FAM96B在Apoptin肿瘤细胞特异性诱导凋亡中的作用及其机制的研究

Apoptin specially induces apoptosis in tumor cells while abolishes cytotoxic effect in normal cells. Apoptin induce apoptosis in a P53-independent way. And it is not inhibited by anti-apoptotic factor Bcl-2、Bcl-xL. Some studies suggest that apoptin-induced apoptosis in tumor cells has closely relation with its nuclear localization and Thr108 phosphorylation. To date, the mechanisms of Apoptin-induced tumor-speci?c cell apoptosis are not clearly understood. As we all know, protein phosphorylation and subcellular localization are closely related with its interacting proteins. At present, a variety of apoptin interaction proteins have been found, for exsample, anaphase promoting complex/cyclosome subunit 1 (APC/C) ，promyelocytic leukemia protein (PML) and so on. These proteins are closely related with apoptin-induced apoptosis in tumor cells. We got apoptin interaction proteins FAM96B by means of Yeast Two-Hybrid system. In addition, we confirmed this interaction by means of immunoprecipitation in both tumor and normal cells. FAM96B is a highly conserved protein. At present about their research reports are very few. Only Tanaka K. et al reported that FAM96B and MMS19, Ciao1, XPD composed of a TFIIH-Independent protein complex, it can regulate the activity of TFIIH, and it plays a role in the normal separation of the chromosomes. FAM96B siRNA knockdown leads to abnormal mitosis and nuclei in HCT116 cells. The above data show that FAM96B may have important biological significance. What's more, FAM96B may play an important role in apoptin-induced apoptosis activity in tumor cells. Therefore, to deep research FAM96B function and its activity in Apoptin-induced apoptosis has an important biological significance. Firstly, we plan to collect some human lung,liver,colon and rectum tumor tissues and its corresponding adjacent normal tissues from clinic. Then we study FAM96B mRNA expression by quantitation Real-time PCR technology and protein expression by western blotting in tumor and normal tissues. Next, we analyze the internal relations between FAM96B expression and tumor invasiveness, tumor differentiation combined with clinical pathology material. Then we construct truncated FAM96B eukaryotic expression vector to study the function of overexpression of FAM96B and truncated FAM96B in tumor and normal cells. We also plan to study the interaction between truncated FAM96B and apoptin or apoptin5ala106(which can not be phosphorylated), apoptinT108E(which mimic phosphorylated apoptin), and the effect of the interaction to the apoptosis induction by apoptin and cellular location of apoptin. Last, we try to know the effect to the apoptosis induction by apoptin and its cellular location if FAM96B gene is knocked down in tumor or normal cells. Meantime, we also want to know the cellular function of FAM96B gene knockdown. This work will offer new information for function study of FAM96B and provide new clues for the mechanism of Apoptin-induced apoptosis.

Apoptin可特异性诱导肿瘤细胞的凋亡，其特异性肿瘤细胞诱导凋亡活性与其细胞核定位和Thr108磷酸化修饰密切相关，其作用的具体机制尚未阐明。我们在前期研究中发现并证实FAM96B是能和Apoptin相互作用的蛋白质；FAM96B能抑制Apoptin在肿瘤细胞中的诱导凋亡效应；且FAM96B在肝癌细胞HepG2中表达明显低于肝正常细胞L02。这提示FAM96B可能在Apoptin的肿瘤特异性诱导凋亡效应中发挥作用。因此我们拟首先比较研究FAM96B在临床人肝癌、肺癌、胃癌、结肠癌、直肠癌组织和相应的癌旁组织中的表达变化；然后研究FAM96B或其缺失重组体与Apoptin的相互作用特点及其对Apoptin的诱导凋亡活性的影响及可能机制。最后研究FAM96B基因沉默对细胞生物学功能以及对Apoptin的诱导凋亡活性及其亚细胞定位的影响。上述研究将为Apoptin的诱导凋亡机制研究提供新的线索

FAM96B是我们在前期研究中发现并证实能和Apoptin相互作用的蛋白质，其功能尚未阐明。本研究收旨在探讨FAM96B的功能及其在Apoptin肿瘤特异性诱导凋亡中的作用。我们首先收集了61例肺癌和42例肝癌患者的肿瘤组织和相应的癌旁正常组织样本，比较FAM96B在肿瘤组织及癌旁正常组织中的表达差异，发现FAM96B在肝癌组织和肺癌组织中的表达均显著低于癌旁组织；结合临床病理资料分析，在肝癌和肺癌组织中FAM96B的表达水平与年龄、性别、分化程度、肿瘤类型没有相关性（P>0.05），而与淋巴结转移、远端转移、肿瘤TNM分期等明显相关（P<0.05）；其次，我们研究了FAM96B及其缺失重组体对Apoptin亚细胞定位的影响。我们构建了FAM96B真核表达载体，将其与Apoptin真核表达载体共转染Hela细胞，IFA显示，FAM96B能使定位于细胞核的Apoptin重新定位于细胞质；MTT或流式细胞术表明共转VP3和FAM96B的HeLa细胞增殖率提高，凋亡率下降。鉴于Apoptin的细胞核定位对其诱导凋亡活性的重要性，上述结果表明FAM96B通过影响Apoptin的细胞核定位抑制其诱导凋亡活性。为进一步明确FAM96B结构和功能的关系，我们用重叠延伸PCR技术构建了三个FAM96B的缺失重组体以研究FAM96B上不同肽段对Apoptin的作用。将FAM96B及各个缺失重组体分别与Apoptin共转染入HEK-293T细胞后，用免疫共沉淀和免疫荧光分别研究缺失重组体和Apoptin的相互作用及其对亚细胞定位的影响。结果发现在HEK-293T细胞中，FAM96B、FAM96B△1和FAM96B△3与Apoptin存在直接的相互作用， FAM96B△2与Apoptin不存在直接的相互作用；免疫荧光表明全长的FAM96B、FAM96B△1能使原本应该定位于细胞核的Apoptin重新定位于细胞质，而FAM96B△2，FAM96B△3不存在上述作用。最后为进一步确认FAM96B在Apoptin诱导凋亡中的作用意义，我们用沉默FAM96B的shRNA和表达Apoptin的质粒共转染正常细胞L02后，结果发现FAM96B基因沉默后对Apoptin的诱导凋亡活性及亚细胞定位没有影响，均定位于细胞质。本研究为FAM96B的结构与功能的研究提供了新的资料。