Dissertation
Dissertation > Medicine, health > Oncology > Genitourinary tumors > Female genital tumors > Uterine tumors

Expression of APRIL and Its Receptors in Cervix Carcinoma Cells and Preparation of Mutant Soluble APRILs

Author ZhengYingRu
Tutor LiLi
School Third Military Medical University
Course Obstetrics and Gynaecology
Keywords Proliferation-inducing ligand Cervical Cancer Acceptor mRNA Immunohistochemistry Clone Mutant Step inverse PCR method The prokaryotic expression Protein Purification
CLC R737.33
Type PhD thesis
Year 2007
Downloads 62
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APRIL (a proliferation-inducing ligand, a proliferation-inducing ligand) is a new member of the tumor necrosis factor (TNF) superfamily. The membrane-bound and soluble forms of the two active. APRIL the 105-250 amino acid region the natural soluble APRIL (soluble APRIL, sAPRIL), its length (i.e. membrane-bound) APRIL having the same biological activity, and can promote the proliferation of a variety of tumor cell lines both in vitro . APRIL expression in normal tissues is very low, but in a variety of tumor cell lines and tumor tissues, such as knot colorectal, breast, lung, blood system cancer, melanoma, bladder cancer, a high level of expression. As a ligand protein, APRIL play and promote tumor effects by binding to its receptor. B lymphocyte maturation antigen (B cell maturation antigen, BCMA) and transmembrane the excited sub and calcium signal modified ring pro-ligand effect sub (transmembrane activator and calcium signal-modulating cyclophilin ligandinteractor, TACI) is currently recognized APRIL receptor. Recently has been reported that mucin polysaccharide (syndecans, S) may also be APRIL receptor. The present study no APRIL with cervical cancer or cell line proliferation between whether the associated reports. The present study was to investigate the expression of sAPRIL whether it has a role to promote cervical cancer cell line HeLa proliferation and APRIL and its receptor mRNA expression in HeLa cells and cervical cancer, precancerous lesions, and APRIL protein in cervical cancer The study prepared sAPRIL two mutant designed on the basis of this study, observed mutants in the inhibition of HeLa cell proliferation in vitro, and the competition of wild-type sAPRIL, so as to further understanding of APRIL in the incidence of cervical cancer development and lay the foundation for the suppression of the the APRIL anti-cervical cancer preparations preparation. In order to understand the role of APRIL in the incidence of cervical cancer in this study using RT-PCR and immunohistochemical detection of cervical cancer, precancerous lesions APRIL mRNA and protein expression levels and found that the mRNA and protein expression in cervical cancer APRIL were significantly higher than the precancerous lesions and normal tissue. At the same time our organization APRIL receptor mRNA was detected by RT-PCR and found that compared with normal tissue and precancerous lesions, cancer tissue BCMA, TACI and mucin polysaccharide -4 (S4) expression levels no significant differences With cell atypia heavier, the expression of mucin polysaccharide -1 (S1) decreased, while the sticky the proteoglycan -2 (S2) of the level gradually increased. After that we detected by RT-PCR method of APRIL and its receptor mRNA expression in HeLa cells, found that HeLa cells express APRIL, S1 and S2, without the express BCMA, TACI and S4. To further understand the role of APRIL in the incidence of cervical cancer in the sAPRIL protein of human cervical cancer cell line HeLa cell proliferation detected by BrdU-ELISA method found sAPRIL protein can promote the proliferation of HeLa cells in a dose-dependent manner. View of APRIL promote cervical cancer cell line HeLa cell proliferation, inhibit the functional activity of APRIL, may offer new avenues for the treatment of cervical cancer. Studies show of APRIL APRIL antibodies and soluble receptors of APRIL can be suppressed to a certain extent, but the human antibody difficulties, while APRIL receptor, a soluble receptor does not play a role in inhibiting APRIL with other receptor combination of the functions of the function. This project intends to start from alterations APRIL molecule itself, building able to bind to the receptor but not triggered APRIL mutant receptor pro-tumor effects, in order to obtain APRIL inhibitors to treat tumors. This topic draw alterations to BLyS (B-Lymphocyte Stimulator B lymphocyte stimulating factor) molecular methods, after BlyS homology sequence and structural analysis of APRIL molecular amino acid Nos. 186, 187, select APRIL as mutation point, as a template to pUC19/sAPRIL step inverse PCR the mutagenic method to build people msAPRIL-1 (186G187Q replacement for 186K187G), and msAPRIL-2 (186G by K replacement, 187Q missing) two mutant DNA sequence; the the sequencing the mutant purpose ligated in the prokaryotic expression vector pQE-80L was successfully constructed recombinant prokaryotic expression the plasmid pQE-80L/msAPRIL-1 pQE-80L/msAPRIL-2; recombinant prokaryotic expression plasmids were transformed into E. coli DH5α, induced by IPTG, a higher level of expression by SDS-PAGE analysis shows that the size of two recombinant proteins of approximately 19kDa, mainly in the form of inclusion body, Western blotting analysis, two proteins in the molecular weight of about 19kDa a single band at the two recombinant proteins have been effective purification and refolding; via Ni 2 -NTA purification system and urea dialysis. Presented a single band of the purified protein on SDS-PAGE. Sephadex G-75 gel filtration chromatography to obtain the two recombinant mutant protein trimer molecules. The biological function analysis revealed that the prepared msAPRIL-1 mutant protein can be well combined with HeLa cells, while msAPRIL-2 only a weak binding with HeLa cells; two mutants were not mitogenic. Further analysis showed msAPRIL-1 can promote the proliferation of HeLa cells inhibit wild-type sAPRIL. The main results of this study are summarized as follows:, APRIL and its receptor expression in cervical squamous cell carcinoma cell lines 1.APRIL cervical squamous cell carcinoma, or both at the mRNA and protein level were significantly higher than the normal squamous epithelium and squamous intraepithelial lesions, with increased cell proliferation and atypia expression levels gradually increased; S1mRNA with cervical squamous cell proliferation and atypia aggravated expression gradually reduced, and the the S2 mRNA expression gradually increased; BCMA TACI and S4 mRNA expression in normal tissue, inflammation, precancerous lesions, carcinoma in situ and invasive; 2. HeLa cells of APRIL, S1 and S2 mRNA expression without express BCMA, TACI and S4, mRNA;, sAPRIL in vitro in a dose-dependent manner to promote the proliferation of human cervical cancer cell line HeLa cells. By the results of these two aspects, the preliminary draw the following conclusions: APRIL and its receptors may play a role in the development of cervical cancer, which APRIL role in cervical cancer may be combined with S1 and / or S2 play; the S1 mRNA reduction and / or S2 mRNA and APRIL expression may be elevated as the progress of the disease early warning indicators. Successfully prepared the sAPRIL two mutant 1. Nos. 186, 187 amino acids APRIL as mutation sites, people msAPRIL-1 DNA (186G187Q replacement 186K187G) step inverse PCR mutagenesis method was successfully constructed and msAPRIL -2 DNA (186G K replacement, 187Q missing) two mutant sequence. 2 mutations purpose of DNA fragments connected to the prokaryotic expression vector pQE-80L successfully constructed prokaryotic expression plasmids were transformed into E. coli reorganization prokaryotic expression plasmid pQE-80L/msAPRIL-1 and pQE-80L/msAPRIL-2. DH5α, induced by IPTG, the recombinant mutant protein was successfully expressed. The 4 Ni 2 -NTA purification system and urea dialysis, two recombinant proteins have been effective purification and refolding; obtained by Sephadex G-75 gel filtration chromatography two recombinant mutant protein trimer molecule. IV the mutants functional analysis 1. Are prepared msAPRIL-1 mutant can be specifically combined with HeLa cells, while msAPRIL-2 and the binding capacity of the HeLa cells is weak. 2.msAPRIL-1 and msAPRIL-2 were lost promote HeLa cell proliferation. 3.msAPRIL-1 mutant of competitive inhibition wild type sAPRIL to promote the proliferation of HeLa cells function to a certain extent, but the role of msAPRIL-2 was not significant. More than three, four two-part Tip msAPRIL-1 and its derivative molecules may have potential applications in the treatment of cervical cancer APRIL tumor.

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