Functional Study Of Splicing Factors, HnRNP A1 And ASF/SF2, Responding To UVB Radiation

Over-exposure of ultraviolet (UV) radiation is known to be one of the risk factors for skin cancer carcinogenesis, while the pathogenic mechanisms are hot topics in biological and clinic research. In recent years, UV radiation-induced gene alternative splicing has drawn more and more concern from researchers, because the variants caused by UV radiation are found benefical to the tumor formation. As an oncogene, the functions of mdm2 are well studied. The results show that mdm2 are often over-expressed in various tumors, accompanied by mdm2 variants, wich play an important role on the tumorigenesis. It has been reported that UVB radiation is capable of inducing mdm2 alternative splicing, but the molecular mechanism is not known yet. Splicing factors are important proteins regulating gene alternative splicing, which leads to the production of gene variants, subsequently influencing physiological processes of cells. However, whether splicing factors are responsible for regulating UVB radiation-induced mdm2 alternative splicing has been less studied. In this thesis, we focus on two well kown splicing facors, hnRNP A1 and ASF/SF2. The effect of hnRNP A1 and ASF/SF2 on regulating UVB radiation-induced mdm2 alternative splicing is analyzed through utilizing the methods of celluar and molecular biology.In addition, hnRNP A1 and ASF/SF2 are often at a high level in tumors and recognized as oncogenes. The study on hnRNP A1 and ASF/SF2 in response to UVB radiation contributes to understanding the mechanism of skin cancer formation. We study the effect of UVB dose and time on the expression of hnRNP A1 and ASF/SF2. Besides, whether ROS, p53, κFκB and Akt signaling induced by UVB radiation could influence the expression of hnRNP A1 and ASF/SF2 is also studied in this project, for achiving a more holistic understanding of their effect on cell survival and tumorigenesis.Autophagy can be caused by UVB and promote cell survival. Autophagy is also playing an important role in tumorigenesis. However, the effect of autophagy on tumorigenesis remains controversial. Since hnRNP A1 and ASF/SF2 much involved with cell survival and carcinogenesis, we try to research on the correlation between splicing factors and autophagy in this study, to get a further understanding of splicing factors and autophagy.This study is mainly divided into three parts:1. To determinate how the splicing factors (hnRNP Al and ASF/SF2) regulating the alterntive splicing process of mdm2 gene induced by UVB radiation.2. To study the activities of hnRNP Aland ASF/SF2 in response to UVB radiation and the effect on cell viability upon UVB radiation.3. To research whether hnRNP A1 and ASF/SF2 involve with UVB-induced autophagy. The results are as follows:① UVB radiation promotes the alternative splicing of mdm2 gene, which increases the expression of mdm2 B and decreases mdm2-FL expression.② UVB radiation up-regulates the expression of hnRNP Al and ASF/SF2 in HaCaT cells, and hnRNP Al is responsible for the regulation of UVB radiation-induced mdm2 alternative splicing by directly binding to mdm2 pre-mRNA, while ASF/SF2 has no effect on this process based on our data.③ The up-regultion of hnRNP A1 and ASF/SF2 expression aroused by UVB radiation is time-dependent, and the two splicing factors are able to shuttle from nucleus to cytoplasm in HaCaT cells after UVB radiation.④ Elimination of ROS by drug further increases the elevation of hnRNP A1 and ASF/SF2 expression induced by UVB radiation, while the transient expression of p53 inhibits UVB radiation induced up-regulation of hnRNP Al and ASF/SF2, suggesting that ROS and p53 probably negatively regulate hnRNP A1 and ASF/SF2 expression in UVB-radiated HaCaT cells.⑤ Inhibiting NF-κB signaling by inhibors depress the intrinsic expression of hnRNP Al and ASF/SF2 in HaCaT cells, but UVB radiation is still capable of up-regulating hnRNP A1 and ASF/SF2 expression in HaCaT cells treated by NF-κB signal pathway inhibitors. While the Akt signal pathway inhibitor decreases the intrinsic expression of hnRNP A1 and ASF/SF2 in HaCaT cells, and also inhibits the up-regulation of hnRNP Al and ASF/SF2 by UVB radiation, indicating that the Akt signaling is responsible for the regulating hnRNP A1 and ASF/SF2 expression in UVB-radiated HaCaT cells.⑥ Knockdown the expression of hnRNP Al and ASF/SF2 by siRNA decrease the cell viability under UVB treatment, which indicates that hnRNP A1 and ASF/SF2 contribute the cell survival of UVB radiated HaCaT cells.⑦ EGFP-LC3B stable expression HaCaT cell line is constructed through lentivirus infection, in which autophagy process is easily detected by fluorescence microscope and western blot. However, transient expression of hnRNP A1 and ASF/SF2 in this cell line has no significant effect on autophagy.