| Cervical cancer is one of the most globally common cancers in women. Thereare more than 40 thousands new cases in which about 80 percent cases are indeveloping countries and about 20 thousands females die from this cancer every yearin world wide. More than 90％of cervical carcinomas are associated with humanpapillomavirus (HPV) infection.The two viral oncogenes E6 and E7 play a major rolein transforming the cells by disrupting p53 and pRb dependent cell cycle checkpoints.Interleukin-18 is an IL-1-like cytokine that was first identified as anIFN-γ-inducing factor (IGIF) based on its ability to induce high level IFN-γsecretionby both NK and T cells.IL-18 mediates other important functions, includingenhancement of NK cell activity and stimulation of proliferation of activated T cells(1-5). Recent reports have suggested that IL-18 affects the development of cellularimmunity (Th1 response).IL-18 does play an important supportive role in Th1development. IL-18 has significant antitumor effects in multiple murine tumormodels.IL-18 can be secreted by many kinds of cells, including Kupffer cells, activatedmacrophages, keratinocytes, and intestinal epithelial cells. Numerous investigationsrevealed importance of IL-18 as a Th1 cytokine, especially in cooperation with IL-12,in antitumor immunity.At the tumor site, locally decreased IFN-γproduction afterdecreased or abolished IL-18 production was correlated with an unfavorable outcome for patients with carcinoma. IL-18 stimulates NK cells, T cells, Bcells, and cells ofthe monocyte lineage to express IFN-γ. at high levels. Furthermore,IL-18 plays animportant role in T-cell proliferation, CTL activation, and enhancement of NK cellactivity primarily through the Fas-FasL mechanism.Recent studies support the notionthat IL-18 plays an important role in the development of cellar immunityfollowingadministration of antigen. This suggests that IL-18 has an important role in localantitumor immune responses.Both E6 and E7 oncoproteins of hrHPV could bind to IL-18Rαand inhibit theIL-18 binding to IL-18Rαby competed with IL-18. So the Th1 cytokine- IFN-γproduction could be inhibited via blockade of IL-18 binding to IL-18Rα. IL-18 isrequired in vivo for induction of IFN-γ.Therefore, the inhibition of IL-18 activity byoncoproteins E6 and E7 of high risk HPV 16 represents a novel immune escapestrategy of the virus and/or the virally induced lesions, including cervical cancer.The cervical carcinomas are associated with human papillomavirus (HPV)infection.But, HPV infection has a transitory pattern, most individuals (70％to 90％)eliminate the virus 12 to 24 months after initial diagnosis. The persistence of HPVonly in 10％to 30％of cases is more common in oncogenic cases and developed tohigh-grade squamous intraepithelial lesions (SIL) and cancer. There is evidence thatthe host's immune response is responsible for the progression of HPV infection, sinceimmune vigilance affects susceptibility to HPV related lesions and their regression.A Single Nucleotide Polymorphism(SNP) is a DNA sequence variation at a singlenucleotide level and is the most abundant resource of genetic variation amongindividuals of a species. It can be expected that these genome-wide SNP associationstudies will identify many SNP alleles related to various complex disorders.Identifying the causative relationships between many disease predisposing alleles andthe corresponding disorders will be a major challenge. SNPs of IL-18 could effect it'sexpression and function. For example, the site-specific IL-18 mutant E42A resulted inabout a 200％increase in the production of IFN-γin the NK0 cell line.And the E6 orE7 did not affect E42A-induced IFN-γproduction in NK0 cells The genotype of rs 1946518 and rs 1946519 in sequence upstream of IL-18 influence the expression ofIL-18 and potentially also of IFN-γ..If the expressing reduced, IL-18 fail to competewith both E6 and E7 oncoproteins of hrHPV, therefore the oncoproteins are morefamiliar to binding the IL-18Rα.Because the signal transmit of IL-18 is blocked, theexpression of the down stream genes on Th1 cytokine are dcreaseed. Persisting ofHPV infections can influnce the function of cellular immunity and lead to endureimmunity. As a result, the cervical epithelia cell transform by the infection of theHPV.For this reason we studied effect of genetic background on cervical cancer casesby researching the relationship between the SNPs in IL-18 gene and cervical cancer.Single nucleotide polymorphisms (SNPs) of IL-18 gene in promoter and exon regionin cervical cancer was screened by direct sequencing.And discuss the relationshipbetween single nucleotide polymorphisms (SNPs) rs1946519 and rs360718 of ILl8and cervical cancer for further study risk forcast of cervical cancer. The relationshipbetween RNAi on HPV 16E6 and biological change of CaSki cells was discussed andinvestigated the possible implication of the haplotypes of rs 1946519 and rs360718 ofPBMC with the expression of IL-18 for researching the relationship IL-18 SNP withthe cervical cancer.Methods:1,Efect of silencing human papillomavirus 16 E6 gene on CaSki cellHPV 16E6 shRNA sequence was designed by on-line designer software on Corp.Invitrogen, after synthesis, double strands oligonucleotide(ds oligo) was cloned intothe pENTRTM/U6 plasmid. Connection product was inverted competent cells andmultiplied. Plasmid was extracted and sequenced. The vectors expressing siRNA ofHPV-16 E6 gene were transfected into CaSki cells. The expression of E6,p53 andIL-18 mRNA was detected by RT-PCR.2,Genotypic frequencies of IL-18 gene from 26 cervical cancer patients werecompared with a control group of 20 female blood donors. Genotypes were determined by direct sequencing. Allele, genotype and haplotype frequencies in IL-18gene and risk of cervical cancer were statistically analyzed by X2 test in both groups.3,Genotypes of SNPs in IL-18 gene from107 cervical cancer patients and 80 femaleblood donors control group. Genotypes both sites rs1946519 and rs360718 weredetermined by YaqMan and MGB probe assays. Allele, genotype and haplotypefrequency in IL-18 and risk of cervical cancer were analyzed by X2 test in bothgroups.4,Study of bioimmunofunction in PBMC with defferent haplotype of IL-18PBMC/ml were isolated by Ficoll-Paque density-gradient centrifugation andsuspended in RPMI 1640 medium.and cultured in the presence of 1μg/ml LPS orcultured with 50μg/ml CaSki cell antigen and cultured for the 24h. PBMCs culturedwithout LPS or CaSki antigen served as a control. Cultured PBMCs were used toassay expression ratio of CD14 positive cells by flow cytometer and IFN-γdetermined by a human IFN-γELISA kit according to the manufacturer's protocol.Results:1,Sequencing results indicated pENTRTM/U6-HPV16 E6-shRNA plasmid is positiveclone. After transfection, the expression of HPV 16 E6 mRNA was reduced in CaS kicell line,while the expression level of IL-18 and P53 were increased.2,We found 7 SNPs in the IL-18 gene in all from our experimental groups, in whichrs1946518 and rs1946519 were significant difference in cervical cancerpatients(P＜0.05), and rs360719,rs360717 and rs360718 appeared to be inverselyassociated with cervical cancer, but this result did not reach at statistical significance(P＞0.05). rs11547404 and an unlanding SNP located in exon 4 were detected in onepatient separately.3,There were no difference in allele and genotype distribution frequency in rs360718between the cervical cancer group and the healthy population (P＞0.05). while alleleand genotype distribution frequency in site rs1946519,and haplotypes of rs1946519, rs360718 (AC+TT) were related to cervical cancer (P＜0.05).4,The expression of CD 14 and IFN-γin PBMC with defferent haplotype of IL-18.There were the significant difference in the expression of CD 14 and IFN-γof PBMCbetween the groups or haplotypes (F=21.317, F=80.327, respectivily, P≤0.001,). Afterstimulating,the expressions of CD 14 and IFN-γof PBMC in the genotypes of AATTand AATG were had significant defference,while there were no significant defferenceamong the genotypes of ACTG,CCTG and ACTT. Before the stimulation, there wereno significant defference in each genotypes, however, after stimulated, there weresignificant defference in each one.Conclusions1,pENTRTM/U6 expression vector of HPV16E6 gene was constructed achievedly.RNAi can inhibite the expression of HPV16 E6 gene in CaSki cells by pENTRTM/U6-HPV16 E6-shRNA plasmid effectively. And HPV E6 could inhibite theexpression of IL-18 and P53.2,We identified IL-18 gene polymorphisms that may be associated with cervicalcancer risk. Direct sequencing assay of small samples can screen the candidate SNPsof IL-18 gene in cervical cancer easily and efficiently.3,We conclude that rs360718 allelic variation in IL18 is unlikely to contribute tocervical cancer in Guangzhou populations and the female population with allelicvariation in site rs1946519 may be susceptible to cervical cancer. The femalepopulation with the genotype of allele AC+TT may be susceptible to cervical cancer.4,Monocytes with the haplotypes of AATT and AATG of rs 1946519 and rs360718in IL-18 could induce immunity response effectively when stimulated by microbes,while,monocytes with the haplotypes of ACTG,CCTG and ACTT have lowerimmunity response to the microbes. The monocytes with variation in site rs 1946519from A to C of the upstream promoter region in gene IL-18 have lower immunityresponse to the microbes, it's expression of IL-18 is decreased, and the IFN-γinduced mainly by IL-18 is also reduced. As a result the host fail to eliminate hrHPV and maybe susceptible to cervical cancer.
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