Department of Translational Research, Lahey Hospital and Medical Center, Burlington, MA, USA.
Department of Urology, Lahey Hospital and Medical Center, Burlington, MA, USA.
Cancer Biomark. 2021;30(3):343-351. doi: 10.3233/CBM-200553.
MicroRNAs (miRNAs), a group of non-coding post-transcriptional regulators of gene expression, are dysregulated in clear cell renal cell carcinoma (ccRCC) and play an important role in carcinogenesis. Our prior work identified a subset of miRNAs in pT1 ccRCC tumors associated with progression to metastatic disease.
To investigate the impact of two of these dysregulated miRNA, miR-15a-5p and -26a-5p, in an effort to elucidate the mechanisms underpinning aggressive forms of stage I ccRCC.
The ccRCC cell line 786-O was transfected with pre-miRs-15a-5p and -26a-5p to rescue expression. Cell proliferation was measured via MT Cell Viability Assay. O-GlcNAc-transferase (OGT), a known protein in ccRCC proliferation, was identified by bioinformatics analysis as a target of both miRNA and validated via luciferase reporter assay to confirm binding of each miR to the 3' untranslated region (UTR). OGT protein expression was evaluated via western blotting.
Luciferase assay confirmed specificity of miR-15a-5p and -26a-5p for the OGT UTR. Western blot analysis for OGT showed reduced expression following co-transfection of both miRNAs compared to negative control or individual transfection. Co-transfection of these miRNAs greatly reduced proliferation when compared to negative control or the individual transfections.
Our results indicate that the dysregulation of miR-15a-5p and -26a-5p contribute cooperatively to the proliferation of ccRCC through their regulation of OGT. These results give insight into the pathogenesis of aggressive early stage ccRCC and suggest potential therapeutic targets for future research.
微小 RNA（miRNA）是一组非编码基因表达的转录后调控因子，在透明细胞肾细胞癌（ccRCC）中失调，在致癌作用中发挥重要作用。我们之前的工作确定了一组与转移性疾病进展相关的 pT1ccRCC 肿瘤中的 miRNA。
研究两种失调的 miRNA（miR-15a-5p 和 -26a-5p）的影响，以阐明支持 I 期 ccRCC 侵袭性形式的机制。
用前 miR-15a-5p 和 -26a-5p 转染 ccRCC 细胞系 786-O 以挽救表达。通过 MT 细胞活力测定法测量细胞增殖。通过生物信息学分析，O-连接的 N-乙酰葡糖胺转移酶（OGT）被确定为两种 miRNA 的已知蛋白质靶点，并通过荧光素酶报告基因测定验证以确认每个 miRNA 与 3'非翻译区（UTR）的结合。通过 Western blot 评估 OGT 蛋白表达。
荧光素酶测定法证实了 miR-15a-5p 和 -26a-5p 对 OGT UTR 的特异性。OGT 的 Western blot 分析显示，与阴性对照或单独转染相比，两种 miRNA 共转染后 OGT 的表达降低。与阴性对照或单独转染相比，这些 miRNA 的共转染大大降低了增殖。
我们的结果表明，miR-15a-5p 和 -26a-5p 的失调通过调节 OGT 共同促进 ccRCC 的增殖。这些结果深入了解侵袭性早期 ccRCC 的发病机制，并为未来的研究提供潜在的治疗靶点。
