Zhang Shiqian, Hou Chunying, Li Guojun, Zhong Yang, Zhang Jie, Guo Xinzhen, Li Baoxin, Bi Zhenggang, Shao Ming
Department of Orthopedic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, P.R. China.
Department of Pharmacology, Harbin Medical University, Harbin, Heilongjiang 150081, P.R. China.
Int J Mol Med. 2016 Sep;38(3):919-26. doi: 10.3892/ijmm.2016.2661. Epub 2016 Jul 4.
The objective of the present study was to explore the molecular mechanism with which a single nucleotide polymorphism (rs61764370) interferes with the interaction between the 3'-untranslated region (3'-UTR) of Kirsten rat sarcoma viral oncogene homolog (KRAS) and let-7a, and its association with the metastasis of osteosarcoma (OS). In this study, we confirmed that KRAS is a target of let-7a in OS cells, and the introduction of rs61764370 minor allele into KRAS 3'-UTR significantly compromised the microRNA (miRNA)/mRNA interaction using a luciferase reporter system. Additionally, a total of 36 OS tissue samples of three different genotypes (TT,22; TG,10; GG,4) were obtained, and the expression of let-7a and KRAS was determined. We showed that let-7a mRNA expression was similar between each group whereas the mRNA and protein expression of KRAS in the TT genotype group was significantly lower than that in the GT or GG genotype groups. Moreover, we identified a negative regulatory relationship between let-7a and KRAS. Furthermore, we demonstrated that let-7a and KRAS interfered with the viability, invasiveness and migration of OS cells genotyped as TT. In the OS cells genotyped as TG, let-7a exerted minimal effects, and the effect of KRAS siRNA remained. Taken together, the findings of the present study demonstrated that the KRAS 3'-UTR rs61764370 polymorphism interfered with miRNA/mRNA interaction, and showed that the minor allele was associated with an elevated risk of developing metastatic disease in OS.
本研究的目的是探讨一种单核苷酸多态性（rs61764370）干扰 Kirsten 大鼠肉瘤病毒癌基因同源物（KRAS）的 3'非翻译区（3'-UTR）与 let-7a 之间相互作用的分子机制，及其与骨肉瘤（OS）转移的关联。在本研究中，我们证实 KRAS 是 OS 细胞中 let-7a 的靶标，并且使用荧光素酶报告系统将 rs61764370 次要等位基因引入 KRAS 3'-UTR 显著损害了微小 RNA（miRNA）/信使核糖核酸（mRNA）的相互作用。此外，共获得了 36 个具有三种不同基因型（TT，22 个；TG，10 个；GG，4 个）的 OS 组织样本，并测定了 let-7a 和 KRAS 的表达。我们发现各组之间 let-7a mRNA 表达相似，而 TT 基因型组中 KRAS 的 mRNA 和蛋白表达显著低于 GT 或 GG 基因型组。此外，我们确定了 let-7a 和 KRAS 之间的负调控关系。此外，我们证明 let-7a 和 KRAS 干扰了基因型为 TT 的 OS 细胞的活力、侵袭性和迁移能力。在基因型为 TG 的 OS 细胞中，let-7a 的作用最小，而 KRAS 小干扰 RNA（siRNA）的作用仍然存在。综上所述，本研究结果表明 KRAS 3'-UTR rs61764370 多态性干扰了 miRNA/mRNA 相互作用，并表明次要等位基因与 OS 发生转移性疾病的风险升高有关。
