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.

Sterol regulatory element binding proteins (SREBPs) have evolved as a focal point for linking lipid synthesis with other pathways that regulate cell growth and survival. Here, we have uncovered a polycistrionic micro-RNA locus that is activated directly by SREBP-2. Two of the encoded miRs, miR-182 and miR-96, negatively regulate expression of Fbxw7 and Insig-2 respectively, and both are known to negatively affect nuclear SREBP accumulation. Direct manipulation of this miR pathway alters nuclear SREBP levels and endogenous lipid synthesis. Thus, we have uncovered a new mechanism for regulation of intracellular lipid metabolism mediated by the concerted action of a pair of miRs that are expressed from the same SREBP-2 regulated miR locus and each targets a different protein of the multi-step pathway that regulates SREBP function. These studies reveal a miR “operon” analogous to the classic model for genetic control in bacterial regulatory systems.

bMicroRNA 211 (miR-211) negatively regulates genes that drive invasion of metastatic melanoma. Compared to normal human melanocytes, miR-211 expression is significantly reduced or absent in nonpigmented melanoma cells and lost during human melanoma progression. To investigate the molecular mechanism of its tumor suppressor function, miR-211 was ectopically expressed in nonpigmented melanoma cells. Ectopic expression of miR-211 reduced hypoxia-inducible factor 1␣ (HIF-1␣) protein levels and decreased cell growth during hypoxia. HIF-1␣ protein loss was correlated with the downregulation of a miR-211 target gene, pyruvate dehydrogenase kinase 4 (PDK4). We present evidence that resumption of miR-211-mediated downregulation of PDK4 in melanoma cells causes inhibition of invasion by nonpigmented melanomas via HIF-1␣ protein destabilization. Thus, the tumor suppressor miR-211 acts as a metabolic switch, and its loss is expected to promote cancer hallmarks in human melanomas. Melanoma, one of the deadliest forms of skin cancer, kills nearly 10,000 people in the United States per year. We had previously shown that a small noncoding RNA, termed miR-211, suppresses invasion and the growth of aggressive melanoma cells. The results presented here support the hypothesis that miR-211 loss in melanoma cells causes abnormal regulation of energy metabolism, which in turn allows cancer cells to survive under low oxygen concentrations-a condition that generally kills normal cells. These findings highlight a novel mechanism of melanoma formation: miR-211 is a molecular switch that is turned off in melanoma cells, raising the hope that in the future we might be able to turn the switch back on, thus providing a better treatment option for melanoma.

Edited by Tamas Dalmay Keywords:Epigenetic regulation MicroRNA miR-375 Cell invasion Human melanoma a b s t r a c tTo identify epigenetically regulated miRNAs in melanoma, we treated a stage 3 melanoma cell line WM1552C, with 5AzadC and/or 4-PBA. Several hypermethylated miRNAs were detected, one of which, miR-375, was highly methylated and was studied further. Minimal CpG island methylation was observed in melanocytes, keratinocytes, normal skin, and nevus but hypermethylation was observed in patient tissue samples from primary, regional, distant, and nodular metastatic melanoma. Ectopic expression of miR-375 inhibited melanoma cell proliferation, invasion, and cell motility, and induced cell shape changes, strongly suggesting that miR-375 may have an important function in the development and progression of human melanomas.