Effect of the Porcine STC-1 Gene on Autophagy and Mitochondrial Function as induced by Serum Starvation&nbsp;
Authors: Wang Hao , Yang Yuefei , Wang Yi , Peng Yuehan , Hu Yuemin , Ju Huiming , Xu Lei 
Published: February 2021

Abstract
Stanniocalcin-1 (STC-1) is a glycoprotein hormone involved in calcium/phosphorus metabolism and direct inhibition of bone and muscle growth. The aim of this study was to investigate the STC-1 gene with respect to the regulatory mechanisms of porcine growth metabolic pathways involving autophagy. PK15 cells were used in this study as control group (CON) cells, and STC-1 gene-knockout PK15 (STC-KO) cells constituted the experimental group. These two groups of cells were subjected to serum starvation to induce autophagy (SCON and SSTC-1 groups). Western blotting was used to detect the expression of autophagy-and mitochondrial function-related proteins, and flow cytometry was used to detect apoptotic cells. Changes in the autophagosome and mitochondrial structure and morphology were observed by electron microscopy. The expression of the autophagy-related proteins LC3B and Parkin was detected by laser scanning confocal microscopy. The results showed that in the CON group and STC-KO group after serum starvation, Pink1 and Parkin expression levels were increased to an extremely high level of significance (P <0.01); LC3B expression was significantly increased (P <0.05); and SQSTM1/P62 expression was increased at an extremely significant level (P < 0.01). Electron microscopy revealed that the cells in the serum starvation treatment group all produced autophagosomes. The fluorescence intensity of GFP-LC3B and GFP-Parkin increased significantly (P<0.05), indicating that autophagy was successfully induced by serum starvation. After serum starvation, compared to the levels in the CON group, the Bax/Bcl-2 ratio, Pink1 and Parkin protein levels were profoundly reduced in the STC-KO group, at an extremely high level of significance (P<0.01). In addition, under serum starvation, the increase in Mfn2, OPA1, and DRP1 proteins was attenuated; the increase in LC3B protein content was significantly attenuated (P<0.05); the decrease in LC3B protein content was significantly attenuated (P<0.05); the increase in the apoptosis rate was significantly attenuated (P<0.05); the decrease in membrane potential was extremely significant (P<0.01); the increased amount of active oxygen was significantly attenuated (P<0.05); the decrease in ATP was reversed, and this increase was extremely significant (P<0.01); the fluorescence intensity of GFP-LC3B was increased; and the increased fluorescence intensity of GFP-Parkin was attenuated significantly (P<0.05). These results indicate that autophagy can be caused by serum starvation. Knocking out the porcine STC-1 gene had an obvious-antiapoptotic effect on cells. The inhibition of serum starvation induced autophagy. This is the first study to show that the porcine STC-1 gene confers self-protection in the absence of nutrients, laying the foundation for studying the related mechanism. Knockout cells were used for further study of STC-1 to provide a theoretical basis for studying the effect of STC-1 on pig growth and development.