assays,and invasion assays.Further,the mechanisms and related signaling pathways were explored via ROS detection,RT-qPCR,and Western blot.Subsequently,a subcutaneous xenograft mouse model was employed to verify the effect of MSRA on ccRCC tumor growth and metastasis at the animal level to explore its potential molecular mechanisms.【Results】The mRNA and protein expressions of MSRA in OS-RC-2 and 786-O cell lines were low(RT-qPCR:0.57±0.09,0.56±0.04;WB:0.26±0.13,0.24±0.09).RT-qPCR and Western blot experiments confirmed the successful construction of OS-RC-2-pLVSO 2-MSRA(RT-qPCR:108.04±1.80;WB:117.01±20.19)and 786-O-pLVSO 2-MSRA(973.45±51.37;WB:190.34±30.13)overexpressed cell lines,with statistically significant differences(P<0.001).Proliferation assays showed reduced proliferation in OS-RC-2-pLVSO 2-MSRA(72 h:1.246±0.003)and 786-O-pLVSO 2-MSRA(72 h:1.468±0.001),with significant differences(P<0.001).Colony formation assays revealed a decrease in colony numbers in OS-RC-2-pLVSO 2-MSRA(0.090±0.002)and 786-O pLVSO 2-MSRA(0.080±0.002),with significant differences(P<0.001).Apoptosis assays demonstrated increased apoptosis rates in OS-RC-2-pLVSO 2-MSRA(2.013±0.116)and 786-O-pLVSO 2-MSRA(4.767±0.199),with significant differences(P<0.001).Wound healing assays indicated less migration distance in OS-RC-2-pLVSO 2-MSRA(0.643±0.028)and 786-O-pLVSO 2-MSRA(0.603±0.034),with significant differences(P<0.001).Transwell assays showed a reduction in the numbers of penetrative cells in OS-RC-2-pLVSO 2-MSRA(16.80±2.28)and 786-O-pLVSO 2-MSRA(21.40±4.78),with significant differences(P<0.001).Fluorescence assays indicated a decreased ROS levels in OS-RC-2-pLVSO 2-MSRA(50.59±6.24)and 786-O-pLVSO 2-MSRA(62.87±5.35),with significant differences(P<0.001).Western blot analysis showed a decrease in the expression of N-cadherin and Vimentin,and an increase in the expression of E-cadherin in OS-RC-2-pLVSO 2-MSRA and 786-O-pLVSO 2-MSRA(P<0.001).Western blot analysis revealed a significant decrease in the expression levels of p-ERK 1/2 and p-SMAD 3 in OS-RC-2-pLVSO 2-MSRA and 786-O-pLVSO 2-MSRA(P<0.001).Animal experiments showed reduced tumor volumes in OS-RC-2-pLVSO 2-MSRA(155.00±50.46),with significant differences(P<0.001).Western blot analysis of tumor tissues from animals confirmed the decreased expression of N-cadherin and Vimentin,and the increased expression of E-cadherin,with significant differences(P<0.001).IHC experiments of OS-RC-2-pLVSO 2-MSRA tumors revealed a decrease in the expression of Ki-67,N-cadherin,and Vimentin,and an increase in the expression of E-cadherin,with significant differences(P<0.001).【Conclusion】MSRA overexpression inhibits ROS expression in ccRCC,suppresses the EMT process,and consequently inhibits the proliferation,invasion,and metastasis of ccRCC.
作者
刘鸿翔
刘锡海
陈敏坚
钟惟德
LIU Hongxiang;LIU Xihai;CHEN Minjian;ZHONG Weide(Department of Urology,The First People's Hospital of Zhaoqing City,Zhaoqing 526000,China;Department of Urology,Guangzhou First People’s Hospital/The Second Affiliated Hospital of South China University of Technology,Guangzhou 510180,China)
出处
《中山大学学报(医学科学版)》
北大核心
2025年第4期639-650,共12页
Journal of Sun Yat-Sen University:Medical Sciences
基金
肇庆市第一人民医院院内基金(YJJ-2023-02-05)。
关键词
肾透明细胞癌
甲硫氨酸亚砜还原酶A
活性氧
上皮-间充质转化
肿瘤转移
clear cell renal carcinoma
methionine sulfoxide reductase A
reactive oxygen species
epithelial mesenchymal transition
tumor metastasis
