| Background and ObjectiveCervical cancer,whose incidence is second only to breast cancer in women,is the most common malignant tumor in female reproductive systems.The persistent infection of high risk human papilloma virus(HR-HPV) is a definite etiological factor for cervical cancer.HPV is divided into two groups according to the carcinogenicity:high risk HPV(which will lead to high levels of cervical intraepithelial neoplasia and invasive cervical cancer) and low risk HPV(which is closely related to some benign lesions,such as genital warts and low levels of cervical intraepithelial neoplasia).We can see that the infected HPV types have direct impact on the outcome of the disease.However,only the HPV infection does not cause the cancer.Thus,there might exist some other pathogenesis co-existing with the HR-HPV infection.Some researches had showed that abnormal DNA methylation had intimate connection with the development of cancer. DNA methylation is a reversible reaction catalyzed by DNA methyltransferase(DNMT) enzymes,adding a methyl group onto carbon 5 of cytosine residues adjacent to guanine residues(5’-Cp G-3’), which mainly occurs in Cp G islands. As we know,there are five DNMTS in the DNMT family so far:including DNMT1,DNMT2, DNMT3 a,DNMT3b and DNMT3 l.DNMT3a and DNMT3 b are de novo methyltransferases, which have tissue specificity and may have abnormal expressions in tumor tissues;DNMT1 is a maintenance methyltransferase that preserves the methylation pattern during each cellular division,and passes the mathylation information to the daughter cells;.DNMT3 l, has no enzyme activity,join the de novo methylation by adjusting the DNMT3 a and DNMT3b’activities;DNMT2 has the same construction as the other DNMTs,however is a t RNA methyltransferase in essence,and has not activity in vitro.So far,the effect of the DNMT2 is no clearly understood.The change of the DNMTs’ expressions and functions are the key factors leading to the change of the DNA methylation pattern.Some studies showed that DNMTs had a higher expression in liver cancer,ovarian cancer,lung cancer, endometrial cancer and so on.There were also some researches suggesting the alteration of the DNMTs’ expressions and functions and the disorder of the functional sites were significant causes for the cervical cancer.Since both the persistent infection of high risk HPV and the alteration of the DNA methylation are the important factors for cervical cancer,then what a re the necessary connection between these two factors?The goal of this study was to test the expressions of the DNMTs(both the m RNA and the protein level) in cervical cancer tissues,to analysis the relationship between DNMTs and the HR-HPV infection.Materials and methodsResearch objects:40 cases of cervical cancer specimen and para cervical cancer tissues were selected as experimental group(which were diagnosed by pathology,38 were squamous-cell carcinoma and 2 were adenocarcinoma),20 cases of normal cervical tissues were picked as control group(which ware diagnosed as uterine fibroids or uterine prolapse,and underwent a hysterectomy).All of the tissue samples came from the Department of Obstetrics and Gynecology, Institute of Surgery Research, Daping Hospital, The Third Military Medical University.Methods:2.1 HPV typing:HPV DNA types were completed by BGI Shenzhen through Matrix Assisted Laser Desorption/ionization Time-of-flight Mass Spectrometry.HR- HPV16、18、31、33、35、39、45、51、52、56、58、59、66、68 were detected in total.2.2 q PCR technique were applied to detect the different expressions of the DNMTs m RNA in three groups.2.3 The protein expressions of the DNMTs were detected by immunohistochemistry and western blotting.Results3.1 Results of infection of HPV typingAmong the 60 cases,40 cases were high risk HPV,38 from cancer tissues and 2 from normal tissues.There were significant differences in the rates of HR-HPV infection(95% in experimental group and 10% in control group).In the cancer group,the types of HPV 16,52,58,31 and 18 were the most common type,and the HPV types in the normal group were both HPV16.Among the infected samples,23 were single HPV types,accounting for 57.5% in all,and17 were multiple infection,accounting for 42.5%.3.2 The m RNA expressions of the DNMTs in the experimental and control groupCompared to the control group,the m RNA expressions of the DNMT1, DNMT3 a,DNMT3b and DNMT3 l were dramatically increased,and the DNMT2 was markedly decreased in the experimental group,P value was less than 0.05.3.3 The protein expressions of the DNMTs in the experimental and control groupThe positive expression rates of DNMT1、DNMT3a、DNMT3b and DNMT3 l in cervical cancer tissues were 90.0%、85.0%、70.0%、80.0%. The positive expression rates of DNMT1、DNMT3a、DNMT3b and DNMT3 l in paracarcinoma tissues were 87.5%、85.0%、72.5%、77.5%.All of them were significantly higher than that in normal cervical tissues(25.0%、15.0%、20.0% and 30.0%).However,the positive expression rate of DNMT2 was 12.5% in cancer tissues,17.5% in paracarcinoma tissues,significantly lower than that in normal cervical tissues(85.0%).3.4 The correlation between the DNMTs’ positive expressions and the HR-HPV infectionThe expressions of DNMT1,DNMT3 a and DNMT3 b were positively correlated with HR-HPV infection(r=0.709、0.538 ' 0.536,P<0.05),the expressions of DNMT2 was negatively related with the HR-HPV infection(r=-0.438, P < 0.05),the expressions of DNMT3 l had no significant relationship with the HR-HPV infection(r=0.163,P>0.05).Conclusion4.1 The rates of HR-HPV infection were 95.0% in cervical cancer tissues,10% in normal samples,indicating that HR-HPV had intimate relation with cervical cancer.4.2 The five genes expression profiles were dramatically changed in the cervical cancer tissues.DNMT1 、 DNMT3 a 、 DNMT3 b and DNMT3 l were significantly increased,DNMT2 was dramatically decreased in cervical cancer,indicating that the change of DNMTs may affect the development of cervical cancer.4.3 HR-HPV infection were positively correlated with the expressions of DNMT1,DNMT3 a and DNMT3 b,negatively related with the expression of DNMT2,but had no relationship with the expression of DNMT3 l,indicating that there might exist one or more mechanisms inducing the HR-HPV to up-regulate the expression of DNMT1, DNMT3 a and DNMT3 b and repress the DNMT2,affecting the DNA methylation,thus leading to the malignant lesions of cervical intraepithelial. |
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