Long non-coding RNA MALAT1 functions as miR-1 sponge to regulate Connexin 43-mediated ossification of the posterior longitudinal ligament

Abstract

Ossification of the posterior longitudinal ligament (OPLL) is the major cause for several deteriorate bone and joint diseases. Its development is a highly organized dynamic process as modulated by various physiological and pathophysiological factors. Both long non-coding RNAs (lncRNAs) and small non-coding RNAs (miRNAs) have been postulated to involve into almost all the biological conditions. Here, we applied high through-put transcriptome screening to unveil lncRNAs highly regulated under OPLL condition. siRNA assay in combination with western blot and quantitative PCR deciphered the lncRNA and miRNA functions in OPLL and their underlying mechanism. Here we identified an lncRNA, named Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) engaged into the development of OPLL by indirectly targeting Connexin 43 (Cx43) gene. As previously reported, Cx43 is one of the main proteins contributing to OPLL partially through enhancing inflammatory signaling. On top of that, we provided another regulatory layer that MALAT1 served as the upstream effector governing the transcription of Cx43 gene. Perturbation of MALAT1 significantly inhibited Cx43 expression, inflammation, and osteogenesis. Mechanistically, in silico analysis and experimental validation both confirmed that microRNA-1 (miR-1) was the mediator connecting MALAT1-Cx43 axis: overexpression of miR-1 diminished Cx43 expression and OPLL process; meanwhile, MALAT1 acted as miR-1 sponge to inhibit its suppressive transcription effect on downstream ossification related genes. Knock-down of MALAT1 released sequestered miR-1, which repressed Cx43 expression and associated OPLL. Likewise, induced OPLL caused by overexpression of MALAT1 can be ameliorated by enhanced miR-1 function, knock-down of Cx43 or inhibition of inflammation. More importantly, further validation using patient ligament samples from non-OPLL and OPLL individuals identified MALAT1-miR-1-Cx43 regulatory axis. Collectively, we found a novel mechanism through lncRNA-miRNA interaction that provides more insights into understanding the development of OPLL.

Keywords: Connexin 43; Long non-coding RNA; MALAT1; Ossification of the posterior longitudinal ligament; miR-1; microRNA.