Orlandi Elisa, De Tomi Elisa, Campagnari Rachele, Belpinati Francesca, Rodolfo Monica, Vergani Elisabetta, Malerba Giovanni, Gomez-Lira Macarena, Menegazzi Marta, Romanelli Maria Grazia
Section of Biology and Genetics, Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Strada Le Grazie, 8, 37134 Verona, Italy.
Section of Biochemistry, Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Strada Le Grazie, 8, 37134 Verona, Italy.
Curr Issues Mol Biol. 2022 Oct 11;44(10):4790-4802. doi: 10.3390/cimb44100326.
Polymorphisms in the ribonuclease L (RNASEL) coding gene and hsa-miR-146a-5p (miR-146a) have been associated with melanoma in a sex-specific manner. We hypothesized that RNASEL and miR-146a expression could be influenced by sex hormones playing a role in the female advantages observed in melanoma incidence and survival. Thus, we explored the effects of testosterone and 17β-estradiol on RNASEL and miR-146a expression in LM-20 and A375 melanoma cell lines. Direct targeting of miR-146a to the 3' untranslated region (3'UTR) of RNASEL was examined using a luciferase reporter system. Our results indicate that RNASEL is a direct target of miR-146a in both melanoma cell lines. Trough qPCR and western blot analyses, we explored the effect of miR-146a mimic transfection in the presence of each hormone either on RNASEL mRNA level or on protein expression of RNase-L, the enzyme codified by RNASEL gene. In the presence of testosterone or 17β-estradiol, miR-146a overexpression did not influence RNASEL transcript level in LM-20 cell line, but it slightly induced RNASEL mRNA level in A375 cells. Remarkably, miR-146a overexpression was able to repress the protein level of RNase-L in both LM-20 and A375 cells in the presence of each hormone, as well as to elicit high expression levels of the activated form of the extracellular signal-regulated kinases (ERK)1/2, hence confirming the pro-tumorigenic role of miR-146a overexpression in melanoma. Thereafter, we assessed if the administration of each hormone could affect the endogenous expression of RNASEL and miR-146a genes in LM-20 and A375 cell lines. Testosterone exerted no significant effect on RNASEL gene expression in both cell lines, while 17β-estradiol enhanced RNASEL transcript level at least in LM-20 melanoma cells. Conversely, miR-146a transcript augmented only in the presence of testosterone in either melanoma cell line. Importantly, each hormone acted quite the opposite regarding the RNase-L protein expression, i.e., testosterone significantly decreased RNase-L expression, whereas 17β-estradiol increased it. Overall, the data show that, in melanoma cells treated with 17β-estradiol, RNase-L expression increased likely by transcriptional induction of its gene. Testosterone, instead, decreased RNase-L expression in melanoma cell lines with a post-transcriptional mechanism in which miR-146a could play a role. In conclusion, the pro-tumor activity of androgen hormone in melanoma cells could be exacerbated by both miR-146a increase and RNase-L downregulation. These events may contribute to the worse outcome in male melanoma patients.
核糖核酸酶L（RNASEL）编码基因和人源微小RNA - 146a - 5p（miR - 146a）中的多态性已被证实与黑色素瘤存在性别特异性关联。我们推测，在黑色素瘤发病率和生存率方面所观察到的女性优势中，性激素可能发挥作用，进而影响RNASEL和miR - 146a的表达。因此，我们研究了睾酮和17β - 雌二醇对LM - 20和A375黑色素瘤细胞系中RNASEL和miR - 146a表达的影响。利用荧光素酶报告系统检测了miR - 146a对RNASEL 3'非翻译区（3'UTR）的直接靶向作用。我们的研究结果表明，在这两种黑色素瘤细胞系中，RNASEL均是miR - 146a的直接靶点。通过定量聚合酶链反应（qPCR）和蛋白质免疫印迹分析，我们探讨了在每种激素存在的情况下，转染miR - 146a模拟物对RNASEL mRNA水平或RNASEL基因编码的核糖核酸酶L（RNase - L）蛋白表达的影响。在睾酮或17β - 雌二醇存在的情况下，miR - 146a过表达在LM - 20细胞系中未影响RNASEL转录水平，但在A375细胞中轻微诱导了RNASEL mRNA水平。值得注意的是，在每种激素存在的情况下，miR - 146a过表达均能够抑制LM - 20和A375细胞中RNase - L的蛋白水平，同时引发细胞外信号调节激酶（ERK）1/2激活形式的高表达，从而证实了miR - 146a过表达在黑色素瘤中的促肿瘤作用。此后，我们评估了每种激素的施用是否会影响LM - 20和A375细胞系中RNASEL和miR - 146a基因的内源性表达。睾酮对两种细胞系中的RNASEL基因表达均无显著影响，而17β - 雌二醇至少在LM - 20黑色素瘤细胞中提高了RNASEL转录水平。相反，仅在睾酮存在的情况下，任一黑色素瘤细胞系中的miR - 146a转录本均增加。重要的是，每种激素对RNase - L蛋白表达的作用恰恰相反，即睾酮显著降低RNase - L表达，而17β - 雌二醇则增加其表达。总体而言，数据表明，在用17β - 雌二醇处理的黑色素瘤细胞中，RNase - L表达可能通过其基因的转录诱导而增加。相反，睾酮通过一种miR - 146a可能参与其中的转录后机制降低了黑色素瘤细胞系中RNase - L的表达。总之，雄激素在黑色素瘤细胞中的促肿瘤活性可能因miR - 146a增加和RNase - L下调而加剧。这些事件可能导致男性黑色素瘤患者预后更差。
