OBJECTIVE:To investigate whether circular RNA circRSF1 regulates radiation-induced inflammatory phenotype of hepatic stellate cells (HSCs) by binding to HuR protein and repressing its function.METHODS:Human HSC cell line LX2 with HuR overexpression or knockdown was exposed to 8 Gy X-ray irradiation, and the changes in the expression of inflammatory factors (IL-1β, IL-6 and TNF-α) were detected by qRT-PCR. The expressions of IκBα and phosphorylation of NF-κB were detected with Western blotting. The binding of circRSF1 to HuR was verified by RNA pull-down assay and RNA-binding protein immunoprecipitation (RIP). The expressions of inflammatory factors, IκBα and the phosphorylation of NF-κB were detected after modifying the interaction between circRSF1 and HuR.RESULTS:Knockdown of HuR significantly up- regulated the expressions of IL-1β, IL-6 and TNF-α, decreased IκBα expression and promoted NF-κB phosphorylation in irradiated LX2 cells, whereas overexpression of HuR produced the opposite changes (P < 0.05). Overexpression or knockdown of circRSF1 did not significantly affect the expression of HuR. RNA pull-down and RIP experiments confirmed the binding between circRSF1 and HuR. Overexpression of circRSF1 significantly reduced the binding of HuR to IκBα and down-regulated the expression of IκBα (P < 0.05). Overexpression of circRSF1 combined with HuR overexpression partially reversed the up-regulation of the inflammatory factors, down-regulated IκBα expression and increased phosphorylation of NFκB in LX2 cells, while the opposite effects were observed in cells with knockdown of both circRSF1 and HuR (P < 0.05).CONCLUSION:circRSF1 reduces IκBα expression by binding to HuR to promote the activation of NF-κB pathway, thereby enhancing radiation- induced inflammatory phenotype of HSCs.

Recently, we revealed how the underlying molecular mechanism of Bone Morphogenetic Protein (BMP) signaling governs the homeostasis of hair follicle stem cells (hfSCs) in vivo. Here, we focused further on identifying the key interactions of Smads in canonical BMP signaling in hfSC regulation, using our recently developed inducible constitutively active Bmpr1a transgenic system. This allowed us to perform ChipSeq analysis in vivo in hfSCsand receive the predicted target lists for Smads for both p22 and p62 telogen hfSCs. Comparison of our previously published global genes expression where BMP signaling has been inhibited in hfSCs to all overlapped in vivo ChipSeq data between p22 v.s p62 revealed that approximately 72% of genes which previously has been 2 fold up or down regulated after BMP ablation in hfSCs were found directly regulated by BMP/Smads pathway. Previously, we identified Id2 gene as a target of BMP signaling in hfSCs with transcriptional downregulation following targeted BMP inhibition. Here, we confirmed that Id2 gene has been consistently present in our recent ChipSeq analysis both at p22 and p62, therefore, we decided to further test the functional role of the BMP effector, Id2 in hfSCs using an in vivo Id2 gain of function approach. Our data demonstrated that Id2 overexpression in hfSCs results in prolonged telogen and a delay in anagen activation, maintaining stem cells quiescence. Thus, we confirmed the functional role of Id2 gene as a direct target and effector of BMP pathway in hfSCs in vivo.