RhoBTB3在肾透明细胞癌中的表达及对肾癌细胞增殖、周期和凋亡的影响

发布时间：2019-05-08 21:47

【摘要】：目的：RhoBTB家族属于RhoGTP酶大家族的一员，与其他典型的Rho家族成员在控制细胞形态、调控细胞增殖、黏附、迁移和膜运输等明确的作用不同，RhoBTB亚族的功能虽然未能完全明确，但是一些研究已经证实RhoBTB蛋白在致瘤过程中是通过降低或消除了某些基因的功能，从而引起蛋白功能失调导致细胞稳态的改变。RhoBTB的研究不仅有利于深入对Rho家族的了解，而且更有助于揭示肿瘤的发病机制和肿瘤靶向药物的研发。本课题旨在从分子，细胞，临床病例三个层面初步探索RhoBTB3在肾透明细胞癌中的生物学效应。方法：通过定量RT-PCR方法，免疫组化以及Western印迹（Western blot法）检测RhoBTB3在肾细胞癌和癌旁组织，以及肾透明细胞癌细胞系和正常肾小管上皮细胞系的表达情况。构建pLVX-IRES-Neo-RhoBTB3和pLVX-IRES-ZsGreen-RhoBTB3慢病毒载体，包装成慢病毒后感染786-o和HKC细胞系。 MTS方法检测过表达和敲除肾透明细胞癌细胞系中RhoBTB3后的生长曲线，通过划痕试验和Transwell法测RhoBTB3对肾透明细胞癌的细胞侵袭和迁移的影响。通过流式细胞仪测RhoBTB3过表达和敲除后对肾透明细胞癌细胞周期和凋亡的影响。结果： RT-PCR，免疫组化以及Western印迹法显示RhoBTB3在肾透明细胞癌组织和细胞株中表达水平明显低于相应的癌旁组织及正常肾小管上皮细胞系（HKC）（P值均＜0.05），过表达RhoBTB3后显著促进肾癌细胞786-O的细胞凋亡，在HKC中敲除RhoBTB3后促进细胞从G1向S期转化，促进细胞增殖，并相应促进了HKC细胞的侵袭和迁移能力。结论：RhoBTB3在肾透明细胞癌组织和体外细胞系中低表达。过表达RhoBTB3促进细胞凋亡抑制细胞增殖，敲除后促进细胞向S转化，，促进细胞增殖。RhoBTB3在肾透明细胞癌的恶性进展中起到抑制作用，可为肾癌的分子靶向治疗提供新的方向。
[Abstract]:Objective: the RhoBTB family is a member of the large family of RhoGTP enzymes. Unlike other typical members of the Rho family in the control of cell morphology, cell proliferation, adhesion, migration and membrane transport, the function of the RhoBTB subfamily is not completely clear. However, some studies have confirmed that RhoBTB protein may cause cell homeostasis by decreasing or eliminating the function of some genes during tumorigenesis. The study of RhoBTB not only contributes to the further understanding of the Rho family, but also leads to the change of cellular homeostasis. Moreover, it is helpful to reveal the pathogenesis of tumor and the development of tumor-targeted drugs. The aim of this study is to explore the biological effects of RhoBTB3 in renal clear cell carcinoma from three aspects: molecular, cellular and clinical. Methods: quantitative RT-PCR, immunohistochemistry and Western blot (Western blot were used to detect the expression of RhoBTB3 in renal cell carcinoma (RCC), paracancerous tissue, renal clear cell carcinoma cell line and normal renal tubular epithelial cell line. PLVX-IRES-Neo-RhoBTB3 and pLVX-IRES-ZsGreen-RhoBTB3 lentiviral vectors were constructed and packaged into lentivirus to infect 786 and HKC cell lines. MTS method was used to detect the growth curve after RhoBTB3 was overexpressed and knocked out in renal clear cell carcinoma cell line. The effect of RhoBTB3 on cell invasion and migration of renal clear cell carcinoma was detected by scratch test and Transwell assay. The effects of RhoBTB3 overexpression and knockout on cell cycle and apoptosis of renal clear cell carcinoma cells were measured by flow cytometry. Results: the expression level of RhoBTB3 in renal clear cell carcinoma tissue and cell line was significantly lower than that in corresponding paracancerous tissue and normal renal tubular epithelial cell line (HKC) (P) by RT-PCR, immunohistochemistry and Western blot. Overexpression of RhoBTB3 significantly promoted apoptosis of renal cancer cell line 786 O, and knockout of RhoBTB3 in HKC promoted cell transformation from G1 to S phase, cell proliferation and invasion and migration ability of HKC cells. Conclusion: the expression of RhoBTB3 is low in renal clear cell carcinoma tissues and cell lines in vitro. Overexpression of RhoBTB3 promotes apoptosis to inhibit cell proliferation, knockout promotes cell transformation to S, and promotes cell proliferation. RhoBTB3 plays an inhibitory role in malignant progression of renal clear cell carcinoma, which may provide a new direction for molecular targeting therapy of renal cell carcinoma.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.11

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