Schroder Araya K, Loy Conor J, Aiala Fernanda, Rafique Jazmyn, Ghosh Arnob, Yoo Lina I
Department of Biology, Denison University, Granville, Ohio, United States of America.
PLoS One. 2024 Dec 2;19(12):e0310315. doi: 10.1371/journal.pone.0310315. eCollection 2024.
MicroRNAs (miRNAs) play an increasingly recognized role in modulating cancer development. Due to their function in regulating gene expression, miRNAs can suppress or promote tumorigenesis. miR-379-5p expression is downregulated in multiple human cancers, including breast and bladder cancers. However, the mRNAs targeted by miR-379-5p that promote cancer development have not been fully identified. Our goal was to identify a gene whose expression is regulated by miR-379-5p, and which may contribute to cancer development in cells where miR-379-5p expression is reduced. Bioinformatics analysis showed the UBE2E3 ubiquitin conjugating enzyme gene to be a potential target for miR-379-5p. To verify that UBE2E3 is a target, we transfected normal human epithelial mammary cells and breast adenocarcinoma cell lines with a miR-379-5p mimic. The mimic reduced UBE2E3 mRNA and protein levels, as would be predicted for a miR-379-5p target. To determine if UBE2E3 is a direct target of miR-379-5p, we engineered two luciferase reporter gene constructs to contain either a wild-type putative miR-379-5p binding sequence isolated from the 3'UTR of the UBE2E3 gene, or a scrambled sequence. The luciferase assay showed that the miR-379-5p mimic suppressed luciferase activity for the WT binding sequence reporter, but not for the scrambled reporter, showing that the effect of miR-379-5p on UBE2E3 expression is likely to be direct. Finally, to determine if the effect of miR-379-5p on UBE2E3 is related to cellular behaviors that play a role in cancer development, we measured cell viability by resazurin assay, cell proliferation by BrdU assay, and apoptosis by caspase 3/7 activation assay. The miR-379-5p mimic and silencing UBE2E3 expression both resulted in significantly diminished cell viability, while silencing UBE2E3 demonstrated both higher proliferation and apoptotic rates. Overall, these results suggest that while the overall effect of miR-379-5p is to inhibit breast cell viability and proliferation, the effect of silencing its target UBE2E3 is more complex because it induces both cell proliferation and apoptosis.
微小RNA（miRNA）在调节癌症发展过程中发挥着越来越被认可的作用。由于其在调控基因表达方面的功能，miRNA可以抑制或促进肿瘤发生。miR-379-5p在包括乳腺癌和膀胱癌在内的多种人类癌症中表达下调。然而，miR-379-5p靶向的促进癌症发展的信使核糖核酸（mRNA）尚未被完全鉴定出来。我们的目标是鉴定一个其表达受miR-379-5p调控的基因，并且该基因可能在miR-379-5p表达降低的细胞中促进癌症发展。生物信息学分析表明泛素结合酶E2E3（UBE2E3）基因是miR-379-5p的一个潜在靶点。为了验证UBE2E3是一个靶点，我们用miR-379-5p模拟物转染正常人乳腺上皮细胞和乳腺癌细胞系。正如对miR-379-5p靶点所预测的那样，该模拟物降低了UBE2E3的mRNA和蛋白质水平。为了确定UBE2E3是否是miR-379-5p的直接靶点，我们构建了两个荧光素酶报告基因构建体，使其分别包含从UBE2E3基因3'非翻译区（UTR）分离的野生型假定miR-379-5p结合序列或一个随机序列。荧光素酶测定表明，miR-379-5p模拟物抑制了野生型结合序列报告基因的荧光素酶活性，但对随机序列报告基因没有抑制作用，这表明miR-379-5p对UBE2E3表达的影响可能是直接的。最后，为了确定miR-379-5p对UBE2E3的影响是否与在癌症发展中起作用的细胞行为相关，我们通过刃天青测定法测量细胞活力，通过溴脱氧尿苷（BrdU）测定法测量细胞增殖，并通过半胱天冬酶3/7激活测定法测量细胞凋亡。miR-379-5p模拟物和沉默UBE2E3表达均导致细胞活力显著降低，而沉默UBE2E3显示出更高水平的增殖和凋亡率。总体而言，这些结果表明，虽然miR-379-5p的总体作用是抑制乳腺细胞活力和增殖，但沉默其靶点UBE2E3的作用更为复杂，因为它既诱导细胞增殖又诱导细胞凋亡。
