RNF111/Arkadia is regulated by DNA methylation and affects TGF-β/Smad signaling associated invasion in NSCLC cells.

RNF111/Arkadia is a critical regulator of TGF-β signaling, being required for SMAD3-mediated responses such as TGF-β-induced repression of E-cadherin. Previous studies show that mutations in RNF111 in human cancers are rare and RNF111 promotes lung tumor metastasis. However, the epigenetic mechanisms underlying the role of RNF111 in non-small cell lung cancer (NSCLC) metastasis remain unknown. Here, we mainly focused on low- (95C) and high-metastatic (95D) NSCLC cell lines, which share a similar genetic background, and investigated the methylation-based regulation of RNF111 expression. Clonal bisulfite sequencing, real-time qRT-PCR, western blot analysis, luciferase reporter assays, RNA interference, chromatin immunoprecipitation (ChIP) assay and transwell migration and invasion assays were performed on human NSCLC cell lines 95C and 95D. RNF111 was high-expressed in 95D cells, which showed low-level methylation at -459CpG site in RNF111 promoter. The opposite results were obtained in 95C cells. Cell-based and biochemical assays revealed that -459CpG methylation can inhibit RNF111 transcriptional expression by interfering with the recruitment of Sp1 to RNF111 promoter. On TGF-β stimulation, siRNA-mediated RNF111 knockdown inhibited TGF-β/Smad signaling activity and Snail (an inducer of metastasis) expression, and enhanced E-cadherin (an epithelial-to-mesenchymal transition marker) expression in 95C and 95D cells. Furthermore, demethylation-induced upregulation of RNF111 enhanced phosphorylation of SMAD3 and Snail expression, and repressed E-cadherin expression in 95C cells expressing low RNF111. Our results suggest that -459CpG methylation in Sp1-binding site of RNF111 promoter transcriptionally decreases RNF111 expression, which inhibits TGF-β/Smad signaling associated invasion in NSCLC cells.

Chen H ，Yang T ，Lei Z ，Wang L ，Yang H ，Tong X ，Yang WT ，Zhao J ，Gu Y ，Chen Y ，Zhang HT ... -  《-》

Methylated +58CpG site decreases DCN mRNA expression and enhances TGF-β/Smad signaling in NSCLC cells with high metastatic potential.

Qian Q ，Shi X ，Lei Z ，Zhan L ，Liu RY ，Zhao J ，Yang B ，Liu Z ，Zhang HT ... -  《-》

MiR-145 and miR-203 represses TGF-β-induced epithelial-mesenchymal transition and invasion by inhibiting SMAD3 in non-small cell lung cancer cells.

MicroRNAs (miRNAs) have been proved to play important role in development of various cancers, including non-small cell lung cancer (NSCLC). Our previous studies have shown that miR-203 and miR-145 are associated with cellular invasion in NSCLC and nasopharyngeal cancer, respectively. However, the mechanistic role of miR-203 and miR-145 in TGF-β-induced epithelial-mesenchymal transition (EMT) has not yet been elucidated in human cancers, including NSCLC. Real-time quantitative reverse transcriptase PCR (qRT-PCR), western blot analysis, luciferase reporter gene assays, small RNA interference and transwell migration and invasion assays were carried on human NSCLC cell lines A549 and 95C. Thirty-six paired NSCLC tissues and adjacent noncancerous lung tissues were collected. Both miR-145 and miR-203 can directly target the 3'-untranslated region (3'-UTR) of SMAD3, and overexpression of the two miRNAs in NSCLC cells inhibited the expression of SMAD3 mRNA and protein, whereas inhibition of endogenous miR-145 or miR-203 caused an increased expression of SMAD3. Moreover, miR-145 and/or miR-203 repressed TGF-β-induced EMT and attenuated cell migration and invasion in A549 and 95C cells. siRNA-mediated knockdown of SMAD3 copied the phenotype of miR-145 and miR-203 overexpression in A549 and 95C cells. MiR-145 and miR-203 inhibited TGF-β-induced EMT and invasion through repression of SMAD3 in NSCLC cells. Our findings provided insights into the miRNA-based mechanism for controlling TGF-β-induced EMT of NSCLC cells and a strategy for targeted therapy of NSCLC.

Hu H ，Xu Z ，Li C ，Xu C ，Lei Z ，Zhang HT ，Zhao J ... -  《-》

TGF-β-activated SMAD3/4 complex transcriptionally upregulates N-cadherin expression in non-small cell lung cancer.

Epithelial-mesenchymal transition (EMT) is a key process in early stage of cancer metastasis. TGF-β-mediated EMT is characterized by repression of E-cadherin and induction of N-cadherin (CDH2) in various cancers. Although many investigations have focused on the regulation of E-cadherin expression, the transcription-mediated events that directly induce N-cadherin expression in TGF-β-induced EMT are not fully clear. Here, we mainly focus on non-small cell lung cancer (NSCLC) cells, in which expression of CDH2 can be activated upon TGF-β stimulation, to investigate the underlying mechanisms of CDH2 expression regulation. Western blot analysis, real-time quantitative reverse transcriptase PCR, luciferase reporter gene assays, RNA interference and in vivo chromatin immunoprecipitation (ChIP) assay were performed on human NSCLC cell lines A549 and SPC-A1. Twenty-six paired NSCLC tissues and adjacent noncancerous lung tissues were collected. Luciferase reporter assay revealed that a functional TGF-β-response element was located at position -1078 to -891 in the CDH2 promoter region. Furthermore, in vivo ChIP experiment indicated that TGF-β-activated SMAD3/4 complex was directly recruited to CDH2 promoter region (-1078 to -891). Upon TGF-β1 stimulation, knockdown of SMAD3 or/and SMAD4 led to a significant reduction in CDH2 promoter activity, and silencing of SMAD3 or SMAD4 significantly inhibited CDH2 mRNA and protein expression in A549 and SPC-A1 cells. In human NSCLC tissues, SMAD3 or SMAD4 mRNA level was positively correlated with CDH2 mRNA level, respectively. We found that TGF-β-activated SMAD3/4 complex may upregulate CDH2 expression by directly interacting with a specific SMAD-binding element in CDH2 promoter. Our findings provide insights into mechanisms underlying the transcriptional regulation of CDH2 expression in TGF-β-induced EMT and SMADs-based therapeutic strategies for NSCLCs.

Yang H ，Wang L ，Zhao J ，Chen Y ，Lei Z ，Liu X ，Xia W ，Guo L ，Zhang HT ... -  《-》

JAK/STAT3 signaling is required for TGF-β-induced epithelial-mesenchymal transition in lung cancer cells.

Liu RY ，Zeng Y ，Lei Z ，Wang L ，Yang H ，Liu Z ，Zhao J ，Zhang HT ... -  《-》