Lack of Period1 accelerates colorectal tumorigeneses in APCmin/+ mice
Description
Clock genes drive the circadian rhythm in each cell, and Period1 (Per1) is one of the core genes in mammals. When the clock genes lose their functions due to deficiency, various behavioral and physiological functions are altered. Although many pathological studies have been conducted on clock genes and cancers, the results could be more consistent. In the present study, we aimed to clarify how the lack of Per1 affects the development and progression of colorectal cancer. We recorded survival days and calculated survival rates, measured the number of polyps, performed histological evaluation, and measured β-catenin expression using ApcMin/+Per1-/- mice. The results showed that loss of Per1 caused variation in the survival rate of mice, increased the number of polyps, and increased β-catenin expression. These results suggest that Per1 plays a role in suppressing the development and progression of colorectal cancer.
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Animals We used ApcMin/+mice, a model animal for familial colorectal adenomatosis, Per1-/-mice lacking the clock gene Per1 (Bae et al., 2001), and ApcMin/+Per1-/-(C57BL/6J background) mice created by crossing them. Recording the number of survival days Survival days of ApcMin/+ and ApcMin/+Per1-/- mice were recorded. The weight of each individual was measured and recorded weekly with an animal balance (Shimadzu Corporation, Kyoto, Japan) starting at 8 weeks of age. Polyp count measurement 150-day-old mice were used, which were close to death due to advanced polyp formation. Mice were euthanized by cervical dislocation, and the intestinal tract from just after the stomach to the anus was removed; after washing with phosphate-buffered saline (PBS), a longitudinal incision was made. The removed intestinal tract was defined as the small intestine from just after the stomach to just before the cecum and colon from just after the cecum to the anus. Polyps less than 2 mm in diameter were defined as small polyps, and polyps greater than 2 mm as large polyps. Histological evaluation Swiss roll is a useful analysis method that allows unbroken observation of the entire intestinal tract on a single slide (Moolenbeek & Ruitenberg, 1981). In the present study, as in the polyp counting, 150-day-old mice were used. After sectioning, each intestine was stained with hematoxylin and eosin (H&E) stain for histological evaluation. In addition, immunofluorescence staining was performed (primary antibody: Beta Catenin Polyclonal antibody, Proteintech Japan, Tokyo, Japan; secondary antibody: Goat anti-Rabbit IgG (H+L) Highly Cross Adsorbed Secondary Antibody, Alexa Fluor 488, Thermo Fisher Scientific). The nuclei were contrast-stained blue with DAPI to confirm the extent of the intestine. Western blotting Western blotting method was used as previously reported (Goto et al., 2020). As in the polyp counting, 150-day-old mice were used. The excised intestine was classified into duodenum, jejunum 1, jejunum 2, ileum, and colon, as in the polyp counting. After homogenization, it was centrifuged at 12,000 g for 15 min (Kubota, Osaka, Japan). The supernatant was collected and the total protein concentration was determined by the Bicinchoninic Acid (BCA) method. used to process and analyze the images. Statistical Analysis Survival days and polyp count for ApcMin/+ mice and ApcMin/+Per1-/- mice are shown as mean ± standard deviation, and Welch's t-test was used for comparison between the two groups. Survival rates of ApcMin/+ mice and ApcMin/+Per1-/- mice were calculated by the Kaplan-Meier method, and survival curves were generated. A log-rank test was performed on the generated survival curves.