| Objective: Cervical cancer(CC)is a one of the most common malignancies affecting women's health.Persistent high-risk human papilloma virus(HPV)infection is considered to be the most important cause of cervical intraepithelial neoplasia(CIN)and CC,especially HPV16,18 type infection.HPV16/18 carcinogenic mechanism is mainly through the virus oncogene E6 and E7 combining with the tumor suppressor gene p53 and retinoblastoma protein p Rb respectively.E6 inhibits p53 activity and E7 inhibits p Rb activity,which will cause these tumor suppressor protein lose their functions,and result in the infinite proliferation and malignant transformation of cells and avoiding apoptosis.However,the occurrence of cancer is a multi-factor,multi-step and multi-stage complex process.Several studis have shown that changes in epigenetics are closely related to the occurrence of various human tumors,and DNA methylation of tumor suppressor genes is the most important form of epigenetic modification in cervical cancer.In contrast to tumor genetics,epigenetic changes are reversible.Epigenetics silencing genes can restore expression by applying DNA demethylation drugs.Therefore,the study of demethylated drugs has a great significance.Epithelial-mesenchymal transition(EMT)refers to the process of transforming epithelial cells into mesenchymal cells with the ability to migrate and invade.It has been confirmed that EMT plays an important role in the progression of CC.Twist is a member of the basic helix-loop-helix(b HLH)transcription factor family,including Twist1(Twist)and Twist2,Twist1 is considered to be a key regulator in EMT processes and plays an important role in tumor invasion and metastasis.In addition,studies have shown that Twist1 also has DNA methylation modification in CC,so Twist1 is an important regulator both in EMT and DNA methylation process.Scholars have found that 5-aza-2'-deoxycytidine(5-Aza-Cd R)can inhibit the proliferation of Si Ha and He La cells,and there is a concentration and time dependent relationship,that is,inhibitory effect on the proliferation of cervical cancer cells is increased with the increase of concentration(0.1-100 umol / L)and prolongation of time(24h,48 h,72h,96h).But the studies about the effect of 5-Aza-Cd R on HPV E6/E7 are inconsistent.Some researchers have found that 5-Aza-Cd R can reduce HPV E6/E7 expression,while others believe that5-Aza-Cd R has no effect on it.There are few studies about the effect of5-Aza-Cd R on P53.We have not found study about its effect on p Rb and Twist1.In this study,HPV16-positive Si Ha cells were used to investigate the effect of DNA transferase inhibitor 5-Aza-Cd R on the expression of HPV16E6/E7?P53?p Rb and Twist1,so to provide the theoretical and experimental basis for the clinical treatment of cervical squamous cell carcinoma.Methods:1 Sqamous cervical cancer cell line Si Ha was cultured.2 The effect of 5-Aza-Cd R on the proliferation of Si Ha cells was analyzed by MTT assay.The concentration of 5-Aza-Cd R in the experimental group was 10?mol / L,20?mol / L and 40?mol / L,respectively.The control group was treated with no regent.The absorbance was measured by a microplate reader and the cell viability was calculated.After investigating the dose-effect relationship of 5-Aza-Cd R on Si Ha.40 ?mol/L 5-Aza-Cd R group was selected to do the following experiment.3 The expression of HPV16 E6/E7,p53,p Rb and Twist1 m RNA in the experimental group and the control group were detected by real-time quantitative PCR and the differences between the two groups were compared.4 The expression levels of p53,p Rb and Twist1 protein were detected by Western-blot and the difference between the two groups was compared.Results:1 After Si Ha cells were treated with 0?mol / L(control group),10?mol /L,20?mol / L and 40?mol/L 5-Aza-Cd R for 72 h,MTT assay showed that the cell viability(A490)of the control group and the experimental group were0.551±0.023,0.488±0.011,0.467±0.012 and 0.437±0.019 respectively.There was significant difference between the experimental group and the control group(F=56.010,P<0.05),the viability of Si Ha cells decreased and the inhibitory effect was gradually increased with the rising of 5-Aza-Cd R concentration within a certain concentration range,which shows the dose-dependent relationship.2 The expression of HPV16 E6/E7,p53,p Rb and Twist1 m RNA in the experimental group and the control group were detected by RT-PCR after treated with Si Ha cells for 72 hours at the final concentration of 40?mol5-Aza-Cd R.The relative expression of HPV16 E6/E7 m RNA was lower than that of the control group(P<0.05).The expression of p53 and p Rb m RNA in the experimental group was higher than that in the control group(P<0.05).The expression of Twist1 m RNA in the experimental group was lower than that in the control group(P<0.05).3 Western-blot analysis showed that the expression of p53 was significantly higher in the experimental group(0.54±0.03)than that of the control group(0.50±0.04),P<0.05,The p Rb protein expression in the experimental group(0.50±0.04)was significantly higher than that in the control group(0.62±0.04),P<0.05.The expression of Twist1(0.50±0.04)in the experimental group was significantly higher than that in the control group(0.62±0.04),P<0.05.Conclusions:1 5-Aza-Cd R can inhibit the proliferation of cervical cancer Si Ha cells,with a dose-dependent relationship;2 5-Aza-Cd R can reduce the expression of HPV16 E6/E7 m RNA in cervical cancer Si Ha cell line;3 5-Aza-Cd R can up-regulate the expression of P53,p Rb m RNA and protein;4 5-Aza-Cd R can reduce the expression of oncogene Twist1 m RNA and protein expression,which may inhibit the EMT process of cervical squamous cell carcinoma. |
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