The Mechanism Of CIRP In Regulation Of STAT3 Phosphorylation Upon UVB Radiation In Keratinocyte

Ultraviolet radiation is the main inducing factor in the occurrence of human skin cancer.About 65%of melanomas and 90%of non-melanoma skin cancers are associated with UV exposure.Long-term UVB exposure can cause DNA damage in skin cells.If not properly repaired in time,these damaged DNA will accumulate and gradually evolve into gene mutations.These gene mutations cause abnormal expression of many proteins and induce the activation of various signaling pathways,which in turn leads to the carcinogenesis of skin cancer.In UVB-irradiated keratinocytes and some melanoma cell lines,the expression of cold-induced RNA-binding protein?CIRP?is up-regulated,suggesting there maybe a positive relationship between CIRP and skin cancer tumorgenesis.Cold-induced RNA-binding protein is a member of the cold shock protein family and its expression can be induced in response to hypothermia conditions.As a stress-regulating protein,CIRP is involved in the regulation of several cellular physiological processes such as cell growth,senescence,apoptosis,and tumourgenesis.In our previous study,we found that CIRP can up-regulate the expression of phosphorylation of signal transduction and activator of transcription 3?Stat3?,prompte the proliferation and survive of HaCaT keratinocytes in response to UVB radiation.The positive correlation between CIRP and p-Stat3 has also been reported in liver cells.However,in HaCaT keratinocytes under UVB radiation,the underlying mechanism of how CIRP regulates Stat3 has not been elucidated.Jaks are intracellular,non-receptor tyrosine kinase receptors that can mediate signaling by a variety of cytokines to activing the stat3 and then induce the development of cancer.What's more,CIRP has been reported that can act as a kinase or kinase regulator.Therefore,we hypothesized that the UVB radiation-induced CIRP may affect the activation of p-Stat3 signaling pathway by regulating Jaks,thereby regulating the tumorgenesis of skin cancer.In addition,some studies also reported CIRP can active the NF-?B signaling pathway and play a significant role in inflammation.So,NF-?B may involov in the regulation of CIRP on p-Stat3.In this paper,we through constructing CIRP overexpressed stable cell line,adding Jak kinase and NF-?B inhibitors or gene silencing to investigate the role of Jaks on the up-regulation of Stat3 phosphorylation induced by CIRP and the regulation of CIRP on the growth and survival of skin cancer cells.In this study,we demonstrate that:?1?In long-term low-dose?3mJ/cm²?UVB-induced transformed HaCaT cells,the expression levels of p-Jak2 and p-Jak3 were up-regulated consistently with the expression of p-Stat3;we also observed that during UVB-induced HaCaT keratinocyte transformation process,the NF-?B signaling pathway is also activated.?2?Low-dose UVB radiation treatment or overexpression of CIRP can up-regulate the expression of p-Jak2 and p-Jak3 in HaCaT cells,which is consistent with the up-regulation of p-Stat3;UVB radiation treatment can up-regulate the expression levels of p-Stat3,p-Jak2 and p-Jak3 in HaCaT^CIRP cells.?3?Low-dose UVB radiation treatment or overexpression of CIRP can up-regulate the expression of p-Akt and p-ERK and activate the NF-?B signaling pathway in HaCaT cells;UVB radiation treatment can up-regulate the expression levels of p-Akt and p-ERK in HaCaT^CIRP cells.?4?In JAK Inhibitor I treated HaCaT^GFPFP cells and HaCaT^CIRP cells,the up-regulated phosphorylation of Stat3 caused by low-dose UVB or CIRP overexpression was completely inhibited;and the expression of Bag-1/S,a downstream Stat3 targeting gene was also inhibited.Bay-11-7085 Inhibited the activation of NF-?B signaling pathway and resulted in decreased expression of phosphorylation of Stat3caused by low dose UVB or CIRP overexpression.?5?The CIRP targeted siRNA sequence silences the expression of CIRP in HaCaTt cells,inhibits the expression of oncogenes in HaCaTt cells and decreases the colony numbers of HaCaT and HaCaTt cells treated with 10 mJ/cm² UVB irradation.?6?The CIRP targeted siRNA sequence silences the expression of CIRP in A375melanoma cells,increases the expression of p53 and p21,inhibits the proliferation of A375 melanoma cells and enhances the sensitivity of A375 melanoma cells to ionizing radiation.The results presented above led us to propose that the CIRP-p-Jak2/3/NF-?B-Stat3-Bag-1/S cascade,which regulates the proliferation of HaCaT keratinocytes and cell survival after UVB radiation.The inhibitory expression of CIRP affects the proliferation and UVB or IR resistance of HaCaT keratinocytes or A375 melanoma cells,suggesting that CIRP can serve as a new target for the prevention or treatment of skin cancer.These findings report the function of CIRP in long-term low dose UVB induced skin cancer tumorgenesis and can enrich our understanding of the progression of skin cancer,provide a research basis for the development of new therapeutics and treatment of skin cancer.