FBXO31基因在肝癌细胞系中的表达水平及功能分析
首发时间:2012-12-28
摘要:摘要:目的: 检测FBXO31在肝癌细胞中的表达模式,并构建FBXO31基因真核表达载体, 利用该载体对FBXO31在肝癌细胞中的功能进行研究。方法:通过荧光实时定量PCR技术和western blot技术检测在肝癌细胞系中FBXO31 mRNA水平和蛋白水平的表达。以 PCR 的方法扩增FBXO31的开放阅读框,把FBXO31开放阅读框插pEGFP-C1载体中以构建EGFP-FBXO31表达载体。将构建的真核表达载体以脂质体介导的方法体外转染肝癌细胞,然后对转染后的肝癌细胞系进行细胞增殖和细胞周期等分析。结果:荧光实时定量PCR技术和western blot结果表明与正常肝上皮细胞Hl7相比,Hep3B, MHCC97L, MHCC97H, HCCLM3, SNU-449细胞系中的FBXO31表达水平降低或极为降低。在HepG2和SNU-449细胞系中表达呈上升趋势(约2.7和2.5倍)。在30/32检测的肝癌样本中,FBXO31的表达水平表达下调。选取Hep3B细胞研究FBXO31在肝癌中表达下调的潜在机制。增强FBXO31的表达水平对细胞衰老,细胞凋亡没有影响,但是可轻微抑制细胞增殖,并且使细胞周期阻滞在G1期。增强FBXO31的表达水平可以是细胞周期素D1的内源表达水平下降。结论: FBXO31可能通过调节细胞周期素D1的内源表达水平来调控细胞周期。
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Expression and function analysis of FBXO31 in hepatocellular carcinoma
Abstract:AIM: To investigate the expression profile of FBXO31 in HCC and the possibility that FBXO31 might function as a tumor suppressor in HCC cell lines. METHODS: Real-time reverse transcription-PCR and western blot were used to detect FBXO31 mRNA and protein expression levels in HCC cell lines. The open reading frame (ORF) of FBXO31 was amplified by PCR. Then the fragment was subcloned into pEGFP-C1, EGFP-FBXO31 was used to investigate the possibility that FBXO31 might function as a tumor suppressor in HCC. RESULTS: FBXO31 is strongly down-regulated in HCC cell lines and specimens. Ectopic expression of FBXO31 inhibited cell proliferation and colony formation in HepG2 and Hep3B cells. The endogenous expression of FBXO31 was fluctuated through cell cycle in the HepG2 cells with maximal expression from late G2 to early G1 phase. Ectopic expression of FBXO31 in HepG2 resulted in the accumulation of cells at the G1 phase of the cell cycle. .Conclusion: Possible mechanism might be cyclin D1 degradation mediate by FBXO31 through ubiquitin ligase pathway.
Keywords: FBXO31 expression profile HCC
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