The Effect And Mechanism Of ECT2 In The Occurrence And Development Of Cervical Cancer

Background and objectiveCervical cancer(CC)is one of the critical factors threatening women’s life and health,which is the fourth leading cause of cancer death in women.In recent years,with the popularity of cervical cancer screening and the inoculation of HPV vaccine,the incidence of CC in developed countries has been dramatically reduced.However,for economically backward developing countries,the incidence of CC is still on the rise.Generally,early CC can be treated by surgery and has a clinically noticeable effect,but the prognosis is significantly worse for advanced or recurrent CC.Radiotherapy combined with platinum-based chemotherapy is considered to be the standard treatment for advanced or recurrent CC.However,the emergence of cisplatin resistance seriously hinders its clinical application.Therefore,it is of great necessity to clarify the mechanism of the malignant development of CC,explore new treatment methods,and improve the sensitivity of cisplatin.Epithelial cell transforming sequence 2(ECT2)is a guanine nucleotide exchange factor(GEF)located in a region of chromosome 3q26 that is frequently altered in human tumor chromosomes.ECT2 is associated with various malignant phenotypes and chemotherapy resistance of human cancers and participates in cell proliferation,metastasis,mitosis,apoptosis,DNA damage repair,and tumorigenesis signal transduction pathways.The expression of ECT2 is increased in many malignancies,but the role of ECT2 in the occurrence,development,and drug resistance of CC is unclear.The primary purposes of this study are as follows:(1)to verify the expression of ECT2 in CC;(2)to explore the effect of ECT2 on the malignant phenotypes of CC;(3)to verify the role of ECT2 on cisplatin resistance of CC;(4)to explain the upstream and downstream mechanisms of ECT2 as a proto-oncogene.Methods1.The differential expression genes(DEGs)closely related to the occurrence and development of CC and cisplatin resistance were obtained from TCGA and GEO databases by bioinformatics analysis,and ECT2 to be studied was determined by literature review.Immunohistochemistry(IHC)was used to verify the expression of ECT2 in cervical cancer tissues and normal cervix tissues.Then,we further detected ECT2 expression in cervical cancer tissues and cell lines by RT-qPCR.Keplan-Meier online website was used to predict the effect of ECT2 on the prognosis of patients with CC.2.Verify the effect of ECT2 on the malignant phenotype of CC.First,construct stable cell lines of Ca Ski and HeLa with ECT2 overexpression(PCMV-ECT2)and ECT2 knockdown(sh-ECT2)or transiently transfect with small interference(si-ECT2)to interfere with the ECT2 expression.Then,the effect of ECT2 on the proliferation of cervical cancer cells was verified by CCK-8 and xenograft model assays,the function of ECT2 on cell migration and invasion was detected by Transwell and wound healing assays,and the effect of ECT2 on apoptosis was proved by flow cytometry and Western Blot.3.Determine the expression of ECT2 in cisplatin-resistant cervical cancer cells.CCK-8 was used to detect the effect of ECT2 knockdown on cell viability after cisplatin and the change of IC50.A colony formation assay was performed to observe the colony forming units to verify the effect of cisplatin on the anti-proliferation after ECT2 knockdown.Flow cytometry was used to prove the effect of ECT2 knockdown on cisplatin-induced apoptosis.The experiment in vivo was used to explore the function of ECT2 knockdown on cisplatin sensitivity of cervical cancer cells.4.Use RNA-SEQ to find the downstream pathway AKT/mTOR in which ECT2 plays a role in promoting tumorigenesis.Western Blot and co-immunoprecipitation(Co-IP)assays were used to verify the regulatory effect of ECT2 on the downstream pathway,and rescue experiments were used to verify the function of AKT knockdown on the proliferation,migration,invasion,and anti-apoptosis induced by ECT2 overexpression.The upstream transcription factors of ECT2 were predicted by the JASPER database.The expression relationship between NFYA and ECT2 was determined by bioinformatics analysis.RT-qPCR,Western Blot,and luciferase experiments verified the regulatory effect of NFYA on ECT2 and further proved the relationship between ECT2 and upstream transcription factor NFYA through rescue experiments.Results1.IHC showed that ECT2 was highly expressed in cervical cancer tissues compared with normal cervix tissues.RT-qPCR not only confirmed this conclusion but also found that the expression of ECT2 was increased in cervical cancer cell lines(P<0.05).In addition,the Keplan-Meier online website demonstrated that cervical cancer patients with high expression of ECT2 have shorter recurrence-free survival(RFS)than those with low expression(P=0.023).2.Experiments in vitro showed that overexpression of ECT2 can promote cell proliferation,invasion and migration,and inhibit cell apoptosis and the expression of apoptotic proteins,while the opposite result could be obtained after the knockdown of ECT2,and the differences are statistically significant.The experiment in vivo found that the ECT2 overexpression group could promote tumor formation of cervical cancer cells in nude mice compared with the control group(P<0.05).3.ECT2 has up-regulated in cisplatin-resistant cells of CC.CCK-8 experiment showed that after gradient administration of cisplatin,the viability of cervical cancer cells was damaged more obviously and rapidly in ECT2 knockdown group.The colony formation assay showed that ECT2 knockdown could enhance the inhibitory effect of cisplatin on cell proliferation,and the flow cytometry experiment found that ECT2 knockdown could promote cell apoptosis induced by cisplatin.In vivo experiments demonstrated that ECT2 knockdown could improve the sensitivity of cervical cancer cells to cisplatin.The differences of the above results are statistically significant.4.RNA-SEQ,Co-IP,and Western Blot showed that ECT2 could directly combine with AKT to form an ECT2/AKT complex to activate the AKT/mTOR pathway to promote tumorigenesis.Down-regulated AKT could rescue the proliferative and anti-apoptosis effects of ECT2 overexpression(P<0.05).The JASPER database predicted the binding site of the transcription factor NFYA in the promoter region of ECT2.Bioinformatic analysis showed that NFYA expression was up-regulated in CC and was positively correlated with ECT2 expression.Then we clarified the regulatory effect of NFYA on ECT2 by RT-qPCR,Western Blot,and luciferase assays.By CCK-8,Transwell,and flow cytometry assays,we proved that knockdown NFYA could offset the impact of promoting proliferation and migration,inhibiting apoptosis caused by overexpression of ECT2(P<0.05).Conclusion1.ECT2 is at high expression in cervical cancer tissues and cells,and ECT2 overexpression is associated with poor prognosis;2.ECT2 knockdown can inhibit the proliferation,migration,and invasion and enhance apoptosis of cervical cancer cells;3.Knocking down the ECT2 expression can improve the sensitivity of cervical cancer cells to cisplatin;4.ECT2 can directly bind to AKT to activate the AKT/mTOR pathway and play a downstream role in promoting tumorigenesis;5.Transcription factor NFYA can promote the malignant phenotype of CC by regulating ECT2 expression.The innovation of this study1.This study confirmed the oncogenic potential of ECT2 in the occurrence and development of CC through in vivo and in vitro experiments,which provided a theoretical basis for finding new therapeutic targets for CC;2.This study innovatively found that ECT2 could induce cisplatin resistance in CC,making ECT2 a new target for treating cisplatin resistance in CC;3.This study clarified that ECT2 could directly bind to AKT and activate AKT/mTOR pathway to promote the occurrence of the malignant phenotype of CC,which provided a new idea for the pathogenesis of CC;4.This study expounded that the transcription factor NFYA could activate ECT2 at the level of transcription and promote the expression of ECT2,thus exerting its biological function.The limitation of this study1.This study can include more cervical cancer tissue samples to analyze the relationship between ECT2 and clinicopathological factors of patients with CC;2.The specific mechanism underlying the induction of cisplatin resistance in CC by ECT2 in this study has not been thoroughly explored,which will serve as a future direction for further research;3.This study is still in preclinical research,and further exploration and experimentation are needed to transform ECT2 from the experimental stage to clinical application.