Epigenetic Mechanisms Underlying The Role Of RNF111 In Controlling TGF-β/Smad Signaling Are Associated With Non-small Cell Lung Cancer Metastasis

Background and Objective: Lung cancer is the fastest growing incidence and mortality, the biggest threat to people health and life of one of the malignant tumor. And due to the lack of early diagnosis and treatment technology, accounts for about 85% of non-small cell lung cancer has as much as 86% of lung cancer mortality, Therefore,in-depth study of the pathogenic mechanism of non-small cell lung cancer research is particularly important. DNA methylation is an important content of epigenetics research,is considered to be the most typical cause gene expression or silent mechanism, and is closely related to the development of lung cancer. TGF-β signaling pathways anomaly closely related to the development and metastasis of a variety of tumor. Arkadia,encoded by gene RNF111, is a positive regulator of TGF-β signaling through degradation of Smad7, SnoN and Ski. This study was performed to detect the expression of RNF111 gene in two cell lines of non small cell lung cancer 95C(low metastasis) and 95D(high metastasis), to explore the potential mechanism between the differential expression of RNF111 and promoter methylation induced regulation change.Methods: Transwell assays to determinate the matastasis ability of 95C、95D and A549 cells; qRT-PCR and Western Blot analysis and was carried out to analysis the expression of RNF111 in two cell lines(95C and 95D), and the expression levels were calculated following normalization to β-actin mRNA levels, and detected the expression of RNF111 in 6 pairs of Lung carcinoma tissues with Stage III or IV NSCLC; we analyzed the methylation status of CpG sites sited in RNF111 promoter region in 95 C and 95 D cell lines through BSP and clone sequencing; Then we analyzed the relationship between the methylation frequencies of CpG sites of which methylation status are significantly different in two cell lines and different mRNA level; in addition,EMSA was used to verify whether the methylated-459 CpG site could affect the abilityof SP1 binding to RNF111 proximal promoter; ChIP assay was tested to explore whether SP1 can bind to-459 CpG site; Construction of luciferase reporter plasmid and luciferase assay were performed to determinate whether SP1 could enhance the luciferase activity of RNF111 promoter, in additionthe. To determinate whether the methylated-459 CpG could diminish the luciferase activity of RNF111 promoter;Western Blot analysis and was carried out to detect the mRNA and protein level of RNF111 after knocking down SP1; Western Blot analysis to detect the level of p-Smad3、Snail and E-cadherin in TGF-β signal pathway after knocking down RNF111 in 95C and 95 D cell lines.Results: After treatment with 5-Aza on 95 C and 95 D cells, RNF111 mRNA and protein expression was increased, the results suggested the involvement of DNA methylation in RNF111 expression; Through TRANSFAC software forecast and CHIP experimental verification,-459 CpG site was a potential functional CpG site, the result of CHIP analysis revealed that RNF111 proximal promoter region containing-459 CpG was bound by SP1; Luciferase assay for the constructs containing the wild-type or deletion and mutation of Sp1-binding site in RNF111 promoter showed that SP1 could enhance the luciferase activity of RNF111 promoter; the results of EMSA and luciferase assay indicated that methylation of-459 CpG can reduce the recruitment of SP1 to RNF111 proximal promoter region and restrain the activity of RNF111 promoter; Knocking down RNF111 related experiments indicate that RNF111 enhances TGF- β /Smad signaling activity and the migratory and invasive abilities of NSCLC cells.Conclusion: Our recent research found that the methylated-459 CpG in RNF111 proximal promoter reduced expression of RNF111 in NSCLC cells. And the deeper research indicate that-459 CpG methylation may be one of mechanisms that reduced recruitment of transcriptional activator SP1 to RNF111 proximal promoter, and reduced the RNF111 expression. And thereby weakened TGF-β/Smad signaling, lead to reduced p-SMAD3 expression level, enhanced expression of E-cadherin.