Dados do Trabalho

Título

INTRONIC VARIANTS OF MITF (RS7623610) AND CREB1 (RS10932201) GENES MAY ENHANCE SPLICING EFFICIENCY IN HUMAN MELANOMA CELL LINE

Introdução

Cutaneous melanoma (CM) is the most aggressive form of skin cancer. MITF and CREB1 pathway is crucial in CM development and progression. We previously reported that single nucleotide variations (SNVs) in MITF (c.938-325G>A, rs7623610) and CREB1 (c.303+373G>A, rs10932201) genes were associated with risk, aggressiveness, and worse prognosis of CM. Those SNVs are located in intronic regions, so they could induce alternative splicing or influence the splicing efficiency. In silico analysis revealed that the SNVs are sited in splicing components binding sites, as hnRNPA1 and SF1.

Objetivo

We aimed to evaluate in what manner the SNVs MITF rs7623610 and CREB1 rs10932201 could influence the splicing process.

Método

We constructed minigenes with wild type and variant alleles from MITF and CREB1 to assess the effect of the SNVs on splicing. The minigenes were transfected in the human melanoma cell line (SK-MEL-28). Reverse transcription PCR (RT-PCR) and DNA sequencing investigated the constructs’ splicing patterns. Minigenes constructs’ splicing efficiency and HNRNPA1 and SF1 splicing genes’ expression were investigated by quantitative PCR (qPCR). Differences between groups were analyzed by t-test and Mann-Whitney test.

Resultado

The PCR products analysis by agarose gel electrophoresis and by DNA sequencing of the transcripts revealed that SK-MEL-28 cells transfected with MITF or CREB1 minigenes presented an identical splicing pattern with the wild type and variant constructs. Although MITF and CREB1 SNVs did not alter the splicing pattern, they influenced the splicing efficiency. MITF-A and CREB1-A minigenes (containing variant alleles) yielded an increase of mRNA generated from the constructions revealed by RT-PCR and qPCR analysis (3.94 arbitrary units (AUs) ± 2.15 standard deviation (SD) vs. 0.98 AUs ± 0.18 SD, P=0.008; 326.79 AUs ± 126.2 SD vs. 1.28 AUs ± 1.22 SD, P<0.001, respectively). Additionally, lower mRNA levels of splicing components HNRNPA1 and SF1 were seen in the variant minigenes MITF-A (0.87 AUs ± SD: 0.19 vs. 1.19 AUs ± 0.21 SD, P=0.04) and CREB1-A (0.10 AUs ± 0.09 SD vs 0.78 AUs ± 0.46 SD, P=0.005).

Conclusão

We described the potential importance of MITF and CREB1 SNVs in splicing efficiency. Once validated in melanoma cells from patients, our results might help to identify individuals with high risk for CM who deserves to receive additional recommendations and could also be the start point for new therapeutic approaches.

Palavras-chave

cutaneous melanoma, minigene assay, single nucleotide variation

Área

Oncologia - Tumores cutâneos e Sarcomas