Mechanism Of HBXIP On Regulating Migration Of Breast Cancer Cells And Investigate Of HBXIP Protein Purification Condition

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.