Expression of STAU1, Klf16, and PPARγ in adipogenesis
In order to verify the expression of STAU1, Klf16, and PPARγ during the induction and differentiation of 3T3-L1 cells, 3T3-L1 preadipocytes were induced using the cocktail method and were analyzed by Oil Red O staining on day 0, 2, 4, 6, and 8 of differentiation. Two days after cell differentiation, stained red lipid droplets were observed, their number was gradually increased, and they accumulated on days 4, 6, and 8 (Fig. 1a, b). After induction, the mRNA and protein levels of STAU1 and Klf16 at days 0, 2, 4, 6, and 8 were determined. After 4 days of induction, the protein (Fig. 1c) and mRNA (Fig. 1d) levels of STAU1 and PPARγ were significantly increased (P < 0.05), whereas the expression of Klf16 protein (Fig. 1c) and mRNA (Fig. 1d) was significantly decreased (P < 0.05). It was observed that in mature 3T3-L1 adipocytes, STAU1 and PPARγ were highly expressed, whereas Klf16 showed low expression.
Overexpression of STAU1 reduces KLF16 expression in adipogenesis
We then examined whether STAU1 overexpression affected the mRNA and protein levels of Klf16 and key SMD factors. 3T3-L1 pre-adipocytes were induced to differentiate into mature adipocytes, and transfected with a STAU1 overexpression plasmid. The mRNA and protein levels of STAU1, Klf16, pUPF1, and UPF1 were then measured at 0, 1, 2, 3, and 4 days. The results of Oil Red O staining showed that lipid droplets were more obvious in the STAU1 overexpression group than in the control group (Fig. 2a, b). The mRNA and protein levels of STAU1 indicated that the STAU1 overexpression efficiency was 65% (Fig. 2c, d). Compared to that in the control group, the mRNA expression of KLF16 in the STAU1 overexpression group showed no significant difference (Fig. 2c), whereas its protein expression was significantly reduced (P < 0.05) (Fig. 2d). Expression of the key SMD pathway factor, pUPF1, was significantly increased at the protein and mRNA levels (P < 0.05), whereas UPF1 showed no difference (Fig. 2d). It was thus observed that STAU1 overexpression did not affect the mRNA expression of KLF16, but affected the protein expression of KLF16, STAU1, and pUPF1.
STAU1 knockdown increases KLF16 expression in adipogenesis
To further examine the mRNA and protein levels of Klf16, pUPF1, and UPF1, 3T3-L1 cells were transfected with Stau1 shRNA1 and Stau1 shRNA2. The mRNA and protein levels of STAU1, Klf16, pUPF1, and UPF1 were measured at 0, 1, 2, 3, and 4 days. The results of Oil Red O staining showed that the STAU1 knockdown group had less lipid droplets than the control group (Fig. 3a, b). The STAU1 knockdown efficiency was about 65%, and there was no significant difference in the Klf16 mRNA levels between the STAU1 knockdown group and the control group (Fig. 3c). However, compared to that in the control group, the protein level of Klf16 in the STAU1 knockdown group was significantly increased (P < 0.05), whereas the STAU1 and pUPF1 levels were significantly decreased (P < 0.05), and the total UPF1 levels remained unchanged (Fig. 3d). The above results jointly indicate that STAU1 affects the stability of Klf16 mRNA at the post-transcriptional level, thereby affecting the production of fat and promoting adipocyte differentiation.
STAU1 specifically binds the 3′ UTR of Klf16 mRNA
In order to further study the binding sites between STAU1 and Klf16 RNA during the differentiation of 3T3-L1 adipocytes, we used RNA co-precipitation experiments to verify the binding of STAU1 and Klf16 mRNA, and PCR confirmed that STAU1 and Klf16 bind at D2 and D4 (Fig. 4a). PAR-CLIP experiments were performed on 3T3-L1 cells to verify the binding of STAU1 to the KLF16 3′UTR. Ten pairs of primers were designed according to the length of the fragment. Each primer pair represents the length of a fragment in the 3'UTR region (Fig. 4b), and qPCR analysis was conducted. We found that Klf16 mRNA contained at least one identified region and was bound by STAU1. ChIP experiments proved that PPARγ could combine with the promoter region of Klf16 and that its binding ability is increasingly strong (Fig. 4c).
After transfection of 3T3-L1 pre-adipocytes with STAU1 overexpression plasmid for 24 h, the cells were treated with actinomycin D for 1 h and then collected at 0, 2, 4, and 6 h. Klf16 mRNA expression was determined by qPCR. The degradation rate of Klf16 mRNA was significantly accelerated after STAU1 overexpression compared to that in the control group (P < 0.05). STAU1 was confirmed to affect the KLF16 mRNA degradation rate (Fig. 4d). These data provide some evidence that STAU1 could specifically bind to KLF16 3′UTR and affect its mRNA degradation rate.
Klf16 regulates Lipin1 to affect adipogenesis
In order to study the downstream target of Klf16, Oil Red O staining was performed after Klf16 overexpression, and morphological changes were observed. Klf16 overexpression resulted in significantly reduced lipid droplets compared to the control group (P < 0.05) (Fig. 5a, b). Compared to that in the control group, the mRNA and protein expression of Lipin1 in the Klf16-overexpression group was significantly downregulated as detected by qPCR and western blotting, respectively (P < 0.5) (Fig. 5c, d). Intracellular triglyceride content was decreased during 3T3-L1 differentiation compared with that in the control group after overexpression of KLF16 (Fig. 5e). This suggests that Klf16 inhibits the gene expression of Lipin1, thereby inhibiting adipogenesis.