Role And Mechanism Of KLF14 In Proliferation And Migration Of Hepatocellular Carcinoma

Background and objectivesKruppel-like factor 14(KLF14)also known as BTEB5,belongs to the Sp/KLF gene family and is a transcription factor.Recently,it has been found that cell metabolism related gene KLF14 may play an important role in the development of tumors,but its role in the proliferation and metastasis of hepatocellular carcinoma remains unclear.Aim:To detect the expression of KLF14 in hepatocellular carcinoma(HCC)by clinical tissue samples,and to observe the effect of up-regulation of KLF14 expression on the growth and migration of HCC cells by over-expression method.Finally,we explore the molecular mechanism of KLF14 inhibiting metastasis of hepatocellular carcinoma,so as to provide more theoretical basis for the research of KLF14 in the treatment of hepatocellular carcinoma.Methods1.Immunohistochemistry was used to detect the localization and expression of KLF14 in 180 samples.2.The relationship between KLF14 expression and survival time was analyzed by KMplotter website,and its prognostic value was predicted.3.Transient KLF14 overexpression plasmid or control plasmid in Bel-7402 and HepG2 cells were detected by Real time PCR and Western blot at the level of mRNA and protein respectively.4.KLF14 was knocked out by crispr-cas9 technology.The knockout effect was verified by sequencing,cruiser Enzyme digestion and Western blot.5.CCK8 assay and clone formation assay were used to explore the effect of KLF14 on the proliferation of hepatocellular carcinoma cells.The effect of overexpression of KLF14 on the proliferation of hepatocellular carcinoma cells was further verified in vivo by nude mice tumorigenesis experiment.6.Scratch test and Transwell test were used to explore the effect of KLF14 on the migration ability of hepatocellular carcinoma cells.7.KLF14 overexpression plasmid or control plasmid was transfected instantaneously in Bel-7402 and HepG2 cells.The effects of KLF14 on the expression levels of EMT epithelial markers E-cadherin,interstitial markers Snail,Vimentin and N-cadherin were explored by Real tume PCR and Western blot,respectively.8.To find the binding site of KLF14 on vimentin promoter through Jaspar website,to explore the effect of KLF14 on the activity of vimentin promoter through double luciferase experiment,and to explore whether KLF14 can bind to vimentin promoter through CHIP experiment.9.KLF14 overexpression plasmid and vimentin overexpression plasmid were co-transfected into Bel-7402 cells and HepG2 cells,and the effect of KLF14 on tumor inhibition through vimentin was explored by response experiment.Results1.Expression of KLF14 in liver cancerKLF14 expression in clinical samples:Statistical analysis of the results of immunohistochemistry showed that KLF14 expression in hepatocellular carcinoma tissues was lower than that in adjacent tissues;KMplotter website was used to analyze the relationship between KLF14 expression and survival.It was found that the total survival time of patients with high expression of KLF14 was higher than that of patients with low expression of KLF14.2.KLF14 can inhibit the proliferation and migration ability of liver cancer cellsEffects of KLF14 on the function of hepatocellular carcinoma cells:CCK8 and clone formation experiments showed that KLF14 inhibited the proliferation of hepatocellular carcinoma cells;subcutaneous tumorigenesis experiments in nude mice showed that the tumorigenesis ability of KLF14 knockout group was stronger than that of control group.Scratch test and Transwell migration test showed that 5 up-regulation of KLF14 expression could inhibit the migration of hepatocellular carcinoma cells.3.KLF14 directly regulates the expression of vimentinKLF14 regulated EMT:Real time PCR results showed that overexpression of KLF14 could enhance the expression of epithelial markers E-cadherin,reduce the expression of interstitial markers vimentin and N-cadherin,and had no effect on Snail expression at RNA level.Western blot assay showed that compared with the control group,the expression of E-cadherin,vimentin and N-cadherin were up-regulated in KLF14 overexpression group,and Snail was down-regulated in protein level.4.KLF14 can work through vimentinDouble luciferase assay confirmed that KLF14 inhibited the activity of vimentin promoter,CHIP assay confirmed that KLF14 could bind to the vimentin promoter region,and reply experiment proved that KLF14 inhibited the migration of hepatocellular carcinoma cells through viemntin.ConclusionsKLF14 inhibits the migration of liver cancer cells by reversing EMT.