目的 研究线粒体转录因子A(TFAM)对宫颈癌HeLa细胞及骨肉瘤U2OS细胞的线粒体功能、自噬、增殖、侵袭、迁移的影响。方法 HeLa、U2OS细胞转染TFAM小干扰片段(si-TFAM)下调TFAM表达，Mito-Tracker Red CMXRos染色结合激光共聚焦显微镜检测线粒体膜电位(MMP)、MitoSOX™ Red标记法检测线粒体活性氧(mtROS)水平、实时定量PCR检测线粒体DNA(mtDNA)的表达，免疫荧光细胞化学染色检测自噬体数量的变化。Western blot法检测TFAM、微管相关蛋白1轻链3A/B(LC3A/B)、自噬相关基因2A(ATG2A)、ATG2B、ATG9A、锌指转录因子Snail、基质金属蛋白酶2(MMP2)和MMP9的表达。CCK-8法、平板集落形成实验检测细胞增殖，TranswellTM实验、划痕愈合实验检测细胞侵袭、迁移的变化。结果 下调TFAM表达导致HeLa及U2OS细胞MMP减少，mtDNA拷贝数减少，mtROS产生量增加。LC3A/B蛋白含量较对照组明显下降，胞质内自噬体数量明显减少，自噬早期阶段蛋白ATG2B、ATG9A表达量明显减少。HeLa及U2OS细胞Snail、MMP2和MMP9蛋白表达均减少。干扰TFAM表达，抑制HeLa及U2OS细胞的增殖、侵袭、迁移能力。结论 下调TFAM表达抑制线粒体功能，延缓自噬进程，降低宫颈癌细胞及骨肉瘤细胞的增殖、侵袭、迁移能力。
Knockdown mitochondrial transcription factor A(TFAM)inhibits autophagy,proliferation,invasion and migration in human cervical cancer and osteosarcoma cells
Objective To investigate the effects of mitochondrial transcription factor A(TFAM)on mitochondrial function,autophagy,proliferation,invasion,and migration in cervical cancer HeLa cells and osteosarcoma U2OS cells.Methods TFAM small-interfering RNA(si-TFAM)was transfected to HeLa and U2OS cells for downregulating TFAM expression.Mito-Tracker Red CMXRos staining combined with laser confocal microscopy was used to detect mitochondrial membrane potential(MMP).MitoSOXTM Red labeling was used to test mitochondrial reactive oxygen species(mtROS)levels.The expression of mitochondrial DNA(mtDNA)was detected by real-time quantitative PCR.Changes in the number of autophagosomes were detected by immunofluorescence cytochemistry.Western blot analysis was used to detect the expressions of TFAM,autophagy microtubule associated protein 1 light chain 3A/B(LC3A/B),autophagy associated protein 2A(ATG2A),ATG2B,ATG9A,zinc finger transcription factor Snail,matrix metalloproteinase 2(MMP2)and MMP9.CCK-8 assay and plate clony formation assay were used to detect cell proliferation,while TranswellTM assay and scratch healing assay were used to detect changes in cell invasion and migration.Results The downregulation of TFAM expression resulted in a decrease in MMP and mtDNA copy number,but an increase in mtROS production.The protein content of LC3A/B decreased significantly compared to the control group and the number of autophagosomes in the cytoplasm decreased significantly.The expressions of ATG2B and ATG9A in the early stage of autophagy were significantly reduced.The expressions of Snail,MMP2 and MMP9 proteins in HeLa and U2OS cells were also decreased.The proliferation,invasion and migration ability of HeLa and U2OS cells were inhibited after being interfered with TFAM expression.Conclusion Downregulation of TFAM expression inhibits mitochondrial function,delays autophagy process and reduces the proliferation,invasion and migration ability of cervical cancer cells and osteosarcoma cells.
cervical cancer cellosteosarcoma cellmitochondrial transcription factor A(TFAM)autophagymigrationinvasion
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