Highly expressed miR-375 is not an intracellular oncogene in Merkel cell polyomavirus-associated Merkel cell carcinoma

Abstract

201-1836945 2 AbstractPurpose. miR-375 is a highly abundant miRNAs in Merkel cell carcinoma. miR-375 may act as a tumor suppressor or oncogene depending on the cell context. While miR-375 is present as circulating free in the serum of patients with advanced MCC and thus serves as surrogate marker for tumor burden, its function within MCC has not been established.Methods. miR-375 knockdown was performed using miR-375 antagomiRs via lipofectamine transfection or nucleofection in classical MCC cell lines WaGa and PeTa. Viability and both changes in growth characteristics as well as morphology were determined. Genes targeted by miR-375 were predicted using ENCORI; based on these miR-375 regulated signaling pathways were determined by genes ontology (GO) and gene set enrichment analysis (GSEA).Expression of these genes was analyzed by multiplexed RT-qPCR to check the effect of miR-375 knockdown on these signaling pathways.Results. Complete knockdown of miR-375 expression by antagomiRs was only achieved by using nucleofection. This knockdown did not affect cell growth pattern, morphology, or proliferative capacity. miR-375 predicted target genes GO analysis revealed that Hippo signaling and focal adhesion-related genes were likely to be regulated by miR-375. GSEA of gene expression data of MCC cell lines further strengthened the regulation of Focal adhesionrelated genes by miR-375. However, gene expression analysis revealed that miR-375 knockdown had only a limited effect on expression of these pathways related genes. Conclusions.Complete miR-375 knockdown did neither change cell viability, morphology, nor oncogenic signaling pathways; these observations render miR-375 unlikely as intracellular oncogene in MCC cells.

Merkel cell carcinoma-derived exosome-shuttle miR-375 induces fibroblast polarization by inhibition of RBPJ and p53

“…In most cancers, miR-375 has been deemed a tumor suppressor [ 36 ], but it also functions as an oncogenic miRNA depending on the cellular context [ 32 , 33 ]. We recently reported that knockdown of miR-375 in MCC cell lines has—at best—minimal effects on cell survival, proliferation and morphology [ 37 ]. Highly efficient knockdown did not alter any of the signaling pathways involving miR-375 target genes [ 37 ], thus posing the question about the functional role of this highly abundant miRNA in MCC.…”

“…We recently reported that knockdown of miR-375 in MCC cell lines has—at best—minimal effects on cell survival, proliferation and morphology [ 37 ]. Highly efficient knockdown did not alter any of the signaling pathways involving miR-375 target genes [ 37 ], thus posing the question about the functional role of this highly abundant miRNA in MCC. Based on our observation that miR-375 is present in MCC conditioned cell culture medium as well as sera of MCC patients [ 27 ], it may function as an exosome-shuttle miRNA.…”

MicroRNAs as Guardians of the Prostate: Those Who Stand before Cancer. What Do We Really Know about the Role of microRNAs in Prostate Biology?

“…For example, two studies argue that miR-375 suppresses growth in PC-3 cells and two studies claim the opposite. Furthermore, the sobering lack of a phenotype from loss of miR-375 expression in MCC cell lines suggests that miR-375 may have functions outside the tumor cells, as suggested by Fan et al [ 99 ] and supported by studies in prostate cancer [ 114 ]. An exosomal function of miR-375 must be explored in more detail, as well as the idea that exosomes loaded with sufficient amounts of miR-375 can be transferred to adjacent cells to mediate an effect in the recipient cells.…”

“…For example, massive un-physiological overexpression of miR-375 may “always” result in a growth-suppressive phenotype in vitro in prostate cancer cells or for that matter any cancer cell (e.g., oral cancer). To explore this issue in a more unbiased fashion and shed light on the unsatisfactory conclusions about miR-375’s role in cancer, we selected ten of the most recent publications [ 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 ] and ten of the most cited publications [ 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 ] that deal with miR-375 and determined the pro- or anti-growth outcomes of its overexpression or inhibition in these studies. Overexpression using a miR-375 mimic produced growth inhibition and/or induction of apoptosis in 16 of the 17 studies, while miR-375 inhibition had either no effect or mild pro-growth effects in 7 of 10 studies.…”

“…The starkest example for the lack of any major effect of miR-375 inhibition comes from Merkel cell carcinoma (MCC) cell lines that express high levels of miR-375. Even almost complete inhibition of miR-375 had almost no effect on the cells in vitro [ 99 ]. Only three studies (two on breast cancer cell lines MDA-MB231 and MCF7 and one on miR375 knockout in mouse pancreas) indicate an oncogenic or at least pro-growth effect of miR-375 [ 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 ].…”