The Role Of Oncogene KrasG12D In ALT Tumorigenesis In The Process Of Senescence

Proto-oncogene ras mutation will cause the tumor suppressor gene p53stress, triggering a series of downstream senescence and apoptotic response. When p53is suppressed or mutated, losing normal monitoring of ras, the cells will proliferate excessively and cause tumor. Some p53mutations ensure their oncogene potential. Interacting with ras mutations makes them easier to cause tumors, and the malignant degree is higher. Rely on "Alternative lengthening of telomere (ALT)" mechanism, some tumors maintain telomere length and ensure unlimited cell divisions. These tumors which do not rely on telomerase activity are known as ALT tumors, most of which are mesenchymal origin. Studies have shown that partial p53deletion will significantly increase epithelial original tumor proportion in telomerase deletion (mTR-/-) mice,which means the mesenechymal-epithelial transition occurred (mesenechymal-epithelial transition, MET). As a close relationship with the p53, the role of oncogene ras activation in ALT tumors occurring is unclear. Accordingly, we intended to introduce the most universal ras mutation (KrasG12D) into mTR-/primary cells and normal ALT tumors without p53mutation, and then changes in these cells were observed.In this study, pAcGFPl-N1-KrasG12D mutant expression vector was constructed by site-specific mutagenesis, and then transfected into three kinds of mesenchymal original cells (mTR-/-(G3) MEF, WT-MEF and SCID666B-1+p21siRNA). In which, mTR-/-telomerase (G3) MEF cells are embryonic fibroblasts from the third generation of mouse with telomerase knockout, SCID666B-1+p21siRNA cells are p53normal expressing ALT cells with P21knockdown through RNA interference, WT-MEF cells are wild type embryonic fibroblast cells which were used as a control. After successfully transfected and stably expressed cells were obtained, Western blotting, real-time fluorescent quantitative PCR, apoptosis dyeing, Ki67dyeing, cell clones and other assays were carried out.We found that when KrasG12D was transfected into mTR-/-(G3) MEF and WT-MEF cells, p53expression increased and apoptosis phenotype occurred (positive apoptosis rate:88.1%vs59.4%); Compared to WT-MEF cells, part of mTR-/-(G3) MEF cells exhibited vacuoles; When KrasG12D was introduced into SCID666B-1+p21siRNA cells, mRNA levels of mesenchymal cell related protein marker (Snail, N-cadherin, etc.) increased, no MET phenomenon was occurred; When Kras-G12D was introduced, enhanced proliferation was observed in SCID666B-1+p21siRNA cells (Ki67positive staining rate:94.03%vs90.3%; clone formation rate:7.75%vs3.75%).As refer to the cell level,these results suggest thatthe activation of oncogenes KrasG12D will causes rapid apoptosis of telomerase deleted primary cells, which may be more severe than normal primary cells due to the lack of telomerase; In ALT tumors cells, ras mutations in ALT tumor cell regulation were different with p53deletion case, KrasG12D activation could not induce MET changes, and cell proliferation increased.In this paper, all the results were obtained from mouse cells, which had a significant difference in vivo. Thus, we breed KrasG12D*mTR-/-mouse model, and the third generation (G3) mice were obtained. With research going deeper, we hope moreresults will be obtained in mice.