Study On The Epigenetic Effect Of MNNG On Kazakh Esophageal Epithelial Cell

Objective:To explore the effect of N-methyl-N’-nitro-N-nitrosogua-nidine (MNNG) on normal Kazakh’s esophageal epithelial cells in general biology and index of epigenetics,so providing the evidences for the prevention and treatment of esophageal cancer in Kazakh nationnality. Method:1. Primary Kazakh esophageal epithelial cells were cultured by combined digestion method, and the cultured cells were identified through their morphological charateristics and immunohistochemical staining.2. Primary Kazakh’s normal esophageal epithelium cells was transfected with SV40-T gene to achieve immortalized cells, the expression of SV40-T gene in immortalized cells was detected by RT-PCR.3. Cell proliferation assay was detected by MTT, cell cycle was detected by flow cytometry and the change of apoptosis was detected by Annexin V-FITC/PI double staining.4. The change of global DNA methylation was detected by HPLC.5. Methylation of p16、 FHIT、LRRFIP1and ALDH1L1were detected by MSP.6. The mRNA and protein level of p16、 FHIT、LRRFIP1、ALDH1L1、DNMT1、DNMT3a、 DNMT3b were detected by RT-PCR and Wersten Blot. Results:(1) Primary cell confluent after cultured8to10d, showed a cobblestone-like growth, and the immunohistochemistry staining result showed that most of the cells were positive.(2) To obtain immortalized Kazak normal esophageal epithelial cells by SV40-T gene transfection, cell morphology and structure remains intact.(3) After exposured the MNNG, compared with the control group (0.00μg/ml), the cell inhibition rate had significantly increased in all concentration groups (P<0.05), the proportion of cells in Go+G1phase had significantly reduced in all concentration groups (P<0.05), the proportion of cells in S and G2+M phase had significantly increased in all concentration groups (P<0.05), the apoptosis rate had significantly increased in middle and high concentration groups (P<0.05).(4) Compared with the control group (0.00μg/ml), the global DNA methylation levels had significantly reduced in middle and high concentration groups (P<0.05).(5)Compared with the control group (0.00μg/ml),the content of DNMT1had significantly increased in high concentration group (P<0.05), the activity of DNMT1had significantly increased in all concentration groups (P<0.05),the content and activity of DNMT3a had significantly increased in all groups (P<0.05),the content of DNMT3b had significantly increased in all groups (P<0.05), the activity of DNMT3b had significantly increased in high concentration group (P<0.05).(6) Compared with the control group, each group of cells with p16、FHIT、LRRFIP1gene methylation status did not change, high concentration group of cells with ALDH1L1gene was trended from partial methylation to completely methylation.(7) Compared with the control group, p16, FHIT, LRRFIP1, ALDH1L1, DNMT1, DNMT3a and DNMT3b mRNA and protein level had significantly increased in each groups (P<0.05). Conclusion:(1)Culture primary Kazak normal esophageal epithelium and successfully establish an immortalized Kazakh normal esophageal epithelial cell line.(2)MNNG can inhibit the proliferation of Kazak normal esophageal epithelial cell, cell cycle arrest in S and G2+M phase and promote apoptosis.(3) MNNG can decrease the Kazakh normal esophageal epithelium global DNA methylation level, ALDH1L1gene trend from partial methylation to completely methylation.(4) MNNG increase the activity of DNMT1, DNMT3a and DNMT3b. MNNG have some effect on Kazakh normal esophageal epithelium general biology and epigenetic index.