The functional characterization of long noncoding RNA SPRY4-IT1 in human melanoma cells

Abstract

Expression of the long noncoding RNA (lncRNA) SPRY4-IT1 is low in normal human melanocytes but high in melanoma cells. siRNA knockdown of SPRY4-IT1 blocks melanoma cell invasion and proliferation, and increases apoptosis. To investigate its function further, we affinity purified SPRY4-IT1 from melanoma cells and used mass spectrometry to identify the protein lipin 2, an enzyme that converts phosphatidate to diacylglycerol (DAG), as a major binding partner. SPRY4-IT1 knockdown increases the accumulation of lipin2 protein and upregulate the expression of diacylglycerol O-acyltransferase 2 (DGAT2) an enzyme involved in the conversion of DAG to triacylglycerol (TAG). When SPRY4-IT1 knockdown and control melanoma cells were subjected to shotgun lipidomics, an MS-based assay that permits the quantification of changes in the cellular lipid profile, we found that SPRY4-IT1 knockdown induced significant changes in a number of lipid species, including increased acyl carnitine, fatty acyl chains, and triacylglycerol (TAG). Together, these results suggest the possibility that SPRY4-IT1 knockdown may induce apoptosis via lipin 2-mediated alterations in lipid metabolism leading to cellular lipotoxicity.

Figure 1. Maturation and Cellular Compartmentalization of SPRY4-IT1 Transcripts

(A) The sequence and location of SPRY4 and SPRY4-IT1. SPRY4-IT1 is embedded in the SPRY4 parent transcript. SPRY4-IT1 starts in the first intron of the SPRY4 gene and extends up to exon 3. 5′ RACE shows the maturation and cleavage of a 244 nt transcript in the 5′ region of SPRY4-IT1. (B) Detection of nuclear and cytoplasmic forms of SPRY4-IT1. (C) Northern blot analysis shows the sizes of SPRY4 (4.9 kb) and SPRY4-IT1 (1.8 kb). The SPRY4 exon 1 probe hybridizes specifically to SPRY4, but the SPRY4 exon 3 probes recognize both SPRY4 and SPRY4-IT. (D) SPRY4 exon 1 siRNA knocks down the expression of SPRY4, but not SPRY4-IT1, in A375 melanoma cells.

Figure 2. SPRY4 and SPRY4-IT1 Are Coordinately Regulated in Melanocytes

Relative expression of SPRY4-IT1 and SPRY4 following induction by FGF2. Melanocytes were treated with 10 ng/ml FGF2 for 1 or 2 h in the presence or absence of FBS, and the fold change in expression of SPRY4 (A) and SPRY4-IT1 (B) was measured by qPCR

Figure 3. SPRY4-IT1 Accumulates in Polysomes and Is Absent from Monosomes

(A) Density gradient fractionation of monosome and polysome peaks. (B) Northern blot analysis showing SPRY4-IT1 probe hybridization to RNA isolated from polysomes but not monosomes. A GAPDH probe served as a control. Separate blots were prepared to probe SPRY4-IT1 and control (GAPDH) due to concerns of overlap in fragment size. L= RNA size marker.

Figure 4. SPRY4-IT1 Knockdown Increases Lipin 2 Protein Accumulation in Melanoma Cells

A) Affinity purification of SPRY4-IT1 from A375 cell lysates with SPRY4-IT1-specific probes followed by qPCR. SPRY4-IT1 is enriched compared to scrambled (control) probes. U1 RNA was used as endogenous control for pull-downs. (B) qPCR validation showing enrichment of SPRY4-IT1 following immunoprecipitation of A375 cell lysates with lipin 2-specific or control IgG antibodies. (C) Relative expression of SPRY4-IT1 in lipin 2 knock-down cells (D) Relative expression of lipin 2 after siRNA-mediated knockdown of SPRY4-IT1, SPRY4 exon 1 or lipin 2. (E) Phosphatidic acid phosphatase assay. (F) Western blot analysis of lipin 2 following siRNA-mediated knockdown of SPRY4-IT1 or lipin 2. All experiments were performed in triplicate.

Figure 5. Expression of Diacylglycerol O-acyltransferase 2 and (DGAT2) Acyl-coA:glycerol-3-phosphate Acyltransferase 3 (GPAT3) Following SPRY4-IT1 Knockdown

Both DGAT2 (A) and GPAT3 (B) mRNA levels are increased upon knockdown of SPRY4-IT1 in A375 cells, which may explain the decrease in DAG levels and increase in TAG levels observed in these cells (Table S2).