A Study On The Mechanism Of TMPyP4 Induced G-quadruplex Formation Leading To Hela Cell Senescence

Objective:G-quadruplex form in both DNA and RNA.These G-quadruplex strctures may affect essential transcription and expression of genomes,and the stablity of genomes studing.Telomeric G-quadruplex structure is an important direction for the treatment of cancer.This study cencents the establishment of the detection method of G-quadruplex of DNA structure.This dissertation studied the mechanism of TMPyP4 induced G-quadruplex formation leading to Hela cell senescence,and laid the theoretical foundation for the study of anti-cancer effect of G-quadruplex stabilizer.Methods:(1)We analyzed the expression and purificaton of G-quadruplex antibody BG4 by pSANG10-BG4.Then identified the relative molecular weight of BG4 antibody by coomassie brilliant blue and Western Blot.In addition,Hela cells were treated with RNase or DNase.We detected red fluorescence signal in cells,immunofluorescence to confirm the activity of BG4,and established a detection method of G-quadruplex in DNA structure.(2)Cells were exposed to TMPyP4 for 24 h,then havested and processed for immunofluorescence.Fluorescence microscopy detected the number of G-quadruplex and telomeric G-quadruplex in Hela cells.TMPyP4 was added to IF-FISH to confirm the effect on DNA damage and telomeric DNA damage through the stablization of G-quadruplex.Then,co-IF showed the number of G-quadruplex DNA damage.(3)Cells were exposed to TMPyP4 for 24 h,havested,and processed for FISH.Fluorescence microscopy detected the number of telomere signals.TMPyP4 was add to SA-β-gal to confirm the effect cell senescence.Results:(1)Expression and purification of BG4 by pSANG10-BG4,coomassie brilliant blue and Western Blot showed that the relative molecular weight of BG4 antibody was 30~40 kD.Besides,the band of BG4 antibody significantly increased by anto-induction media.We conclude that the method is feasible of expression and purification of BG4 antibody.RNase treatment had noticeable effects on nuclear staining,whereas treanment with DNase was completely effective on cytoplasm staining in Hela cells.Above all,BG4 antibody was successful expression and purification,meanwhile BG4 antibody had a high biological activity in vivo,and the establishment of the detection method of G-quadruplex of DNA structure.(2)We treated Hela cells with TMPyP4 that stabilize G-quadruplex DNA would increase the number of G-quadruplex and telomeric G-quadruplex detected by BG4.Then cells were exposed to TMPyP4 for 24 h,havested,and processed for IF-FISH experiment.After we detected an increased number of DNA damage and telomeric DNA damage.In addition,co-immunofluorescence revealed that TMPyP4 induced the number locations where G-quadruplex DNA damage was present.(3)We detected an increase MTS with increaing amounts of TMPyP4 at 24 h in Hela cells.Taken together,our results confirmed chromosome integrity and there was no present chromosome break.In addition,the number of cell senescence was slightly increased by TMPyP4 at 24 h in Hela cells by SA-β-gal.Conclusion:BG4 antibody of expression and purification was a tool for studies on the role of G-quadruplex in cells,and established the detection method of G-quadruplex.We used this method to study the mechanism of TMPyP4 induced G-quadruplex formation leading to Hela cells senescence.We treated Hela cells with TMPyP4,and we detected an increased number of G-quadruplex,telomeric G-quadruplex,DNA damage,telomeric damage and G-quadruplex DNA damage.To further investigated that the number of MTS and cell senescence was increased by TMPyP4.In this study,we investigated the mechanism of cell senescence of TMPyP4 in Hela cells.Therefore,G-quadruplex structure may be a potential therapeutic target in cancer.