| Objective: To detect the methylation of long non-coding RNA maternally expressed gene 3(MEG3)promoter in Hirschprung’s disease(HSCR)and regulate the p53/p21 signaling pathway in a cell model,to explore the mechanism of the regulation of MEG3promoter’s methylation on p53/p21 signaling pathway in the pathogenesis of HSCR.Methods: Expression of p53 gene and methylation of MEG3 in narrow and anastomotic section of 30 HSCR-confirmed cases were detected by immunohistochemical staining(IHC),fluorescence quantitative polymerase chain reaction(q RT-PCR),immunoblotting(Western blot),methylation specific polymerease chain reaction(MSP)and direct sequencing method.MEG3 overexpression and silencing plasmid were used to transfect SK-N-BE(2)cell line,and overexpression of MEG3 group,knockdownof MEG3 group and blank group were established,methylation inhibitor azacytidine(5-aza-Cd R)was added to each group.The proliferation and apoptosis of each groups were detected by cell counting kit-8(CCK-8)and flow cytometry.The expression of p21 were determined by q RT-PCR and Western blot.Results:(1)IHC: immunoreactivity of p53 was detected in the nucleus of submucosal plexus,myenteric plexus and glandular epithelial cells in the intestine of HSCR.The positive expression was pale yellow to tan,and the stenosis was higher than the intestinal tube of the anastomosis(10.21 ±2.40&5.47 ±0.99,P<0.05).(2)q RT-PCR: the relative expression of p53 m RNA in the stenosis segment was significantly higher than that in the anastomotic tissue(10.56±0.37&2.24±0.3,P<0.05).(3)Western blot: p53 protein in the stenosis segment was significantly higher than the anastomosis seg-ment(1058.5 ±106.9&583.6 ± 87.6,P<0.05).(4)MSP and direct sequencing results: the incidence of MEG3 methylation in the stenotic tissue of children with HSCR was higher than that of the anastomosis(58.33 ± 4.5%&33.30 ± 1.3%,P<0.05).(5)successfully constructed MEG3-MEG3-OE and MEG3-KD SK-N-BE(2)cellgroup,compared wi-th the NC group,the expression of MEG3 m RNA in the MEG3-OE group was significantly higher than the NC group and the MEG3-KD group was significantly lower(4.70±2.62&0.89±0.15&0.20±0.10,P<0.05).(6)CCK-8: in negative control group: the proliferative capacity of MEG3-KD group was higher than NC group and MEG3-OE group(72h absorbance value1.83±0.07&1.56± 0.34&1.30±0.15,P<0.05),in 5-aza-Cd R group: MEG3-OE group was higher than MEG3-KD group and NC group(72h absorbance value 0.516 ±0.06&0.505±0.01&0.466±0.05,P<0.05).(7)flow c-ytometry: in negative control group:the apoptotic rate of MEG3-KD group was lower than NC group and MEG3-OE group(8.98% ± 2.37%&4.24% ± 0.85%&1.84% ± 0.82%,P<0.05),in 5-aza-Cd R group:MEG3-OE group was lower apoptotic rate than MEG3-KD group and NC group(7.59%±3.87%&4.08% ± 0.57%&2.22% ± 1.12%,P<0.05).(8)q RT-PCR: in negative control group: The relative expression of p21 m RNA in cells were higher in MEG3-OE group than in NC group and MEG3-KD group(6.53±0.88&3.33±0.58&2.01 ±0.29,P<0.05),in5-aza-Cd R group: the MEG3-OE gr-oup was higher than in MEG3-KD group and NC group(4.54 ± 2.13&0.83 ± 0.16&0.06 ± 0.04,P<0.05).(9)Western blot: in negative control group: the rela-tive ex-pression of p21 protein in cells were higher in MEG3-KD group than NC group and MEG3-OE group(56.31 ±0.88&23.09 ±0.78&12.13±0.79,P<0.05),in 5-aza-Cd R group: MEG3-OE group was higher than MEG3-KD group and NC group(7.09±0.95&6.21±0.34&3.24±1.4,P<0.05).Conclusion: The MEG3 promoter is highly methylated in HSCR and involved in the pathogenesis of HSCR by up-regulating the p53/p21 signaling pathway to mediate the proliferation and apoptosis of neural crest cells. |
