Cellular Localization,Expression Patterns of CCAR1 Gene,and Its Effect on Cell Proliferation in Pig |
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| Authors: | ZHANG Ningfang WU Yiqi CHENG Zhimin YANG Xiaowei LI Meng YANG Yang LIU Hong GAO Pengfei CAI Chunbo GUO Xiaohong LI Bugao CAO Guoqing |
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| Institution: | 1. College of Animal Science, Shanxi Agricultural University, Taigu 030801, China;2. Shanxi Academy of Advanced Research and Innovation, Taiyuan 030032, China;3. Datong Customs Technology Center, Datong 037000, China;4. Datong Pig Breeding Farm, Datong 037000, China |
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| Abstract: | The aim of this study was to obtain the complete coding sequence (CDS) of CCAR1 gene, and to explore its subcellular localization, expression profile and its effects on cell prolification and action mechanism in pig. In this study, the cDNA from kidney tissue of 1-day-old Mashen pigs were used as the template to obtain the full-length CDS of CCAR1 gene by RT-PCR and sequencing. The cellular immunofluorescence staining was used to explore the subcellular localization of CCAR1 in PK15 cells. The temporal and spatial expression profile of CCAR1 was investigated by qRT-PCR in this study. The CCAR1 gene in PK15 cells was knocked out by using CRISPR/Cas9 gene editing technology, and the effects of CCAR1 gene on cell proliferation and expression of cell proliferation and apoptosis related genes were investigated by qRT-PCR, Western blot and CCK8 (cell counting kit 8) technologies in this experiment. The results showed that the complete CDS region of pig CCAR1 gene was 3 459 bp in length (MH301308.1). CCAR1 protein was localized in both cytoplasm and nucleus of PK15 cells. The expression profiles of CCAR1 mRNA between Large White and Mashen pigs was similar, which was expressed in all detected tissues, with the highest expression in kidney and small intestine, middle expression in spleen, liver, cerebellum and muscle, and the lowest expression in heart and subcutaneous fat. Temporal expression results showed that CCAR1 was expressed in both psoas muscle and longissimus dorsi muscle at 3 developmental stages both in Mashen and Large White pigs. The CRISPR/Cas9 gene editing system effectively reduced the expression of CCAR1. CCK8 results showed that after 48 hours of transfection, compared with the control group, the proliferation of cells in the experimental groups were extremely significantly inhibited (P<0.01). After CCAR1 gene knocked out, the expression level of Mki67, a marker of cell proliferation, was significantly decreased (P<0.05). There was no significant difference in the expression level of Caspase3 and the core protein β-catenin in Wnt pathway between control group and experimental groups, and the expression level of downstream target gene C-myc of Wnt pathway was decreased significantly(P<0.05). CCAR1 gene was expressed almost in all tissues at different developmental stages, and affected cell proliferation by regulating the expression levels of Mki67 and C-myc, and played an important role in growth and development of pig. |
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| Keywords: | pig CCAR1 gene cellular localization expression patterns gene knockout cell proliferation |
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