The Effect Of Folic Acid，DNMT1 On Cervical Cancer Cells Growth And The Relationship With HPV16 Infection

ObjectiveEpidemiological studies show that: cervical cancer onset and relevant by HPV infection, butit is not the only factor for cervical cancer happen. Some research suggests that folic aciddeficiency can increase the occurrence of cervical cancer risk. Folic acid as a carrier of carbonunit, participate in DNA methylation. In recent years, it has found that DNA methylation andhuman multiple tumors of the disease take relevant. Among them, as the key enzyme, DNMT1methylation plays an important role in the process of cancer occurring.The study found thatDNMT1 of Cervical cancer cells is abnormal in high expression, but after DNMT1 inhibitioncan reverse the cervical cancer malignant phenotype. Folic acid, whether through the impact ofDNA methylation in cervical cancer development and progression of work, and this process isthe existence of HPV synergy and there is a limited area concerning such research at present. Inour previous study found that folic acid, DNMT1 expression and the occurrence of abnormalcervical cancer related,this topic to HPV negative C33A and HPV16 positive Caski cell as theresearch object, through Folic acid intervention,RNA interference method to discuss theinfluence of Folic acid and DNMT1 to cervical cell growth and the relationship with HPV16 ,analysis of the interaction between folic acid and DNMT1 in the occurrence of cervical cancerchanged.MethodsThe recombinant plasmid and empty plasmid were introduced into the cervical cancer cellsC33A and Caski by using the transient transfection method, and the establishment ofnon-transfected group was the control, were added to different concentrations of folic acid,through MTT assay cervical cancer cell activity,flow cytometry analysis of cell proliferationand apoptosis, using RT-PCR detection of DNMT1 of HPV16 oncogene E6, the E7 mRNA inthe expression, western blotting detection of expression of DNMT1 protein.Results1.Identification of recombinant plasmid：The recombinant plasmid and empty plasmid afterrestriction enzyme digestion, get two recombinant fragment of 291bp and 290bp. Therecombinant plasmid and empty plasmid successfully transfected stained cervical cancer cellsC33A and Caski, get 65% and 63% transfection efficiency and 53% and 52% of the DNMT1gene suppression rate. 2.The impact of folate on cervical cancer cell growth：(1) Folic acid can inhibit the activityof cervical cancer cells C33A and Caski, in addition to the concentration of 10.0μg/ml folic acidgroup and control group,the differences not statistically significant. With folic acidconcentration increased, two kinds of cells in other groups compared with the control group,cell activity decreased (P<0.05). The same folic acid concentration, the stronger inhibitoryeffect of folic acid on the activity of Caski cells in C33A cells; (2) folic acid inhibits cellproliferation, in addition to the concentration of 10.0μg/ml folic acid group and control groupwith no significant differences in two types of cells in the other groups. Other folateconcentrations all contribute to cell proliferation decreased (P<0.05), and with folic acidconcentration increased, the proliferation inhibition enhanced;(3) Folic acid to promoteapoptosis of cervical cancer cells C33A and Caski, in each experimental group, the apoptosisrate was higher than that in of the control group, and with the increase in the concentration offolic acid, folic acid also enhanced the two cell apoptosis. (4) Different concentrations of folicacid of HPV16 oncogenes E6 in Caski cells express no significant differences. Folateconcentrations of 1000.0μg/ml when the E7 oncogene mRNA expression compared with thecontrol group, reduced expression（F= 3.248,P=0.044）.3.The influence of DNMT1 to cervical cancer cell growth：before and after DNMT1interference cervical cancer cells C33A and Caski are different.DNMT1 interference comparedwith untransfected and empty plasmid rent comparison, cell activity decreased (P<0.001), cellproliferation was inhibited (P< 0.001), apoptosis increased. (P<0.001). The relative expressionlevels of the HPV16 oncogenes E6 mRNA in Caski cells after DNMT1 interference with theuntransfected and empty plasmid group difference was not statistically significant; HPV16 E7oncogene mRNA of DNMT1 interference untransfected and empty plasmid group, theexpression was decreased（F=9.989,P=0.012）.4.Folic acid to cervical cancer cells in the influence of DNMT1 expression:Folic acid oncervical cancer cells C33A and the Caski,DNMT1mRNA expression and protein expressioninhibited.In C33A group, folic acid concentration as the 500.0,1000.0μg/ml,the DNMT1mRNAand protein expression compared with the control group decreased expression（DNMT1 mRNAexpression:F=34.728,P＜0.001；Protein expression：F=28.735，P＜0.001）; In Caski groupfolate concentrations of 100.0, 500.0,1000.0μg/ml,compared with the control group, mRNAexpression level of reduced.Folate concentrations of 250.0,500.0,1000.0μg/ml of DNMT1protein expression compared with control group, reduced expression（DNMT1mRNAexpression:F=38.242,P＜0.001；Protein expression：F=92.807,P＜0.001）.5.The influence of Folic acid, DNMT1 to cervical cancer cell growth:(1) folic acidintervention,DNMT1 interference to inhibit the activity of cervical cancer cells C33A and Caski. In C33A cells, no transfection group to group and control group 10.0μg/ml concentrationdifference was not statistically significant, the rest of the group with the folic acid concentrationincreases, the cell activity is suppressed (F=184.735,P<0.001); empty plasmid group folateconcentrations 250.0, 500.0, 1000.0μg/ml, compared with the control group, cell activitydecreased(F=482.942,P<0.001); folic acid concentration in the restructuring group500.0,1000.0μg/ml,the cell activity with the control group was decreased(F=87.841,P<0.001).InCaski cells, not transfected group compared addition to 10.0μg/ml concentration group andcontrol group with no significant differences, the rest of the group and control group cellactivity were lower(F=68.185,P<0.001);empty plasmid group with the folic acid concentrationincreased, cell activity is suppressed(F=210.516,P<0.001). Folic acid concentration in therestructuring group group for 250.0、500.0、1000.0μg/ml,compared with control group,cellactivity is suppressed(F=147.821,P<0.001). The same folic acid concentration, the restructuringgroup in the two cells and the untransfected and empty plasmid groups were statisticallysignificant lower cell activity of the restructuring group, cell activity is suppressed. Caski cellsuntransfected, empty vector group and the reorganization of group cell activity lower thanC33A cells. (2) Folic acid,DNMT1 interference to inhibit the proliferation of cervical cancercells C33A and Caski.In C33A cells, the non-transfected group and control group of 10.0μg/mlconcentration with no significant difference in the group, the rest of the group compared withcontrol group, value-added index lower (F=21.499,P<0.001). Empty plasmid group in additionto 10.0,100.0μg/ml group compared with control group was no significant difference.In theother groups compared with the control group, the proliferation index decreased(F=5.134,P=0.010); restructuring group in folic acid concentrations for 50 0.0,1000.0μg/ml, cellproliferation index compared with control group, the proliferation index decreased(F=12.139,P<0.001).In Caski cells, not transfected group, empty plasmid group in addition togroup and control group of 10.0μg/ml concentration difference was not statistically significant,the rest of the group with the folic acid concentration increases, the proliferation indexlower(P<0.001); restructuring group folate concentrations 500.0,1000.0μg/ml,cell proliferationcompared with control group decreased (F=12.607,P<0.001).The same folic acid concentration,the restructuring group in the two cells untransfected and empty plasmid group werestatistically significant lower proliferative index of the restructuring group. (3) folic acid,DNMT1 interfere with the promotion of apoptosis of cervical cancer cells C33A and Caski, twocells, untransfected, empty vector group, as well as restructuring group compared with thecontrol group within the respective group, the difference was statistically significant , that is,with the folic acid concentration increased, the rate of apoptosis is on the rise. The same folicacid concentrations, restructuring group apoptosis rate is higher than that in the latter two groups. (4) folic acid, DNMT1 interference in non-transfected group, empty vector group andthe reorganization of the group E6mRNA expression compared with the control group, nosignificant differences; the E7mRNA the expression level of folic acid in the three treatmentgroups, both with the concentration increases, reduced expression of the folic acidconcentration for 1000.0μg/ml.Conclusions1.Folic acid inhibit the growth of cervical cancer cells. Two cervical cancer cell inhibitiondifferences with HPV16 infection.Tip: lack of folic acid change caused by cervical cancer andHPV infection have synergistic effect.2.The DNMT1 interference inhibits cervical cancer cell growth.And the differences ininhibition of cervical cancer cells is associated with HPV16 infection, suggesting that DNMT1promote the growth of cervical cancer cells and synergy with HPV16 infection.3.Folic acid suppression of DNMT1 expression in cervical cancer cells, folic acid supplementscan make DNMT1 expression decreased.4.Folic acid intervention, DNMT1 interference has the inhibition on cervical cancer cellgrowth,and suppress is a collaborative. The difference of suppression between C33A and Caskiis due to the HPV16 infection.Suggestive of folate deficiency in cervical carcinogenesis processassociated with the DNMT1.