The Effect Of LncRNA POU6F2-AS2 On Growth And Radiotherapeutic Efficacy In Hunman Oesophageal Squamous Cell Carcinoma

Objective In this study,the effect of lncRNA POU6F2-AS2 on growth,apoptosis,DNA damage repair,radiotherapeutic efficacy,and its accurate mechanism of radiosensitization were investigated in human oesophageal squamous cell carcinoma,in vitro and in vivo.Methods To identify genes specific expressed in OSCC,we performed a meta-analysis based on the GENEVESTIGATOR platform which included 73971 samples.POU6F2-AS2 is the only noncoding gene that is not highly expressed in either OAC or normal esophageal tissues,but overexpressed in OSCC.In vitro,real-time PCR was used to check the expression profile of POU6F2-AS2 in 6 OSCC cell lines and 2 OAC cell lines;Knockdown,stable knockdown and over-expressing of POU6F2-AS2 were transfected by Lipofectamin 2000,lentiviral constructs and pLenti6.3 vector,respectively;The cell viability was detected by MTT assay;Clonogenic survival data was obtained to assess effects of treatment on radiosensitization;Changes of cell apoptosis was analyzed by flow cytometry assay;Single-cell gel electrophoresis,cytokinesis block micronucleus method and immunofluorescencewere assay were used to assess ability of DNA damage repair;POU6F2-AS2 associated protein and binding region of POU6F2-AS2 to the associated protein were identified and confirmed by RNA pulldown,mass spectrometry analysis,western-blot,RIP and clonogenic survival assay;Whether POU6F2-AS2 could affect its associated protein's chromatin binding was explored by immunofluorescencewere,ChIP-Seq and qPCR assays.To determine in vivo radiotherapeutic efficacy,tumor growth delay was analyzed in an OSCC tumor-bearing xenograft mouse model after repeated treatment of shPOU6F2-AS2 and/or IR.Results POU6F2-AS2 is the only noncoding gene that is not highly expressed in either OAC or normal esophageal tissues,but overexpressed in OSCC;POU6F2-AS2 expressions were high in OSCC cell lines and low in OAC cell lines;Stable knockdown cell lines and stable over-expression cell lines were generated for functional study;POU6F2-AS2 knockdown alone did not affect OSCC cell growth(P>0.05);POU6F2-AS2 knockdown enhanced the radiation-induced cytotoxicity of OSCC cells,in a radiation dose-dependent manner(P<0.01);POU6F2-AS2 knockdown and over-expressing in combination with radiation resulted in increased and decreased radiosensitivity of OSCC cells,respectively;POU6F2-AS2 knockdown enhanced radiation-induced apoptosis in OSCC cells(P<0.01):POU6F2-AS2 knockdown increased DNA damage by radiation(P<0.01):Western blot showed that POU6F2-AS2 sense RNA binded Ybxl protein,vice versa,we immunoprecipitated the endogenous Ybxl proteins and analyzed the bound RNAs,POU6F2-AS2 was abundantly found in the real time PCR results;The enhanced expression of Ybxl in POU6F2-AS2 knockdown cells partially rescued the cells from clonogenic inhibition,on The contrary,reduced expression of Ybx1 expression in POU6F2-AS2 over-expressing cells partially reversed the resistance;The catRAPID program was used to predict the binding region,series of truncations of POU6F2-AS2 were generated based on the prediction to perform the pull-down assay.The western blot results showed that the N-terminal 1-800 nucleotide region of POU6F2-AS2 is important for the binding;Knockdown POU6F2-AS2 expression abrogated the Ybxl's localization to DNA damage sites after laser micro-irradiating,decreased Ybxl's signal on chromatin,attenuated the Ybxl binding to its target genes promoter and down-regulating the expression of Ybxl's target genes;Tumor growth in OSCC xenograft mice was markedly delayed by POU6F2-AS2 knockdown combined with IR(P<0.05).Conclusions We reported an uncharacterized IncRNA POU6F2-AS2.It is highly expressed in OSCC.POU6F2-AS2 is also involved in the growth,apoptosis,DNA repair process and radiotherapeutic efficacy.POU6F2-AS2 is indispensable for Ybxl's chromatin localization and Ybx1 could compensate or reverse the function of POU6F2-AS2 in DNA damage response and radiotherapeutic efficacy.We suggest that POU6F2-AS2 could represent a novel biomarker candidate for the prognosis ESCC cancers and a promising new component to the biological complexity of esophageal tumor biology.