The Research On The Mechanism Of LncRNA ANRIL Promoting The Proliferation Of Head And Neck Squamous Cell Carcinoma

Background and objective: Long non-coding RNA is involved in the generation and development of many diseases.Experiments show that long non-coding RNA ANRIL can promote the proliferation,invasion and migration of prostate cancer cells in vitro and in vivo,and can be used as a predictor of the prognosis of cancer patients.However,mechanism of long non-coding RNA ANRIL in head and neck squamous cell carcinomas is still not clear.Our studies mainly search the biological role of long non-coding RNA ANRIL in head and neck squamous cell carcinoma and its related molecular mechanism of the proliferation of cancer in order to find a potential new target for the treatment of head and neck squamous cell carcinoma patients.Methods: 1.The expression of lncRNA ANRIL in 49 pairs of HPV-negative head and neck squamous cell carcinomas and normal tissues was detected by RT-PCR.2.Construction of lentivirus-mediated silencing ANRIL vector,establishment of stable transfected cell lines,and validation of knockdown efficiency.CCK8,plate cloning and flow cytometry were used to study the effect of ANRIL on the proliferation of head and neck squamous cell carcinoma cells.3.Bioinformatics software predicted targeted microRNAs,immunoprecipitation and luciferase reporter experiments verified the correlation between ANRIL and microRNAs.4.Construction of the overexpression and knockdown microRNA plasmid,establishment of transfected cell lines,and validation of transfected efficiency.CCK8 and plate cloning were used to detect the effect of microRNAs on the proliferation of head and neck squamous cell carcinoma cells.5.RT-PCR was used to identify targeted genes of microRNAs,and luciferase reporter experiment was used to validate target genes.6.SiRNA that knockdown of target genes was transfected into head and neck squamous cell carcinoma cell lines.The knockdown efficiency was verified by Western blot and RT-PCR.CCK8,plate cloning and flow cytometry were used to study the effects of target genes on proliferation.7.Investigating the effect of target genes on the MAPK signaling pathway by Western blot.8.Xenograft tumor assay and observing the effects of ANRIL and target genes on proliferation of head and neck squamous cell carcinoma by intraperitoneal injection.Res?lts 1.The expression of ANRIL in head and neck squamous cell carcinomas was detected by RT-PCR.It was found that ANRIL was highly expressed in head and neck squamous cell carcinomas and was associated with poor pathological differentiation.The worse differentiation and higher expression,the worse prognosis.2.Construction of lentiviral stable ANRIL knockdown cell model,and the knockdown efficiency is more than 85%.Knockdown of lncRNA ANRIL inhibited the proliferation of HNSCC cancer cell(CAL27,HN6),decreased the tumorigenicity of platelets and decreased S-phase cells.3.Expression of lncRNA ANRIL is negatively correlated with the expression of downstream microRNA-125a-3p in vivo.LncRNA ANRIL competitively inhibits the expression of microRNA-125a-3P in cells.4.Construction of miR-125a-3p overexpression and knockdown plasmid were verified by qRT-PCR,and the efficiency of overexpression and knockdown were more than 60%.The knockdown and recovery experiments showed that lncRNA ANRIL promoted the proliferation of head and neck squamous cell carcinoma cells by competitively inhibiting the expression of microRNA-125a-3p.5.Expression of miR-125a-3p was negatively correlated with the expression of downstream target gene FGFR1.The small interfering efficiency of FGFR1 knockdown is up to 60%.After knocking down FGFR1,it can effectively inhibit the proliferation of head and neck squamous cell carcinoma cells and reduce S phase cells.6.Wersten blot showed that the expression of p-p38,p-ERK and p-AKT decreased(increased)after the knockdown(over-expression)of FGFR1.7.Xenograft tumor assay showed that the tumorigenesis and proliferation ability of head and neck squamous cell carcinoma cells decreased after ANRIL knockdown,and further decreased after combining with FGFR1 inhibitor.Conclusions 1.Compared with normal tissues,the expression of ANRIL in head and neck squamous cell carcinoma is higher,which co?ld be used as a candidate target for dignosis of head and neck squamous carcinoma.2.ANRIL activates the MAPK signaling pathway and further promotes the proliferation of head and neck squamous cell carcinomas by miR-125a-3p/FGFR1/MAPK signalling axis.3.We can inhibit the proliferation of head and neck squamous cell carcinoma by inhibiting the expression of ANRIL or using small molec?le inhibitor of FGFR1,and it may provide a new targeted therapy of head and neck squamous cell carcinoma.