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1.
Figure 2

Figure 2. Increased hepatocyte proliferation and apoptosis in Dicer1-deficient hepatocytes. From: Disruption of Dicer1 induces dysregulated fetal gene expression and promotes hepatocarcinogenesis.

A, D: Immunohistochemistry for phospho-histone H3 and cleaved caspase-3 in 3- (A) and 6-week-old (D) mouse livers. Dotted lines in 6-week-old Albumin-Cre;Dicer1loxP/loxP mouse livers indicate borders between Dicer1-positive hepatocyte nodules (left side) and Dicer1-negative areas (right side). B, C, E, F: Quantification of hepatocyte proliferation and apoptosis in Albumin-Cre;Dicer1loxP/loxP mouse livers (n=4-7, each group). At least 1000 cells were counted for quantification of phospho-histone H3 and cleaved caspase-3-positive hepatocytes. For 6-week-old Albumin-Cre;Dicer1loxP/loxP mice, hepatocytes in Dicer1-positive (grey columns) and –negative (black columns) areas were separately examined. *, P<0.05

Shigeki Sekine, et al. Gastroenterology. ;136(7):2304-2315.e1-4.
2.
Figure 4

Figure 4. Expression of liver-enriched transcription factors, developmentally regulated genes and mir-122 target genes in Dicer1-deficient livers. From: Disruption of Dicer1 induces dysregulated fetal gene expression and promotes hepatocarcinogenesis.

A: Relative expression levels of liver-enriched transcription factors. Quantitative PCR analysis of liver samples from Albumin-Cre;Dicer1loxP/loxP and control littermates at 3 weeks after birth (n=4, each group). B: The expression of developmentally regulated genes in control and Albumin-Cre;Dicer1loxP/loxP mice at 3 weeks (n=5, each group). The expression levels of wild-type mouse livers at different developmental stages were used as references (n=3, each group). C: Expression of putative direct targets of mir-122 in Albumin-Cre;Dicer1loxP/loxP mouse livers (n=4, each group). Values are shown as mean ± S.D. *, P<0.05. Ctrl: Control; Mut: mutant (Albumin-Cre;Dicer1loxP/loxP mice); Fetus: embryonic day 14 fetus; Neo: postnatal day 0 neonate; Adult: postnatal day 42 adult

Shigeki Sekine, et al. Gastroenterology. ;136(7):2304-2315.e1-4.
3.
Figure 5

Figure 5. Development of hepatocellular carcinoma in Albumin-Cre;Dicer1loxP/loxP mice. From: Disruption of Dicer1 induces dysregulated fetal gene expression and promotes hepatocarcinogenesis.

A: Hepatocellular carcinomas in Albumin-Cre;Dicer1loxP/loxP mice at 6 and 12 months after birth. A single tumor was observed in a 6-month-old Albumin-Cre;Dicer1loxP/loxP mouse liver (left, arrow). A 12-month-old Albumin-Cre;Dicer1loxP/loxP mouse liver carried multiple tumors that had replaced the liver parenchyma (right). B: Histological spectrum of hepatocellular carcinomas included thick trabecular arrangement with mild steatosis (top, left), prominent steatosis (top, right) as highlighted by oil red O staining (inset), poorly differentiated tumors with a solid growth pattern (bottom, left) and a pseudoglandular pattern (bottom, right). C: Summary of tumor incidence in Albumin-Cre;Dicer1loxP/loxP mice. Note that 12-month-old mice with tumors include 3 mice that had succumbed to HCCs at 9-11 months of age.

Shigeki Sekine, et al. Gastroenterology. ;136(7):2304-2315.e1-4.
4.
Figure 3

Figure 3. Impaired lipid and glucose metabolism in Albumin-Cre;Dicer1loxP/loxP mice. From: Disruption of Dicer1 induces dysregulated fetal gene expression and promotes hepatocarcinogenesis.

A: Histology and ultrastructure of control and Albumin-Cre;Dicer1loxP/loxP mouse livers. Hepatocytes of control mice displayed uniform eosinophilic cytoplasm and were arranged in a trabecular pattern. The liver architecture was preserved in the Albumin-Cre;Dicer1loxP/loxP mice, but the hepatocytes exhibited small cytoplasmic vacuoles. Electron microscopy analysis of Dicer1-deficient hepatocytes revealed a lack of glycogen granules that are readily observed in control hepatocytes (Gy). Instead, the Dicer1-deficient hepatocytes contained an abundance of lipid droplets (Ld). Lipid accumulation was barely detectable in the control liver, whereas numerous lipid droplets were visible in Dicer1-deficient liver by oil red O staining. PAS staining highlighted glycogen in the control liver, but not in Dicer1-deficient liver. B-E: Lipid analysis of the liver. The lipid composition of the liver tissue was determined following methanol/chloroform extraction. F: Blood glucose analysis. Tail vein blood glucose was measured in a fed condition or after 6 hours of starvation. n=4-5, each group. *, P<0.05; **, P<0.0001

Shigeki Sekine, et al. Gastroenterology. ;136(7):2304-2315.e1-4.
5.
Figure 1

Figure 1. Efficient deletion of Dicer1 in young Albumin-Cre;Dicer1loxP/loxP mouse liver and repopulation with Dicer1-expressing hepatocytes. From: Disruption of Dicer1 induces dysregulated fetal gene expression and promotes hepatocarcinogenesis.

A: Gross morphology of control and Albumin-Cre;Dicer1loxP/loxP mouse livers at 3, 6 and 12 weeks after birth. At 3 weeks, the Albumin-Cre;Dicer1loxP/loxP mouse liver was pale compared with the control. At 6 weeks, the Albumin-Cre;Dicer1loxP/loxP liver had become yellowish, with the appearance of normal-colored spots. The normal-colored areas had expanded at 12 weeks. B: Quantitative PCR analysis of Cre transgene expression in Albumin-Cre;Dicer1loxP/loxP mouse liver. C: Quantitative PCR analysis of Dicer1 expression. Dicer1 expression recovered with age. D: Northern blotting for mir-122. Ethidium bromide staining of 5S RNA was used as a loading control. C: control; M: mutant (Albumin-Cre;Dicer1loxP/loxP). E: In situ hybridization for mir-122. Mir-122 expression was determined in control and Albumin-Cre;Dicer1loxP/loxP mouse livers at 3 and 6 weeks old. Livers from control mice showed diffuse expression of mir-122. Hepatocytes in the 3-week-old Albumin-Cre;Dicer1loxP/loxP mouse liver did not express mir-122 except for a few cells (arrow heads). Nodules of mir-122-positive hepatocytes (arrowheads) appeared in the 6-week-old Albumin-Cre;Dicer1loxP/loxP mouse liver. F: Expression of microRNAs in 3-week-old Albumin-Cre;Dicer1loxP/loxP mouse livers. Relative expression levels of mouse microRNAs were determined in comparison with control mouse livers. MicroRNAs with significantly altered expression (FDR<0.5) are represented by open and black triangles. Four previously reported liver-specific microRNAs are indicated by black triangles. Black dots indicate microRNAs without significant alteration.

Shigeki Sekine, et al. Gastroenterology. ;136(7):2304-2315.e1-4.
6.
Figure 6

Figure 6. Hepatocellular carcinomas in Albumin-Cre;Dicer1loxP/loxP mice are derived from Dicer1-deficient hepatocytes. From: Disruption of Dicer1 induces dysregulated fetal gene expression and promotes hepatocarcinogenesis.

A: Expression of Dicer1 in non-neoplastic and tumor samples of control and Albumin-Cre;Dicer1loxP/loxP mice (n=5-6, each group). mRNA expression was determined by quantitative PCR. Values are mean ± S.D. *, P<0.05. T: Tumor; N: Non-neoplastic liver tissue. B: RT-PCR analysis of Cre transgene expression. Actb served as a positive control. C: Northern blot for mir-122. Ethidium bromide staining of 5S RNA served as a loading control. D: Quantitative PCR analysis of oncofetal genes (n=5-6, each group). E: Expression of phospho-Erk and phospho-Akt in HCCs and Dicer1-deficient livers. Protein samples from HCCs, corresponding non-neoplastic liver, 3-week-old Dicer1-deficient and control livers were treated with the indicated antibodies. F: Model of hepatocarcinogenesis in Albumin-Cre;Dicer1loxP/loxP mice. Albumin-Cre;Dicer1loxP/loxP mice exhibit the efficient deletion of Dicer1 at 3 weeks after birth. However, Dicer1-deficient hepatocytes (red cells) gradually undergo apoptosis and Dicer1-expressing wild-type hepatocytes (pink cells) that have escaped Cre-mediated recombination repopulate the entire liver over time. When a Dicer1-deficient hepatocyte acquires secondary oncogenic stimuli presumably through additional genetic alterations, the absence of Dicer1 cooperatively promotes the development of hepatocellular carcinoma. spectrum of hepatocellular carcinomas included thick trabecular arrangement with mild steatosis (top, left), prominent steatosis (top, right) as highlighted by oil red O staining (inset), poorly differentiated tumors with a solid growth pattern (bottom, left) and a pseudoglandular pattern (bottom, right). C: Summary of tumor incidence in Albumin-Cre;Dicer1loxP/loxP mice. Note that 12-month-old mice with tumors include 3 mice that had succumbed to HCCs at 9-11 months of age.

Shigeki Sekine, et al. Gastroenterology. ;136(7):2304-2315.e1-4.

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