miR-214-3p对慢性社会挫败应激所致小鼠抑郁样行为的作用及机制
发布时间：2018-08-30 18:45
【摘要】：第一部分慢性社会挫败应激对小鼠前额皮层miRNAs及P-catenin蛋白表达水平的影响 目的：建立小鼠慢性社会挫败应激模型,检测前额皮层区小RNA (microRNA, miRNA)的差异化表达及蛋白表达变化。 方法：采用慢性社会挫败应激和慢性不可预知性应激建立抑郁症模型；糖水偏好测试,悬尾测试和社会接触测试等检测抑郁样行为；Elisa检测模型小鼠血清皮质酮水平；实时荧光定量PCR定量分析前额皮层区miRNAs的差异表达；Western Blotting检测前额皮层区p-连环素(β-catenin)的表达。 结果：(1)慢性社会挫败应激模型鼠前额皮层miR-214-3p表达相对于对照组上调4.249±0.816,miR-690相对于在对照组上调2.527±0.292；(2)慢性社会挫败应激模型小鼠的糖水偏好率下降到40.577±4.81%,社会接触时间缩短至60.85±13.17s；(3)慢性社会挫败应激模型小鼠血清皮质酮含量升高到17.943±0.86μg/L；(4)慢性社会挫败应激和慢性不可预知性应激模型鼠前额皮层β-catenin表达相对于对照组降低至54.7±11.2%。 结论：慢性社会挫败应激模型小鼠均可作为抑郁症研究的理想模型,前额皮层区miR-214-3p表达上调,β-catenin表达降低。 第二部分miR-214-3p和miR-690的作用靶点研究 目的：明确miR-214-3p和miR-690以ctnnb1mRNA作为靶点。 方法：采用实时荧光定量PCR的方法检测miR-214-3p和miR-690的表达水平；Western Blotting检测前额皮层区β-catenin的表达；双荧光素酶酶报告系统检测miR-690与ctnnb1mRNA的3'-UTR序列的结合位点。 结果：(1)神经元转染miR-690的模拟物后miR-690的表达量相对于对照增加44.2±0.44；(2)神经元转染miR-690的模拟物后β-catenin蛋白表达水平相对于对照组降低到60±12%；(3)双荧光素酶报告实验证明miR-690以ctnnbl的3'UTR区作为靶点；(4)第三脑室在体注射miR-214-3p和miR-690的模拟物后相对于对照组miR-214-3p的表达增加到2.465±0.032,miR-690的表达增加到1.21±0.05；(5)第三脑室在体注射miR-214-3p模拟物后,相对于对照组降低β-catenin蛋白表达至64.6±4.35,而注射miR-690不影响β-catenin蛋白表达；(6)在体沉默ctnnbl区的3'UTR区相对于对照组可以有效降低mRNA的表达至0.37±0.07,同时降低P-catenin蛋白的表达量至24±3.5%。 结论：miR-214-3p以ctnnb1的3'-UTR区为靶点调控β-catenin蛋白表达。 第三部分调控前额皮层区miR-214-3p表达对慢性社会挫败应激所致小鼠抑郁样行为的影响及机制 目的：研究调控前额皮层区miRNA-214-3p对小鼠抑郁样行为的影响及机制。 方法：糖水偏好测试,悬尾测试和社会接触测试检测小鼠抑郁样行为的改变；实时荧光定量PCR分析前额皮层区miR-214-3p的表达；Western Blotting检测前额皮层区β-catenin的表达；脑片钳记录前额皮层区的微小兴奋性突触后电流(miniature excitatory postsynaptic current, mEPSC)。 结果：(1)相对于对照组,antagomir-214明显降低miR-214-3p表达至1.046±0.05,增加β-catenin蛋白表达至91.3±4.7%；(2)antagomir-214明显降低模型小鼠悬尾不动时间至27.954±4.06s,并增加社会接触时间至64.283±10.7s；(3)antagomir-214能增加模型小鼠前额皮层区mEPSC幅度；(4)慢病毒包被miR-214-3p抑制物(lentivirus-miR-214, LV-miR-214)明显降低miR-214-3p表达水平至1.013±0.046,增加β-catenin蛋白表达至227±59.8%；(5)LV-miR-214降低模型小鼠悬尾不动时间至45.7±12.08,并增加社会接触时间至53.6±10.051s；(6)过表达ctnnb1降低模型小鼠悬尾不动时间至37.5±6.91s,并增加社会接触时间至46.833±6.921s；(7)与对照组相比,经鼻腔给予antagomir-214降低miR-214-3p表达至0.4±0.1,增加β-catenin蛋白表达至110±9.3%；(8)经鼻腔给予antagomir-214降低模型小鼠悬尾不动时间至67.83±14.35s,增加社会接触时间至50.55±7.44s；(9)antagomir-214, LV-miR-214和过表达ctnnbl均不影响模型小鼠的糖水偏好率。 结论：抑制miR-214-3p表达可以逆转慢性社会挫败应激所致小鼠的抑郁样行为。
[Abstract]:Part I Effects of chronic social frustration stress on the expression of microRNAs and P-catenin in prefrontal cortex of mice
Objective: To establish a chronic social frustration stress model in mice and detect the differential expression and protein expression of small RNA (microRNA, microRNA) in prefrontal cortex.
Methods: Depression model was established by chronic social frustration stress and chronic unpredictable stress; depression-like behavior was detected by sugarwater preference test, tail suspension test and social contact test; serum corticosterone level was detected by Elisa; the expression of microRNAs in prefrontal cortex was quantitatively analyzed by real-time fluorescence quantitative PCR. Blotting was used to detect the expression of p- catenin (-catenin) in the prefrontal cortex.
Results: (1) Compared with the control group, the expression of microRNA-214-3p in the prefrontal cortex was up-regulated by 4.249 (+ 0.816) and that of microRNA-690 was up-regulated by 2.527 (+ 0.292) in the chronic social frustration stress model mice; (2) The sugar preference rate of the chronic social frustration model mice decreased to 40.577 (+ 4.81%) and the social contact time shortened to 60.85 (+ 13.17s); and (3) in the chronic society. The content of serum corticosterone in frustration stress model mice increased to 17.943
Conclusion: Chronic social frustration stress model mice can be used as an ideal model for depression study. The expression of microRNA214-3p is up-regulated and the expression of beta-catenin is down-regulated in prefrontal cortex.
The second part is about the target of miR-214-3p and miR-690.
Objective: to make clear that miR-214-3p and miR-690 take ctnnb1mRNA as the target.
Methods: Real-time fluorescence quantitative PCR was used to detect the expression of microRNAs-214-3p and microRNAs-690, Western Blotting was used to detect the expression of beta-catenin in prefrontal cortex, and double luciferase reporter system was used to detect the 3'-UTR binding sites of microRNAs-690 and CTNNB1 mRNA.
Results: (1) The expression of microRNA-690 in neurons transfected with mimics of microRNA-690 increased by 44.2 (+ 0.44) compared with the control; (2) The expression of beta-catenin protein in neurons transfected with mimics of microRNA-690 decreased to 60 (+ 12%); (3) Diluciferase reporter assay showed that microRNA-690 targeted the 3'UTR region of ctnnbl; (4) The third brain. Compared with the control group, the expression of Mi-214-3p and Mi-690 mimics increased to 2.465 (+ 0.032) and the expression of Mi-690 increased to 1.21 (+ 0.05) after intraventricular injection of Mi-214-3p mimics, the expression of beta-catenin protein decreased to 64.6 (+ 4.35) after intraventricular injection of Mi-214-3p mimics, while the expression of Mi-690 did not affect the expression of beta-catenin protein. (6) Compared with the control group, the 3'UTR region of ctnnbl silenced in vivo could effectively reduce the expression of mRNA to 0.37 [0.07] and the expression of P-catenin protein to 24 [3.5].
Conclusion: miR-214-3p regulates the expression of beta -catenin protein in CTNNB1 3'-UTR region.
The third part is about the effect and mechanism of regulating the expression of microRNA-214-3p in prefrontal cortex on depression-like behavior induced by chronic social frustration stress in mice
Objective: To study the effect and mechanism of prefrontal cortex miRNA-214-3p on depressive behavior in mice.
Methods: Sugar preference test, tail suspension test and social contact test were used to detect depression-like behavior changes in mice; real-time fluorescence quantitative PCR was used to analyze the expression of microRNA-214-3p in prefrontal cortex; Western Blotting was used to detect the expression of beta-catenin in prefrontal cortex; and miniature excitatory postsynaptic currents (miniature ex) in prefrontal cortex were recorded by brain patch clamp. Citatory postsynaptic current, mEPSC).
Results: (1) Compared with the control group, antagomir-214 significantly decreased the expression of microRNAs-214-3p to 1.046 [0.05] and increased the expression of beta-catenin protein to 91.3 [4.7%]; (2) antagomir-214 significantly decreased the tail-suspension immobility time to 27.954 [4.06s] and increased the social contact time to 64.283 [10.7s]; (3) antagomir-214 significantly increased the forehead skin of the model mice. Layered mEPSC amplitude; (4) Lentivirus-coated microRNA-214-3p inhibitor (LV-microRNA-214) significantly decreased the expression of microRNA-214-3p to 1.013 (+0.046) and increased the expression of beta-catenin protein to 227 (+59.8%); (5) LV-microRNA-214 decreased the tail immobility time to 45.7 (+12.08) and increased the social contact time to 53.6 (+10.051s); (6) Over The expression of CTNNB1 decreased the immobility time of mice tail suspension to 37.5 65 (9) Ant agomir-214, LV-miR-214 and over-expression of ctnnbl did not affect the sugar preference rate of the model mice.
Conclusion: inhibition of miR-214-3p expression can reverse the depressive behavior of mice with chronic social stress.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R749.4
[Abstract]:Part I Effects of chronic social frustration stress on the expression of microRNAs and P-catenin in prefrontal cortex of mice
Objective: To establish a chronic social frustration stress model in mice and detect the differential expression and protein expression of small RNA (microRNA, microRNA) in prefrontal cortex.
Methods: Depression model was established by chronic social frustration stress and chronic unpredictable stress; depression-like behavior was detected by sugarwater preference test, tail suspension test and social contact test; serum corticosterone level was detected by Elisa; the expression of microRNAs in prefrontal cortex was quantitatively analyzed by real-time fluorescence quantitative PCR. Blotting was used to detect the expression of p- catenin (-catenin) in the prefrontal cortex.
Results: (1) Compared with the control group, the expression of microRNA-214-3p in the prefrontal cortex was up-regulated by 4.249 (+ 0.816) and that of microRNA-690 was up-regulated by 2.527 (+ 0.292) in the chronic social frustration stress model mice; (2) The sugar preference rate of the chronic social frustration model mice decreased to 40.577 (+ 4.81%) and the social contact time shortened to 60.85 (+ 13.17s); and (3) in the chronic society. The content of serum corticosterone in frustration stress model mice increased to 17.943
Conclusion: Chronic social frustration stress model mice can be used as an ideal model for depression study. The expression of microRNA214-3p is up-regulated and the expression of beta-catenin is down-regulated in prefrontal cortex.
The second part is about the target of miR-214-3p and miR-690.
Objective: to make clear that miR-214-3p and miR-690 take ctnnb1mRNA as the target.
Methods: Real-time fluorescence quantitative PCR was used to detect the expression of microRNAs-214-3p and microRNAs-690, Western Blotting was used to detect the expression of beta-catenin in prefrontal cortex, and double luciferase reporter system was used to detect the 3'-UTR binding sites of microRNAs-690 and CTNNB1 mRNA.
Results: (1) The expression of microRNA-690 in neurons transfected with mimics of microRNA-690 increased by 44.2 (+ 0.44) compared with the control; (2) The expression of beta-catenin protein in neurons transfected with mimics of microRNA-690 decreased to 60 (+ 12%); (3) Diluciferase reporter assay showed that microRNA-690 targeted the 3'UTR region of ctnnbl; (4) The third brain. Compared with the control group, the expression of Mi-214-3p and Mi-690 mimics increased to 2.465 (+ 0.032) and the expression of Mi-690 increased to 1.21 (+ 0.05) after intraventricular injection of Mi-214-3p mimics, the expression of beta-catenin protein decreased to 64.6 (+ 4.35) after intraventricular injection of Mi-214-3p mimics, while the expression of Mi-690 did not affect the expression of beta-catenin protein. (6) Compared with the control group, the 3'UTR region of ctnnbl silenced in vivo could effectively reduce the expression of mRNA to 0.37 [0.07] and the expression of P-catenin protein to 24 [3.5].
Conclusion: miR-214-3p regulates the expression of beta -catenin protein in CTNNB1 3'-UTR region.
The third part is about the effect and mechanism of regulating the expression of microRNA-214-3p in prefrontal cortex on depression-like behavior induced by chronic social frustration stress in mice
Objective: To study the effect and mechanism of prefrontal cortex miRNA-214-3p on depressive behavior in mice.
Methods: Sugar preference test, tail suspension test and social contact test were used to detect depression-like behavior changes in mice; real-time fluorescence quantitative PCR was used to analyze the expression of microRNA-214-3p in prefrontal cortex; Western Blotting was used to detect the expression of beta-catenin in prefrontal cortex; and miniature excitatory postsynaptic currents (miniature ex) in prefrontal cortex were recorded by brain patch clamp. Citatory postsynaptic current, mEPSC).
Results: (1) Compared with the control group, antagomir-214 significantly decreased the expression of microRNAs-214-3p to 1.046 [0.05] and increased the expression of beta-catenin protein to 91.3 [4.7%]; (2) antagomir-214 significantly decreased the tail-suspension immobility time to 27.954 [4.06s] and increased the social contact time to 64.283 [10.7s]; (3) antagomir-214 significantly increased the forehead skin of the model mice. Layered mEPSC amplitude; (4) Lentivirus-coated microRNA-214-3p inhibitor (LV-microRNA-214) significantly decreased the expression of microRNA-214-3p to 1.013 (+0.046) and increased the expression of beta-catenin protein to 227 (+59.8%); (5) LV-microRNA-214 decreased the tail immobility time to 45.7 (+12.08) and increased the social contact time to 53.6 (+10.051s); (6) Over The expression of CTNNB1 decreased the immobility time of mice tail suspension to 37.5 65 (9) Ant agomir-214, LV-miR-214 and over-expression of ctnnbl did not affect the sugar preference rate of the model mice.
Conclusion: inhibition of miR-214-3p expression can reverse the depressive behavior of mice with chronic social stress.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R749.4
【共引文献】
相关期刊论文 前10条
1 王姬;王晓斌;;MicroRNA与抑郁症[J];国际精神病学杂志;2013年01期
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