Interaction Between Abnormal Methylation And Expression Of FHIT,P16and HPV16Infection In Cervical Carcinogenesis

ObjectiveCervical cancer is a kind of malignant tumors of reproductive system which seriously hazard tothe health of women. It has been proved that persistent infection of HR-HPV is the main causeof cervical cancer. CpG islands hypermethylation of tumor-suppressor genes is the importantmechanism in causing inactivation of tumor-suppressor genes. There is a close relationshipbetween the two suppressor genes p16and FHIT in cervical cancer, but the expression levelsshowed differently in cervical cancer. The controversy is still existence whether the differentexpression of p16and FHIT genes relates with HPV16infection in cervical cancer and therelated studies about the interactive effect between them are very few at home and abroad. Thisstudy aimed to investigate the relationship between abnormal expression of FHIT, p16protein,CpG islands aberrant methylation and HPV16infection and the interaction effects among theabove factors in cervical carcinogenesis.Methods108cervical inflammation patients and142cases of cervical intraepithelialneoplasia(CIN)(including72cases of CINⅠand70cases of CINⅡ/Ⅲ) were chosen from theSecond Hospital affiliated to Shanxi Medical University and Maternal and Child Health Centerin Taiyuan and Jiexiu and100new cases of cervical squamous cell carcinoma diagmosed bypathology of Shanxi Tumor Hosiptal during September2009to March2011. Demographiccharacteristics, behavior and health of sexual, reproductive factors were collected usingstructured questionnaire. Cervical tissue was collected by surgery and biopsy. HPV16wasdetected by PCR.p16and FHIT gene methylation was detected by methylation specific PCR(MSP).Western blotting was used to detect the expression of p16and FHIT protein. Real-timequantitative PCR (real time PCR) was used to examine the levels of mRNA of HPV16E6, E7,p16and FHIT. Using SPSS13.0, Measurement data was analyzed by ANOVA and Welch; countdata was examined using the χ~2test and χ~2trend test; related factors were analyzed by Logisticregression analysis; the interaction effects were analyzed by additive model.Results(1) The rate of HPV16infection in CINⅠ(45.8%),CINⅡ/Ⅲ(68.6%) and cervical cancer(73.0%) were all significantly higher than that in cervical inflammation (28.7%)(χ~2=5.452,P=0.020; χ~2=25.607,P<0.001; χ~2=37.788,P<0.001).The rate of HPV16infectionincreased gradually with the increasing of cervical lesions (χ~2trend=47.623, P<0.001). Expression of HPV16E6mRNA in cervical cancer were significantly higher than that incervical inflammation and in CINⅠ(P<0.001,P=0.004); Expression of HPV16E7mRNA incervical cancer was significantly higher than that in cervical inflammation (P<0.001), CINⅠ(P<0.001) and CINⅡ/Ⅲ(P=0.018).(2) Methylation rates of FHITCpG island is3.7%in cervicitis group,13.9%in CINⅠgroup,21.4%in CIN Ⅱ/Ⅲgroup and38.0%in cervical cancer group, with the increasing of cervicallesions, methylation rates of FHITCpG island are gradually increasing (χ~2trend=40.147, P＜0.001), expression of FHIT protein in CIN Ⅰgroup (1.184±0.172), CIN Ⅱ/Ⅲgroup (1.133±0.126) and in cervical cancer group (1.099±0.148) are all lower than that in cervicitis group(1.255±0.130(P=0.001, P <0.001and P <0.001). Expression of FHIIT mRNA in cervicalcancer group and CIN Ⅱ/Ⅲgroup are both lower than that in cervicitis group (P＜0.001, P＝0.002), and in cerical cancer group is lower than that in CIN Ⅱ/Ⅲgroup (P=0.020).There is apositive additive effect between methylation of FHITCpG island and infection ofHPV16.Expression of mRNA is negatively correlated beween HPV16E6andFHIT(r=-0.142,P=0.008).(3) Methylation rates ofp16CpG island is0.9%in cervicitis group,11.1%in CINⅠgroup,18.6%in CIN Ⅱ/Ⅲ group and35.0%in cervical cancer group, with the increasing of cervicallesions, methylation rates of p16CpG island are gradually increasing (χ~2trend=44.976, P＜0.001),expression of p16protein in CIN Ⅰgroup (1.231±0.410), CIN Ⅱ/Ⅲgroup (1.967±0.617) and incervical cancer group (2.123±0.524) are all higher than that in cervicitis group (0.780±0.333)(P<0.001). Expression of p16mRNA in cervical cancer group and CIN Ⅱ/Ⅲ group are bothhigher than that in cervicitis group (P＜0.001, P＝0.030), and in cervical cancer group is higherthan that in CIN Ⅱ/Ⅲgroup and CIN Ⅰgroup (P=0.029, P＜0.001).There is a positive additiveeffect between methylation of p16CpG island and infection of HPV16.Expression of mRNA isnegatively correlated between HPV16E7and p16(r=0.397, P＜0.001).(4) Take the factors which are statistical significance into Logistic regression model,including related factors of cervical cancer,methylation of p16and FHIT CpG islands, theexpression of p16mRNA and protein, the expression of FHIT mRNA and protein, HPV16,andso on. Risk factors of CINⅠwere low expression of FHIT mRNA and protein, high expressionof p16protein, hypermethylation of FHIT CpG islands, low frequency of cleaning pudendum,no cleaning aftersexual life, multiple parity, and tumor familial history; Risk factors ofCINⅡ/Ⅲ were HPV16infection, low expression of FHIT mRNA and protein, high expressionof p16mRNA and protein, hypermethylation of FHIT CpG islands, farmers, low frequency ofcleaning pudendum, multiple pregnancy, history of abortion, gynecological diseases historyand tumor familial history; Risk factors of cervical cancer were HPV16infection, low expression of FHIT mRNA and protein, high expression of p16mRNA and protein,hypermethylation of FHIT CpG islands, farmers, low frequency of washing and cleaningpudendum, multiple pregnancy and parity, history of abortion, gynecological diseases historyand tumor familial history.Conclusions(1) Low expression of FHIT protein can increase the risk of cervical carcinogenesis, boththe methylation of FHIT CpG island and infection of HPV16can reduce the expression of FHITprotein. Maybe there is a synergistic effect between the methylation of FHITCpG island, lowexpression of FHIT protein and infection of HPV16in cervical carcinogenesis.(2) High expression of p16protein can increase the risk of cervical carcinogenesis. Highlyexpression of p16protein may related to oncogene of HPV16E7. Maybe there is a synergisticeffect beween methylation of p16CpG island, high expression of p16protein and infection ofHPV16in cervical carcinogenesis.(3) The occurrence of cervical cancer attributes to multiple factors. This study shows thatthe cervical carcinogenesis is closely related to HPV16infection once more, and highexpression of oncogenes of HPV16E6and E7are not only the risk factors of cervical cancerbut also related to the development of cervical cancer, farming, poor sexual health habits,pregnant and more productive, abortion history, gynecological history and family history ofcancer are all risk factors of cervical cancer.