Department of Internal Medicine, Shandong Rongjun Hospital, Jinan, P.R. China.
Department of General Surgery, Shandong Rongjun Hospital, Jinan, P.R. China.
J Cell Biochem. 2018 Jul;119(7):5233-5242. doi: 10.1002/jcb.26581. Epub 2018 Apr 6.
The objective of this study was to explore the role of rs4705342 located in the miR-143 promoter in relation to the control of HBV positive HCC and the underlying molecular mechanism. A luciferase assay was performed to explore the factors which influenced miR-143 transcription activity and the target gene of miR-143. This would further be confirmed by ChIP assay. Western blot and real-time PCR were performed to identify the relationship between miR-143 and ORP8. Luciferase activity of miR-143 SNP was increased with the presence of C allele. The presence of T allele partially restored the transcription ability. NF-κB displayed a much higher degree of luciferase activity in relation to the cells transfected with vectors containing either T or C allele rather than control cells with a greater extent in C allele group than T allele group. At the same time, ChIP assay indicated that the affinity of NF-ΚB in the miR-143 promoter was higher in C/C cells. The over-expression of HBX promotes NF-kB expression thus increasing the extent of binding of NF-kB on the CC allele of the miR-143 promoter. The binding is also abolished by NF-kB siRNA. ORP8 was proven to be a target gene of miR-143 using bioinformatics algorithm analysis. It was further confirmed by the luciferase assay that miR-143 substantially inhibited luciferase activities of wild-type ORP8. However, it did not affect the mutant ORP8. HBx induced by HBV infection up-regulated miR-143 expression. NF- kB can partially abolish the promotion effect of HBx on the miR-143 level in cells genotyped as CC but not in cells genotyped as TT. Tissues derived from participants genotyped as CC exhibited a higher level of miR-143, but a lower level of ORP8. The presence of the minor allele of rs4705342 in the promoter of miR-143 attenuated the transcription ability. This promoted ORP8 expression and could be a factor contributing to the oncogenesis in HBV positive HCC.
本研究旨在探讨位于 miR-143 启动子中的 rs4705342 多态性与乙型肝炎病毒（HBV）阳性 HCC 发生的关系及其潜在的分子机制。通过荧光素酶报告基因检测，我们探讨了影响 miR-143 转录活性的因素及其靶基因。通过 ChIP 实验进一步证实了这一点。Western blot 和实时定量 PCR 检测 miR-143 与 ORP8 之间的关系。rs4705342 多态性的 C 等位基因可增加 miR-143 的荧光素酶活性。T 等位基因的存在部分恢复了转录能力。NF-κB 在转染含 T 或 C 等位基因载体的细胞中的荧光素酶活性明显高于对照组，C 等位基因组的活性高于 T 等位基因组。同时，ChIP 实验表明，C/C 细胞中 NF-ΚB 在 miR-143 启动子上的亲和力更高。HBX 的过表达促进 NF-κB 表达，从而增加 NF-κB 与 miR-143 启动子 CC 等位基因的结合程度。NF-κB siRNA 可消除这种结合。通过生物信息学算法分析，证明 ORP8 是 miR-143 的靶基因。荧光素酶报告基因实验进一步证实，miR-143 显著抑制野生型 ORP8 的荧光素酶活性，但不影响突变型 ORP8。HBV 感染诱导的 HBx 上调 miR-143 的表达。NF-κB 可部分消除 HBx 对 CC 基因型细胞中 miR-143 水平的促进作用，但不能消除 TT 基因型细胞中的促进作用。CC 基因型患者组织中 miR-143 水平较高，而 ORP8 水平较低。miR-143 启动子中 rs4705342 的次要等位基因的存在减弱了转录能力。这促进了 ORP8 的表达，可能是 HBV 阳性 HCC 发生的一个因素。
