Functional And Mechanism Research Of Micropeptide Encoded By Y Chromosome Linked LncRNA In Tumorigenesis And Progression Of Male Esophageal Carcinoma

Background:Esophageal squamous cell carcinoma(ESCC),a major subtype of esophageal cancer,is the third most prevalent and highly aggressive form of esophageal cancer in China.ESCC is two to four times more common in men than in women worldwide.However,the mechanism of this gender difference is still unclear.Previous studies suggest that several male-specific factors,including cigarette smoking and sexual hormone,contribute to such gender disparity,and it is still unclear how these factors lead to gender differences in esophageal squamous cell carcinoma.In human genome,only 1-2%of the region could transcript to protein-coding mRNA,most of the transcripts are non-coding RNA.LncRNA(long non-coding RNA)is a subtype of non-coding RNA,which contains more than 200 nucleotides.LncRNA is involved in multiple human diseases,including cancer.Numerous lncRNAs have been shown to regulate cancer progression through chromosomal modification,epigenetic modification,transcriptional regulation,and alternative splicing regulation.However,whether lncRNA with different expressional pattern between male and female causes the gender difference of ESCC still unknown.Therefore,it is necessary to explore the mechanism of lncRNA with gender-differential expression in the occurrence and development of ESCC.Methods:(1)We obtained Y-linked lncRNAs with gender-differential expression by mining microarray data that derived from 179 pairs of ESCC tissues and validated in ESCC tissues that collected from eastern and southern of China.(2)Using bioinformatic analysis and western blot,we predicted and validated the coding ability of lncRNA.(3)We generated cell models by CRISPR/Cas9 and Lentiviral transduction,and explored the function of lncRNA and micropeptide in ESCC by xenograft models.(4)We investigated the molecular mechanism of cigarette smoking and m6A modification of lncRNA in ESCC by MeRIP,RNA Pulldown,RNA EMSA,western blot and DNA methylation analysis.(5)Using Co-IP and LC-MS/MS,we investigated the target and mechanism of micropeptidein ESCC.(6)Through analyzed the ChIP-seq data and performed ChIP and luciferase reporter assays,we explored the role of micropeptide in the downsteam signaling pathway.(7)We explored the role of micropeptide in ESCC using IHC,Flow cytometry,TUNEL and CCK-8 assays,as well as using cell and xenograft models.Results:In this study,we characterized a Y-linked lncRNA,LINC00278,which was downregulated in male ESCC.LINC00278 encoded a Yin Yang 1(YY1)-binding micropeptide,designated YY1BM.YY1BM was involved in the ESCC progression and inhibited the interaction between YY1 and androgen receptor(AR),which in turn decreased expression of eEF2K through the AR signaling pathway.Downregulation of YY1BM significantly upregulated eEF2K expression and inhibited apoptosis,thus conferring ESCC cells more adaptive to nutrient deprivation.Cigarette smoking decreased m6A modification of LINC00278 and YY1BM translation.Furthermore,the synthetic YY1BM also could inhibit ESCC.Conclusion:Our study provided a novel mechanistic link between cigarette smoking and AR signaling in male ESCC progression.