Relationship Of Folate And P16, FHIT Expression In Cervical Carcinogenesis

ObjetiveCervical cancer is the second most common cancer which seriously hazard to the health of women. Infection of high risk human papillomavirus (HR-HPV) is the established major cause for cervical cancer, but not the only. Based on the critical role played by folate deficiency and DNA methylation in carcinogenesis, and the transcriptional inactivation mechanism of tumor suppressor genes, hypothesis was brought up that folate may participate in the carcinogenesis through regulating tumor suppression genes P16and FHIT transcription or expression. The aim of the study is to explore the role of folate and P16, FHIT expression in the cervical carcinogenesis, so as to pave up the ground for the further research etiology and pathogenesis of cervical cancer.MethodsAll the samples were collected from women attending the Shanxi Tumor Hosiptal, the Second Hospital of Shanxi Medical University, Maternal and Child Health Center in Taiyuan and Jiexiu from2009to2011. Total254women with cervical inflammation (CI,80), low-grade cervical intraepithelial neoplasia (CIN1,55), high-grade cervical intraepithelial neoplasia (CIN2/3,55) and cervical squamous cell carcinoma (SCC,64) were recruited. Relevant information of subjects was collected through structural questionnaire including demographic characteristics, behavior and health of sexual, reproductive factors, and cervical tissues and3ml fast intravenous blood before the systemic therapy were collected from each participant. HPV16was detected by PCR. Microbiological assay was adopted to test the level of serum folate and RBC folate.Methyl-specific PCR (MSP), real-time PCR and Western blot were used to detect the methylation status, levels of mRNA and protein expression of P16and FHIT. The data were analyzed by SPSS17.0. For comparison among histology groups categorical variables were examined using the%2test and χ2trend test, continuous variables were examined by ANOVA and Welch. Pearson and spearman rank method were used to analy the realationship. Logistic regression was used to analy the related factors and OR values were used to explain the strength of association between cervical carcinogenesis and risk factors. The interaction effects were analyzed by additive model and RERI, AP, S.Results(1) HPV16infection ratio in CIN1(41.8%), CIN2/3(56.4%) and SCC(64.1%) were all significantly higher than that in CI (17.5%) group. Rates of HPV16infection increased gradually with the increase of cervical lesions (χ2trend=34.962, P<0.001). Expression levels of HPV16E6and E7mRNA in different cervical lesions were significantly different (F=8.717, P<0.001; F=13.528, P<0.001). The SCC group is highest, and CI group is the lowest. LSD-t showed difference was not significant only between the CIN1and CIN2/3groups.(2)Population distribution of serum and RBC folate was different for the subjects of the four groups(H=59.08, P<0.001; H=43.68, P<0.001. The results showed that levels of serum and RBC folate were descended with the severity of the cervix lesions. Bonferroni test suggested that serum folate contents differed significantly in any two of them (P<0.0083) except the groups between Cland CIN1, CFN2/3and SCC. And for RBC folate levels, The difference was significant between CI and CIN2/3、CI and SCC、CIN1and SCC(P<0.0083). There is a positive correlation between serum and RBC folate (rs=0.405, P<0.001)(3)Methylation rates of p16is1.3%,12.7%,18.2%and29.7%, respectively, from CI group to SCC group. The trend of p46methylation increased gradually with the severity of the cervix lesions (χ2trend=23.46, P<0.001). Population mean of P16mRNA and protein expression was different for the subjects (F=10.628, P<0.001; F=86.944, P<0.001). All showed an uptrend with the severity of the cervix lesions. Expression of P16mRNA in CIN2/3and SCC groups are higher than CI group, and in SCC group is higher than that in CIN1group. Expression of p16protein in SCC, CIN2/3and CIN1group are all higher than that in CI group, and in SCC and CIN2/3group is higher than that in CIN1and CI group. There was negative correlation between folate and P16methylation, P16protein. Maybe there is a synergistic effect among methylation of P16CpG island, high expression of P16protein and the deficiency of RBC folate. There is an possitive association between the expression of P16mRNA HPV16E7(r=0.383, P<0.001)(4)Methylation rates of FHIT is5.0%,16.4%,25.5%and40.6%, respectively, from CI group to SCC group. The trend of FHIT methylation increased gradually with the severity of the cervix lesions (χ2trend=28.34, P<0.001). Population mean of FHITmRNA and protein expression was different for the subjects (F=19.676, P<0.001; F=18.656, P<0.001). Both showed downtrend with the severity of the cervix lesions. Expression of FHIT mRNA and protein is similar in the four groups. Expression of FHIT in SCC, CIN2/3and CIN1group are all lower than that in CI group, and in SCC group is lower than that in CIN1group. There was negative correlation between folate and FHIT methylation, and the positive correlation was existed between folate and FHIT protein. Maybe there is a synergistic effect among methylation of FHIT CpG island, low expression of FHIT protein and the deficiency of RBC folate.(5) From the results analyzed by multinomial Logistic regression model, multiple parity, the aberrant expression of p16and FHIT protein were the risk factors for CIN1; Risk factors of CIN2/3were HPV16infection, farmers, low frequency of cleaning pudendum, multiple parity, deficiency of serum and RBC folate, high expression of p16mRNA and protein, low expression of FHIT protein; Risk factors of cervical cancer were HPV16infection, farmers, low frequency of cleaning pudendum, multiple pregnancy,multiple parity, hypermethylation of FHIT CpG islands, farmers, multiple pregnancy, deficiency of serum and RBC folate, high expression of p16mRNA and protein, low expression of FHIT mRNA and protein.Conclusions(1) The results showed deficiency of serum and RBC folate could increase the risk of cervical cancer:both the serum and RBC folate levels descend with the severity of the cervix lesions, and there is a positive correlation between them. Maybe there is a synergistic effect between the deficiency of RBC folate and HPV16infection in cervical carcinogenesis.(2) Methylation of P16CpG island, high expression of P16mRNA and protein can increase the risk of cervical cancer. Results showed that methylation of P16CpG island, expression of P16protein and mRNA increased gradually with the severity of the cervix lesions. The high expression of P16maybe associated with the expression of HPV16E7. Maybe there is a synergistic effect among methylation of P16CpG island, high expression of P16protein and the deficiency of RBC folate.(3) Methylation of FHIT CpG island, low expression of FHIT mRNAand protein can increase the risk of cervical cancer. Results showed that expression of FHIT protein and mRNA decended gradually with the severity of the cervix lesions, whereas changes were opposite for methylation of FHIT CpG island. Maybe there is a synergistic effect among methylation of FHIT CpG island, low expression of FHIT protein and the deficiency of RBC folate.(4) Our study suggesents that HPV16infection, poor sexual health habits, pregnant and more productive are all risk factors of cervical cancer. What’s more, the index which can reflect social status such as career and education level are all related to the incidence of cervical cancer.