The Role And Mechanism Of PRDX2 In Enhancing Radiosensitivity Of Cervical Cancer

Purpose:Cervical cancer is the fourth most common cause of cancer-related death in women all over the world.Radiotherapy(RT)is an important treatment method for the treatment and management of cervical cancer.More than 60%of cervical cancer patients need radiotherapy.However,due to tumor heterogeneity,some tumor cells have natural or acquired radio-resistance,resulting in local uncontrolled or recurrence after radiotherapy.Therefore,it is of great significance to explore the molecular mechanism of radiotherapy resistance and improve the radiotherapy sensitivity of cervical cancer patients.PRDX2 is a member of the peroxiredoxins family who are reactive oxygen species scavengers that hydrolyze H₂O₂through catalytic cysteine.The expression of PRDX2 is increased in some human cancer cells and tissues,and affects a variety of cellular processes including survival,proliferation and apoptosis.More and more studies have shown that PRDX2 promotes or inhibits tumorigenesis by interacting with other proteins in cancer signaling pathway^[2].However,there are few reports on the effect of PRDX2 expression on the radiosensitivity of cervical cancer cells.Methods:1)R-Siha cell lines were constructed using radiation.Siha cell lines were employed as parent cells,and then received fractionation radiation to the total dose 80Gy.To screen the survival cells then named as R-Siha,which should be completed by passing verification and validation for further experiments.To verify whether the R-Siha model was successfully established is mainly through the cloning formation ability at different doses and DNA double strand break markers(?-H₂AX),the expression of apoptosis related protein cleaved-caspase 3,and ratio of apoptosis,cell cycle distribution by flow cytometry after irradiation were detected.Western blot,Scratch wound healing and Transwell assays were used to determine EMT phenotype of R-Siha cells.2)R-Siha cell lines and Siha cell lines were sequenced and analyzed for differential gene expression(proteomics).The identified proteins/genes were validated at m RNA level and proteins level by q PCR and Western Blot.3)Retrospective analysis was performed on a group of cervical cancer,all the enrolled patient were diagnosed as cervical squamous carcinoma,and the patients were all at locally advanced stage,and received the full course of radical radiotherapy treatment(with or without chemotherapy).The efficacy was evaluated according to Response Evaluation Criteria in Solid Tumors(RECIST)and defined as CR group and NCR group.The tissues before treatment of two group patients were also sequenced and analyzed for differential gene expression(proteomics)to verify the differential genes.Next step was to using the GEO dataset,the proteomic sequencing results of patients with CR and NCR after radical radiotherapy(±chemotherapy)were used to verify the expression of differential genes again.2)Cervical squamous cell carcinoma cell lines ME180,MS751,HT-3,C33a,Si Ha,R-siha,Caski were selected.The basal expression of PRDX2 was measured by Western Blot.We found that the relatively high expression of PRDX2 in cell lines were C33a,Siha and ME180,lower expression levels were in Caski,HT-3 and R-Siha.The expression of PRDX2 in C33a and Siha was knocked down by lentivirus transfection technology,and then the clone formation ability at different doses,the expression of?-H₂AX at 0h,4h,8h and 12h after irradiation,the expression of apoptosis indicator protein Cleaved-Caspase3,and the incidence of apoptosis of flow cytometry were detected in order to judge the effect of knockdown of PRDX2 on the radiosensitivity of cervical cancer cells.Our next steps were overexpressed PRDX2 in R-Siha cells by lentivirus transfection technology.The effect of overexpression of PRDX2 on radiosensitivity of R-Siha cells was also detected by the above methods.A natural compound gliotoxin(GT),which is known to exert a PRDX-like activity,was introduced.We first examined the IC50 of gliotoxin in Siha cell lines of24h,and then determine the action concentration of GT.The effect of introduction of GT on the radiosensitivity of R-Siha cells was detected by the above methods.3)The changes of phosphorylation of STAT3 at different sites after knockdown of PRDX2 were detected,that is,the downstream key proteins after knockdown of PRDX2 were found and verified.After knockout of PRDX2 and the changes of EMT marker protein detected by Western Blot.Then knock down the key protein to detect whether it can reverse the radiosensitivity and EMT phenotype.4)We have further verified these results in vivo.A Siha-sh PRDX2 tumor subcutaneous model was established in nude mice.To verify the change of radiosensitivity of subcutaneous xenograft tumors after knockdown of PRDX2,and whether the addition of PRDX2 analogues can reverse the effect of knockdown of PRDX2 on radiosensitivity of cervical cancer.Results:1)The acquired radioresistance cells R-Siha of cervical cancer was successfully constructed.Cloning formation experiment confirmed that the clonogenic ability of R-Siha cells was higher than that of Siha cells at different doses.Flow cytometry showed that the proportion of early apoptosis+late apoptosis of R-Siha cells after radiation was significantly lower than that of Siha.At the same time,Western Blot showed that the anti apoptotic protein Bcl2 of R-Siha was significantly higher than that of Siha.The cell cycle distribution of R-Siha in G2/M phase was significantly higher than that of Siha cells.Compared with Siha cells,R-Siha cells have stronger migration and invasion ability and more EMT phenotype.2)Cell sequencing and tissue sequencing confirmed that PRDX2 was down regulated in radio-resistant cells and NCR group.Through q PCR and Western blot,it was confirmed that PRDX2 were down regulated in R-Siha cells at m RNA and protein levels.After knockdown of PRDX2 in cell lines with high expression of PRDX2(C33a and Siha cells),Clone formation experiment confirmed that Siha-sh PRDX2 had stronger clone formation ability at different doses;After irradiation,Siha-sh PRDX2 was expressed?-H₂AX is slower,and it decreases in a short time.Meanwhile,the results of flow cytometry and Western blot confirmed that Siha-sh PRDX2 had a lower incidence of apoptosis.That is,knockdown of PRDX2 can reduce the radiosensitivity of cervical cancer cells.PRDX2 was successfully overexpressed in R-Siha cells.Clone formation experiment confirmed that the clone formation ability of overexpression group decreased at different doses;Overexpression group after radiation increased?-H₂AX expression rapidly and at a high level,which was significantly higher than that in the control group.At the sa me time,the results of flow cytometry and Western blot confirmed that overexpression of PRDX2 had a higher incidence of apoptosis,which confirmed that overexpression of PRDX2 could promote the radiosensitivity of cervical cancer.The introduction of gliotoxin,an active analogue of PRDX2,is consistent with the overexpression of PRDX2 and promotes the radiosensitivity of cervical cancer.3)At the drug level and gene level,we inhibited the expression of STAT3 and P-STAT3,and the EMT phenotype of cervical cancer Siha-sh PRDX2 cells was reversed.At the same time,clone formation experiment also confirmed that the radiosensitivity of cervical cancer Siha-sh PRDX2 cells was increased after inhibiting the expression of STAT3.5)In vivo experiments confirmed that knockdown of PRDX2 inhibited the radiosensitivity of cervical cancer,and the active analog of PRDX2,gliotoxin,could reverse this radiation resistance without obvious toxic and side effects.Conclusion:PRDX2 can regulate the radiosensitivity of cervical cancer squamous cell carcinoma cells.Overexpression of PRDX2 or addition of gliotoxin can reverse the radiation resistance and promote the radiosensitivity.PRDX2 mainly regulates the level of phosphorylation and dephosphorylation of STAT3,to change the radiosensitivity of cervical cancer,and effects EMT phenotype.Gliotoxin may become a radiation sensitizer to inhibit the phosphorylation of STAT3 in cervical cancer,which is helpful to control the invasion and metastasis of tumor.