Expression Of APRIL And Its Receptors In Cervix Carcinoma Cells And Preparation Of Mutant Soluble APRILs

APRIL(a proliferation-inducing ligand) is a new member of the tumor necrosis factor (TNF) family,which exists in two forms,cytomembrane APRIL and soluble APRIL(sAPRIL).It is expressed in small amounts in normal tissues but produced abundantly in many kinds of malignant tumors,such as colorectal carcinoma,breast cancer, lung cancer,malignancy of hematological system,bladder cancer and melanoma.APRIL and sAPRIL can not only promote growth of many malignant tumors in vivo and in vitro, but also prolong the life-span of the tumors.As a ligand,APRIL plays its roles by binding to its receptors,BCMA(B cell maturation antigen) and TACI(transmembrane activator and calcium signal-modulating cyclophilin ligand interactor).Besides the above two receptors, recent studies indicate that proteoglycan syndecans(S) may also be the receptors of APRIL.Although it is affirmed that APRIL is highly expessed in a lot of malignant tumors and can promote growth of the tumors,there is few reprorts about its roles on cervix carcinoma. So in this study we want to primarily figure out the relationship between APRIL and cervix carcinoma.Firstly,mRNA expression profiles of APRIL and its receptors were analyzed by semi-quantitive RT-PCR in HeLa cells and cervix carcinoma tissues,taking the tissues from nomal cervix,chronic cervicitis and cervix introepithelium neoplasia as controls.APRIL protein expression profiles were detected by immunohistochemistry in cervix carcinoma tissues and the controls.Secondly,The proliferation-stimulating effect of sAPRIL to HeLa cells was detected by BrdU-ELISA assay.Thirdly,taking 186G and 187Q of human sAPRIL as mutation sites,one-step opposite orientation PCR was used to construct the two mutant sAPRIL(msAPRIL) DNAs,namely msAPRIL-1 DNA(in which 186G187Q replaced by 186K187G) and msAPRIL-2 DNA(in which 186G replaced by K and 187Q deleted). After inserting the two msAPRIL DNAs into pQE-80L vector respectively,the recombinant msAPRIL proteins were expressed in E.coli DH5αand purified by Ni²⁺-NTA chromatography and the homotrimers of msAPRILs were isolated by Sephadex G-75. Finally,the binding profiles of the two homotrimers of msAPRILs to HeLa cells were analyzed by indirect immunofluorescence assay.The stimulating function of the two msAPRIL homotrimers(alone or with wild type sAPRIL) to HeLa cells were detected by BrdU-ELISA assay.The major results and conclusions are summarized as follows:1.With the development of the proliferation and heteromorphism of cervix squamous cells,the mRNA expression of APRIL and S2 were increased gradually,whereas the expression of S1 mRNA was decreased,and the mRNA level of S4,BCMA and TACI had no difference among the tissues from cervix carcinoma,nomal cervix,chronic cervicitis and cervix introepithelium neoplasia.2.The mRNAs of APRIL and S1 and S2,but not S4,BCMA and TACI,were expressed in HeLa cells.3.With the development of the proliferation and heteromorphism of squamous cells, the expression of APRIL protein was up-regulated in cervix.4.sAPRIL could dose-dependent promote the growth of HeLa cells in vitro.Based on the above results,it might be concluded that there was a positive correlation between APRIL and cervix carcinoma.5.The two recombinant msAPRIL proteins(msAPRIL-1 and msAPRIL-2) were successfully expressed in E.coli DH5αand purified effectively by Ni²⁺-NTA chromatography,and the functional homotrimers of msAPRILs were isolated by Sephadex G-75.6.The prepared msAPRIL-1 homotrimer,not msAPRIL-2 homotrimer,could strongly bind to HeLa cells.Both of the two msAPRIL proteins lost the function of stimulating growth of HeLa cells,and msAPRIL-1 could inhibit the stimulating activity of wild type sAPRIL to HeLa cells,which indicated that msAPRIL-1 and its derivates might have a therapeutic potential in treatment of APRIL-associated cervix carcinoma and other tumors.