EZH2 promotes invasion and metastasis of laryngeal squamous cells carcinoma via epithelial-mesenchymal transition through H3K27me3.

Enhancer of Zeste Homolog 2(EZH2), which can change chromatin structure by tri-methylation of the 27th lysine of H3 in nucleosome histone (H3K27me3), is involved in different types of cancers. However, the role and mechanism underlying aberrant EZH2 expression in laryngeal squamous cells carcinoma (LSCC) remain unclear. In the present study, we found that down-regulation of EZH2 and H3K27me3 in LSCC cells (Hep-2 and SCC10A) resulted in an mesenchymal-epithelial transition(MET) like cell morphology and lower invasion in vitro, weakened tumor growth, intrahepatic and pulmonary metastasis in vivo. Furthermore, EZH2 promoted the epithelial-mesenchymal transition(EMT) process through down-regulation of Ca2+ dependent cell adhesion molecule E (E-cadherin) and up-regulation of H3K27me3 in vitro and in vivo. Moreover, E-cadherin was transcriptionally induced upon stable knockdown of EZH2, and quantitative chromatin immunoprecipitation(qChIP) analysis confirmed the depletion of H3K27me3 enrichment on E-cadherin promoter upon EZH2 knockdown in Hep-2 and SCC10A cells. In addition, the expression of EZH2 was positively correlated with that of H3K27me3 and both of them were inversely correlated with E-cadherin expression in human LSCC tissues. In summary, this study indicated that EZH2 promoted invasion and metastasis of LSCC via EMT through H3K27me3.

Aberrant methylation­mediated decrease of lncRNA HNF1A­AS1 contributes to malignant progression of laryngeal squamous cell carcinoma via EMT.

Aberrant methylation is one of the most frequent epigenetic alterations that regulate the expression levels of genes, including long non­coding RNAs (lncRNAs), in tumors. However, to the best of our knowledge, the expression and function of hepatic nuclear factor 1α antisense RNA 1 (HNF1A­AS1) and its methylation condition have not yet been reported in the development and progression of laryngeal squamous cell carcinoma (LSCC). In the present study, the expression and methylation of HNF1A­AS1 were first examined by reverse transcription­quantitative PCR, bisulfite genomic sequencing and methylation­specific polymerase chain reaction in samples from patients with LSCC, which were based on the in silico analysis using The Cancer Genome Atlas data, and were then further verified in LSCC cell lines with and without 5­Aza­2'­deoxycytidine (5­Aza­dC) treatment. Subsequently, proliferation, cell cycle distribution, migration and invasion of LSCC cells following either knockdown or overexpression of HNF1A­AS1 were determined in vitro. Furthermore, the characteristic of HNF1A­AS1 on epithelial­mesenchymal transition (EMT) changes was investigated in vitro and in vivo. The associations between the expression levels of HNF1A­AS1 and tumorigenicity and cervical lymph node metastasis were assessed in a xenograft model in nude mice. In the present study, downregulation and hypermethylation in CpG sites of HNF1A­AS1 were detected in LSCC tissues as well as metastatic cervical lymph nodes samples when compared with those in the adjacent non­tumor tissues. Additionally, HNF1A­AS1 inhibited proliferation, migration and invasion of LSCC cells in vitro by regulating the process of EMT. Furthermore, HNF1A­AS1 inhibited tumor growth and metastasis by regulating EMT in vivo. Additionally, the migration and invasion abilities, and the expression levels of HNF1A­AS1 and EMT markers in LSCC cells were significantly reversed by treatment with 5­Aza­dC. In summary, HNF1A­AS1 was downregulated by hypermethylation in LSCC and laryngeal cancer cells. These findings suggested that HNF1A­AS1 could serve as a tumor suppressor lncRNA in LSCC by regulating the EMT process, leading to the discovery of novel therapeutic targets and strategies for the treatment of patients with LSCC.