KLF4 Inhibits Radiosensitivity And Play The Role Of Molecular Mechanism In Cervical Cancer

ObjectiveCervical cancer is one of the three major malignant tumors of gynecology,the latest statistics show that about 12360 new cases of new cervical cancer were projected to occur a year in the United States.The treatment of cervical cancer includes surgery,radiotherapy,chemotherapy and comprehensive treatment.Radiotherapy is one of the most important methods in the treatment of cervical cancer.However,radiotherapy resistance is still considered as a big problem.There are a lot of factors of radiation resistance,such as the apoptosis,cyclo-oxygenases,angiogenesis,hypoxia and temperature.Kruppel-like factor 4(Krüppel-like factor 4,KLF4)is widely expressed in a variety of human tissue as a transcription factor,and in different physiological,play an important role in growth and development,mutation and homeostasis.KLF4 played an anti-apoptotic role in response to irradiation injury and might also aid in cell survival secondary to irradiation through either its association with DNA repair,reduced cell death,increased regeneration,or all three processes.KLF4 played a critical role in modulating the BMI1+ intestinal stem cell fate during both homeostasis and the regenerative response to radiation injury in vivo.At present,studies have shown that KLF4 can lead to cell cycle arrest,inhibition of BAX expression,and ultimately reduce the ability of apoptosis.KLF4 also showed the anti-apoptotic effect after radiotherapy in mice.Furthermore,in pre-experiments,we found that KLF4 expression was also associated with radiotherapy sensitivity in cervical cancer cells(HeLa).So we hypothesized whether KLF4 is also associated with radiotherapy sensitivity of cervical cancer.If we regulate the expression level of KLF4,what kind response would happened to cervical cancer cells on the sensitivity of radiotherapy,and What important role does KLF4 have in the process of radiotherapy sensitivity? In order to confirm this hypothesis,we will study from the clinical data retrospective research,molecules,cells,and animal models.This will lay the foundation for the study of KLF4’s mechanism of radiotherapy sensitivity,and provide potential drug targets for improving clinical radiotherapy.Methods1.Immunohistochemistry was used to detect the expression of KLF4 in cervical cancer tissues.The relationship between KLF4 expression and clinical parameters was analyzed statistically.2.The expression of molecular was detected by Immunofluorescence staining,qPCR and western-blot.RNAi technique and overexpression technique was used to construct lentiviral vector.Gene chip was analyzed to detect the downstream genes regulated by KLF4.The dual luciferase reporter assay was used to detect the transcriptional activity of the promoter.3.Cell cycle and apoptosis were measured by flow cytometry.Clone formation assay was used to measure radiotherapy sensitivity.4.At the animal level,a cervical cancer model of nude mice was established.The effect of KLF4 on radiotherapy of cervical cancer was further observed according to the cervical cancer xenografts in nude mice.HE was used to observe the histomorphology.Molecular expression of apoptosis and proliferation was analyzed by immunohistochemistry.Results1.In radiotherapy-sensitive and resistant groups,FIGO stage,tumor diameter,and tumor differentiation was associated with radiotherapy.The expression level of KLF4 was correlated with FIGO staging and tumor differentiation.The expression level of KLF4 was correlated with radiotherapy sensitivity and was higher in resistant group than in sensitive group.The high expression of KLF4 in the resistant group may be associated with no-response to radiotherapy,local recurrence,and distant metastasis after radiotherapy.Survival analysis showed that the PFS and OS of KLF4 high expression was significantly shorter than the low expression group.The univariate/ multivariate Cox regression analysis revealed that KLF4 expression level,FIGO stage,tumor diameter size and tumor differentiation degree may be the high risk factors for PFS and OS.2.The highest level of KLF4 expression was HeLa and the lowest was C33 A in four cervical cancer cells.The sensitivity of C33 A after radiotherapy was more sensitive than that of HeLa.We successfully constructed the RNAi KLF4 and over expression lentivirus.Overexpression KLF4 promotes the radioresistance in cervical cancer cells.Knockdown KLF4 enhances the radiosensitivity in cervical cancer cells.3.Successful establishment of cervical cancer xenografts in nude mice.Overexpression of KLF4 increases radiation resistance of cervical carcer xenografts.Knockdown KLF4 enhances the radiosensitivity in cervical cancer xenografts.4.Overexpression of KLF4 up-regulated IGF2 gene.The expression level of KLF4 was positively correlated with the activity of IGF2 promoter.High expression of KLF4 involved in the radiation resistance of cervical cancer through IGF2/PI3K-AKT pathway.ConclusionsIn summary,high expression of KLF4 inhibits cervical cancer radiotherapy sensitivity.Intervention of KLF4 expression level can affect the sensitivity of cervical cancer radiotherapy.KLF4 involved in the radiation resistance of cervical cancer through IGF2/PI3K-AKT pathway.The study laid the foundation for the study of KLF4’s mechanism of radiotherapy sensitivity,and provided potential drug target for improving clinical radiotherapy.