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Research Article
Runjie Sun
Shandong University of Traditional Chinese Medicine Affiliated Hospital
Corresponding Author
ORCiD: https://orcid.org/0000-0001-6924-4140
License:
This work is licensed under a CC BY 4.0 License. Read Full License
A patient was admitted to the hospital with thymoma, branchio-oto syndrome and pure red aplastic anemia. He had both Six1 and mitochondrial mutations. The Six1 gene has been linked to branchio-oto-renal spectrum disorder and malignant tumors. Additionally, mitochondrial mutations are known to cause anemia as well. To this end, we designed experiments to explore the relationship between Six1 mutations, mitochondrial function and these diseases.
Whole-exome sequencing (WES) was used for gene detection, and transfected 293T cells were used for in vitro experiments. CCK-8 and flow cytometry were used to detect cell proliferation, cell cycle stage and apoptotic populations. Caspase-3 activity was used as an apoptosis indicator. The effects on mitochondria were examined by ROS and JC-1. Western blotting was used to detect the expression of Bcl-2, Bax and cleaved caspase-3.
WES revealed a Six1c.374A > G point mutation in the proband and deaf members of his family, as well as, a mitochondrial mutation (ND3) in the proband. 293T cells transfected with Six1 were used as the WT group, the Six1c.374A > G transfection group was used as the MUT group, and the untransfected 293T cells were used as the NC group. A CCK-8 test found that the cell proliferation rates were reduced both in the WT group and the MUT group, and MUT group cells were found to be blocked in S-phase. Due to these findings, apoptosis was investigated. Following investigation, increased levels of ROS, caspase-3 activity and cleaved caspase-3 were detected in WT and MUT groups. Following these results, mitochondrial function was measured. The level of mitochondrial membrane potential in WT and MUT groups was decreased significantly. Western blot showed that and the Bcl-2/Bax ratio decreased.
Six1c.374A > G point mutation can affect cell proliferation through increased apoptosis controlled by the mitochondrial apoptotic pathway. This could be the cause of the patient's pure red cell aplasia.
Keywords
mutation, SIX1, mitochondrial, Branchio-oto syndrome, thymoma, pure red cell aplasia
Figure 1
Figure 2
Figure 3
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFigure1.tif
Supplementary Figure1. Full-length blots of Figure 2.c
- SupplementaryFigure2.tif
Supplementary Figure2 Full-length blots of Figure 3.d
- SupplementaryFigure3.tif
Supplementary Figure3 Cell were incubated with DCFH-DA for 30 min, and DCF fluorescence was detected at 0h, 12h, 24h and 48h.
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research Article
Runjie Sun
Shandong University of Traditional Chinese Medicine Affiliated Hospital
Corresponding Author
ORCiD: https://orcid.org/0000-0001-6924-4140
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 03 May, 2021
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Molecular Biology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
A patient was admitted to the hospital with thymoma, branchio-oto syndrome and pure red aplastic anemia. He had both Six1 and mitochondrial mutations. The Six1 gene has been linked to branchio-oto-renal spectrum disorder and malignant tumors. Additionally, mitochondrial mutations are known to cause anemia as well. To this end, we designed experiments to explore the relationship between Six1 mutations, mitochondrial function and these diseases.
Whole-exome sequencing (WES) was used for gene detection, and transfected 293T cells were used for in vitro experiments. CCK-8 and flow cytometry were used to detect cell proliferation, cell cycle stage and apoptotic populations. Caspase-3 activity was used as an apoptosis indicator. The effects on mitochondria were examined by ROS and JC-1. Western blotting was used to detect the expression of Bcl-2, Bax and cleaved caspase-3.
WES revealed a Six1c.374A > G point mutation in the proband and deaf members of his family, as well as, a mitochondrial mutation (ND3) in the proband. 293T cells transfected with Six1 were used as the WT group, the Six1c.374A > G transfection group was used as the MUT group, and the untransfected 293T cells were used as the NC group. A CCK-8 test found that the cell proliferation rates were reduced both in the WT group and the MUT group, and MUT group cells were found to be blocked in S-phase. Due to these findings, apoptosis was investigated. Following investigation, increased levels of ROS, caspase-3 activity and cleaved caspase-3 were detected in WT and MUT groups. Following these results, mitochondrial function was measured. The level of mitochondrial membrane potential in WT and MUT groups was decreased significantly. Western blot showed that and the Bcl-2/Bax ratio decreased.
Six1c.374A > G point mutation can affect cell proliferation through increased apoptosis controlled by the mitochondrial apoptotic pathway. This could be the cause of the patient's pure red cell aplasia.
Figure 1
Figure 2
Figure 3
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFigure1.tif
Supplementary Figure1. Full-length blots of Figure 2.c
- SupplementaryFigure2.tif
Supplementary Figure2 Full-length blots of Figure 3.d
- SupplementaryFigure3.tif
Supplementary Figure3 Cell were incubated with DCFH-DA for 30 min, and DCF fluorescence was detected at 0h, 12h, 24h and 48h.
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