Effect On WEE1 Isoforms In Interaction Between E6 And P53 Of HPV

Cervical cancer is the second female most common cancer with the morbidity and mortality in the world. Human papilloma virus (HPV) is a major virus which causes cervical cancer, including high risk HPV 16 and HPV 18. High-risk type HPV encoding two important oncogene E6 and E7, its main function is to start cell transformation, and many steps involved in virus and cell protein interaction, participation in the process of transformation of HPV infection and cancer. Tumor suppressor gene p53 corrects point monitoring in the process of cell cycle of G1 and G2/M phase, closely associated with transcriptional activation. In HPV positive cervical cancer cells, p53 protein can be antagonized by E6 on the function, the E6 protein can be combined with p53 protein, and induced the mediated via the ubiquitin-proteasome pathway degradation. E6 promote ubiquitin regulation of the degradation of p53 to inhibit apoptosis. WEE1 kinase is a kind of cell cycle regulatory proteins and its mRNA 3’end and 5’end of each have different length of the two kinds of the translation section, including four different isoforms. The 3’long untranslated region (3N),3’short untranslated region (3X),5’long untranslated region (5L) and 5’ short untranslated region (5S). The 5’end sequences have two phosphorylation sites, can be the ubiquitin-proteasome system recognition and degradation, affect the half-life of the protein; WEE1 kinase of G2/M checkpoint and S phase plays an important role in regulating, p53 mutation or the lack of cancer cells can cause mitotic catastrophe.This study selected samples of cervical cancer cell lines. The SiHa cell contains integrated 1-2 copies of HPV 16, E6 expression level is lower and the p53 expression level is higher than CaSki cell. CaSki cell contains 600 copies of HPV 16, E6 expression level is high, the expression level of p53 is low. C33A cell contains mutant p53 and no HPV, the expression level of p53 is particularly high in C33A cell. Due to oxidation molecules can damage DNA in the cell, so use the hydrogen peroxide (H2O2) deal with the three kinds of cells can cause DNA damage response, to observe the oxidative DNA damage under pressure after WEE1 iso forms of reaction. By fluorescence quantitative PCR to detect H2O2 treatment three different concentration gradient cell changes in mRNA level, found that WEE1 3X and 5S had changed, then use the half lethal dose and timecourse to treat cells.Due to the different levels of E6 and p53 genes in cervical cancer cell lines, adjust their expression level vision to affect the changes of WEE1 isoforms. Some people had reported that p38MAPK signaling pathways is a major repair regulation in cell p53 defects, we need to cut MK2 gene to determine whether affected WEE1 isoforms of regulation.This experiment mainly use the E6 gene and p53 gene associated with HPV, study the interaction on WEE1 isoform, the influence of the research of cervical cancer has important significance.The results are as follows:First, the fluorescence quantitative PCR to detect H2O2 treatment three mRNA changes in cervical cancer cells, found that WEE1 3X and 5S changed more obvious. The MTS method to detect the half lethal dose of SiHa cell is 0.8 mM, CaSki cell’s half lethal dose is 0.8 mM, C33A cell’s half lethal dose of is 0.5 mM.The timecourse results found WEE1 3X and 5S changed obviously at 2.5 hours. The other isoforms 3N,5L changed weak, the CaSki cells increased the ratio was the highest, second was C33 A cell, SiHa cells increase ratio was low relatively.Seond, the corresponding changes after each gene to handle each cell line. Raised the p53 gene in CaSki cell, WEE1 3X and 5S has increased; Down-regulated MK2 gene, WEE1 5S significantly down. Raised E6 and E6* genes indirectly p53 protein and WEE1 3X drops,5S changes smaller in SiHa cell. Directly by p53 gene, WEE1 3X had down,5S change was not obvious. WEE1 3X rised,5S changed weak after raised E6 and E6* gene; Down-regulated the p53, WEE1 3X droped,5S changed weak in C33A cell.The conclusions are as follows:Firstly, the content of p53 mainly affects the WEE13X level change. Higher levels of p53 was raised, WEE1 3X has increased, p53 content is low, WEE1 3X is lower. Under oxidative stress p53can affect WEE1 3’end shorter untranslation region, regulate the splicing change, and affect cell cycle progression.Secondly, raising the E6 and E6*, down-regulate p53 indirectly, including wild-type p53 cell, WEE1 3X will also decline, but in containing mutant cell, it rised, this may be associated with the status of p53.Thirdly, down-regulate MK2 genes, WEE1 5S decreased significantly, explain p38MAPK signaling pathways only regulate WEE1 5’end short untranslation region, associated with degradation WEE1 5’end. This shows that the change of different WEE1 isoforms are regulated by different signaling pathways.