LncRNA-Six1 Encodes a Micropeptide to Activate Six1 in Cis and Is Involved in Cell Proliferation and Muscle Growth

Abstract

Long non-coding RNAs (lncRNAs) play important roles in epigenetic regulation of skeletal muscle development. In our previous RNA-seq study (accession number GSE58755), we found that lncRNA-Six1 is an lncRNA that is differentially expressed between White Recessive Rock (WRR) and Xinghua (XH) chicken. In this study, we have further demonstrated that lncRNA-Six1 is located 432 bp upstream of the gene encoding the protein Six homeobox 1 (<i>Six1</i>). A dual-luciferase reporter assay identified that lncRNA-Six1 overlaps the <i>Six1</i> proximal promoter. In lncRNA-Six1, a micropeptide of about 7.26 kDa was found to play an important role in the lncRNA-Six1 <i>in cis</i> activity. Overexpression of lncRNA-Six1 promoted the mRNA and protein expression level of the <i>Six1</i> gene, while knockdown of lncRNA-Six1 inhibited <i>Six1</i> expression. Moreover, tissue expression profiles showed that both the lncRNA-Six1 and the <i>Six1</i> mRNA were highly expressed in chicken breast tissue. LncRNA-Six1 overexpression promoted cell proliferation and induced cell division. Conversely, its loss of function inhibited cell proliferation and reduced cell viability. Similar effects were observed after overexpression or knockdown of the <i>Six1</i> gene. In addition, overexpression or knockdown of <i>Six1</i> promoted or inhibited, respectively, the expression levels of muscle-growth-related genes, such as <i>MYOG, MYHC, MYOD, IGF1R</i>, and <i>INSR</i>. Taken together, these data demonstrate that lncRNA-Six1 carries out <i>cis</i>-acting regulation of the protein-encoding <i>Six1</i> gene, and encodes a micropeptide to activate <i>Six1</i> gene, thus promoting cell proliferation and being involved in muscle growth.