FILIP-1L Protein Antibody Preparation And Study On Its Biological Function

BackgroundHepatocellular carcinoma is a global high lethality cancer in the wold. It is also the one of most lethalcancers that occurs in our country. Clinical and laboratory research results show HBV and HVC infection–induced chronic hepatitis and cicrosis, the food contaminated with inflatoxin and other carcinogeniccompounds and gene mutations are the majors pathological causes. These carcinogens can induce multiplegenes’ expression and activation, which leads to the malignant transformation of hepatocyte, e.g. c-myc,pRB，p53and heat shock proteins.Heat shock proteins can regulate not only the normal cellular homeostasis, but also the occurrence、metastasis and drug resistance of various organ cancers （including hepatocellular carcinoma）. Hsf1is thekey transcription factor to regulate the expression of heat shock proteins, and has been found to playimportant regulatory roles in some specific tissues’ tumorigenesis. Hsf1has been reported to be upregulatedin most of hepatocellular carcinoma tissues. knock-out mouse Hsf1Gene can inhibit the DMBP-inducedskin cancer, P53mutant-induced lymphoma and DEN-induced hepatocellular carcinoma. Recent reportindicates that Hsf1can interact with FILIP-1L （filamin A interacting protein1like, also know asdown-regulated ovarian cancer protein1）. FILIP-1L can inhibit the protein expression of Hsf1andHsf1-mediated heat shock response. FILIP-1L is a nevel protein with unknown biological function. Therecent studies indicate FILIP-1L is low expressed in ovarian cancer cell lines and HPV E7-immortalizedprostate epithelial cells. Overexpression of FILIP-1L can inhibit cancer cell proliferation and migration.Those data imply that FILIP-1L has potential tumor suppressing characteristics. However, It is still notclear the expression and function of FILIP-1L in hepatocellaular carcinoma. In present study we willinvestigate the biological function of FILIP-1L in the hepatocellular carcinoma deveoplment.AimsWe will study of FILIP-1L’s expression in hepatocellular carcinoma and its biological effect, anddiscuss proteins which can interact with FILIP-1L and their function.Methods 1.Making the anti-FILIP1L antibody: The construct pGEX-4T₃-FILIP-1L（1-288） was transformed intothe competent E. coli BL21to induce the expression of fusion protein GST-FILIP-1L（1-288） under theIPTG stimulation.GST-FILIP-1L（1-288） fusion proteins, which are purified with GST affinitychromatography, are subjected to immunized Rabbit to make the polyclonal antibody agansit FILIP1L.After purification with Ammonium sulfate precipitation and affinity chromatography, the antibody is usedin our furthere experiments;2. RT-PCR and Immunoblotting technique were used to detecting FILIP-1LDNA and protein’s expression in different hepatocellular carcinoma cell lines;3.We establish the FILIP1L-over-expression and shRNA silencing hepatocellular carcinoma cell lines with Liposome transfectiontechnique, and test the cell proliferation, migration and tumorigenesis with MTT assay, wound healingassay and soft agar cloning experiments;4.with immunoblotting and co-immunoprecipitation techniqueswe find the interaction between FILIP-1L and Rb.ResultsUsing receptor bacteria E.coli BL21, we successfully induced the expression of GST-FILIP-1L（1-288）fusion protein, and used the purified protein to immunize animals and obtained polyclonal antibodies withhigh potency, by RT-PCR and immunoblotting technique we verified FILIP-1L protein expression indifferent hepatoma cells and hepatocellular carcinoma,and FILIP-1L protein in hepatocellular carcinomatissues’ expression is higher than adjacent tissues. We established expression and silencing of FILIP-1L inhepatocellular carcinoma cell lines successfully,and by MTT, cell migration assays, soft agar cloningexperiments we found hepatocellular carcinoma cells with FILIP-1L protein’s over-expression grewslower,migrated shorter distances and had fewer clone numbers than the mock transfected hepatomacells;and the hepatoma cells with FILIP-1L protein’s silencing grew faster,migrated longer distances andhad more clone numbers.By immunoblotting technique, this study found that in hepatocellular carcinomacells PLC/PRF/5and human embryonic kidney cell lines293T with over-expression of FILIP-1L, Rbprotein’s expression is lower,but in cells with silencing of FILIP-1L,it’s higher. And byco-immunoprecipitation experiments, we knew FILIP-1L and Rb are combined in the hepatocellularcarcinoma cells PLC/PRF/5.ConclusionsWe obtained GST-FILIP-1L（1-288） fusion protein,and prepared FILIP-1L ’s polyclonal antibody successfully. FILIP-1L protein expressed in different hepatoma cells and hepatocellularcarcinoma.FILIP-1L protein can inhibit hepatic cancer cell PLC/PRF/5’s proliferation,migration andtumor formation.In the hepatocellular carcinoma cells PLC/PRF/5,FILIP-1L binds with Rb,and inhibitRb’s expression.