Effects of the CDC10 (Septin 7) Gene on the Proliferation and Differentiation of Bovine Intramuscular Preadipocyte and 3T3-L1 Cells

Simple Summary Intramuscular fat content and marbling affecting meat quality are important economic traits in beef cattle. CDC10 (cell division cycle 10 or Septin 7), a member of the septin family involved in cellular proliferation, was considered as a functional and positional candidate gene for beef marbling. In the present study, we found that overexpression of CDC10 could promote the differentiation of bovine intramuscular preadipocyte and 3T3-L1 cells, whereas knockdown of CDC10 resulted in the opposite consequences. Our results provide new insights into the regulatory roles of CDC10 in adipocytes in animals. Abstract Intramuscular fat content and marbling affecting meat quality are important economic traits in beef cattle. CDC10 (cell division cycle 10 or Septin 7), a member of the septin family involved in cellular proliferation, was considered as a functional and positional candidate gene for beef marbling. In a previous study, we revealed that the expression levels of CDC10 were also positively correlated with marbling scores in Japanese Black cattle. However, the regulatory mechanism of the CDC10 gene on IMF deposition in cattle remains unclear. In the present study, flow cytometry, EdU proliferation assays, and Oil Red O staining results showed that overexpression of CDC10 could promote the differentiation of bovine intramuscular preadipocyte (BIMP) and 3T3-L1 cells, whereas knockdown of CDC10 resulted in the opposite consequences. Furthermore, quantitative PCR and Western blotting results showed that overexpression of CDC10 could promote the expression levels of adipogenic marker genes PPARγ and C/EBPα at both mRNA and protein levels in BIMP and 3T3-L1 cells, whereas knockdown of CDC10 resulted in the opposite consequences. Our results provide new insights into the regulatory roles of CDC10 in adipocytes in animals.


Introduction
Intramuscular fat (IMF) content and marbling are important economic traits in the beef cattle industry. IMF is the streak of visible fat intermixed with the lean within a muscle fiber of different muscle types and locations [1]. It was found that the sensory quality traits of beef, such as tenderness, juiciness, taste, and flavor, were related to IMF content and marbling [2]. Moreover, IMF content is a complex quantitative trait affected by multiple genetic components, cellular signals, environmental conditions, and hormones [3,4]. To date, a number of studies have demonstrated that many candidate genes and mutations play of DMEM/F12 with 10% FBS at 37 • C with 5% CO 2 . For BIMP and 3T3-L1 differentiation, when the confluence of cells reached 90%, they were induced to differentiate using a differentiation cocktail (DMEM/F12 with 10% FBS, 0.5 mmol/L 3-Isobutyl-1-methyxanthine (IBMX), 10 µg/mL insulin, 1 µmol/mL dexamethasone (Sigma-Aldrich, MO, USA)) for 3 d. Cells were then cultured in DMEM/F12 with 10% FBS and 10 µg/mL insulin for 3 d. Finally, cells were cultured in DMEM/F12 with 10% FBS and the culture medium was changed every 3 d until day 15. Cells were incubated at 37 • C with 5% CO 2 .

Transfection of siRNA and Lentivirus Infection
The bovine and murine small-interfering RNA (siRNA) used in this study were designed by RIBOBIO (RIBOBIO, Guangzhou, China) and contained three CDC10 gene siRNA sequences (Table 1) and an siRNA negative control (NC). Cells were cultured in 6-well plates to reach about 50% confluency prior to transfection and then siRNA was transfected using the ribioFECT TM CP (RIBOBIO, Guangzhou, China) transfection reagent according to the manufacturer's protocol. Table 1. Bovine and murine CDC10 siRNA target sequences.

Name
Target Sequence ( For overexpression, we constructed two lentiviral overexpression vectors by introducing the coding region sequence of the bovine CDC10 gene (GenBank accession: BC148893.1) and of the murine CDC10 gene (GenBank accession: BC058587.1) available on GenBank into the virus plasmid. Constructions of bovine and murine overexpression vectors were completed by Gemma gene (Shanghai, China). In order to establish the stable cell line, the preadipocyte were inoculated with the lentivirus for 96 h and then stable cell lines were screened by flow cytometry assay.

RNA Isolation and Quantitative Real-Time PCR
Total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and then the reverse transcription kit (Takara, Kusatsu, Japan) was used to synthesize cDNA. Quantitative real-time PCR (RT-PCR) was carried out with the use of an SYBR Green master mix and specific primers ( Table 2) on a BioRad system (Bio-Rad, Hercules, CA, USA). The relative mRNA abundance of each gene was analyzed by the 2 −∆∆CT method.

EdU and Flow Cytometry Assay
We used the Cell-Light™ EdU Apollo ® 488 In Vitro Imaging Kit (RIBOBIO, Guangzhou, China) and configured the mixed solution according to the instructions. The preadipocyte in the normal growth stage were treated with 50 µmol/L EdU medium for 2 h. After the cells were fixed with 4% paraformaldehyde, they were stained with the Apollo reaction solution and then the cell nucleus was stained with Hoechst. A Nikon TE2000 microscope (Nikon, Tokyo, Japan) was used to take pictures and the data were analyzed using ImageJ.
Preadipocyte were seeded in 6-well culture plates at a density of 2 × 10 5 cells per well. Cells were transfected with CDC10-siRNA or a CDC10 overexpression vector. After washing three times with PBS, cells were fixed with 75% alcohol overnight at 4 • C, followed by treatment with 1 mg/mL RNase at 37 • C for 30 min, and were then stained with 50 mg/mL propidium iodide (PI) at 37 • C for 30 min. The samples were detected using a CytoFLEX flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The proliferation index shows the proportion of mitotic cells among the cells examined.

Oil Red O Staining
The CDC10-siRNAor CDC10 overexpression vector-treated cells were matured for 9 d and then washed with PBS, fixed with 4% paraformaldehyde (PFA) for 0.5 h at room temperature, and washed again three times with PBS. After staining with Oil Red O (Sigma-Aldrich, MO, USA) solution for 1 h, the results were visualized on a fluorescence microscope (Nikon, Tokyo, Japan). Oil Red O was extracted with 100% isopropanol and its relative concentration was determined by measuring the absorbance at 510 nm.

Statistical Analysis
All charts were created using GraphPad Prism 7.0 and Origin 2019b. The data represent the mean ± SEM. The significance of differences between the groups was assessed using the Student's t-test (*, p < 0.05; **, p < 0.01).

Growth Curve of Primary Cultured BIMP and Identification of Adipogenic Marker Genes
In order to identify the growth of primary cultured BIMP and whether they can stably differentiate into adipocytes, we measured the growth curve of BIMP and also detected the expression trends of adipogenic marker genes during their differentiation. The growth curve of BIMP was drawn by trypan blue staining and the results are shown in Figure 1A. The growth curve of BIMP showed an "S" shape ( Figure 1A). We found that the expression of PPARγ and C/EBPα increased first and then decreased during the differentiation in BIMP and 3T3-L1 cells ( Figure 1B-E). The levels of PPARγ and C/EBPα proteins in the preadipocyte followed a similar trend as the mRNA expression ( Figure 1F,G).

Statistical Analysis
All charts were created using GraphPad Prism 7.0 and Origin 2019b. Th sent the mean ± SEM. The significance of differences between the groups using the Student's t-test (*, p < 0.05; **, p < 0.01).

Growth Curve of Primary Cultured BIMP and Identification of Adipogenic Ma
In order to identify the growth of primary cultured BIMP and whether bly differentiate into adipocytes, we measured the growth curve of BIMP tected the expression trends of adipogenic marker genes during their differe growth curve of BIMP was drawn by trypan blue staining and the results Figure 1A. The growth curve of BIMP showed an "S" shape ( Figure 1A). W the expression of PPARγ and C/EBPα increased first and then decreased du ferentiation in BIMP and 3T3-L1 cells ( Figure 1B-E). The levels of PPARγ proteins in the preadipocyte followed a similar trend as the mRNA expre 1F,G).

Expression of the CDC10 Gene in the Proliferation and Differentiation of BIMP and 3T3-L1 Cells
In order to unveil the function of CDC10 in preadipocyte in animals, both 3T3-L1 and BIMP cells were used to analyze CDC10 expression during preadipocyte proliferation and differentiation. We found that CDC10 expression increased during proliferation and differentiation in both cell types. Then, CDC10 mRNA levels peaked at day 8 during BIMP proliferation and peaked at hour 60 during 3T3-L1 proliferation (Figure 2A,C). Meanwhile, CDC10 mRNA levels continued to rise until day 15 in BIMP and 3T3-L1 differentiation ( Figure 2B,D). The levels of CDC10 protein in the preadipocyte followed a similar trend as the mRNA expression ( Figure 2E,F).

Knockdown of CDC10 Had No Significant Effect on the Proliferation of BIMP and 3T3-L1 Cells
The interference efficiencies of the six siRNAs were detected by RT-PCR. The results indicated that CDC10-siRNA1 in BIMP had the strongest interference efficiency, with 81% and 82% compared to control and NC, respectively ( Figure 3A), and CDC10-siRNA3 in 3T3-L1 had the strongest interference efficiency, with 74% and 77% compared to control and NC, respectively ( Figure 3B). The interference efficiency in both cell types was greater than 70% and could be used for subsequent experiments. Compared with the control group, knockdown of CDC10 had no significant effect on the expression of CyclinD1 and CyclinB1 in both BIMP and 3T3-L1 cells ( Figure 3C,D). Similarly, knockdown of CDC10 had no significant effect on the protein levels of CyclinD1 and CyclinB1 in both BIMP and 3T3-L1 cells ( Figure 3E-H). Knockdown of CDC10 had no significant effect in the percentage of EdU-positive BIMP and 3T3-L1 cells compared with the control groups ( Figure 4A-D). Moreover, we found using flow cytometry that the proportion of G1, S, and G2 phase cells was not significantly changed in both BIMP and 3T3-L1 cells following knockdown of CDC10 ( Figure 4E-H).

Expression of the CDC10 Gene in the Proliferation and Differentiation of BIMP Cells
In order to unveil the function of CDC10 in preadipocyte in animals, bot BIMP cells were used to analyze CDC10 expression during preadipocyte pro differentiation. We found that CDC10 expression increased during prolifera ferentiation in both cell types. Then, CDC10 mRNA levels peaked at day 8 proliferation and peaked at hour 60 during 3T3-L1 proliferation (Figure 2 while, CDC10 mRNA levels continued to rise until day 15 in BIMP and 3T3ation ( Figure 2B,D). The levels of CDC10 protein in the preadipocyte follow trend as the mRNA expression ( Figure 2E,F).

Knockdown of CDC10 Had No Significant Effect on the Proliferation of BIMP Cells
The interference efficiencies of the six siRNAs were detected by RT-PCR indicated that CDC10-siRNA1 in BIMP had the strongest interference efficien and 82% compared to control and NC, respectively ( Figure 3A), and CDC1 3T3-L1 had the strongest interference efficiency, with 74% and 77% compar and NC, respectively ( Figure 3B). The interference efficiency in both cell type than 70% and could be used for subsequent experiments. Compared wit group, knockdown of CDC10 had no significant effect on the expression of CyclinB1 in both BIMP and 3T3-L1 cells ( Figure 3C,D). Similarly, knockdow had no significant effect on the protein levels of CyclinD1 and CyclinB1 in bo 3T3-L1 cells ( Figure 3E-H). Knockdown of CDC10 had no significant effect in age of EdU-positive BIMP and 3T3-L1 cells compared with the control group D). Moreover, we found using flow cytometry that the proportion of G1, S, a cells was not significantly changed in both BIMP and 3T3-L1 cells following

Overexpression of CDC10 Had No Significant Effect on the Proliferation of BIMP and 3T3-L1 Cells
The titer of overexpressed virus was detected by gradient dilution method. The results show that lentivirus titers of BIMP, 3T3-L1, and NC were 1.1 × 10 −6 , 3.1 × 10 −6 , and 1.6 × 10 −7 , respectively. Therefore, these lentiviruses could be used in subsequent tests ( Figure 5A). BIMP and 3T3-L1 cells were infected with lentivirus for 96 h. GFP fluorescence was found throughout the field of view and the results showed that BIMPs were effectively transfected ( Figure 5B). Compared with the control group, overexpression of CDC10 had no significant effect on the expression of CyclinD1 and CyclinB1 in BIMP and 3T3-L1 cells ( Figure 5C,D). Similarly, overexpression of CDC10 had no significant effect on the protein levels of CyclinD1 and CyclinB1 in BIMP and 3T3-L1 cells ( Figure 5E-H). Stable cells were screened by flow cytometry after infection. Overexpression of CDC10 had no significant effect in the percentage of EdU-positive BIMP and 3T3-L1 cells compared with the control group ( Figure 6A-D). Moreover, we found using flow cytometry that the proportion of G1, S, and G2 phase cells were not significantly changed in BIMP and 3T3-L1 cells following overexpression of CDC10 ( Figure 6E-H

Overexpression of CDC10 Had No Significant Effect on the Proliferation o 3T3-L1 Cells
The titer of overexpressed virus was detected by gradient dilut results show that lentivirus titers of BIMP, 3T3-L1, and NC were 1.1 × 1 1.6 × 10 −7 , respectively. Therefore, these lentiviruses could be used in ( Figure 5A). BIMP and 3T3-L1 cells were infected with lentivirus

Knockdown of CDC10 Inhibits Differentiation of BIMP and 3T3-L1 Cells
Oil Red O staining revealed that knockdown of CDC10 led to significantly reduced lipid droplets produced by BIMP and 3T3-L1 cells compared with the control groups ( Figure 7A,B). The results of Oil Red O extraction showed that knockdown of CDC10 led to decreases in the intracellular lipid content of BIMP and 3T3-L1 cells compared with the control groups (p < 0.01) ( Figure 7C,D). RT-PCR results showed that the expression of CDC10 was significantly lower than that of the control groups in BIMP and 3T3-L1 cells (p < 0.01) ( Figure 8A,B). The levels of adipogenic marker genes PPARγ, C/EBPα, and fatty acid-binding protein (FABP4) were significantly reduced in BIMPs at the mRNA level following knockdown of CDC10 (p < 0.01, p < 0.01, and p < 0.01, respectively) ( Figure 8A), while PPARγ, C/EBPα, and fatty acid synthetase (FASN) were significantly reduced in 3T3-L1 at the mRNA level following knockdown of CDC10 (p < 0.01, p < 0.01, and p < 0.05, respectively) ( Figure 8B). However, expression of adipose triglyceride lipase (ATGL) was increased in BIMP (p < 0.05) and 3T3-L1 (p < 0.01) after knockdown of CDC10 ( Figure 8A,B). Similarly, the protein levels of PPARγ and C/EBPα were significantly reduced in BIMP and 3T3-L1 cells after knockdown of CDC10 ( Figure 8C-F).

Knockdown of CDC10 Inhibits Differentiation of BIMP and 3T3-L1 Cells
Oil Red O staining revealed that knockdown of CDC10 led to significantly r lipid droplets produced by BIMP and 3T3-L1 cells compared with the control (Figure 7A,B). The results of Oil Red O extraction showed that knockdown of CD to decreases in the intracellular lipid content of BIMP and 3T3-L1 cells compared w control groups (p < 0.01) ( Figure 7C,D). RT-PCR results showed that the expres CDC10 was significantly lower than that of the control groups in BIMP and 3T3-(p < 0.01) ( Figure 8A,B). The levels of adipogenic marker genes PPARγ, C/EBPα, an acid-binding protein (FABP4) were significantly reduced in BIMPs at the mRNA le lowing knockdown of CDC10 (p < 0.01, p < 0.01, and p < 0.01, respectively) (Figu while PPARγ, C/EBPα, and fatty acid synthetase (FASN) were significantly red 3T3-L1 at the mRNA level following knockdown of CDC10 (p < 0.01, p < 0.01, and p respectively) ( Figure 8B). However, expression of adipose triglyceride lipase (ATG

Overexpression of CDC10 Promotes Differentiation of BIMP and 3T3-L1 Cells
Oil Red O staining revealed that overexpression of CDC10 led to significantly increased lipid droplets produced by BIMP and 3T3-L1 cells compared with the control groups ( Figure 9A,B). The results of Oil Red O extraction showed that overexpression of CDC10 led to increases in the intracellular lipid content of BIMP and 3T3-L1 cells compared with the control group (p < 0.01) ( Figure 9C,D). RT-PCR results showed that the expression of CDC10 was significantly higher than that of the control group (p < 0.01) ( Figure 10A,B). The levels of adipogenic marker genes PPARγ, C/EBPα, FABP4, and FASN were significantly increased in BIMPs at the mRNA level following overexpression of CDC10 (p < 0.05, p <0.01, p < 0.01, and p < 0.01, respectively) ( Figure 10A), while PPARγ, C/EBPα, and FABP4 were significantly increased in 3T3-L1 at the mRNA level following overexpression of CDC10 (p < 0.01, p < 0.05, and p < 0.01, respectively) ( Figure 10B). However, expression of ATGL was decreased in BIMP (p < 0.01) and 3T3-L1 cells (p < 0.05) after overexpression of CDC10 ( Figure 10A,B). Similarly, the protein levels of PPARγ and C/EBPα were significantly increased in BIMP and 3T3-L1 cells after overexpression of CDC10 ( Figure 10C-F).

Discussion
Marbling is an important determinant of beef quality and nutrient value. Therefore, it is essential to understand the mechanism of IMF deposition in beef cattle [4,28]. Intramuscular fat deposition involves a series of events that initiate and maintain preadipocyte proliferation, differentiation of preadipocyte into adipocytes, and adipocyte maturation in muscle [4,29]. In this study, we first used 3T3-L1 and BIMP cells as experimental materials, interfered with the CDC10 gene expression by siRNA, overexpressed the CDC10 gene by constructing a CDC10 gene lentiviral overexpression vector, and detected the effects on proliferation and differentiation of 3T3-L1 and BIMP cells after interfering and overexpressing the CDC10 gene. Eventually, we first demonstrated that CDC10 could promote the differentiation of BIMP and 3T3-L1 cells, providing new clues to reveal the mechanism of intramuscular fat formation.

Effect of the CDC10 Gene on Proliferation of BIMP and 3T3-L1 Cells
CDC10 could be considered as a centrosome protein that plays an important role in maintaining cell proliferation [23], and knockdown of the CDC10 gene significantly reduced the proliferation rate of breast cancer cells [22]. Studies on C2C12 cells found that the CDC10 gene is the direct target of miR-127-3p and overexpression of miR-127-3p could significantly inhibit the proliferation of C2C12 cells, whereas interference with miR-127-3p could significantly promote the proliferation of C2C12 cells by regulating the expression of CDC10 [30]. However, there was no effect of CDC10 on the proliferation of 3T3-L1 and BIMP cells by the EdU proliferation assay and the cell cycle-related assay, and also no influence on the expressions of CyclinD1 and CyclinB1 in this study. Thus, we speculate that CDC10 may differentially regulate proliferation in different cell types.

Effect of the CDC10 Gene on the Differentiation of BIMP and 3T3-L1 Cells
Adipocyte differentiation refers to the accumulation of fat and morphological changes achieved through the expression of a series of lipogenesis-related genes under the regulation of multiple transcription factors. CDC10 can form a reciprocal relationship by binding to the FIT2 gene, which is a key gene for packing fat into lipid droplets and is highly expressed in adipose tissue. During adipocyte differentiation, CDC10 and FIT2 are transiently enriched in lipid droplet formation sites, and the CDC10 gene can influence the formation of new lipid droplets by promoting the expression of FIT2 [27]. This is consistent with our findings that CDC10 can promote the differentiation of 3T3-L1 cells and bovine adipocytes that we observed through the Oil Red O stain assay, which showed that overexpression of CDC10 could increase the formation of lipid droplets, whereas knockdown of CDC10 could decrease formation of lipid droplets in BIMP and 3T3-L1 cells.
In addition, the expression levels of adipogenesis-related transcription factors and their activity could crucially determine the differentiation process of adipocytes. Among them, PPARγ plays an important role in the regulation of adipocyte differentiation, glucolipid metabolism, and insulin signaling. As reported, PPARγ could directly affect a number of genes related to lipid storage in adipocytes and is necessary for adipocyte differentiation [31]. C/EBPα is induced in the late stage of adipogenesis and is most abundant in mature adipocytes [32]. C/EBPα could initiate transcription of adipocyte-specific genes and promotes adipocytes to the terminal differentiation stage [33]. In this study, we first found that the expression levels of CDC10 had an upward trend during the differentiation of BIMP and 3T3-L1 cells. We also found that overexpression of CDC10 could increase the expression levels of PPARγ and C/EBPα, whereas knockdown of CDC10 could decrease the expression levels of PPARγ and C/EBPα in BIMP and 3T3-L1 cells, suggesting that the CDC10 gene has an effect on adipogenesis. Meanwhile, ATGL is the key rate-limiting enzyme involved in the breakdown of fat cells [34]. The main function of ATGL in adipocytes is to regulate lipid droplet size and fat degradation in adipocytes by catalyzing the hydrolysis of triglycerides in adipocytes [35,36]. In this study, overexpression of CDC10 could decrease the expression of ATGL, whereas knockdown of CDC10 could increase the expression of ATGL in BIMP and 3T3-L1 cells. Since PPARγ and C/EBPα are key genes involved in adipogenesis and ATGL is a key gene involved in adipocyte catabolism, we speculate that the promotion of 3T3-L1 and BIMP cell differentiation by CDC10 is achieved by promoting the expressions of the PPARγ and C/EBPα genes and suppressing the expression of the ATGL gene.
Besides, CDC10 depletion could specifically impair ERK1/2 phosphorylation and CDC10 is required for MEK/ERK activation [22]. The ERK1/2 pathway activation could accelerate preadipocyte proliferation and promote their differentiation, and ERK1/2 is required for PPARγ and C/EBPα gene transcription during adipocyte differentiation [37]. Similarly, the expression levels of CDC10 were also positively correlated with marbling scores in Japanese Black cattle [38]. In summary, we speculate that the CDC10 gene could regulate adipogenesis through promoting the expression of PPARγ and C/EBPα by regulating the MEK/ERK signaling pathway in IMF of marbling in beef cattle.

Conclusions
In conclusion, this is the first study to demonstrate the regulatory mechanism of the CDC10 gene in the process of preadipocyte proliferation and differentiation. We found that overexpression of CDC10 could promote the differentiation of BIMP and 3T3-L1 cells, whereas knockdown of CDC10 resulted in the opposite consequences. These findings provide new opportunities for improving the meat quality of beef by regulating intramuscular fat deposition and help us to better understand the role and regulatory mechanism of the CDC10 gene in IMF adipogenesis.