Folic Acid And FHIT Gene DNMT1 Of Cervical Cells In Rats

Objective:Cervical cancer is a common female malignant tumor, epidemiological and experimental studies show that persistent infection of type high-risk human papilloma virus (HPV) is one of the causes of cervical cancer, but not the only factor. The role of folic acid in cervical cancer was concerned increasingly for it associated with DNA synthesis and DNA methylation. DNA methylation is one of the most widely studied epigenetic events. DNMT1, a key enzyme of DNA methylation, had been confirmed that it is related to a variety of tumor. Fragile Histidine Triad gene(FHIT) is a tumor suppressor gene, its inactivation is may be related to hypermethylation of CpG island. Studies reported that folate can affect the acivity of DNMT1and the supplementation of folate can elevate the expression of FHIT protein. But whether folate affects the expression of FHIT through DNA methylation in cervical cancer cells is unreported now, and this study will study on them in vitro.Methods:The recombinant plasmid and empty plasmid were introduced into the cervical cancer cells C33A and Caski by transient transfection.Choosing the non-transfected group as the control, and adding folic acid of different concentrations to the group of non-transfected、empty plasmid d and recombinant plasmi. The grouth situation of cervical cancer cells was detected by cell counting assay and the proliferation and apoptosis of cervical cancer cells was analyzed by flow cytometry.Using real-time PCR to detecte the expression level of mRNA of DNMT1、FHIT、E6、E7and using Western blot to detecte the expression level of protein of DNMT1and FHIT.Result:1.The impact of folate on cervical cancer cell growth and the expression of DNMT1and FHIT gene:(1) Compared with control group,Folic acid of different concentrations groups can inhibit the growth of cervical cancer cells significantly (P <0.05) except10.0μg/ml group. With folic acid concentration increased, inhibition rate increased (C33A:r=0.976,P<0.001;Caski:r=0.952,P<0.001).(2) With folic acid concentration increased, proliferation index of cell cycle reduced(C33A:r=-0.736, P=0.001; Caski:r=-0.864, P<0.001) and apoptosis rate increased gradually (C33A: r=0.776, P<0.001; Caski:r=0.679, P=0.002).(3)Folate can inhibit the expression of DNMT1. the expression of DNMT1mRNA and protein in500μg/ml and1000μg/ml group was lower than control group (4)Folate can influence the methylation of FHIT. In the C33A cells, the methylation of FHIT in10μg/ml group is positive, while the methylation of FHIT in10,100and250μg/ml groups appear partly. In the Caski cells. FHIT methylation is negative in500and1000g/ml group while the methylation of FHIT appear partly in250μg/ml group and the remaining set of FHIT methylation is positive.(5)Folate can inhibit the expression of FHIT. With the increasing of folic acid concentration, the expression of mRNA (C33A:r=0.853, P<0.001, Caski: r=0.919. P<0.001) and protein of FHIT (C33A:r=0.721, P=0.001, Caski:r=0.801. P<0.001) are decreasing, the expression of FHIT in500、1000μg/ml group are lower significantly than control group.(6)There is no effect on E6’mRNA expression in groups of different concentrations folic acid in Caski cells.Compared with control group, the expression of E7’mRNA is lower in the group of1000.0μg/ml.2. The influence of DNMT1on cervical cancer cell growth and the expression of FHIT gene:(1) Recombinant plasmid and empty plasmid get two recombinant fragment of291bp and290bp after identification by restriction enzyme digestion. The recombinant plasmid and empty plasmid successfully transfected in C33A and Caski. transfcction efficiency are65%and63%. suppression rate of DNMT1are53%and52%respectively.(2)After DNMT1interference, compared with untransfected group. living cell counts decreased in recombinant plasmid (P<0.05);compared with untransfected and empty plasmid group, proliferation of cell was inhibited, apoptosis increased in recombinant plasmid(P<0.05).(3)After DNMT1interference, FHIT methylation was positive in untransfected and empty plasmid group while non-methyaltion was negative. transfected group appear non-methyaltion band.(4)After transfecting. compared with untransfected group, FHIT mRNA and protein expression of transfected group were increased(P<0.001), and the empty plasmid group was no significant differences;(5) There was no effect on E6’ expression of DNMT1interference; compared with the control group.the expression of E6is lower of transfected group (P<0.05).3.The influence of Folic acid. DNMT1to cervical cancer cell growth and the expression of FHIT gene:(1)With the increasing of folic acid concentrations,the living cell counts of the three groups of C33A and Caski decreasing.The difference of living cell counts is significant except10.0μg/ml of untransfected and recombinant plasmid group.At the same concentration of folic acid,compared with recombinant plasmid group.the living cell counts of empty plasmid and untransfected group are lower(P<0.05).(2) The interference of Folic acid and DNMT1can inhibit the proliferation of cervical cancer cells C33A and Caski.In recombinant plasmid group of C33A. with the increasing of folic acid concentrations,cell proliferation index decreasing gradually.compared with control group.the differences of cell proliferation index in500.0and1000.0μg/ml group are significant. In empty plasmid and recombinant plasmid group of Caski, with the increasing of folic acid concentrations,cell proliferation index decreasing gradually.and cell proliferation indexs of500.0and1000.0μg/ml group are lower than control group.The cell proliferation indexs of untransfected and recombinant plasmid group are lower than recombinant plasmid group at the same concentration of folic acid.With folic acid concentrations increased, apoptosis rate of C33A and Caski of recombinant plasmid and empty plasmid group increased gradually.Compared with1.0μg/ml group,the apoptosis rates of the rest groups of folic acid expect10μg/ml group are higher.At the same concentration of folic acid,the apoptosis rates of recombinant plasmid group of C33A and Caski are higher than untransfected and empty plasmid group.(3) Folic acid. DNMT1interference can change the methylation status of the FHIT gene. In C33A cells, of1000μg/ml group of empty plasmid group.the methylation of FHIT is partial of500μg/ml group and FHIT methylation are positive of the rest groups of folic acid; FHIT methylation are negative in the groups of folate of recombinant plasmid group. In Caski cells, FHIT methylation are negative in the groups of250μg/ml.500μg/ml and1000μg/ml and FHIT methylation are positive in the rest folate groups of empty plasmid group; the methylation of FHIT is negative in recombinant plasmid group.(4). In untransfected and empty plasmid groups of C33A and Caski, with the increasing of folate concentrations, the expression of FHIT mRNA increasing. Compared with lμg/ml group, defferences of500μg/ml and1000p.g/ml groups are significant (P<0.05). In recombinant plasmid group,compared with control group, the expression of FHIT mRNA are higher of all experment groups. At the same concentration of folate, the expression of FHIT mRNA of recombinant plasmid group is higher than untransfected and empty plasmid group((P<0.05)).(5)In C33A cell, the expression of FHIT protein increased with folate’concentration increased.Compared with1μg/ml group, the differences of the expression of FHIT protein are significant in the250μg/ml.500μg/ml and1000μg/ml of untransfected and recombinant plasmid group while the the differences are significant of500μg/ml and1000μg/ml in empty plamid group.In Caski cell. compared with1μg/ml group, the differences of FHIT protein’ s expression are significant of500μg/ml and1000μg/ml in untransfected and empty plasmid group.while the differences are significant of 250μg/ml,500μg/ml and1000μg/ml in recombinant plasmid group.At the same concentration of folate,compared with untransfected and empty plasmid group,FHIT protein’s expression are higher of the rest folate groups in recombinant plasmid group expect250μg/ml of C33A and10μg/ml of Caski.(6) The effect of folic acid and DNMT1interference on E6is unsignificant;the expression of E7mRNA in untransfected、empty plasmid and recombinant plasmid group are decreasing with the increasing of folate concention.Conc lusions:1.Folic acid can inhibit the growth of cervical cancer cells and the expression of DNMT1, change the methylation status of FHIT and elevate its expression. It is suggested that the impact of folic acid on FHIT maybe associated with the process of DNA methylation.2.The interference of DNMT1could inhibit the growth of cervical cancer cells, it can cause demethylating and restoring expression of FHIT.It suggest that the methylation status and expression of FHIT could be affected by DNMT1.3.The interference of folic acid and DNMT1can inhibit the growth and proliferation of cervical cancer cells. and there is a synergistic effect of both4. The interference of folic acid and DNMT1can cause demethylating and restoring expression of FHIT. interference on cervical cancer cells. Interference folic acid is applied after DNMT1. the expression of FHIT is further increased, suggesting that folate may affect DNMT1to elevate FHIT expression.5. In Caski cells, the interference of folic acid and DNMT1have no effect on the expression of E6while they can reduce the expressio of E7,but the specific mechanism needs further research.