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Mitochondrial damage and cholesterol storage in human hepatocellular carcinoma cells with silencing of UBIAD1 gene expression

View Article: PubMed Central - PubMed

ABSTRACT

Heterozygous mutations in the UBIAD1 gene cause Schnyder corneal dystrophy characterized by abnormal cholesterol and phospholipid deposits in the cornea. Ubiad1 protein was recently identified as Golgi prenyltransferase responsible for biosynthesis of vitamin K2 and CoQ10, a key protein in the mitochondrial electron transport chain. Our study shows that silencing UBIAD1 in cultured human hepatocellular carcinoma cells causes dramatic morphological changes and cholesterol storage in the mitochondria, emphasizing an important role of UBIAD1 in mitochondrial function.

No MeSH data available.


Related in: MedlinePlus

Mitochondrial damage in UBIAD1-silent HepG2 cells.(A) Non-transfected control cells showing normal morphology; the mitochondria are relatively abundant and elongated (inset). (B) Control cells transfected with scrambled RNA construct show normal morphology similar to that of non-transfected control cells; the inset shows a mitochondrion at higher magnification. (C), (D) and (E) UBIAD1 shRNA-transfected cell lines (sh65, sh66 and sh67) show an increase in number and a change in shape of the mitochondria, which have signs of swelling (inset). Bar is equal to 2 μm and applies to all figures. Panels show representative images of at least 30 studied for each cell line.
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f0005: Mitochondrial damage in UBIAD1-silent HepG2 cells.(A) Non-transfected control cells showing normal morphology; the mitochondria are relatively abundant and elongated (inset). (B) Control cells transfected with scrambled RNA construct show normal morphology similar to that of non-transfected control cells; the inset shows a mitochondrion at higher magnification. (C), (D) and (E) UBIAD1 shRNA-transfected cell lines (sh65, sh66 and sh67) show an increase in number and a change in shape of the mitochondria, which have signs of swelling (inset). Bar is equal to 2 μm and applies to all figures. Panels show representative images of at least 30 studied for each cell line.

Mentions: The phenotypes of the wild type cells and those with silenced UBIAD1 were examined by light and electron microscopy. Cultured cells were detached from the culture dishes with a rubber policeman, washed with Hanks' balanced salt solution, fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 48 h at 4 °C, post-fixed with potassium ferrocyanide-reduced osmium tetroxide, dehydrated in ethanol and embedded in Epon. Semi-thin (1 μm thick) sections were cut, mounted on slides, stained with toluidine blue, and studied with a Leica DMS light microscope, which revealed extensive vacuolization in the cells treated with shRNA expressing vector, but not in the scrambled RNA-treated cells, or untransfected HepG2 cells (Supplementary Fig. 2). For high-resolution analysis ultrathin (120 nm) sections were mounted on 200-mesh copper grids (Polysciences) and stained with uranyl acetate for 5 min, followed by lead citrate for 2 min. The grids were analyzed on a Tecnai FEI electron microscope. Examination of control non-transfected cells showed round or elongated nuclei, with one or two nucleoli. The double nuclear membrane and the sparse profiles of endoplasmic reticulum (ER) had a normal appearance. The elongated mitochondria had a typical double membrane arrangement with infolded inner membrane. The mitochondria were often seen associated with ER profiles (Fig. 1A). Similar morphology was observed in the scrambled RNA-transfected cells (Fig. 1B).


Mitochondrial damage and cholesterol storage in human hepatocellular carcinoma cells with silencing of UBIAD1 gene expression
Mitochondrial damage in UBIAD1-silent HepG2 cells.(A) Non-transfected control cells showing normal morphology; the mitochondria are relatively abundant and elongated (inset). (B) Control cells transfected with scrambled RNA construct show normal morphology similar to that of non-transfected control cells; the inset shows a mitochondrion at higher magnification. (C), (D) and (E) UBIAD1 shRNA-transfected cell lines (sh65, sh66 and sh67) show an increase in number and a change in shape of the mitochondria, which have signs of swelling (inset). Bar is equal to 2 μm and applies to all figures. Panels show representative images of at least 30 studied for each cell line.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5121353&req=5

f0005: Mitochondrial damage in UBIAD1-silent HepG2 cells.(A) Non-transfected control cells showing normal morphology; the mitochondria are relatively abundant and elongated (inset). (B) Control cells transfected with scrambled RNA construct show normal morphology similar to that of non-transfected control cells; the inset shows a mitochondrion at higher magnification. (C), (D) and (E) UBIAD1 shRNA-transfected cell lines (sh65, sh66 and sh67) show an increase in number and a change in shape of the mitochondria, which have signs of swelling (inset). Bar is equal to 2 μm and applies to all figures. Panels show representative images of at least 30 studied for each cell line.
Mentions: The phenotypes of the wild type cells and those with silenced UBIAD1 were examined by light and electron microscopy. Cultured cells were detached from the culture dishes with a rubber policeman, washed with Hanks' balanced salt solution, fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 48 h at 4 °C, post-fixed with potassium ferrocyanide-reduced osmium tetroxide, dehydrated in ethanol and embedded in Epon. Semi-thin (1 μm thick) sections were cut, mounted on slides, stained with toluidine blue, and studied with a Leica DMS light microscope, which revealed extensive vacuolization in the cells treated with shRNA expressing vector, but not in the scrambled RNA-treated cells, or untransfected HepG2 cells (Supplementary Fig. 2). For high-resolution analysis ultrathin (120 nm) sections were mounted on 200-mesh copper grids (Polysciences) and stained with uranyl acetate for 5 min, followed by lead citrate for 2 min. The grids were analyzed on a Tecnai FEI electron microscope. Examination of control non-transfected cells showed round or elongated nuclei, with one or two nucleoli. The double nuclear membrane and the sparse profiles of endoplasmic reticulum (ER) had a normal appearance. The elongated mitochondria had a typical double membrane arrangement with infolded inner membrane. The mitochondria were often seen associated with ER profiles (Fig. 1A). Similar morphology was observed in the scrambled RNA-transfected cells (Fig. 1B).

View Article: PubMed Central - PubMed

ABSTRACT

Heterozygous mutations in the UBIAD1 gene cause Schnyder corneal dystrophy characterized by abnormal cholesterol and phospholipid deposits in the cornea. Ubiad1 protein was recently identified as Golgi prenyltransferase responsible for biosynthesis of vitamin K2 and CoQ10, a key protein in the mitochondrial electron transport chain. Our study shows that silencing UBIAD1 in cultured human hepatocellular carcinoma cells causes dramatic morphological changes and cholesterol storage in the mitochondria, emphasizing an important role of UBIAD1 in mitochondrial function.

No MeSH data available.


Related in: MedlinePlus