Abstract 1825: The inhibitory effect of long non-coding RNA MALAT1 on Smads signaling pathway.

Abstract Long non-coding RNA (lncRNA) is arbitrarily defined as RNA longer than 200 bases that does not encode mRNA, rRNA or tRNA. LncRNAs are involved in almost every aspect of cellular and molecular biology. In most cases, lncRNAs assemble with proteins as RNA-protein complexes to execute their functions. We hypothesize some important molecular in critical signaling pathway, such as Smad2 and Smad3 in TGF-β/Activin pathway, might have their lncRAN partner/partners to fine regulate their function. Based on the hypothesis, we carried on little scale screen and finally identified lncRAN MALAT1 can exclusively bind to pSmad2 and pSmad3 in hepatocellular carcinoma cell lines Hep3B, SK-Hep1 and cholangiocarcinoma cell line CCLP1 by methods of RNA Immunoprecipitation (RIP). After quantifying by qRT-PCR, we found that MALAT1 enriched by anti-pSmad2/3 antbodies is >15 times more than that by IgG control. Co-localization of MALAT1 and pSmad2 and 3 in nuclear speckles of Hep3B cells were also proved by methods of combining FISH and immunofluorescence staining. Further bioinformatics analysis and immunoprecipitation reveal that pSmad2/3 binding to MALAT1 through SET domain containing 2 (SETD2) indirectly. Accordingly, if SETD2 was knockdown by siRNA, the binding between pSmad2/3 with MALAT1 was proved decreasing dramatically by methods of RIP. We also found that MALAT1 knockdown by shRNA or antisense oligonucleotide will increase the Smad2/3 signaling activity significantly, which was proved by Smad2/3 luciferase reporter constructs, DNA pull-down using synthesized oligonucleotide containing consensus sequence of Smads binding elements (SBE) and enhanced inhibition effect of TGF-β on the proliferation of Hep3B and CCLP1 cells. Next, Protein Phosphatase 1A (PPMA1), which act as a Smad phosphatase to terminate pSmad2/3 activity, was proved to bind to MALAT1 and MALAT1 knockdown decreased the binding of pSmad2/3 to PPM1A. Therefore, MALAT1 (together with SETD2) might act as scaffold to facility the interaction between pSmad2/3 and PPM1A, which lead to the dephosphorylation of pSmad2/3. This kinetically decrease of pSmad2/3 dephosphorylation might be the reason of increased Smads signaling activity in MALAT1 knockdown cells. This work was supported by National Institutes of Health grants CA102325, CA106280, CA134568, DK077776 (to T.W.) and CA137729 (to C.H.) Citation Format: Jinqiang Zhang, Chang Han, Tong Wu. The inhibitory effect of long non-coding RNA MALAT1 on Smads signaling pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1825. doi:10.1158/1538-7445.AM2013-1825