| Objective: To detect the gene expression of the MEG3 and Rac1/Limk1/Cofilin signal pathway in Hirschsprung’s disease(HSCR)transitional segment,stenosis segment tissues and normal intestinal tissues,and discussing the significance of MEG3 and Rac1,Limk1 and Cofilin in the pathogenesis of HSCR.Methods: To dectect The m RNA expression of MEG3 and Rac1/Limk1/Cofilin in 30 cases of HSCR transitional segment,stenosis segment tissues and 14 cases of normal intestinal tissues by q RT-PCR technology,and test Rac1,Limk1 and Cofilin protein expression and phosphorylation leves by Western Blot technology.Analysis the differences of MEG3 and Rac1/Limk1/Cofilin gene expression in HSCR stenosis segments,transitional segments and normal intestinal tissues.Results: q RT-PCR:(1)The relative expression of MEG3 in stenosis,transitional and normal intestinal tissues(0.35±0.07,0.51±0.07,1.19±0.20),Comparison of stenosis and transitional segments with normal intestinal tissues,the difference is statistically significant(P<0.05),the difference is not statistically significant between the stenosis segments and the transitional segments(P>0.05).(2)The relative expression of Rac1 in stenosis segment,transitional segment and normal intestinal tissues(0.57±0.06,0.80±0.07,1.18± 0.12),The differences between the two groups was statistically significant(P﹤0.05).(3)The relative expression of Limk1 in the stenosis segments,transition segments and normal intestinal tissues(0.56±0.09,0.65±0.08,1.28±0.19),Comparison of stenosis and transitional segments with normal intestinal tissues,the difference is statistically significant(P<0.05).There is no statistically significant difference between the stenosis segments and the transitional segments(P>0.05).(4)The relative expression of Cofilin in stenosis,transitional and normal intestinal tissues(0.77±0.07,0.78±0.05,1.19±0.08),Comparison of stenosis and transitional segments with normal intestinal tissues,the difference is statistically significant(P<0.05).the difference is not statistically significant between the stenosis segments and the transitional segments(P>0.05).Western Blot:(1)The expression of Rac1,Limk1 and Cofilin in the stenosis and transitional segment of HSCR is significantly lower than normal intestinal tissues(Rac1: 0.39±0.06 vs.0.37±0.05 vs.2.12±0.62.Limk1:0.40±0.04 vs.0.46±0.05 vs.1.52±0.17.Cofilin:0.94±0.08 vs.0.86±0.07 vs.2.37±0.23),the difference is statistically significant(P<0.05),the difference is not statistically significant between the stenosis segments and the transitional segments(P>0.05).(2)The expression of p-Rac1,p-Limk1 and p-Cofilin in the stenosis and transitional segments were significantly lower than normal intestinal tissues(p-Rac1:0.94±0.14 vs.0.84±0.09 vs.3.39±0.66.p-Limk1:0.47±0.08 vs.0.34±0.09 vs.1.24±0.26.p-Cofilin:0.44±0.06 vs.0.31±0.60 vs.1.47±0.23),the difference is statistically significant(P<0.05),and the difference is not statistically significant between the stenosis segments and the transitional segments(P>0.05).Conclusion: MEG3 and Rac1,Limk1 and Cofilin are low expressed in Hirschsprung disease,which may be related to the pathogenesis of HSCR.Objective:Through the use of MEG3 to regulate the Rac1 pathway in the SH-SY5 Y cell models,and explore the biological functions of MEG3 and Rac1,Limk1,Cofilin in the HSCR cell models in vitro.Methods:To transfect SH-SY5 Y cells with MEG3 overexpression plasmid and Lipofectamin2000 to construct MEG3 overexpression group,solvent group.To construct Rac1,Limk1,Cofilin inhibitor group and Rac1 agonist Group(PDGF-BB group),Rac1agonist+Limk1 inhibitor group(P-B group)and Rac1 agonist group+Cofilin inhibitor group(P-C group)by Rac1,Limk1,Cofilin inhibitor and Rac1 agonist.q RT-PCR detects the relative expression of MEG3 and Rac1/Limk1/Cofilin in each group.To detect the expression of Rac1/Limk1/Cofilin pathway protein and the level of phosphorylation by Western Blot,and test the proliferation and migration ability of each groups by CCK-8 and Transwell.Results:(1)The expression level of MEG3,Rac1 and Limk1 m RNA in the MEG3 overexpression group were significantly higher than control groups and solvent group(MEG3:8.28±0.60 vs.1.00±0.03 vs.1.54±0.15.Rac1:2.97±0.14 vs.1.00±0.01 vs.1.26±0.02,Limk1:1.73±0.20 vs.1.00±0.01 vs.0.96±0.02),the difference is statistically significant(P<0.05).The expression of Cofilin m RNA in the MEG3 overexpression group was compared with the control group and the solvent group(1.11±0.03 vs.1.00±0.05 vs.1.04±0.04),the difference is not statistically significant(P>0.05).(2)The expression of Rac1,Limk1 and Cofilin in the MEG3 overexpression group is significantly higher than the solvent group and the control group(Rac1:1.29±0.11 vs.0.47±0.05 vs.0.53±0.06.Limk1:1.43±0.30 vs.0.58±0.23 vs.0.64±0.04.Cofilin:4.53±1.09 vs.0.77±014vs.1.07±0.16),the difference is statistically significant(P<0.05).(3)The expression of p-Rac1,p-Limk1 and p-Cofilin in the MEG3 overexpression group is significantly higher than the solvent group and the control group(p-Rac1:0.96±0.31 vs.0.33±0.03 vs.0.18±0.02.p-Limk1:0.90±0.09 vs.0.63±0.06 vs.0.28±0.09.p-Cofilin:2.80±0.54 vs.0.80±0.23 vs.0.99±0.13),the difference is statistically significant(P<0.05).(4)The cells proliferation rate of MEG3 overexpression group at 24 h,48h,72 h and 96 h is significantly higher than the solventgroup(24h:151.32±4.36 vs.131.56±5.31.48h:118.23±2.07 vs.82.33±5.47.72h:129.92±8.45 vs.78.53±4.02.96h:147.92±10.01 vs.125.52±6.48),the difference is statistically significant(P<0.05).(5)The number of cells migration in the MEG3 overexpression group is significantly higher than the control and solvent groups(86.80±3.40 vs.53.87±3.30 vs.50.87±3.24),the difference is statistically significant(P<0.05).(6)The protein expression of Rac1,Limk1 and Cofilin in the control group are significantly higher than the Rac1,Limk1 and Cofilin inhibitor groups(Rac1:1.30±0.42 vs.0.51±0.03 vs.0.41±0.05 vs.0.25±0.07.Limk11.02±0.20 vs.0.24±0.03 vs.0.41±0.12 vs.0.50±0.07.Cofilin: 2.01±0.43 vs.0.67±0.11 vs.0.93±0.18 vs.0.70±0.10),the difference is statistically significant(P<0.05).(7)The protein expression of p-Rac1,p-Limk1,p-Cofilin in control group is significantly higher than the Rac1,Limk1,and Cofilin inhibitor group(p-Rac1:1.37±0.26 vs.0.68±0.07 vs.0.66±0.05 vs.0.60±0.05.p-Limk1:2.49±0.74 vs.85±0.04 vs.0.73±0.01 vs.0.72±0.01.p-Cofilin:1.23±0.22 vs.0.65±0.07 v s.0.62±0.05 vs.0.58±0.16),the difference is statistically significant(P<0.05).(8)The inhibitory effect of Limk1 and Cofilin inhibitor group gradually increased,and the effect was most obvious at the 96 hour(Limk1 inhibitor group:37.60±5.43 vs.65.30±5.23 vs.65.88±1.61 vs.91.98±1.21.cofilin inhibitor group:34.87±5.00 vs.46.48±6.77 vs.44.09±5.20 vs.89.53±1.79),the Rac1 inhibitor group had the most obvious inhibitory effect at 72 hours,and the inhibitory effect on cells decreased after 72 hours(23.27±3.63 vs.42.77±6.01 vs.47.22±2.40 vs.24.91±4.38).(10)The protein expression of Rac1,Limk1 and Cofilin in PDGF-BB group was significantly higher than the control group,P-B group and P-C group(Rac1:1.18±0.06 vs.0.82±0.02 vs.0.46±0.06 vs.0.42±0.08.Limk1:1.07±0.02 vs.0.57±0.10 vs.0.52±0.13 vs.0.61±0.06.Cofilin:1.46±0.20 vs.0.61±0.20 vs.0.59±0.18 vs.0.43±0.20),the difference was statistically significant(P ﹤ 0.05).(11)The protein expression of p-Rac1,p-Limk1,and p-Cofilin in PDGF-BB group are significantly higher than the control group,P-B group and P-C group(p-Rac1:1.10±0.21 vs.0.58±0.07 vs.0.36±0.08 vs.0.35±0.08.p-Limk1:1.39±0.34 vs.0.63±0.10 vs.0.63±0.10 vs.0.52±0.08.p-Cofilin:1.37±0.18 vs.0.86±0.21 vs.0.77± 0.06 vs.0.77±0.09),the difference It is statistically significcant(P﹤0.05).(12)The cells proliferation ability of PDGF-BB group was significantly higher than the control group P-B group P-C group after 72h(72h:0.35±0.10 vs.0.25±0.05 vs.015±0.01 vs.0.17±0.03.96h:0.44±0.05 vs.0.30±0.03 vs.0.11±0.00 vs.0.12 ±0.02),the difference is statistically significan(P ﹤0.05).(13)The number of cells migration in PDGF-BB group is significantly higher than the control group,P-B group and P-C group(107.93±5.54 vs.51.77±1.92 vs.15.57±1.75 vs.16.54 ±1.31),the difference was statistically significant(P﹤0.05).Conclusion:MEG3 Overexpression can promote the expression of Rac1 signaling pathway and promote the migration and proliferation of neural crest cells.The low expression of MEG3 may be related to the pathogenesis of Hirschsprung’s disease by inhibiting the Rac1 signaling pathway. |
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