Effect Of Folate Of Different Levels On FHIT And MeCP2 In The Cervical Cancer Cells Of CaSki And C33A

ObjectiveCervical cancer (CC) remains to be one of the greatest killers of women worldwide. Though HPV infection, especially high-risk HPV16 infection, is a necessary cause of CC, there are many other cofactors facilitating the development of CC. There are not enough epidemiological evidence to prove and make clear the role of folate in the development of CC though some research showed that The shortage of folate can increase probility of contracting cervical cancer. Up to now, gene FHIT prohibiting cancer is closely associated with cervical cancer, because protein FHIT covers the fragile point FRA3B of the chromesome 3p, and HPV16 often integrites with the fragile point on the gene FHIT. MeCP2 protein may reflect the state of DNA CpG island methylation and its abnormal expression relates to a variety of tumors. The relevant reports have not been seen about its exact role in the course of cervical lesions. In this study, HPV16-positive cervical cancer cell and HPV-negative cervical cancer cell were cultured with different concentrations of folic acid in vitro so as to evaluate effect of folate on the cell proliferation and cell apoptosis, examine the expression of FHIT and MeCP2, and analyze the relationship among these factors in the advance of cervical cancer.MethodsThe C33A and CaSki cells were cultured routinely in vitro. Cells collected in the logarithm growth stage were treated with the different concentrations of folate. The effect on the activity and function of two cell lines was evaluated by MTT assay. Suppression of cell growth of two cell lines was assessed by Cell Proliferation Curve Analysis. The mRNA levels of HPV16 E2 and E6 gene and FHIT and MeCP2 were detected by Q-PCR. the protein expression conditions of FHIT and MeCP2 in the two cell lines were detected by Western Blotting. Using SPSS16.0, normally distributed data was analyzed by the T-test,F-test and Pearson correlation, skewed distribution of data by the Spearman correlation.Results(1)Folate might effect on the activity and proliferation of two cell lines. The results of MTT suggested that increasing concentration of folate inhibited the activity and function of C33A and CaSki. Result obtained from the method of MTT, revealed that folate of different levels suppressed the activity of two kinds of cervical cancer cells, and difference between control and experient groups was significant except the levels of 100μg/ml,500μg/ml of C33A cells and 10μg/ml,50μg/ml,750μg/ml of Caski cells.The growing concentration of folate inhibited cell proliferation obviously. The effects of folate on the cell growth suppression were increased gradually with the concentration of folate increasing and cultured duration prolonging. The two types of cells growed normally, under the situation that the supply of folate was insufficient, and the difference between control and experient groups was of no significance at the point of the 72nd hour.It showed there was no significant difference (P>0.05)on activity and proliferation between HPV negative C33A cells and HPV positive CaSki cells.(2)There was no significant difference on expression of HPV16 E2 and E6 genes by Q-PCR when CaSki cells were cultured with different concentration of FL.(3)In C33A cells, relative expression levels of FHIT mRNA in different concentrations of folate were statistically differen(tF=23.089,P=0.00),while relative expression levels of MeCP2 mRNA were not significantly different(F=2.308 P=0.079). In Caski cells, the result was just opposite.The result from regression analysis revealed that in C33A cells, various levels of foalte had no significantly different effect on the relative expression levels of FHIT mRNA, while having significant different effect on the levels of MeCP2 mRNA. In Caski cells, conclusion was just opposite.The result from the correlational analysis showed that in C33A cells, relative expression ratio of FHIT mRNA was of positive correlation to the relative expression ratio of MeCP2 mRNA, and such correlation was of significance(r=0.953 P=0.000), while in Caski cells, correlation of the relative expression ratios between FHIT mRNA and MeCP2 mRNA was of no significance(r=-0.621,P=0.451).In C33A and Caski cells, different levels of folate had siginaficantly different effects on the relative expression levels of protein FHIT (F=62.676 P=0.000,F=81.307P=0.000)and protein MeCP2(F=40.594 P=0.000,F=37.805 P=0.000)..The result from the regressional analysis demonstrated that in two types of cells, folate of different levels had siginificant influence on the relative expression levels of protein FHIT and protein MeCP2.The result from the correlational analysis revealed that in two types of cells, positive correlation of the relative expression levels between protein FHIT and protein MeCP2 was siginificant(C33A:r=0.954 P=0.000;CaSki:r=0.918 P=0.000).Conclusions(1)The adding different concentration of FL inhibited cell proliferation. And with the concentration of FL increasing and cultured duration prolonging, the effects of folate on the cell growth suppression, the percentage of G0/G1 cells and cell apoptosis rate increased gradually. But FD had no effects on cell proliferation and apoptosis of two cervical cnacer cell lines. (2)The effects of folate on expression of mRNA of CaSki HPV16 E2 and E6 genes had no obvious changes, which means effect of FL on transcription of CaSki cells may be slight.(3) Folate has a siginificant effect on the expression of gene FHIT supressing cancer in the cancer cells, which is based on the fact that shortage of folate led to relatively low expression levels of FHIT mRNA and high expression levels of protein FHIT, that when folate concentration ranged from 10ug/ml to 100ug/ml, the expression levels of FHIT mRNA increased correspondingly, that when folate concentrations range from 500ug/ml to 1000ug/ml, the expression levels decreased grudually, and that with the gradual increase of the folate concentrations, the expression levels of protein FHIT decreased gradually.(4)Folate has a siginificant effect on the expression of MeCP2 in cervical cancer cells. Such conclusion is evidenced by the result that insufficient folate led to relatively low level of MeCP2 mRNA and high expression level of protein MeCP2, that when the folate concentrations ranged from 10ug/ml to 1000ug/ml, the expression levels of FHIT mRNA increased correspondingly, that when the folate concentrarions ranged from 500ug/ml to 1000ug/ml, the expression levels decreased gradually, and that with the increase of folate concentrations, the expression levels of protein MeCP2 decreased.