【作者】 张建丽；

【导师】 叶丽虹； 张晓东；

【作者基本信息】 南开大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 乙肝病毒X蛋白结合蛋白(Hepatitis B X-interacting protein, HBXIP)因可与乙肝病毒X蛋白(hepatitis B virus x protein, HBx)的羧基端特异性结合而得名,在细胞凋亡及有丝分裂等细胞生命活动过程中发挥重要作用。我们发现,HBXIP也能促进乳腺癌细胞迁移,但其分子机制尚不清楚。MicroRNA(miRNAs)作为一类小分子非编码RNA,可以参与肿瘤发生、发展等诸多生命活动。本室前期研究发现,在不同转移潜能的乳腺癌细胞系中,HBXIP和miR-520b的表达水平均存在差异,且二者在表达量上呈负相关。本研究以不同转移能力的人乳腺癌细胞系MCF-7、LM-MCF-7及MDA-MB-231为材料,探讨了HBXIP促进乳腺癌细胞迁移的分子机制,并对HBXIP的纯化条件进行了初步探索,主要研究内容如下：1. HBXIP调节乳腺癌细胞迁移的分子机制在前期研究基础上,应用western blot及实时定量PCR法分别检测了三种不同转移能力乳腺癌细胞系中HBXIP和miR-520b的表达水平,发现具有高转移潜能的LM-MCF-7及MDA-MB-231细胞中的HBXIP表达水平明显高于低转移的MCF-7细胞,而miR-520b的表达水平则明显低于MCF-7细胞。这表明HBXIP可能促进乳腺癌细胞迁移,而miR-520b可能抑制乳腺癌细胞迁移。近年来,HBXIP一直是本室的研究重点,为了阐释HBXIP促进乳腺癌细胞迁移的分子机制,我们应用生物信息学方法进行了预测,发现HBXIP是miR-520b的靶基因之一。接下来,我们克隆了HBXIP 3’UTR及其3’UTR突变体的报告基因载体,双荧光素酶报告基因实验及western blot检测结果显示,miR-520b可以靶向HBXIP的3’UTR,影响HBXIP的表达水平。在LM-MCF-7细胞系中共转染miR-520b及pCMV-hbxip-3’UTR(或pCMV-hbxip)后检测细胞的迁移能力,发现,当HBXIP的表达水平被miR-520b下调后,LM-MCF-7细胞系的迁移能力也随之降低,表明HBXIP促进乳腺癌细胞迁移的过程受miR-520b的调控。本实验对HBXIP调节乳腺癌细胞迁移的机理进行了探索,其分子机制是：miR-520b靶向HBXIP的3’UTR,抑制HBXIP的翻译,从而调节乳腺癌细胞迁移。2.HBXIP蛋白纯化条件的探索HBXIP全长173个氨基酸,分子量约19 kDa,几乎表达于高等动物的各种组织中,但在肌肉组织和恶性肿瘤组织中高表达。HBXIP可以参与细胞凋亡中心体复制、细胞迁移、细胞周期等过程,揭示HBXIP的蛋白晶体结构有助于进一步了解HBXIP在细胞生命活动中所发挥的作用。实验中,我们构建了含HBXIP基因全长522bp的原核表达载体pet-28a-HBXIP1和含其C端273bp的原核表达载体pet-28a-HBXIP2,利用亲和层析、分子筛层析、离子交换层析等方法,对His-HBXIP1及His-HBXIP2的纯化条件进行了初步探索。发现,His-HBXIP1经分子筛层析纯化后,吸收峰单一,但蛋白浓度较低；经离子交换层析纯化后,His-HBXIP1穿透明显,蛋白弥散分布。His-HBXIP2的纯化效果与His-HBXIP1情况类似。本实验对HBXIP的纯化条件进行了初步探索,发现His-HBXIP的分子筛层析纯化效果优于离子交换层析。更多还原

【Abstract】 Hepatitis B X-interacting protein(HBXIP),which plays important roles in many cellular activities,such as apoptosis and mitosis,was named according to that it can bind to the C terminal of hepatitis B virus x protein(HBx) specificly. Besides,we found that HBXIP can involve in promoting breast cancer cell migration. However, the molecular mechanism is unclear. MicroRNAs (miRNAs) are small non-coding RNA, participating in many cellular activities during the carcinogenesis and tumor progression. Our previous studies showed that both miR-520b and HBXIP were differently expressed in breast cancer cell lines with different metastatic potential,representing inverse relationship in expression level. In the present study,we investigated the molecular mechanism of HBXIP on regulating breast cancer cell migration,using human breast cancer cell lines MCF-7,LM-MCF-7 and MDA-MB-231 as cell models. In addition, we explored the purification conditions of HBXIP fusion protein. The investigation contains two parts as follows:Part I:Mechanism of HBXIP on regulating migration of breast cancer cellsOn the basis of previous research, firstly, we examined the expression level of HBXIP and miR-520b in MCF-7, LM-MCF-7 and MDA-MB-231 cell lines by western blot and quantitative real-time PCR respectively, founding that the expression level of HBXIP was much higher in LM-MCF-7 and MDA-MB-231 cell lines than its in MCF-7.On the contrary, the level of miR-520b is higher in MCF-7 than in LM-MCF-7 and MDA-MB-231.This indicates that HBXIP may promote the migration of breast cancer cells, and miR-520b plays a opposite role in regulating breast cancer cell migration. To investigate the mechanism of HBXIP which has been a research focus of our lab for many years on regulating breast cancer cell migration, bioinformatics was used, founding that HBXIP was one of the target genes of miR-520b. Then,we constructed the dual-luciferase reporter gene vectors containing HBXIP 3’UTR or its 3’UTR mutant. The results of dual-luciferase reporter gene assay and western blot detection indicated that miR-520b can target the 3’UTR of HBXIP directly, regulating the expression of HBXIP. The cell migration abilities of LM-MCF-7 was detected after co-transfecting with miR-520b and pCMV-hbxip-3’UTR plasmid (or pCMV-hbxip),founding that the migration ability of LM-MCF-7 was decreased while the expression of HBXIP was down-regulated by miR-520b,which indicated that the role of HBXIP on regulating breast cancer cell migration was regulated by miR-520b.In this study,we explored the molecular mechanism of HBXIP on regulating breast cancer cell migration,and found that HBXIP was regulated by miR-520b when it plays roles in regulating the migration of breast cancer cells.Part II:Purification conditions exploration of HBXIPThe molecular weight of HBXIP is about 19 kDa,with 173 amino acids in all. HBXIP was expressed in almost all tissues of higher animals, with over-expression in muscle tissue and malignant tumors. HBXIP can involve in many celluar activities,such as apoptosis, centrosome replication,cell migration,cell cycle and so on. So, revealing the crystal structure of HBXIP may help to understand the roles of HBXIP further. In the present study, we constructed pet-28a-HBXIP1 prokaryotic gene expression vector, containing full-length(522bp) of HBXIP gene,and pet-28a-HBXIP2 prokaryotic gene expression vector,with C terminal 273bp of the HBXIP gene, and explored the purification conditions of His-HBXIP1 and His-HBXIP2.We found that after molecular sieve chromatography using Superdex G200, the map peak of His-HBXIP1 was single, but the protein concentration was low. The results of ion exchange chromatography showed that His-HBXIP1 hardly binded to the Q columns. So did His-HBXIP2.This study explored the purification conditions of His-HBXIP and found that His-HBXIP was more suitable for molecular sieve chromatography.更多还原