The Role Of Folic Acid On The Expression Of P16 And DNMT1 In The Caski And C33A Cervical Cancer Cells

ObjectiveCervical cancer(CC) is one of the female reproductive system cancer, high risk HPV infection (HR HPV) is the major reason of CC, but not the unique cause. Studies have shown that folic acid deficiency increases the risk of CC. Folate as one donor of the SAM participates in DNA methylation. Studies found that abnormal DNA methylation is closely related with the tumor, hypermethylation of promoter region CpG island of P16 gene is an important reason that causes CC. DNMTs plays the primary role in the process of DNA methylation,the abnormal expression of DNMT was found in a variety of tumor cells.The above events were linked in CC,there was few research about this area. In this study, HPV16 positive cervical cancer cell and HPV negative cervical cancer cell were selected as research subjects,under the different folic acid concentrations,observing the proliferation of the cells, examining the expression of p16 and DNMT1, and analysising the relationship of these events occurred in cervical cancer. MethodsSelecting HPV16 positive cells Caski and HPV negative cells C33A were cultured routinely in vitro.Cells collected in the logarithm growth stage were treated by groups with different intervention levels of folate.The effect of the activity and function of 2 cell lines were observed by MTT;Cell growth suppression of two CC cell lines was assessed by Cell proliferation curve analysis; the mRNA levels of HPV16 E2, E6 gene, p16 and DNMT1 were detected by real timePCR;the protein expression of p16 and DNMT1 in the two cell lines were detected by Western blotting. Using SPSS16.0, normally distributed data was analyzed by the T test,F test, Pearson correlation and Linear regression, skewed distribution of data with the Spearman correlation.Results1,The activity of the two cervical cancer cell lines is inhibited with different concentrations of folic acid, in addition to 50ug/ml and 100ug/ml of folic acid concentrations in cell lines C33A, the differences between other experimental groups and control groups are statistically significant (P < 0.05), at the same concentration of folic acid, the inhibition of activity between Caski cells and C33A cells is significantly different (P <0.05); and the inhibition of the folic acid on the Caski cells are stronger than that on C33A.Cell counting found that when folic acid concentration is 50ug/ml and above, compared with the control group, the total number of cells in experimental groups is significantly decreased , and this difference is statistically significant (P <0.05); with the folic acid concentration increased, the inhibition ratio of 2 cell lines'proliferation is increased.2,The expression of p16 In C33A cells and Caski cells, giving different concentrations of folic acid, the differences of p16 mRNA expression levels and the differences of p16 protein expression in different groups are both statistically significant (p16mRNA expression:C33A F=2.969,P=0.034;Caski F=3.186,P=0.026;p16 protein expression:C33A F=68.821,P<0.001;Caski F=36.674,P<0.001), and with the rising of folate levels, the expression levels of p16 mRNA increased(P=0.006),but p16 protein expression decreased(P<0.001)in C33A;in Caski, p16 protein expression decreased(P<0.001), the expression levels of p16 mRNA decreased(P=0.496); on the same levels of folic acid,the expressions of p16 mRNA and protein in Caski were higher than that in C33A( P<0.05).3,The expression of DNMT1In Caski cells, giving different concentrations of folic acid, the differences of DNMT1 mRNA expression levels and the differences of DNMT1 protein expression in different groups are both statistically significant (DNMT1mRNA expression:F=30.633,P<0.001;DNMT1 protein expression:F=32.214,P<0.001), and with the rising of folate levels, the expression levels of DNMT1 mRNA and its protein expression decreased(P<0.05);in C33A, giving different concentrations of folic acid, the differences of DNMT1 protein expression in different groups are statistically significant (F=73.67,P<0.001), and with the rising of folate levels, the expression levels of DNMT1 protein expression decreased(P<0.05);on the same levels of folic acid,the expressions of DNMT1 mRNA and protein in Caski were higher than that in C33A( P<0.05).4,The expression of E2 and E6Folate had no siginificant effect on the expression of E2 mRNA and E6 mRNA(E2:F=3.246,P=0.111;E6:F=2.641,P=0.150).Conclusions1,Folic acid could inhibit CC cells'growth, and with the level of folic acid increased, the inhibition is stronger; on the same levels of folic acid,the inhibition of the folic acid on the Caski cells are stronger than that on C33A.2,Folic acid may affect the expressions of p16 mRNA and protein in CC cells, adding folic acid , p16 protein expression will decrease, and p16mRNA expression will increase; HPV16 infection may increase the expression of p16 in CC cell. 3,Folate deficiency may affect the expression of DNMT1 mRNA and protein in CC cells, adding folic acid can make the expression of DNMT1 decrease. HPV16 infection may have the certain impact on expression of DNMT1.4,The effect of FL on transcription of HPV16 in Caski cells may be slight.