Molecular Mechanism Of Transcription Factor KLF5 In Regulation Of Epithelial Mesenchymal Transition In Cervical Cancer Cells

Cervical cancer accounts for nearly 12% of all female cancers worldwide,making it the fourth most common female cancer in the world and a major global health challenge.According to the WHO report,there are an estimated 570,000 new cases of cervical cancer in 2018,with approximately 310,000 deaths.According to the degree of pathological development of cervical cancer,the treatment can be performed by surgery or chemotherapy,or both.However,for patients with metastatic or recurrent disease,the overall prognosis is poor,and further research is needed.Epithelial-mesenchymal transition(EMT)is an important biological process for the migration and invasion of epithelial-derived malignant cells.The purpose of this experiment was to study the molecular mechanism of Krüppel-like factor 5(KLF5)regulate the epithelial-mesenchymal transition(EMT)process in cervical cancer cells.Firstly,the effect of overexpression of KLF5 on cervical cancer cell lines was tested.Based on the expression of KLF5 gene in several cervical cancer cell lines,the cell line HeLa and SiHa cell lines were selected in this study.The effects of KLF5 on the proliferation,invasion and metastasis of cervical cancer cell lines were detected by MTT,Cell Scratch,Transwell methods after overexpression of KLF5 gene in HeLa cells.As a results,MTT assay showed that the cell proliferation ability did not change significantly;however,Cell Scratch and Transwell experiments showed that KLF5 can significantly inhibit the migration and invasion of HeLa cells.After silencing KLF5 with siRNA in SiHa cells,Cell Scratch and Transwell experiments showed that KLF5 can significantly promote the migration and invasion.Secondly,the effect of the transcription factor KLF5 on the expression of EMT-related molecules was studied.HeLa cells and SiHa cells were selected as the research objects,and the expression of EMT-related molecules was detected by real-time PCR and Western blot.The results showed that overexpression of KLF5 promoted the expression of E-cadherin in HeLa cells,inhibited the expression of N-cadherin and MMP9,and down-regulated the expression of SNAI1.Silencing KLF5 with siRNA inhibited the expression of E-cadherin in SiHa cells,promoted the expression of N-cadherin,and up-regulated the expression of the upstream transcription factor SNAI1.We investigated the activity of KLF5 to regulate SNAI1 promoter activity.The KLF5 binding site of the SNAI1 promoter region was analyzed by the database JASPAR,and multiple binding sites were found.Based on this,SNAI1 promoter truncated plasmids of different lengths were constructed,and KLF5 was detected by luciferase reporter gene for SNAI1 initiation.The effect of sub-truncated activity was analyzed.KLF5 may bind to the-200 ~-100 region of the SNAI1 promoter region to inhibit SNAI1 expression.Thirdly,KLF5 regulates the activity of the SNAI1 promoter assay.The KLF5 binding site of the SNAI1 promoter region was analyzed by the database JASPAR,and multiple binding sites were found.Then,SNAI1 promoter truncated plasmids of different lengths were constructed,and KLF5 was detected by luciferase reporter gene for SNAI1 initiation.The effect of sub-truncated activity was analyzed.KLF5 may bind to the-200 ~-100 region of the SNAI1 promoter region to inhibit SNAI1 expression.Finally,to further verify that KLF5 mediates the cell EMT process through SNAI1 to regulate the metastasis and invasion of cervical cancer cells,SNAI1 was overexpressed in HeLa cells,and the expression of EMT-related genes was detected by real-time PCR and Western blot.The results showed that overexpression of KLF5 could inhibit the expression of E-cadherin in HeLa cells,promote the expression of N-cadherin and MMP9,and up-regulate the expression of transcription factors SLUG,ZEB1/2 and TWIST1.Therefore,it is demonstrated that SNAI1 can regulate the EMT process of cervical cancer cells in cervical cancer.