Department of Oncology, the Second Affiliated Hospital, Nanchang University, Nanchang 330006, China ; Key Laboratory of Molecular Medicine Jiangxi Province, Nanchang 330006, China.
Department of Pathology, the Second Affiliated Hospital, Nanchang University, Nanchang 330006, China ; Key Laboratory of Molecular Medicine Jiangxi Province, Nanchang 330006, China.
Cancer Cell Int. 2014 Jun 19;14:55. doi: 10.1186/1475-2867-14-55. eCollection 2014.
Bone morphogenetic protein receptor II (BMPR-II) plays an important role in tumor's invasion and proliferation. In this study, we observed the effects of small interfering RNA (siRNA) targeting bone morphogenetic protein receptor II (BMPR-II) on the biological activities of human liver cells and explore its mechanism.
The molecular sequences of three siRNA targeting BMPR-IIwere designed and synthesized. In this study, there were 6 groups including group I (normal control), group II (blank control), group III (negative control) and group IV-VI (BMPR-II-siRNA-a, siRNA-b and siRNA-c-transfected cells, respectively). The levels of mRNA and protein of BMPR-II were determined to select the best sequence for BMPR-II silence. After liver cancer cells were transfected with the best sequence, proliferation and invasion of transfected cells were assessed, and apoptosis and cell cycle were detected. The expressions of mitogen-activated protein kinases (MAPKs) signal pathway-related VEGF-C protein were observed after BMPR-II silence and BMPR-II silence combined with inhibiting MAPKs signal pathway, respectively.
RT-PCR and Western blot indicated that BMPR-II expression was the highest in HepG2 among the three liver cancer lines (P < 0.01) and the lowest in group IV among the six groups (P < 0.01). MTT assay and transwell assay revealed that the numbers of cell growth and cell transmembrane were significantly lower in group IV than in control groups 48 h after cells were transfected (P < 0.05). Flow cytometer showed that apoptosis was the highest and cells were significantly blocked in S phase 48 h after cells were transfected in group IV (P < 0.01). Western blot indicated that the protein levels of p-P38 (P < 0.01) and vascular endothelial growth factor-C (VEGF-C) (P < 0.01) were significantly decreased after BMPR-II silence. The protein level of VEGF-C was significantly decreased in PD98059 + siRNA-BMPR-II-a and SB203580 + siRNA-BMPR-II-a groups (P < 0.01), especially in SB203580 + siRNA-BMPR-II-a group (P < 0.01).
siRNA targeting BMPR-IIcan markedly inhibit HepG2 proliferation and invasion, promote apoptosis and block HepG2 in S phase. Its mechanism may be that BMPR-II silence down-regulates VEGF-C expression through MAPK/P38 and MAPK/ERK1/2 pathways, especially MAPK/P38. This study provides a new targeted therapy for liver cancer.
骨形成蛋白受体 II（BMPR-II）在肿瘤的侵袭和增殖中发挥重要作用。本研究观察了针对骨形成蛋白受体 II（BMPR-II）的小干扰 RNA（siRNA）对人肝细胞生物学活性的影响，并探讨其机制。
设计并合成了针对 BMPR-II 的三个 siRNA 的分子序列。本研究共分为 6 组，包括 I 组（正常对照组）、II 组（空白对照组）、III 组（阴性对照组）和 IV-VI 组（分别为 BMPR-II-siRNA-a、siRNA-b 和 siRNA-c 转染细胞组）。测定 BMPR-II 的 mRNA 和蛋白水平，以选择 BMPR-II 沉默的最佳序列。转染肝癌细胞后，评估转染细胞的增殖和侵袭能力，检测细胞凋亡和细胞周期。观察 BMPR-II 沉默后及 BMPR-II 沉默联合抑制 MAPKs 信号通路后，丝裂原活化蛋白激酶（MAPKs）信号通路相关 VEGF-C 蛋白的表达。
RT-PCR 和 Western blot 结果表明，在三种肝癌细胞系中，HepG2 中 BMPR-II 的表达最高（P<0.01），而在 6 组中，IV 组的表达最低（P<0.01）。MTT 检测和 Transwell 检测结果显示，转染后 48 h 时，IV 组细胞的生长和穿膜细胞数量明显低于对照组（P<0.05）。流式细胞仪结果显示，转染后 48 h 时，IV 组细胞凋亡率最高，且细胞明显阻滞于 S 期（P<0.01）。Western blot 结果表明，BMPR-II 沉默后，p-P38（P<0.01）和血管内皮生长因子-C（VEGF-C）（P<0.01）的蛋白水平显著降低。PD98059+siRNA-BMPR-II-a 和 SB203580+siRNA-BMPR-II-a 组的 VEGF-C 蛋白水平显著降低（P<0.01），尤其是 SB203580+siRNA-BMPR-II-a 组（P<0.01）。
针对 BMPR-II 的 siRNA 可显著抑制 HepG2 的增殖和侵袭，促进凋亡并阻滞 HepG2 于 S 期。其机制可能是 BMPR-II 沉默通过 MAPK/P38 和 MAPK/ERK1/2 通路下调 VEGF-C 的表达，尤其是 MAPK/P38 通路。本研究为肝癌的靶向治疗提供了新的策略。
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