RNA结合蛋白RBM43对肝癌细胞增殖的影响及机制研究

RBM43 (RNA-binding motif protein 43) residues at chromosome 2q23.3, contains an RNA binding domain. Although the biological roles of RBM43 is still unknown, recent GWAS studies has suggested that RBM43 has a pathological role in human diseases.. In order to illustrate the physical function of RBM43, especially in the progroess of cancer, the association relationship between RBM43 and HCC was investigated. In our previously work , we found out that RBM43 exhibited differential expression pattern in paired clinical HCC tissues for the first time. Over-expression of RBM43 in HCC cells inhibited the growth rate of HCC cell lines. Knocking down of RBM43 in HCC cells promoted cell growth. Additionally, over-expression of RBM43 resulted in a decreased population of HCC cells in S phase that was accompanied by an increased population in the G2/M phase. Conversely, suppression of RBM43 led to a increase in the population of HCC cells in S phase. This was accompanied by a modest but consistent decreased cell population in G2/M phase.. To understand how RBM43 may affect cell proliferation, we set out to screen the target genes of RBM43. A global post-transcriptional procedures like RIP- seq has been used to identify RNAs. The results showed that RBM43 was associated with approximately 1629 mRNAs, many of which was invovled in the regulation of S phase and G2/M cell-cycle regulation . In comparison with control IP complexes, RIP-qPCR analysis of RBM43-expressing cells showed that CCNA2 and CCNB1 mRNAs exhibited the most dramatic enrichment in the RBM43-IP complexes, consistent with most genes were the S phase and G2/M cell-cycle regulatory factors in the RIP-seq analysis. Real-time RT–PCR assay and Western blot analysis showed that the mRNAs levels and the protein levels of cyclins A2 and B1 were significantly elevated in RBM43 konckdown cells.. The aim of this project is to explore the clinico-pathological correlation of RBM43 down-regulation in HCC tissues and the molecular mechanism of RBM43 in CCNA2 and CCNB1 regulation.

RBM43是RNA结合蛋白，已有研究提示其可能参与多种疾病发生，但其生理功能与肿瘤的关系未知。我们前期研究发现RBM43在肝癌组织中下调表达；过量表达RBM43抑制肝癌细胞生长，降低RBM43表达促进肝癌细胞生长；RBM43通过影响细胞周期调节肝癌细胞生长。分子机制上，通过RIP-seq，发现RBM43结合很多细胞周期相关基因的mRNA；进一步证实RBM43与重要周期蛋白CCNA2和CCNB1 mRNA相结合，通过降低它们的mRNA稳定性，调节它们的蛋白水平。本课题拟在前期工作基础上，进一步明确RBM43下调表达和肝癌患者临床指标及生存预后的相关性、 RBM43对肝癌细胞增殖表型的影响、RBM43与CCNA2、CCNB1 mRNA结合并调节它们稳定性的分子机制。构建RBM43缺陷型小鼠，分析其生理功能。探讨其作为肝癌诊疗分子靶标的可能性。

肝癌的发生是一个多基因参与、多种环境因素共同作用的结果，找到与肝癌发生相关的基因，并发现其导致肝癌发生的分子机制，对于肝癌的预防和治疗都具有重要意义。RNA结合蛋白在细胞代谢过程中起着重要作用，其家族的许多成员都与肿瘤的发生有关。RBM43（RNA binding motif protein 43）属于RNA结合蛋白，位于第2号染色体，编码357个氨基酸，关于RBM43的生理功能未见报道。. 我们发现RBM43在肝癌样本中低表达；分析相应的临床病理和随访数据，发现RBM43表达水平与肝癌患者预后负相关，并与肝癌临床分期、肿瘤大小、肝硬化程度负相关。这些结果表明RBM43在肝癌中低表达，可作为一个独立的肝癌预后评估因子。. 在肝癌细胞中过表达RBM43抑制肝癌细胞生长及裸鼠体内成瘤；与之相反，抑制RBM43表达则促进肝癌细胞生长和裸鼠体内成瘤，提示RBM43可能是一个抑癌基因。分子机制上，我们发现RBM43结合细胞周期蛋白B1(cyclin B1)的3’-UTR，促进其降解，从而降低cyclinB1的表达并抑制肝癌细胞周期进程。. 为了深入探究RBM43在肝癌发生发展中的作用，我们构建了Rbm43基因缺陷型小鼠，并通过化学诱导肝癌的方法构建了肝癌小鼠模型。结果显示，与WT小鼠相比，Rbm43-/-小鼠肝脏肿瘤数目显著增加、肝脏病灶更明显、细胞增殖指标表达增强、基因缺陷型小鼠的生存期缩短；这些结果表明Rbm43缺陷促进了药物诱导小鼠肝癌的发生和发展。. 综上所述，本项目的主要结果表明RBM43在肝癌组织中下调表达，且其表达水平与肝癌患者预后负相关；RBM43通过靶向抑制cyclinB1的表达，抑制肝癌细胞的生长；Rbm43缺陷促进了小鼠肝癌（药物诱导）发生发展，并显著缩短了小鼠的生存期；说明RBM43作为一个抑癌基因，在肝癌的发生发展中发挥重要的抑制作用，有望成为肝癌诊断和治疗的一个新的靶点。