Long Noncoding RNA HOTAIR Promotes Metastasis Of Oral Squamous Cell Cacinoma By Regulating Epithelial-to-Mesenchymal Transition

Objective: Long non-coding RNA(lnc RNA)plays an important role in tumor development and invasion at epigenetic,transcriptional and post-transcriptional levels.HOX transcription of antisense RNA(HOTAIR),regarded as an oncogene,play a critical role in the epigenetic regulation in human cancer.However,the functional mechanisms of HOTAIR are unclear in oral squamous cell carcinoma(OSCC).This study firstly investigated the relationship between HOTAIR expression and clinicopathological parameters as well as prognosis of oral squamous cell carcinoma.Secondly,the study focused on HOTAIR mediated regulation on proliferation,apoptosis,invasion and metastasis of OSCC cells in vitro.Thirdly,we elucidated the molecular mechanisms involved in HOTAIR included EMT and tumor metastasis in OSCC.Finally,a tumor-bearing mouse models were gengreted to investigate the HOTAIR mediated EMT and anti-tumor activity of HOTAIR target therapy in vivo.Methods: 1.Both OSCC tissues and adjacent histological normal tissues were obtained from 76 patients who were admitted to the Department of Maxillofacial Surgery and Otorhinolaryngology Head and Neck Surgery of Tianjin Medical University between January 2001 and March 2009.None of the patients received treatment prior to radical surgical treatment.The relationship between HOTAIR expression and clinicopathological characteristics as well as patient's prognosis.2.We performed a q PCR analysis to examine the expression level of HOTAIR in different 2 high expression OSCC cell lines,and HOTAIR are efficiently knocked down by si RNA.MTT assay,colony assay,flow cytometry cytometry assay,Wound healing scratch assay and Matrigel invasion assay were used to detect the proliferation,colony formation ability,apoptosis cells,invasion and metastasis of HOTAIR-knocked down cell lines.3.We performed real-time PCR and Western blot to detect the expression of EMT markers,PRC2 and HOTAIR.The correlation between HOTAIR and E-cadherinand as well as EZH2,H3K27me3 were compared.Ch IP assay was performed to detect the binding activity between E-cadherin promoter and EZH2/H3K27me3 after HOTAIR knockdown.4.Xenograft model was generated to observe tumor growth and lymphnode metastasis of OSCC cells.Furthermore,the expression lecel of HOTAIR,EZH2,H3K27me3 and E-cadherin were detected by real-time PCR and immunohistochemistry.Results: 1.The expression level of HOTAIR was detected in 76 OSCC samples and adjacent normal tissues by q RT-PCR and normalized by GAPDH.HOTAIR was upregulated in OSCC tumor compared with non-tumor tissues(p<0.001),and especially overexpressed in tumors with regional lymph nodes metastasis(N1)compared to tumors without lymph nodes metastasis(N0)(p<0.001).The univariate analysis revealed that HOTAIR expression strongly correlated with the clinical stage,lymph node metastasis,and histological differentiation in patients with OSCC.Kaplan–Meier survival analysis showed that HO TAIR overexpression patients had significantly shorter disease free survival(DFS)and overall survival(OS)compared to HOTAIR low-expression patients.2.Both TSCCA and Tca8113 cells expressed significantly higher levels of HO TAIR.HOTAIR expression was effectively knocked down in TSCCA and Tca8113 cells by si RN A method.MTT assay reveaed that knockdown of HOTAIR notably repressed the proliferation of OSCC cells.Colony formation assay indicated that the silence of HOTAIR expression significantly reduced the numbers colony of both OSCC cells.Flow-cytometric analysis assay indicated that HO TAIR knockdown promoted both early apoptosis and later apoptosis of OSCC cells Tca8113 and TSCCA.Wound healing scratch assay and matrigel invasion assay showed that downregulation of HOTAIR decreased the migration and invasion ability of both OSCC cells.3.A significant negative correlation is observed between the E-cadherin m RNA levels and the HOTAIR expression in OSCC tissues(r2=-0.327,p= 0.004). Silence of HOTAIR significantly increased E-cadherin expression at both transcript and protein levels in TSCCA and Tca8113 cells.Bsides that,we observed expression/recruitment of EZH2 and H3K27me3 significantly decreased after HOTAIR knockdown,accompanied by significantly increased expression of E-cadherin in OSCC cells.Ch IP-arrays showed that HOTAIR silence decreased the binding of EZH2 and H3K27me3 with the E-cadherin promoter in OSCC cel s.4.The tumor growth of OSCC xenograft was suppressed by silence of HOTAIR in vivo.At the same time,the expression of EZH2 and H3K27me3 was downregulated and E-cadherin was upregulated along with HO TAIR silence in xenograft tissues.Conclusion: 1.HOTAIR overexpression is observed in oral squamous cell carcinoma,and significantly correlates lymph node metastasis and poor prognosis.2.HOTAIR promotes proliferation,colony formation,invasion and metastasis,as well as inhibits apoptosis in oral squamous cell carcinoma cel s.3.HOTAIR represses E-cadherin expression by interfering the expression/recruitme nt of EZH2 and the level of H3K27me3 in OSCC cells.4.HOTAIR promotes metastasis of ora l squa mous cell carcinoma by induc ing EMT and HOTAIR target therapy can significantly inhibit tumor growth and metastasis in vivo.